Evidence for an Inactivating System of Nitrate Reductase in Hordeum vulgare L. during Darkness That Requires Protein Synthesis 1

PubMed Central

Travis, R. L.; Jordan, W. R.; Huffaker, R. C.

1969-01-01

The disappearance of nitrate reductase activity in leaves of Hordeum vulgare L. during darkness was inhibited by cycloheximide, actinomycin D, and low temperature. Thus, protein synthesis was probably required for the disappearance of nitrate reductase in the dark. Since chloramphenicol did not affect the rate of loss of activity, the degradation or inactivation apparently required protein synthesis by the cytoplasmic ribosomal system. Consistent with this observation, nitrate reductase is also reportedly located in the cytoplasm. Thus, the amount of nitrate reductase activity present in leaves of barley may be controlled by a balance between activating and inactivating systems. PMID:16657182

Resistance genes in barley (Hordeum vulgare L.) and their identification with molecular markers.

PubMed

Chełkowski, Jerzy; Tyrka, Mirosław; Sobkiewicz, Andrzej

2003-01-01

Current information on barley resistance genes available from scientific papers and on-line databases is summarised. The recent literature contains information on 107 major resistance genes (R genes) against fungal pathogens (excluding powdery mildew), pathogenic viruses and aphids identified in Hordeum vulgare accessions. The highest number of resistance genes was identified against Puccinia hordei, Rhynchosporium secalis, and the viruses BaYMV and BaMMV, with 17, 14 and 13 genes respectively. There is still a lot of confusion regarding symbols for R genes against powdery mildew. Among the 23 loci described to date, two regions Mla and Mlo comprise approximately 31 and 25 alleles. Over 50 R genes have already been localised and over 30 mapped on 7 barley chromosomes. Four barley R genes have been cloned recently: Mlo, Rpg1, Mla1 and Mla6, and their structures (sequences) are available. The paper presents a catalogue of barley resistance gene symbols, their chromosomalocation and the list of available DNA markers useful in characterising cultivars and breeding accessions.

Water uptake by seminal and adventitious roots in relation to whole-plant water flow in barley (Hordeum vulgare L.).

PubMed

Knipfer, Thorsten; Fricke, Wieland

2011-01-01

Prior to an assessment of the role of aquaporins in root water uptake, the main path of water movement in different types of root and driving forces during day and night need to be known. In the present study on hydroponically grown barley (Hordeum vulgare L.) the two main root types of 14- to 17-d-old plants were analysed for hydraulic conductivity in dependence of the main driving force (hydrostatic, osmotic). Seminal roots contributed 92% and adventitious roots 8% to plant water uptake. The lower contribution of adventitious compared with seminal roots was associated with a smaller surface area and number of roots per plant and a lower axial hydraulic conductance, and occurred despite a less-developed endodermis. The radial hydraulic conductivity of the two types of root was similar and depended little on the prevailing driving force, suggesting that water uptake occurred along a pathway that involved crossing of membrane(s). Exudation experiments showed that osmotic forces were sufficient to support night-time transpiration, yet transpiration experiments and cuticle permeance data questioned the significance of osmotic forces. During the day, 90% of water uptake was driven by a tension of about -0.15 MPa.

Water uptake by seminal and adventitious roots in relation to whole-plant water flow in barley (Hordeum vulgare L.)

PubMed Central

Knipfer, Thorsten; Fricke, Wieland

2011-01-01

Prior to an assessment of the role of aquaporins in root water uptake, the main path of water movement in different types of root and driving forces during day and night need to be known. In the present study on hydroponically grown barley (Hordeum vulgare L.) the two main root types of 14- to 17-d-old plants were analysed for hydraulic conductivity in dependence of the main driving force (hydrostatic, osmotic). Seminal roots contributed 92% and adventitious roots 8% to plant water uptake. The lower contribution of adventitious compared with seminal roots was associated with a smaller surface area and number of roots per plant and a lower axial hydraulic conductance, and occurred despite a less-developed endodermis. The radial hydraulic conductivity of the two types of root was similar and depended little on the prevailing driving force, suggesting that water uptake occurred along a pathway that involved crossing of membrane(s). Exudation experiments showed that osmotic forces were sufficient to support night-time transpiration, yet transpiration experiments and cuticle permeance data questioned the significance of osmotic forces. During the day, 90% of water uptake was driven by a tension of about –0.15 MPa. PMID:20974734

Cytogenetic effect of low dose gamma-radiation in Hordeum vulgare seedlings: non-linear dose-effect relationship.

PubMed

Geras'kin, Stanislav A; Oudalova, Alla A; Kim, Jin Kyu; Dikarev, Vladimir G; Dikareva, Nina S

2007-03-01

The induction of chromosome aberrations in Hordeum vulgare germinated seeds was studied after ionizing irradiation with doses in the range of 10-1,000 mGy. The relationship between the frequency of aberrant cells and the absorbed dose was found to be nonlinear. A dose-independent plateau in the dose range from about 50 to 500 mGy was observed, where the level of cytogenetic damage was significantly different from the spontaneous level. The comparison of the goodness of the experimental data fitting with mathematical models of different complexity, using the most common quantitative criteria, demonstrated the advantage of a piecewise linear model over linear and polynomial models in approximating the frequency of cytogenetical disturbances. The results of the study support the hypothesis of indirect mechanisms of mutagenesis induced by low doses. Fundamental and applied implications of these findings are discussed.

Genetic Transformation of Hordeum vulgare ssp. spontaneum for the Development of a Transposon-Based Insertional Mutagenesis System.

PubMed

Cardinal, Marie-Josée; Kaur, Rajvinder; Singh, Jaswinder

2016-10-01

Domestication and intensive selective breeding of plants has triggered erosion of genetic diversity of important stress-related alleles. Researchers highlight the potential of using wild accessions as a gene source for improvement of cereals such as barley, which has major economic and social importance worldwide. Previously, we have successfully introduced the maize Ac/Ds transposon system for gene identification in cultivated barley. The objective of current research was to investigate the response of Hordeum vulgare ssp. spontaneum wild barley accessions in tissue culture to standardize parameters for introduction of Ac/Ds transposons through genetic transformation. We investigated the response of ten wild barley genotypes for callus induction, regenerative green callus induction and regeneration of fertile plants. The activity of exogenous Ac/Ds elements was observed through a transient assay on immature wild barley embryos/callus whereby transformed embryos/calli were identified by the expression of GUS. Transient Ds expression bombardment experiments were performed on 352 pieces of callus (3-5 mm each) or immature embryos in 4 genotypes of wild barley. The transformation frequency of putative transgenic callus lines based on transient GUS expression ranged between 72 and100 % in wild barley genotypes. This is the first report of a transformation system in H. vulgare ssp. spontaneum.

Genome-Wide Association Mapping of Acid Soil Resistance in Barley (Hordeum vulgare L.)

PubMed Central

Zhou, Gaofeng; Broughton, Sue; Zhang, Xiao-Qi; Ma, Yanling; Zhou, Meixue; Li, Chengdao

2016-01-01

Genome-wide association studies (GWAS) based on linkage disequilibrium (LD) have been used to detect QTLs underlying complex traits in major crops. In this study, we collected 218 barley (Hordeum vulgare L.) lines including wild barley and cultivated barley from China, Canada, Australia, and Europe. A total of 408 polymorphic markers were used for population structure and LD analysis. GWAS for acid soil resistance were performed on the population using a general linkage model (GLM) and a mixed linkage model (MLM), respectively. A total of 22 QTLs (quantitative trait loci) were detected with the GLM and MLM analyses. Two QTLs, close to markers bPb-1959 (133.1 cM) and bPb-8013 (86.7 cM), localized on chromosome 1H and 4H respectively, were consistently detected in two different trials with both the GLM and MLM analyses. Furthermore, bPb-8013, the closest marker to the major Al3+ resistance gene HvAACT1 in barley, was identified to be QTL5. The QTLs could be used in marker-assisted selection to identify and pyramid different loci for improved acid soil resistance in barley. PMID:27064793

PM19, a barley (Hordeum vulgare L.) gene encoding a putative plasma membrane protein, is expressed during embryo development and dormancy.

PubMed

Ranford, Julia C; Bryce, James H; Morris, Peter C

2002-01-01

A barley (Hordeum vulgare L.) cDNA, PM19, encoding a putative plasma membrane protein was isolated through differential screening of a dormant wild oat embryo library. PM19 is expressed in barley embryos from mid-embryogenesis up to maturity. PM19 mRNA levels decline upon germination, whereas dormant embryos retained high levels of message for up to 72 h of imbibition. PM19 mRNA levels also remained high or were reinduced in non-dormant embryos by treatments that prevented germination (250 mm NaCl, 10% sorbitol, or 50 microm ABA). The PM19 protein sequence is highly conserved in monocotyledonous and dicotyledonous plants.

Farmers without borders—genetic structuring in century old barley (Hordeum vulgare)

PubMed Central

Forsberg, N E G; Russell, J; Macaulay, M; Leino, M W; Hagenblad, J

2015-01-01

The geographic distribution of genetic diversity can reveal the evolutionary history of a species. For crop plants, phylogeographic patterns also indicate how seed has been exchanged and spread in agrarian communities. Such patterns are, however, easily blurred by the intense seed trade, plant improvement and even genebank conservation during the twentieth century, and discerning fine-scale phylogeographic patterns is thus particularly challenging. Using historical crop specimens, these problems are circumvented and we show here how high-throughput genotyping of historical nineteenth century crop specimens can reveal detailed geographic population structure. Thirty-one historical and nine extant accessions of North European landrace barley (Hordeum vulgare L.), in total 231 individuals, were genotyped on a 384 single nucleotide polymorphism assay. The historical material shows constant high levels of within-accession diversity, whereas the extant accessions show more varying levels of diversity and a higher degree of total genotype sharing. Structure, discriminant analysis of principal components and principal component analysis cluster the accessions in latitudinal groups across country borders in Finland, Norway and Sweden. FST statistics indicate strong differentiation between accessions from southern Fennoscandia and accessions from central or northern Fennoscandia, and less differentiation between central and northern accessions. These findings are discussed in the context of contrasting historical records on intense within-country south to north seed movement. Our results suggest that although seeds were traded long distances, long-term cultivation has instead been of locally available, possibly better adapted, genotypes. PMID:25227257

Farmers without borders-genetic structuring in century old barley (Hordeum vulgare).

PubMed

Forsberg, N E G; Russell, J; Macaulay, M; Leino, M W; Hagenblad, J

2015-02-01

The geographic distribution of genetic diversity can reveal the evolutionary history of a species. For crop plants, phylogeographic patterns also indicate how seed has been exchanged and spread in agrarian communities. Such patterns are, however, easily blurred by the intense seed trade, plant improvement and even genebank conservation during the twentieth century, and discerning fine-scale phylogeographic patterns is thus particularly challenging. Using historical crop specimens, these problems are circumvented and we show here how high-throughput genotyping of historical nineteenth century crop specimens can reveal detailed geographic population structure. Thirty-one historical and nine extant accessions of North European landrace barley (Hordeum vulgare L.), in total 231 individuals, were genotyped on a 384 single nucleotide polymorphism assay. The historical material shows constant high levels of within-accession diversity, whereas the extant accessions show more varying levels of diversity and a higher degree of total genotype sharing. Structure, discriminant analysis of principal components and principal component analysis cluster the accessions in latitudinal groups across country borders in Finland, Norway and Sweden. FST statistics indicate strong differentiation between accessions from southern Fennoscandia and accessions from central or northern Fennoscandia, and less differentiation between central and northern accessions. These findings are discussed in the context of contrasting historical records on intense within-country south to north seed movement. Our results suggest that although seeds were traded long distances, long-term cultivation has instead been of locally available, possibly better adapted, genotypes.

Nitrate transport is independent of NADH and NAD(P)H nitrate reductases in barley seedlings

NASA Technical Reports Server (NTRS)

Warner, R. L.; Huffaker, R. C.

1989-01-01

Barley (Hordeum vulgare L.) has NADH-specific and NAD(P)H-bispecific nitrate reductase isozymes. Four isogenic lines with different nitrate reductase isozyme combinations were used to determine the role of NADH and NAD(P)H nitrate reductases on nitrate transport and assimilation in barley seedlings. Both nitrate reductase isozymes were induced by nitrate and were required for maximum nitrate assimilation in barley seedlings. Genotypes lacking the NADH isozyme (Az12) or the NAD(P)H isozyme (Az70) assimilated 65 or 85%, respectively, as much nitrate as the wild type. Nitrate assimilation by genotype (Az12;Az70) which is deficient in both nitrate reductases, was only 13% of the wild type indicating that the NADH and NAD(P)H nitrate reductase isozymes are responsible for most of the nitrate reduction in barley seedlings. For all genotypes, nitrate assimilation rates in the dark were about 55% of the rates in light. Hypotheses that nitrate reductase has direct or indirect roles in nitrate uptake were not supported by this study. Induction of nitrate transporters and the kinetics of net nitrate uptake were the same for all four genotypes indicating that neither nitrate reductase isozyme has a direct role in nitrate uptake in barley seedlings.

Nitrate Transport Is Independent of NADH and NAD(P)H Nitrate Reductases in Barley Seedlings 1

PubMed Central

Warner, Robert L.; Huffaker, Ray C.

1989-01-01

Barley (Hordeum vulgare L.) has NADH-specific and NAD(P)H-bispecific nitrate reductase isozymes. Four isogenic lines with different nitrate reductase isozyme combinations were used to determine the role of NADH and NAD(P)H nitrate reductases on nitrate transport and assimilation in barley seedlings. Both nitrate reductase isozymes were induced by nitrate and were required for maximum nitrate assimilation in barley seedlings. Genotypes lacking the NADH isozyme (Az12) or the NAD(P)H isozyme (Az70) assimilated 65 or 85%, respectively, as much nitrate as the wild type. Nitrate assimilation by genotype (Az12;Az70) which is deficient in both nitrate reductases, was only 13% of the wild type indicating that the NADH and NAD(P)H nitrate reductase isozymes are responsible for most of the nitrate reduction in barley seedlings. For all genotypes, nitrate assimilation rates in the dark were about 55% of the rates in light. Hypotheses that nitrate reductase has direct or indirect roles in nitrate uptake were not supported by this study. Induction of nitrate transporters and the kinetics of net nitrate uptake were the same for all four genotypes indicating that neither nitrate reductase isozyme has a direct role in nitrate uptake in barley seedlings. PMID:11537465

Variability in Proline-Accumulating Ability of Barley (Hordeum vulgare L.) Cultivars Induced by Vapor Pressure Deficit 1

PubMed Central

Naidu, Bodapati P.; Aspinall, Donald; Paleg, Leslie G.

1992-01-01

This work was undertaken in an effort to reconcile the conflicting proline-accumulating responses of the barley (Hordeum vulgare L.) cultivars, Excelsior and Proctor, reported by Singh et al. (1972) and Hanson et al. (1976). It deals with the effects of different vapor pressure deficits (VPD) during growth and subsequent drought stress on several barley cultivars. A higher VPD (1.2 kilopascals) during Clipper seedling growth resulted in higher solute-accumulating ability, seemingly independently of leaf water potential, than a lower VPD (0.12 kilopascals). The higher VPD during stress also resulted in higher solute contents, and this response may be more closely related to leaf water potential. When the responses of Excelsior and Proctor were examined in detail, it was found that the relative proline-accumulating ability of the two cultivars was dependent upon the VPD under which they were grown. At low VPD, Proctor accumulated significantly more proline than did Excelsior; whereas at higher VPD, Excelsior accumulated more proline than did Proctor. The crossover occurred at a VPD of about 0.72 kilopascals. This reversal of cultivar response was enhanced by multiplying seed under the two VPD extremes. Glycinebetaine accumulation did not demonstrate the crossover effect, although the concentration of this compound in all cultivars also depended on the VPD prevailing during growth and/or stress. Solute levels, in general, were more closely related to the decrease in relative water content than to a decrease in leaf water potential. It is concluded that the conflicting proline-accumulating responses of Excelsior and Proctor could be explained by these findings. PMID:16668700

Enhanced Pb Absorption by Hordeum vulgare L. and Helianthus annuus L. Plants Inoculated with an Arbuscular Mycorrhizal Fungi Consortium.

PubMed

Arias, Milton Senen Barcos; Peña-Cabriales, Juan José; Alarcón, Alejandro; Maldonado Vega, María

2015-01-01

The effect of an arbuscular mycorrhizal fungi (AMF) consortium conformed by (Glomus intraradices, Glomus albidum, Glomus diaphanum, and Glomus claroideum) on plant growth and absorption of Pb, Fe, Na, Ca, and (32)P in barley (Hordeum vulgare L.) and sunflower (Helianthus annuus L.) plants was evaluated. AMF-plants and controls were grown in a substrate amended with powdered Pb slag at proportions of 0, 10, 20, and 30% v/v equivalent to total Pb contents of 117; 5,337; 13,659, and 19,913 mg Pb kg(-1) substrate, respectively. Mycorrhizal root colonization values were 70, 94, 98, and 90%, for barley and 91, 97, 95, and 97%, for sunflower. AMF inoculum had positive repercussions on plant development of both crops. Mycorrhizal barley absorbed more Pb (40.4 mg Pb kg(-1)) shoot dry weight than non-colonized controls (26.5 mg Pb kg(-1)) when treated with a high Pb slag dosage. This increase was higher in roots than shoots (650.0 and 511.5 mg Pb kg(-1) root dry weight, respectively). A similar pattern was found in sunflower. Plants with AMF absorbed equal or lower amounts of Fe, Na and Ca than controls. H. vulgare absorbed more total P (1.0%) than H. annuus (0.9%). The arbuscular mycorrizal consortium enhanced Pb extraction by plants.

Characterization of the association of nitrate reductase with barley (Hordeum vulgare L.) root membranes

NASA Technical Reports Server (NTRS)

Meyerhoff, P. A.; Fox, T. C.; Travis, R. L.; Huffaker, R. C.

1994-01-01

The nature of the association between nitrate reductase (NR) and membranes was examined. Nitrate reductase activity (NRA) associated with the microsomal fraction of barley (Hordeum vulgare L.) roots amounted to 0.6 to 0.8% of soluble NRA following sonication in the presence of 250 mM KI and repeated osmotic shock. This treatment removed all contaminating soluble NRA from microsomes of uninduced barley roots that had been homogenized in a soluble extract from roots of NO3(-)-induced plants. On continuous sucrose gradients, NRA co-migrated specifically with VO4(-)-sensitive ATPase activity, a plasma membrane (PM) marker; activity of glucose-6-phosphate dehydrogenase, assayed as cytosolic marker, co-migrated with NRA. Microsomal NRA was absent in barley deficient in soluble NR. Perturbation and trypsinolysis experiments with PM vesicles isolated by aqueous two-phase partitioning indicated that NR is associated with the periphery of the cytoplasmic face of the bilayer. These results demonstrate that PM and soluble NRs are essentially the same protein but that the membrane-associated form is tightly bound. Although it is possible that PM-associated NR exists in vivo, unequivocal evidence for this has yet to be shown. However, PM NR is definitely present in vitro.

Localization and pattern of graviresponse across the pulvinus of barley Hordeum vulgare

NASA Technical Reports Server (NTRS)

Brock, T. G.; Lu, C. R.; Ghosheh, N. S.; Kaufman, P. B.

1989-01-01

Pulvini of excised stem segments from barley (Hordeum vulgare cv Larker') were pretreated with 1 millimolar coumarin before gravistimulation to reduce longitudinal cell expansion and exaggerate radial cell enlargement. The cellular localization and pattern of graviresponse across individual pulvini were then evaluated by cutting the organ in cross-section, photographing the cross-section, and then measuring pulvinus thickness and the radial width of cortical and epidermal cells in enlargements of the photomicrographs. With respect to orientation during gravistimulation, we designated the uppermost point of the cross-section 0 degrees and the lowermost point 180 degrees. A gravity-induced increase in pulvinus thickness was observable within 40 degrees of the vertical in coumarin-treated pulvini. In upper halves of coumarin-treated gravistimulated pulvini, cells in the inner cortex and inner epidermis had increased radial widths, relative to untreated gravistimulated pulvini. In lower halves of coumarin-treated pulvini, cells in the central and outer cortex and in the outer epidermis showed the greatest increase in radial width. Cells comprising the vascular bundles also increased in radial width, with this pattern following that of the central cortex. These results indicate (a) that all cell types are capable of showing a graviresponse, (b) that the graviresponse occurs in both the top and the bottom of the responding organ, and (c) that the magnitude of the response increases approximately linearly from the uppermost point to the lowermost. These results are also consistent with models of gravitropism that link the pattern and magnitude of the graviresponse to graviperception via statolith sedimentation.

Increased yield stability of field-grown winter barley (Hordeum vulgare L.) varietal mixtures through ecological processes

PubMed Central

Creissen, Henry E.; Jorgensen, Tove H.; Brown, James K.M.

2016-01-01

Crop variety mixtures have the potential to increase yield stability in highly variable and unpredictable environments, yet knowledge of the specific mechanisms underlying enhanced yield stability has been limited. Ecological processes in genetically diverse crops were investigated by conducting field trials with winter barley varieties (Hordeum vulgare), grown as monocultures or as three-way mixtures in fungicide treated and untreated plots at three sites. Mixtures achieved yields comparable to the best performing monocultures whilst enhancing yield stability despite being subject to multiple predicted and unpredicted abiotic and biotic stresses including brown rust (Puccinia hordei) and lodging. There was compensation through competitive release because the most competitive variety overyielded in mixtures thereby compensating for less competitive varieties. Facilitation was also identified as an important ecological process within mixtures by reducing lodging. This study indicates that crop varietal mixtures have the capacity to stabilise productivity even when environmental conditions and stresses are not predicted in advance. Varietal mixtures provide a means of increasing crop genetic diversity without the need for extensive breeding efforts. They may confer enhanced resilience to environmental stresses and thus be a desirable component of future cropping systems for sustainable arable farming. PMID:27375312

Chlorophyll Fluorescence as a Possible Tool for Salinity Tolerance Screening in Barley (Hordeum vulgare L.).

PubMed Central

Belkhodja, R.; Morales, F.; Abadia, A.; Gomez-Aparisi, J.; Abadia, J.

1994-01-01

The application of chlorophyll fluorescence measurements to screening barley (Hordeum vulgare L.) genotypes for salinity tolerance has been investigated. Excised barley leaves were cut under water and incubated with the cut end immersed in water or in a 100-mM NaCl solution, either in the dark or in high light. Changes in rapid fluorescence kinetics occurred in excised barley leaves exposed to the saline solution only when the incubation was carried out in the presence of high light. Fluorescence changes consisted of decreases in the variable to maximum fluorescence ratio and in increases in the relative proportion of variable fluorescence leading to point I in the Kautsky fluorescence induction curve. These relative increases in fluorescence at point I appeared to arise from a delayed plastoquinone reoxidation in the dark, since they disappeared after short, far-red illumination, which is known to excite photosystem I preferentially. We show that a significant correlation existed between some fluorescence parameters, measured after a combined salt and high-light treatment, and other independent measurements of salinity tolerance. These results suggest that chlorophyll fluorescence, and especially the relative fluorescence at point I in the Kautsky fluorescence induction curve, could be used for the screening of barley genotypes for salinity tolerance. PMID:12232117

High-throughput transcriptome analysis of barley (Hordeum vulgare) exposed to excessive boron.

PubMed

Tombuloglu, Guzin; Tombuloglu, Huseyin; Sakcali, M Serdal; Unver, Turgay

2015-02-15

Boron (B) is an essential micronutrient for optimum plant growth. However, above certain threshold B is toxic and causes yield loss in agricultural lands. While a number of studies were conducted to understand B tolerance mechanism, a transcriptome-wide approach for B tolerant barley is performed here for the first time. A high-throughput RNA-Seq (cDNA) sequencing technology (Illumina) was used with barley (Hordeum vulgare), yielding 208 million clean reads. In total, 256,874 unigenes were generated and assigned to known peptide databases: Gene Ontology (GO) (99,043), Swiss-Prot (38,266), Clusters of Orthologous Groups (COG) (26,250), and the Kyoto Encyclopedia of Genes and Genomes (KEGG) (36,860), as determined by BLASTx search. According to the digital gene expression (DGE) analyses, 16% and 17% of the transcripts were found to be differentially regulated in root and leaf tissues, respectively. Most of them were involved in cell wall, stress response, membrane, protein kinase and transporter mechanisms. Some of the genes detected as highly expressed in root tissue are phospholipases, predicted divalent heavy-metal cation transporters, formin-like proteins and calmodulin/Ca(2+)-binding proteins. In addition, chitin-binding lectin precursor, ubiquitin carboxyl-terminal hydrolase, and serine/threonine-protein kinase AFC2 genes were indicated to be highly regulated in leaf tissue upon excess B treatment. Some pathways, such as the Ca(2+)-calmodulin system, are activated in response to B toxicity. The differential regulation of 10 transcripts was confirmed by qRT-PCR, revealing the tissue-specific responses against B toxicity and their putative function in B-tolerance mechanisms. Copyright © 2014. Published by Elsevier B.V.

Evolutionary History of Wild Barley (Hordeum vulgare subsp. spontaneum) Analyzed Using Multilocus Sequence Data and Paleodistribution Modeling

PubMed Central

Jakob, Sabine S.; Rödder, Dennis; Engler, Jan O.; Shaaf, Salar; Özkan, Hakan; Blattner, Frank R.; Kilian, Benjamin

2014-01-01

Studies of Hordeum vulgare subsp. spontaneum, the wild progenitor of cultivated barley, have mostly relied on materials collected decades ago and maintained since then ex situ in germplasm repositories. We analyzed spatial genetic variation in wild barley populations collected rather recently, exploring sequence variations at seven single-copy nuclear loci, and inferred the relationships among these populations and toward the genepool of the crop. The wild barley collection covers the whole natural distribution area from the Mediterranean to Middle Asia. In contrast to earlier studies, Bayesian assignment analyses revealed three population clusters, in the Levant, Turkey, and east of Turkey, respectively. Genetic diversity was exceptionally high in the Levant, while eastern populations were depleted of private alleles. Species distribution modeling based on climate parameters and extant occurrence points of the taxon inferred suitable habitat conditions during the ice-age, particularly in the Levant and Turkey. Together with the ecologically wide range of habitats, they might contribute to structured but long-term stable populations in this region and their high genetic diversity. For recently collected individuals, Bayesian assignment to geographic clusters was generally unambiguous, but materials from genebanks often showed accessions that were not placed according to their assumed geographic origin or showed traces of introgression from cultivated barley. We assign this to gene flow among accessions during ex situ maintenance. Evolutionary studies based on such materials might therefore result in wrong conclusions regarding the history of the species or the origin and mode of domestication of the crop, depending on the accessions included. PMID:24586028

Differential heat sensitivity index in barley cultivars (Hordeum vulgare L.) monitored by chlorophyll a fluorescence OKJIP.

PubMed

Oukarroum, Abdallah; El Madidi, Saïd; Strasser, Reto J

2016-08-01

The objective of this study was to differentiate the heat tolerance in ten varieties of barley (Hordeum vulgare L.) originating from Morocco. Five modern varieties and five landraces (local varieties) collected at five different geographical localities in the south of Morocco were investigated in the present study. After two weeks of growth, detached leaves were short term exposure to various temperatures (25, 30, 35, 40, and 45 °C) for 10 min in the dark. Two chlorophyll a fluorescence parameters derived from chlorophyll a fluorescence transient (OKJIP) (performance index (PIABS) and relative variable fluorescence at the K-step (VK)) were analysed. Heat treatment had a significant effect on the PIABS and VK at 45 °C treatment and the analysis of variance for PIABS and VK is highly significant between all varieties. The slope of the relationship between logPIABS and VK named heat sensitivity index (HSI) was used to evaluate the thermotolerance of photosystem II (PSII) between the studied barley varieties. According to this approach, barley varieties were screened and ranked for improving heat tolerance. HSI was found to be a new indicator with regard to distinguishing heat tolerance of different barley cultivars. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Response of Microcystis aeruginosa BCCUSP 232 to barley (Hordeum vulgare L.) straw degradation extract and fractions.

PubMed

Mecina, Gustavo Franciscatti; Dokkedal, Anne Lígia; Saldanha, Luiz Leonardo; Chia, Mathias Ahii; Cordeiro-Araújo, Micheline Kézia; do Carmo Bittencourt-Oliveira, Maria; da Silva, Regildo Márcio Gonçalves

2017-12-01

The eutrophication of aquatic ecosystems is a serious environmental problem that leads to increased frequency of cyanobacterial blooms and concentrations of cyanotoxins. These changes in aquatic chemistry can negatively affect animal and human health. Environment-friendly methods are needed to control bloom forming cyanobacteria. We investigated the effect of Hordeum vulgare L. (barley) straw degradation extract and its fractions on the growth, oxidative stress, antioxidant enzyme activities, and microcystins content of Microcystis aeruginosa (Kützing) Kützing BCCUSP232. Exposure to the extract significantly (p<0.05) inhibited the growth of M. aeruginosa throughout the study, whereas only the highest concentration of fractions 1 and 2 significantly (p<0.05) reduced the growth of the cyanobacterium on day 10 of the experiment. The production of reactive oxygen species (ROS), lipid peroxidation and antioxidant enzyme activities were significantly (p<0.05) altered by the extract and fractions 1 and 2. Phytochemical profiling of the extract and its fractions revealed that the barley straw degradation process yielded predominantly phenolic acids. These results demonstrate that barley straw extract and its fractions can efficiently interfere with the growth and development of M. aeruginosa under laboratory conditions. Copyright © 2017 Elsevier B.V. All rights reserved.

Direct Effects of Physcion, Chrysophanol, Emodin, and Pachybasin on Germination and Appressorium Formation of the Barley ( Hordeum vulgare L.) Powdery Mildew Fungus Blumeria graminis f. sp. hordei (DC.) Speer.

PubMed

Hildebrandt, Ulrich; Marsell, Alexander; Riederer, Markus

2018-04-04

Several anthraquinone derivatives are active components of fungicidal formulations particularly effective against powdery mildew fungi. The antimildew effect of compounds such as physcion and chrysophanol is largely attributed to host plant defense induction. However, so far a direct fungistatic/fungicidal effect of anthraquinone derivatives on powdery mildew fungi has not been unequivocally demonstrated. By applying a Formvar-based in vitro system we demonstrate a direct, dose-dependent effect of physcion, chrysophanol, emodin, and pachybasin on conidial germination and appressorium formation of Blumeria graminis f. sp. hordei (DC.) Speer, the causative agent of barley ( Hordeum vulgare L.) powdery mildew. Physcion was the most effective among the tested compounds. At higher doses, physcion mainly inhibited conidial germination. At lower rates, however, a distinct interference with appressorium formation became discernible. Physcion and others may act by modulating both the infection capacity of the powdery mildew pathogen and host plant defense. Our results suggest a specific arrangement of substituents at the anthraquinone backbone structure being crucial for the direct antimildew effect.

The heterotoxicity of Hordeum vulgare L. extracts in four growth stages on germination and seedlings growth of Avena ludoviciana.

PubMed

Kolahi, M; Peivastegan, B; Hadizade, I; Abdali, A

2008-07-15

Phytotoxicity of barley extracts (Hordeum vulgare L.) on wild oat (Avena ludoviciana Durieu) was investigated. Water extracts five varieties of barley were bioassayed on germination and seedling growth of wild-oat to test the heterotoxicity of barley on wild-oat, study the dynamics of allelopathic potential over four growth stages and identify the most allelopathic plant part of barley in each stage. Whole barley plants were extracted at growth stage 4 (stems not developed enough), whilst for the following growth stages roots, stems, panicles and leaves were extracted separately. Seedling growth bioassays demonstrated that the wild-oat responded differently to the allelopathic potential of barley. For wild-oat radical growth and coleoptile growth were more depressed than germination, though. The allelopathic potential of barley plant parts was not stable over its life cycle for wild-oat. Leaves and stems were the most phytotoxic barley plant parts for wild-oat in the all stages. Among the varieties Eizeh appeared as the best one showing toxicity to seed germination of wild oat at its stage 4 and 8. Results suggested that the response by wild-oat varied depending on the source of allelochemicals (plant part) and the growth stage of the barley plant and kind of variety. The results leaded to conclude that Eizeh variety of barley was good to grow as it has good check on seed germination of wild oat plants as well as it also retarded the growth of root and shoot length of oat.

Abiotic stresses modulate expression of major intrinsic proteins in barley (Hordeum vulgare).

PubMed

Ligaba, Ayalew; Katsuhara, Maki; Shibasaka, Mineo; Djira, Gemechis

2011-02-01

In one of the most important crops, barley (Hordeum vulgare L.), gene expression and physiological roles of most major intrinsic proteins (MIPs) remained to be elucidated. Here we studied expression of five tonoplast intrinsic protein isoforms (HvTIP1;2, HvTIP2;1, HvTIP2;2, HvTIP2;3 and HvTIP4;1), a NOD26-like intrinsic protein (HvNIP2;1) and a plasma membrane intrinsic protein (HvPIP2;1) by using the quantitative real-time RT-PCR. Five-day-old seedlings were exposed to abiotic stresses (salt, heavy metals and nutrient deficiency), abscisic acid (ABA) and gibberellic acid (GA) for 24 h. Treatment with 100 mM NaCl, 0.1 mM ABA and 1 mM GA differentially regulated gene expression in roots and shoots. Nitrogen and prolonged P-deficiency downregulated expression of most MIP genes in roots. Intriguingly, gene expression was restored to the values in the control three days after nutrient supply was resumed. Heavy metals (0.2 mM each of Cd, Cu, Zn and Cr) downregulated the transcript levels by 60-80% in roots, whereas 0.2 mM Hg upregulated expressions of most genes in roots. This was accompanied by a 45% decrease in the rate of transpiration. In order to study the physiological role of the MIPs, cDNA of three genes (HvTIP2;1, HvTIP2;3 and HvNIP2;1) have been cloned and heterologous expression was performed in Xenopus laevis oocytes. Osmotic water permeability was determined by a swelling assay. However, no water uptake activity was observed for the three proteins. Hence, the possible physiological role of the proteins is discussed. Copyright © 2010 Académie des sciences. Published by Elsevier SAS. All rights reserved.

Uptake, degradation and chiral discrimination of N-acyl-D/L-homoserine lactones by barley (Hordeum vulgare) and yam bean (Pachyrhizus erosus) plants.

PubMed

Götz, Christine; Fekete, Agnes; Gebefuegi, Istvan; Forczek, Sándor T; Fuksová, Kvetoslava; Li, Xiaojing; Englmann, Matthias; Gryndler, Milan; Hartmann, Anton; Matucha, Miroslav; Schmitt-Kopplin, Philippe; Schröder, Peter

2007-11-01

Bacterial intraspecies and interspecies communication in the rhizosphere is mediated by diffusible signal molecules. Many Gram-negative bacteria use N-acyl-homoserine lactones (AHLs) as autoinducers in the quorum sensing response. While bacterial signalling is well described, the fate of AHLs in contact with plants is much less known. Thus, adsorption, uptake and translocation of N-hexanoyl- (C6-HSL), N-octanoyl- (C8-HSL) and N-decanoyl-homoserine lactone (C10-HSL) were studied in axenic systems with barley (Hordeum vulgare L.) and the legume yam bean (Pachyrhizus erosus (L.) Urban) as model plants using ultra-performance liquid chromatography (UPLC), Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) and tritium-labelled AHLs. Decreases in AHL concentration due to abiotic adsorption or degradation were tolerable under the experimental conditions. The presence of plants enhanced AHL decline in media depending on the compounds' lipophilicity, whereby the legume caused stronger AHL decrease than barley. All tested AHLs were traceable in root extracts of both plants. While all AHLs except C10-HSL were detectable in barley shoots, only C6-HSL was found in shoots of yam bean. Furthermore, tritium-labelled AHLs were used to determine short-term uptake kinetics. Chiral separation by GC-MS revealed that both plants discriminated D-AHL stereoisomers to different extents. These results indicate substantial differences in uptake and degradation of different AHLs in the plants tested.

Genome-Wide Association Mapping of Stem Rust Resistance in Hordeum vulgare subsp. spontaneum

PubMed Central

Sallam, Ahmad H.; Tyagi, Priyanka; Brown-Guedira, Gina; Muehlbauer, Gary J.; Hulse, Alex; Steffenson, Brian J.

2017-01-01

Stem rust was one of the most devastating diseases of barley in North America. Through the deployment of cultivars with the resistance gene Rpg1, losses to stem rust have been minimal over the past 70 yr. However, there exist both domestic (QCCJB) and foreign (TTKSK aka isolate Ug99) pathotypes with virulence for this important gene. To identify new sources of stem rust resistance for barley, we evaluated the Wild Barley Diversity Collection (WBDC) (314 ecogeographically diverse accessions of Hordeum vulgare subsp. spontaneum) for seedling resistance to four pathotypes (TTKSK, QCCJB, MCCFC, and HKHJC) of the wheat stem rust pathogen (Puccinia graminis f. sp. tritici, Pgt) and one isolate (92-MN-90) of the rye stem rust pathogen (P. graminis f. sp. secalis, Pgs). Based on a coefficient of infection, the frequency of resistance in the WBDC was low ranging from 0.6% with HKHJC to 19.4% with 92-MN-90. None of the accessions was resistant to all five cultures of P. graminis. A genome-wide association study (GWAS) was conducted to map stem rust resistance loci using 50,842 single-nucleotide polymorphic markers generated by genotype-by-sequencing and ordered using the new barley reference genome assembly. After proper accounting for genetic relatedness and structure among accessions, 45 quantitative trait loci were identified for resistance to P. graminis across all seven barley chromosomes. Three novel loci associated with resistance to TTKSK, QCCJB, MCCFC, and 92-MN-90 were identified on chromosomes 5H and 7H, and two novel loci associated with resistance to HKHJC were identified on chromosomes 1H and 3H. These novel alleles will enhance the diversity of resistance available for cultivated barley. PMID:28855281

Genome-Wide Association Mapping of Stem Rust Resistance in Hordeum vulgare subsp. spontaneum.

PubMed

Sallam, Ahmad H; Tyagi, Priyanka; Brown-Guedira, Gina; Muehlbauer, Gary J; Hulse, Alex; Steffenson, Brian J

2017-10-05

Stem rust was one of the most devastating diseases of barley in North America. Through the deployment of cultivars with the resistance gene Rpg1 , losses to stem rust have been minimal over the past 70 yr. However, there exist both domestic (QCCJB) and foreign (TTKSK aka isolate Ug99) pathotypes with virulence for this important gene. To identify new sources of stem rust resistance for barley, we evaluated the Wild Barley Diversity Collection (WBDC) (314 ecogeographically diverse accessions of Hordeum vulgare subsp. spontaneum ) for seedling resistance to four pathotypes (TTKSK, QCCJB, MCCFC, and HKHJC) of the wheat stem rust pathogen ( Puccinia graminis f. sp. tritici , Pgt ) and one isolate (92-MN-90) of the rye stem rust pathogen ( P. graminis f. sp. secalis , Pgs ). Based on a coefficient of infection, the frequency of resistance in the WBDC was low ranging from 0.6% with HKHJC to 19.4% with 92-MN-90. None of the accessions was resistant to all five cultures of P. graminis A genome-wide association study (GWAS) was conducted to map stem rust resistance loci using 50,842 single-nucleotide polymorphic markers generated by genotype-by-sequencing and ordered using the new barley reference genome assembly. After proper accounting for genetic relatedness and structure among accessions, 45 quantitative trait loci were identified for resistance to P. graminis across all seven barley chromosomes. Three novel loci associated with resistance to TTKSK, QCCJB, MCCFC, and 92-MN-90 were identified on chromosomes 5H and 7H, and two novel loci associated with resistance to HKHJC were identified on chromosomes 1H and 3H. These novel alleles will enhance the diversity of resistance available for cultivated barley. Copyright © 2017 Sallam et al.

Detection of QTLs for salt tolerance in Asian barley (Hordeum vulgare L.) by association analysis with SNP markers

PubMed Central

Sbei, Hanen; Sato, Kazuhiro; Shehzad, Tariq; Harrabi, Moncef; Okuno, Kazutoshi

2014-01-01

Two hundred ninety-six Asian barley (Hordeum vulgare L.) accessions were assessed to detect QTLs underlying salt tolerance by association analysis using a 384 single nucleotide polymorphism (SNP) marker system. The experiment was laid out at the seedling stage in a hydroponic solution under control and 250 mM NaCl solution with three replications of four plants each. Salt tolerance was assessed by leaf injury score (LIS) and salt tolerance indices (STIs) of the number of leaves (NL), shoot length (SL), root length (RL), shoot dry weight (SDW) and root dry weight (RDW). LIS was scored from 1 to 5 according to the severity of necrosis and chlorosis observed on leaves. There was a wide variation in salt tolerance among Asian barley accessions. LIS and STI (SDW) were the most suitable traits for screening salt tolerance. Association was estimated between markers and traits to detect QTLs for LIS and STI (SDW). Seven significant QTLs were located on chromosomes 1H (2 QTLs), 2H (2 QTLs), 3H (1 QTL), 4H (1 QTL) and 5H (1 QTL). Five QTLs were associated with LIS and 2 QTLs with STI (SDW). Two QTLs associated with LIS were newly identified on chromosomes 3H and 4H. PMID:25914593

Metabolism of hydroxypyruvate in a mutant of barley lacking NADH-dependent hydroxypyruvate reductase, an important photorespiratory enzyme activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Murray, A.J.S.; Blackwell, R.D.; Lea, P.J.

1989-09-01

A mutant of barley (Hordeum vulgare L.), LaPr 88/29, deficient in NADH-dependent hydroxypyruvate reductase (HPR) activity has been isolated. The activities of both NADH (5%) and NADPH-dependent (19%) HPR were severely reduced in this mutant compared to the wild type. Although lacking an enzyme in the main carbon pathway of photorespiration, this mutant was capable of CO{sub 2} fixation rates equivalent to 75% of that of the wild type, in normal atmospheres and 50% O{sub 2}. There also appeared to be little disruption to the photorespiratory metabolism as ammonia release, CO{sub 2} efflux and {sup 14}CO{sub 2} release from L-(U-{supmore » 14}C)serine feeding were similar in both mutant and wild-type leaves. When leaves of LaPr 88/29 were fed either ({sup 14}C)serine or {sup 14}CO{sub 2}, the accumulation of radioactivity was in serine and not in hydroxypyruvate, although the mutant was still able to metabolize over 25% of the supplied ({sup 14}C)serine into sucrose. After 3 hours in air the soluble amino acid pool was almost totally dominated by serine and glycine. LaPr 88/29 has also been used to show that NADH-glyoxylate reductase and NADH-HPR are probably not catalyzed by the same enzyme in barley and that over 80% of the NADPH-dependent HPR activity is due to the NADH-dependent enzyme. We also suggest that the alternative NADPH activity can metabolize a proportion, but not all, of the hydroxypyruvate produced during photorespiration and may thus form a useful backup to the NADH-dependent enzyme under conditions of maximal photorespiration.« less

Response of the rhizosphere prokaryotic community of barley (Hordeum vulgare L.) to elevated atmospheric CO2 concentration in open-top chambers.

PubMed

Szoboszlay, Márton; Näther, Astrid; Mitterbauer, Esther; Bender, Jürgen; Weigel, Hans-Joachim; Tebbe, Christoph C

2017-08-01

The effect of elevated atmospheric CO 2 concentration [CO 2 ] on the diversity and composition of the prokaryotic community inhabiting the rhizosphere of winter barley (Hordeum vulgare L.) was investigated in a field experiment, using open-top chambers. Rhizosphere samples were collected at anthesis (flowering stage) from six chambers with ambient [CO 2 ] (approximately 400 ppm) and six chambers with elevated [CO 2 ] (700 ppm). The V4 region of the 16S rRNA gene was PCR-amplified from the extracted DNA and sequenced on an Illumina MiSeq instrument. Above-ground plant biomass was not affected by elevated [CO 2 ] at anthesis, but plants exposed to elevated [CO 2 ] had significantly higher grain yield. The composition of the rhizosphere prokaryotic communities was very similar under ambient and elevated [CO 2 ]. The dominant taxa were Bacteroidetes, Actinobacteria, Alpha-, Gamma-, and Betaproteobacteria. Elevated [CO 2 ] resulted in lower prokaryotic diversity in the rhizosphere, but did not cause a significant difference in community structure. © 2017 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

Limitation of Cell Elongation in Barley (Hordeum vulgare L.) Leaves Through Mechanical and Tissue-Hydraulic Properties.

PubMed

Touati, Mostefa; Knipfer, Thorsten; Visnovitz, Tamás; Kameli, Abdelkrim; Fricke, Wieland

2015-07-01

The aim of the present study was to assess the mechanical and hydraulic limitation of growth in leaf epidermal cells of barley (Hordeum vulgare L.) in response to agents which affect cellular water (mercuric chloride, HgCl(2)) and potassium (cesium chloride, CsCl; tetraethylammonium, TEA) transport, pump activity of plasma membrane H(+)-ATPase and wall acidification (fusicoccin, FC). Cell turgor (P) was measured with the cell pressure probe, and cell osmotic pressure (π) was analyzed through picoliter osmometry of single-cell extracts. A wall extensibility coefficient (M) and tissue hydraulic conductance coefficient (L) were derived using the Lockhart equation. There was a significant positive linear relationship between relative elemental growth rate and P, which fit all treatments, with an overall apparent yield threshold of 0.368 MPa. Differences in growth between treatments could be explained through differences in P. A comparison of L and M showed that growth in all except the FC treatment was co-limited through hydraulic and mechanical properties, though to various extents. This was accompanied by significant (0.17-0.24 MPa) differences in water potential (ΔΨ) between xylem and epidermal cells in the leaf elongation zone. In contrast, FC-treated leaves showed ΔΨ close to zero and a 10-fold increase in L. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Identification of two key genes controlling chill haze stability of beer in barley (Hordeum vulgare L).

PubMed

Ye, Lingzhen; Huang, Yuqing; Dai, Fei; Ning, Huajiang; Li, Chengdao; Zhou, Meixue; Zhang, Guoping

2015-06-11

In bright beer, haze formation is a serious quality problem, degrading beer quality and reducing its shelf life. The quality of barley (Hordeum vulgare L) malt, as the main raw material for beer brewing, largely affects the colloidal stability of beer. In this study, the genetic mechanism of the factors affecting beer haze stability in barley was studied. Quantitative trait loci (QTL) analysis of alcohol chill haze (ACH) in beer was carried out using a Franklin/Yerong double haploid (DH) population. One QTL, named as qACH, was detected for ACH, and it was located on the position of about 108 cM in chromosome 4H and can explain about 20 % of the phenotypic variation. Two key haze active proteins, BATI-CMb and BATI-CMd were identified by proteomics analysis. Bioinformatics analysis showed that BATI-CMb and BATI-CMd had the same position as qACH in the chromosome. It may be deduced that BATI-CMb and BATI-CMd are candidate genes for qACH, controlling colloidal stability of beer. Polymorphism comparison between Yerong and Franklin in the nucleotide and amino acid sequence of BATI-CMb and BATI-CMd detected the corresponding gene specific markers, which could be used in marker-assisted selection for malt barley breeding. We identified a novel QTL, qACH controlling chill haze of beer, and two key haze active proteins, BATI-CMb and BATI-CMd. And further analysis showed that BATI-CMb and BATI-CMd might be the candidate genes associated with beer chill haze.

Purification and characterization of soluble (cytosolic) and bound (cell wall) isoforms of invertases in barley (Hordeum vulgare) elongating stem tissue

NASA Technical Reports Server (NTRS)

Karuppiah, N.; Vadlamudi, B.; Kaufman, P. B.

1989-01-01

Three different isoforms of invertases have been detected in the developing internodes of barley (Hordeum vulgare). Based on substrate specificities, the isoforms have been identified to be invertases (beta-fructosidases EC 3.2.1.26). The soluble (cytosolic) invertase isoform can be purified to apparent homogeneity by diethylaminoethyl cellulose, Concanavalin-A Sepharose, organo-mercurial Sepharose, and Sephacryl S-300 chromatography. A bound (cell wall) invertase isoform can be released by 1 molar salt and purified further by the same procedures as above except omitting the organo-mercurial Sepharose affinity chromatography step. A third isoform of invertase, which is apparently tightly associated with the cell wall, cannot be isolated yet. The soluble and bound invertase isoforms were purified by factors of 60- and 7-fold, respectively. The native enzymes have an apparent molecular weight of 120 kilodaltons as estimated by gel filtration. They have been identified to be dimers under denaturing and nondenaturing conditions. The soluble enzyme has a pH optimum of 5.5, Km of 12 millimolar, and a Vmax of 80 micromole per minute per milligram of protein compared with cell wall isozyme which has a pH optimum of 4.5, Km of millimolar, and a Vmax of 9 micromole per minute per milligram of protein.

Long-term agricultural fertilization alters arbuscular mycorrhizal fungal community composition and barley (Hordeum vulgare) mycorrhizal carbon and phosphorus exchange.

PubMed

Williams, Alwyn; Manoharan, Lokeshwaran; Rosenstock, Nicholas P; Olsson, Pål Axel; Hedlund, Katarina

2017-01-01

Agricultural fertilization significantly affects arbuscular mycorrhizal fungal (AMF) community composition. However, the functional implications of community shifts are unknown, limiting understanding of the role of AMF in agriculture. We assessed AMF community composition at four sites managed under the same nitrogen (N) and phosphorus (P) fertilizer regimes for 55 yr. We also established a glasshouse experiment with the same soils to investigate AMF-barley (Hordeum vulgare) nutrient exchange, using carbon ( 13 C) and 33 P isotopic labelling. N fertilization affected AMF community composition, reducing diversity; P had no effect. In the glasshouse, AMF contribution to plant P declined with P fertilization, but was unaffected by N. Barley C allocation to AMF also declined with P fertilization. As N fertilization increased, C allocation to AMF per unit of P exchanged increased. This occurred with and without P fertilization, and was concomitant with reduced barley biomass. AMF community composition showed no relationship with glasshouse experiment results. The results indicate that plants can reduce C allocation to AMF in response to P fertilization. Under N fertilization, plants allocate an increasing amount of C to AMF and receive relatively less P. This suggests an alteration in the terms of P-C exchange under N fertilization regardless of soil P status. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

Effects of Substituted Pyridazinones (San 6706, San 9774, San 9785) on Glycerolipids and Their Associated Fatty Acids in the Leaves of Vicia faba and Hordeum vulgare1

PubMed Central

Khan, Mobashsher-Uddin; Lem, Nora W.; Chandorkar, Kashinath R.; Williams, John P.

1979-01-01

The fatty acids of the major glycerolipids from the leaves of Vicia faba and Hordeum vulgare plants treated with three different concentrations of pyridazinone derivatives were analyzed. These compounds showed multiple effects on the levels of lipids and pigments. At low concentrations, the primary effect of San 9785 was on the level of linolenic acid (18:3) in the galactolipids of V. faba, whereas the effect of San 6706 was primarily on the trans-Δ3-hexadecenoic acid (16:1) content in phosphatidylglycerol. At higher concentrations, the two compounds reduced the content of both fatty acids in the leaves. The results appear to indicate a differential effect of these herbicides on fatty acid accumulation and a difference in susceptibility of two fatty acids in the species examined. Electron microscopic studies revealed that two herbicides caused different abnormalities in V. faba chloroplast ultrastructure. Images PMID:16660953

Interactions and Toxicity of Cu-Zn mixtures to Hordeum vulgare in Different Soils Can Be Rationalized with Bioavailability-Based Prediction Models.

PubMed

Qiu, Hao; Versieren, Liske; Rangel, Georgina Guzman; Smolders, Erik

2016-01-19

Soil contamination with copper (Cu) is often associated with zinc (Zn), and the biological response to such mixed contamination is complex. Here, we investigated Cu and Zn mixture toxicity to Hordeum vulgare in three different soils, the premise being that the observed interactions are mainly due to effects on bioavailability. The toxic effect of Cu and Zn mixtures on seedling root elongation was more than additive (i.e., synergism) in soils with high and medium cation-exchange capacity (CEC) but less than additive (antagonism) in a low-CEC soil. This was found when we expressed the dose as the conventional total soil concentration. In contrast, antagonism was found in all soils when we expressed the dose as free-ion activities in soil solution, indicating that there is metal-ion competition for binding to the plant roots. Neither a concentration addition nor an independent action model explained mixture effects, irrespective of the dose expressions. In contrast, a multimetal BLM model and a WHAM-Ftox model successfully explained the mixture effects across all soils and showed that bioavailability factors mainly explain the interactions in soils. The WHAM-Ftox model is a promising tool for the risk assessment of mixed-metal contamination in soils.

Dehydration induced transcriptomic responses in two Tibetan hulless barley (Hordeum vulgare var. nudum) accessions distinguished by drought tolerance.

PubMed

Liang, Junjun; Chen, Xin; Deng, Guangbing; Pan, Zhifen; Zhang, Haili; Li, Qiao; Yang, Kaijun; Long, Hai; Yu, Maoqun

2017-10-11

The harsh environment on the Qinghai-Tibetan Plateau gives Tibetan hulless barley (Hordeum vulgare var. nudum) great ability to resist adversities such as drought, salinity, and low temperature, and makes it a good subject for the analysis of drought tolerance mechanism. To elucidate the specific gene networks and pathways that contribute to its drought tolerance, and for identifying new candidate genes for breeding purposes, we performed a transcriptomic analysis using two accessions of Tibetan hulless barley, namely Z772 (drought-tolerant) and Z013 (drought-sensitive). There were more up-regulated genes of Z772 than Z013 under both mild (5439-VS-2604) and severe (7203-VS-3359) dehydration treatments. Under mild dehydration stress, the pathways exclusively enriched in drought-tolerance genotype Z772 included Protein processing in endoplasmic reticulum, tricarboxylic acid (TCA) cycle, Wax biosynthesis, and Spliceosome. Under severe dehydration stress, the pathways that were mainly enriched in Z772 included Carbon fixation in photosynthetic organisms, Pyruvate metabolism, Porphyrin and chlorophyll metabolism. The main differentially expressed genes (DEGs) in response to dehydration stress and genes whose expression was different between tolerant and sensitive genotypes were presented in this study, respectively. The candidate genes for drought tolerance were selected based on their expression patterns. The RNA-Seq data obtained in this study provided an initial overview on global gene expression patterns and networks that related to dehydration shock in Tibetan hulless barley. Furthermore, these data provided pathways and a targeted set of candidate genes that might be essential for deep analyzing the molecular mechanisms of plant tolerance to drought stress.

Adaptation and diversity along an altitudinal gradient in Ethiopian barley (Hordeum vulgare L.) landraces revealed by molecular analysis

PubMed Central

2010-01-01

Background Among the cereal crops, barley is the species with the greatest adaptability to a wide range of environments. To determine the level and structure of genetic diversity in barley (Hordeum vulgare L.) landraces from the central highlands of Ethiopia, we have examined the molecular variation at seven nuclear microsatellite loci. Results A total of 106 landrace populations were sampled in the two growing seasons (Meher and Belg; the long and short rainy seasons, respectively), across three districts (Ankober, Mojanawadera and Tarmaber), and within each district along an altitudinal gradient (from 1,798 to 3,324 m a.s.l). Overall, although significant, the divergence (e.g. FST) is very low between seasons and geographical districts, while it is high between different classes of altitude. Selection for adaptation to different altitudes appears to be the main factor that has determined the observed clinal variation, along with population-size effects. Conclusions Our data show that barley landraces from Ethiopia are constituted by highly variable local populations (farmer's fields) that have large within-population diversity. These landraces are also shown to be locally adapted, with the major driving force that has shaped their population structure being consistent with selection for adaptation along an altitudinal gradient. Overall, our study highlights the potential of such landraces as a source of useful alleles. Furthermore, these landraces also represent an ideal system to study the processes of adaptation and for the identification of genes and genomic regions that have adaptive roles in crop species. PMID:20565982

Differential responses of two Egyptian barley (Hordeum vulgare L.) cultivars to salt stress.

PubMed

Elsawy, Hayam I A; Mekawy, Ahmad Mohammad M; Elhity, Mahmoud A; Abdel-Dayem, Sherif M; Abdelaziz, Maha Nagy; Assaha, Dekoum V M; Ueda, Akihiro; Saneoka, Hirofumi

2018-06-01

Although barley (Hordeum vulgare L.) is considered a salt tolerant crop species, productivity of barley is affected differently by ionic, osmotic, and oxidative stresses resulting from a salty rhizosphere. The current study was conducted to elucidate the mechanism of salt tolerance in two barley cultivars, Giza128 and Giza126. The two cultivars were exposed to 200 mM NaCl hydroponically for 12 days. Although both cultivars accumulated a large amount of Na + in their leaves with similar concentrations, the growth of Giza128 was much better than that of Giza126, as measured by maintaining a higher dry weight, relative growth rate, leaf area, and plant height. To ascertain the underlying mechanisms of this differential tolerance, first, the relative expression patterns of the genes encoding Na + /H + antiporters (NHX) and the associated proton pumps (V-PPase and V-ATPase) as well as the gene encoding the plasma membrane PM H + -ATPase were analyzed in leaf tissues. Salt stress induced higher HvNHX1 expression in Giza128 (3.3-fold) than in Giza126 (1.9-fold), whereas the expression of the other two genes, HvNHX2 and HvNHX3, showed no induction in either cultivar. The expression of HvHVP1 and HvHVA was higher in Giza128 (3.8- and 2.1-fold, respectively) than in Giza126 (1.6- and 1.1-fold, respectively). The expression of the PM H + -ATPase (ha1) gene was induced more in Giza128 (8.8-fold) than in Giza126 (1.8-fold). Second, the capacity for ROS detoxification was assessed using the oxidative stress biomarkers electrolyte leakage ratio (ELR) and the concentrations of malondialdehyde (MDA) and hydrogen peroxide (H 2 O 2 ), and these parameters sharply increased in Giza126 leaves by 66.5%, 42.8% and 50.0%, respectively, compared with those in Giza128 leaves. The antioxidant enzyme (CAT, APX, sPOD, GR, and SOD) activities were significantly elevated by salt treatment in Giza128 leaves, whereas in Giza126, these activities were not significantly altered. Overall, the

Effects of Cerium and Titanium Oxide Nanoparticles in Soil on the Nutrient Composition of Barley (Hordeum vulgare L.) Kernels

PubMed Central

Pošćić, Filip; Mattiello, Alessandro; Fellet, Guido; Miceli, Fabiano; Marchiol, Luca

2016-01-01

The implications of metal nanoparticles (MeNPs) are still unknown for many food crops. The purpose of this study was to evaluate the effects of cerium oxide (nCeO2) and titanium oxide (nTiO2) nanoparticles in soil at 0, 500 and 1000 mg·kg−1 on the nutritional parameters of barley (Hordeum vulgare L.) kernels. Mineral nutrients, amylose, β-glucans, amino acid and crude protein (CP) concentrations were measured in kernels. Whole flour samples were analyzed by ICP-AES/MS, HPLC and Elemental CHNS Analyzer. Results showed that Ce and Ti accumulation under MeNPs treatments did not differ from the control treatment. However, nCeO2 and nTiO2 had an impact on composition and nutritional quality of barley kernels in contrasting ways. Both MeNPs left β-glucans unaffected but reduced amylose content by approximately 21%. Most amino acids and CP increased. Among amino acids, lysine followed by proline saw the largest increase (51% and 37%, respectively). Potassium and S were both negatively impacted by MeNPs, while B was only affected by 500 mg nCeO2·kg−1. On the contrary Zn and Mn concentrations were improved by 500 mg nTiO2·kg−1, and Ca by both nTiO2 treatments. Generally, our findings demonstrated that kernels are negatively affected by nCeO2 while nTiO2 can potentially have beneficial effects. However, both MeNPs have the potential to negatively impact malt and feed production. PMID:27294945

Improving phenolic bioactive-linked anti-hyperglycemic functions of dark germinated barley sprouts (Hordeum vulgare L.) using seed elicitation strategy.

PubMed

Ramakrishna, Ramnarain; Sarkar, Dipayan; Manduri, Avani; Iyer, Shreyas Ganesan; Shetty, Kalidas

2017-10-01

Sprouts of cereal grains, such as barley ( Hordeum vulgare L.), are a good source of beneficial phenolic bioactives. Such health relevant phenolic bioactives of cereal sprouts can be targeted to manage chronic hyperglycemia and oxidative stress commonly associated with type 2 diabetes (T2D). Therefore improving phenolic bioactives by stimulating plant endogenous defense responses such as protective pentose phosphate pathway (PPP) during sprouting has significant merit. Based on this metabolic rationale, this study aimed to enhance phenolic bioactives and associated antioxidant and anti-hyperglycemic functions in dark germinated barley sprouts using exogenous elicitor treatments. Dark-germinated sprouts of two malting barley cultivars (Pinnacle and Celebration), treated with chitosan oligosaccharide (COS) and marine protein hydrolysate (GP), were evaluated. Total soluble phenolic content (TSP), phenolic acid profiles, total antioxidant activity (TA) and in vitro inhibitory activities of hyperglycemia relevant α-amylase and α-glucosidase enzymes of the dark germinated barley sprouts were evaluated at day 2, 4, and 6 post elicitor treatments. Overall, TSP content, TA, and α-amylase inhibitory activity of dark germinated barley sprouts decreased, while α-glucosidase inhibitory activity and gallic acid content increased from day 2 to day 6. Among barley cultivars, high phenolic antioxidant-linked anti-hyperglycemic bioactives were observed in Celebration. Furthermore, GP and COS seed elicitor treatments in selective doses improved T2D relevant phenolic-linked anti-hyperglycemic bioactives of barley spouts at day 6. Therefore, such seed elicitation approach can be strategically used to develop bioactive enriched functional food ingredients from cereal sprouts targeting chronic hyperglycemia and oxidative stress linked to T2D.

Molecular dynamics simulations revealed structural differences among WRKY domain-DNA interaction in barley (Hordeum vulgare).

PubMed

Pandey, Bharati; Grover, Abhinav; Sharma, Pradeep

2018-02-12

The WRKY transcription factors are a class of DNA-binding proteins involved in diverse plant processes play critical roles in response to abiotic and biotic stresses. Genome-wide divergence analysis of WRKY gene family in Hordeum vulgare provided a framework for molecular evolution and functional roles. So far, the crystal structure of WRKY from barley has not been resolved; moreover, knowledge of the three-dimensional structure of WRKY domain is pre-requisites for exploring the protein-DNA recognition mechanisms. Homology modelling based approach was used to generate structures for WRKY DNA binding domain (DBD) and its variants using AtWRKY1 as a template. Finally, the stability and conformational changes of the generated model in unbound and bound form was examined through atomistic molecular dynamics (MD) simulations for 100 ns time period. In this study, we investigated the comparative binding pattern of WRKY domain and its variants with W-box cis-regulatory element using molecular docking and dynamics (MD) simulations assays. The atomic insight into WRKY domain exhibited significant variation in the intermolecular hydrogen bonding pattern, leading to the structural anomalies in the variant type and differences in the DNA-binding specificities. Based on the MD analysis, residual contribution and interaction contour, wild-type WRKY (HvWRKY46) were found to interact with DNA through highly conserved heptapeptide in the pre- and post-MD simulated complexes, whereas heptapeptide interaction with DNA was missing in variants (I and II) in post-MD complexes. Consequently, through principal component analysis, wild-type WRKY was also found to be more stable by obscuring a reduced conformational space than the variant I (HvWRKY34). Lastly, high binding free energy for wild-type and variant II allowed us to conclude that wild-type WRKY-DNA complex was more stable relative to variants I. The results of our study revealed complete dynamic and structural information

Replacing Escherichia coli NAD-dependent glyceraldehyde 3-phosphate dehydrogenase (GAPDH) with a NADP-dependent enzyme from Clostridium acetobutylicum facilitates NADPH dependent pathways.

PubMed

Martínez, Irene; Zhu, Jiangfeng; Lin, Henry; Bennett, George N; San, Ka-Yiu

2008-11-01

Reactions requiring reducing equivalents, NAD(P)H, are of enormous importance for the synthesis of industrially valuable compounds such as carotenoids, polymers, antibiotics and chiral alcohols among others. The use of whole-cell biocatalysis can reduce process cost by acting as catalyst and cofactor regenerator at the same time; however, product yields might be limited by cofactor availability within the cell. Thus, our study focussed on the genetic manipulation of a whole-cell system by modifying metabolic pathways and enzymes to improve the overall production process. In the present work, we genetically engineered an Escherichia coli strain to increase NADPH availability to improve the productivity of products that require NADPH in its biosynthesis. The approach involved an alteration of the glycolysis step where glyceraldehyde-3-phosphate (GAP) is oxidized to 1,3 bisphophoglycerate (1,3-BPG). This reaction is catalyzed by NAD-dependent endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) encoded by the gapA gene. We constructed a recombinant E. coli strain by replacing the native NAD-dependent gapA gene with a NADP-dependent GAPDH from Clostridium acetobutylicum, encoded by the gene gapC. The beauty of this approach is that the recombinant E. coli strain produces 2 mol of NADPH, instead of NADH, per mole of glucose consumed. Metabolic flux analysis showed that the flux through the pentose phosphate (PP) pathway, one of the main pathways that produce NADPH, was reduced significantly in the recombinant strain when compared to that of the parent strain. The effectiveness of the NADPH enhancing system was tested using the production of lycopene and epsilon-caprolactone as model systems using two different background strains. The recombinant strains, with increased NADPH availability, consistently showed significant higher productivity than the parent strains.

Quantitative flux analysis reveals folate-dependent NADPH production

NASA Astrophysics Data System (ADS)

Fan, Jing; Ye, Jiangbin; Kamphorst, Jurre J.; Shlomi, Tomer; Thompson, Craig B.; Rabinowitz, Joshua D.

2014-06-01

ATP is the dominant energy source in animals for mechanical and electrical work (for example, muscle contraction or neuronal firing). For chemical work, there is an equally important role for NADPH, which powers redox defence and reductive biosynthesis. The most direct route to produce NADPH from glucose is the oxidative pentose phosphate pathway, with malic enzyme sometimes also important. Although the relative contribution of glycolysis and oxidative phosphorylation to ATP production has been extensively analysed, similar analysis of NADPH metabolism has been lacking. Here we demonstrate the ability to directly track, by liquid chromatography-mass spectrometry, the passage of deuterium from labelled substrates into NADPH, and combine this approach with carbon labelling and mathematical modelling to measure NADPH fluxes. In proliferating cells, the largest contributor to cytosolic NADPH is the oxidative pentose phosphate pathway. Surprisingly, a nearly comparable contribution comes from serine-driven one-carbon metabolism, in which oxidation of methylene tetrahydrofolate to 10-formyl-tetrahydrofolate is coupled to reduction of NADP+ to NADPH. Moreover, tracing of mitochondrial one-carbon metabolism revealed complete oxidation of 10-formyl-tetrahydrofolate to make NADPH. As folate metabolism has not previously been considered an NADPH producer, confirmation of its functional significance was undertaken through knockdown of methylenetetrahydrofolate dehydrogenase (MTHFD) genes. Depletion of either the cytosolic or mitochondrial MTHFD isozyme resulted in decreased cellular NADPH/NADP+ and reduced/oxidized glutathione ratios (GSH/GSSG) and increased cell sensitivity to oxidative stress. Thus, although the importance of folate metabolism for proliferating cells has been long recognized and attributed to its function of producing one-carbon units for nucleic acid synthesis, another crucial function of this pathway is generating reducing power.

General approach to reversing ketol-acid reductoisomerase cofactor dependence from NADPH to NADH

DOE PAGES

Brinkmann-Chen, Sabine; Flock, Tilman; Cahn, Jackson K. B.; ...

2013-06-17

To date, efforts to switch the cofactor specificity of oxidoreductases from nicotinamide adenine dinucleotide phosphate (NADPH) to nicotinamide adenine dinucleotide (NADH) have been made on a case-by-case basis with varying degrees of success. Here we present a straightforward recipe for altering the cofactor specificity of a class of NADPH-dependent oxidoreductases, the ketol-acid reductoisomerases (KARIs). Combining previous results for an engineered NADH-dependent variant of Escherichia coli KARI with available KARI crystal structures and a comprehensive KARI-sequence alignment, we identified key cofactor specificity determinants and used this information to construct five KARIs with reversed cofactor preference. Additional directed evolution generated two enzymesmore » having NADH-dependent catalytic efficiencies that are greater than the wild-type enzymes with NADPH. As a result, high-resolution structures of a wild-type/variant pair reveal the molecular basis of the cofactor switch.« less

Constitutive NADPH-dependent electron transferase activity of the Nox4 dehydrogenase domain.

PubMed

Nisimoto, Yukio; Jackson, Heather M; Ogawa, Hisamitsu; Kawahara, Tsukasa; Lambeth, J David

2010-03-23

NADPH oxidase 4 (Nox4) is constitutively active, while Nox2 requires the cytosolic regulatory subunits p47(phox) and p67(phox) and activated Rac with activation by phorbol 12-myristate 13-acetate (PMA). This study was undertaken to identify the domain on Nox4 that confers constitutive activity. Lysates from Nox4-expressing cells exhibited constitutive NADPH- but not NADH-dependent hydrogen peroxide production with a K(m) for NADPH of 55 +/- 10 microM. The concentration of Nox4 in cell lysates was estimated using Western blotting and allowed calculation of a turnover of approximately 200 mol of H(2)O(2) min(-1) (mol of Nox4)(-1). A chimeric protein (Nox2/4) consisting of the Nox2 transmembrane (TM) domain and the Nox4 dehydrogenase (DH) domain showed H(2)O(2) production in the absence of cytosolic regulatory subunits. In contrast, chimera Nox4/2, consisting of the Nox4 TM and Nox2 DH domains, exhibited PMA-dependent activation that required coexpression of regulatory subunits. Nox DH domains from several Nox isoforms were purified and evaluated for their electron transferase activities. Nox1 DH, Nox2 DH, and Nox5 DH domains exhibited barely detectable activities toward artificial electron acceptors, while the Nox4 DH domain exhibited significant rates of reduction of cytochrome c (160 min(-1), largely superoxide dismutase-independent), ferricyanide (470 min(-1)), and other electron acceptors (artificial dyes and cytochrome b(5)). Rates were similar to those observed for H(2)O(2) production by the Nox4 holoenzyme in cell lysates. The activity required added FAD and was seen with NADPH but not NADH. These results indicate that the Nox4 DH domain exists in an intrinsically activated state and that electron transfer from NADPH to FAD is likely to be rate-limiting in the NADPH-dependent reduction of oxygen by holo-Nox4.

Low-Resolution Structure of the Full-Length Barley (Hordeum vulgare) SGT1 Protein in Solution, Obtained Using Small-Angle X-Ray Scattering

PubMed Central

Taube, Michał; Pieńkowska, Joanna R.; Jarmołowski, Artur; Kozak, Maciej

2014-01-01

SGT1 is an evolutionarily conserved eukaryotic protein involved in many important cellular processes. In plants, SGT1 is involved in resistance to disease. In a low ionic strength environment, the SGT1 protein tends to form dimers. The protein consists of three structurally independent domains (the tetratricopeptide repeats domain (TPR), the CHORD- and SGT1-containing domain (CS), and the SGT1-specific domain (SGS)), and two less conserved variable regions (VR1 and VR2). In the present study, we provide the low-resolution structure of the barley (Hordeum vulgare) SGT1 protein in solution and its dimer/monomer equilibrium using small-angle scattering of synchrotron radiation, ab-initio modeling and circular dichroism spectroscopy. The multivariate curve resolution least-square method (MCR-ALS) was applied to separate the scattering data of the monomeric and dimeric species from a complex mixture. The models of the barley SGT1 dimer and monomer were formulated using rigid body modeling with ab-initio structure prediction. Both oligomeric forms of barley SGT1 have elongated shapes with unfolded inter-domain regions. Circular dichroism spectroscopy confirmed that the barley SGT1 protein had a modular architecture, with an α-helical TPR domain, a β-sheet sandwich CS domain, and a disordered SGS domain separated by VR1 and VR2 regions. Using molecular docking and ab-initio protein structure prediction, a model of dimerization of the TPR domains was proposed. PMID:24714665

Variation between Ethiopian and North American barley varieties (Hordeum vulgare) in response to Russian wheat aphid (Diuraphis noxia) populations.

PubMed

Araya, Alemu; Belay, Tesfay; Hussein, Temam

2014-03-15

The Russian wheat aphid, Diuraphis noxia (Mordvilko) (Hemiptera: Aphididae), causes severe damage to barley, Hordeum vulgare L. (Poales: Poaceae), in the highlands of Ethiopia. Little information is available on the control of this pest in Ethiopia. An experiment aimed at evaluating the resistance of barley varieties from the USA to D. noxia populations and determining biotypic variation between Ethiopian and North American D. noxia populations was conducted. The D. noxia-resistant barley varieties Burton and RWA-1758 from the USA, the resistant barley line 3296-15 from Ethiopia, and a local Ethiopian susceptible variety were included in a randomized design in a greenhouse under natural light conditions. There were highly significant differences (P < 0.001) in the mean D. noxia population, leaf chlorosis, leaf rolling, plant stunting, number of tillers per plant, and the percentage of infested tillers per plant between the resistant and susceptible varieties. The aphid population per tiller was lower on the resistant barley plants than on the susceptible plants. Severe plant damage was observed on the local barley variety, while the least damage was observed on Burton, followed by RWA-1758. Burton and RWA-1758 were therefore highly resistant and moderately resistant, respectively, to the northern Ethiopian D. noxia populations, indicating similarities in biotypes between the United States and northern Ethiopian D. noxia populations. The damage to variety 3296-15 was greater than to Burton and RWA-1758. Leaf chlorosis scores and leaf rolling scores for variety 3296-15 upon treatment with the north Ethiopian D. noxia population indicate likely biotypic variation between D. noxia populations of northern and central Ethiopia. This is an open access paper. We use the Creative Commons Attribution 3.0 license that permits unrestricted use, provided that the paper is properly attributed.

Population-genetic analysis of HvABCG31 promoter sequence in wild barley (Hordeum vulgare ssp. spontaneum)

PubMed Central

2012-01-01

Background The cuticle is an important adaptive structure whose origin played a crucial role in the transition of plants from aqueous to terrestrial conditions. HvABCG31/Eibi1 is an ABCG transporter gene, involved in cuticle formation that was recently identified in wild barley (Hordeum vulgare ssp. spontaneum). To study the genetic variation of HvABCG31 in different habitats, its 2 kb promoter region was sequenced from 112 wild barley accessions collected from five natural populations from southern and northern Israel. The sites included three mesic and two xeric habitats, and differed in annual rainfall, soil type, and soil water capacity. Results Phylogenetic analysis of the aligned HvABCG31 promoter sequences clustered the majority of accessions (69 out of 71) from the three northern mesic populations into one cluster, while all 21 accessions from the Dead Sea area, a xeric southern population, and two isolated accessions (one from a xeric population at Mitzpe Ramon and one from the xeric ‘African Slope’ of “Evolution Canyon”) formed the second cluster. The southern arid populations included six haplotypes, but they differed from the consensus sequence at a large number of positions, while the northern mesic populations included 15 haplotypes that were, on average, more similar to the consensus sequence. Most of the haplotypes (20 of 22) were unique to a population. Interestingly, higher genetic variation occurred within populations (54.2%) than among populations (45.8%). Analysis of the promoter region detected a large number of transcription factor binding sites: 121–128 and 121–134 sites in the two southern arid populations, and 123–128,125–128, and 123–125 sites in the three northern mesic populations. Three types of TFBSs were significantly enriched: those related to GA (gibberellin), Dof (DNA binding with one finger), and light. Conclusions Drought stress and adaptive natural selection may have been important determinants in the observed

Characterization of plant growth promoting traits of bacterial isolates from the rhizosphere of barley (Hordeum vulgare L.) and tomato (Solanum lycopersicon L.) grown under Fe sufficiency and deficiency.

PubMed

Scagliola, M; Pii, Y; Mimmo, T; Cesco, S; Ricciuti, P; Crecchio, C

2016-10-01

Plant Growth Promoting Bacteria (PGPB) are considered a promising approach to replace the conventional agricultural practices, since they have been shown to affect plant nutrient-acquisition processes by influencing nutrient availability in the rhizosphere and/or those biochemical processes determining the uptake at root level of nitrogen (N), phosphorus (P), and iron (Fe), that represent the major constraints for crop productivity worldwide. We have isolated novel bacterial strains from the rhizosphere of barley (Hordeum vulgare L.) and tomato (Solanum lycopersicon L.) plants, previously grown in hydroponic solution (either Fe deficient or Fe sufficient) and subsequently transferred onto an agricultural calcareous soil. PGPB have been identified by molecular tools and characterized for their capacity to produce siderophores and indole-3-acetic acid (IAA), and to solubilize phosphate. Selected bacterial isolates, showing contemporarily high levels of the three activities investigated, were finally tested for their capacity to induce Fe reduction in cucumber roots two isolates, from barley and tomato plants under Fe deficiency, significantly increased the root Fe-chelate reductase activity; interestingly, another isolate enhanced the reduction of Fe-chelate reductase activity in cucumber plant roots, although grown under Fe sufficiency. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Detection of QTLs for seedling characteristics in barley (Hordeum vulgare L.) grown under hydroponic culture condition.

PubMed

Wang, Qifei; Sun, Genlou; Ren, Xifeng; Wang, Jibin; Du, Binbin; Li, Chengdao; Sun, Dongfa

2017-11-07

Seedling characteristics play significant roles in the growth and development of barley (Hordeum vulgare L.), including stable stand establishment, water and nutrients uptake, biotic resistance and abiotic stresses, and can influence yield and quality. However, the genetic mechanisms underlying seedling characteristics in barley are largely unknown and little research has been done. In the present work, 21 seedling-related characteristics are assessed in a barley double haploid (DH) population, grown under hydroponic conditions. Of them, leaf age (LAG), shoot height (SH), maximum root length (MRL), main root number (MRN) and seedling fresh weight (SFW) were investigated at the 13th, 20th, 27th, and 34th day after germination. The objectives were to identify quantitative trait loci (QTLs) underlying these seedling characteristics using a high-density linkage map and to reveal the QTL expression pattern by comparing the QTLs among four different seedling growth stages. A total of 70 QTLs were distributed over all chromosomes except 4H, and, individually, accounted for 5.01%-77.78% of phenotypic variation. Out of the 70 detected QTLs, 23 showed a major effect on 14 seedling-related characteristics. Ten co-localized chromosomal regions on 2H (five regions), 3H (two regions) and 7H (three regions) involved 39 QTLs (55.71%), each simultaneously influenced more than one trait. Meanwhile, 9 co-localized genomic regions involving 22 QTLs for five seedling characteristics (LAG, SH, MRL, MRN and SFW) at the 13th, 20th, 27th and 34th day-old seedling were common for two or more growth stages of seedling. QTL in the vicinity of Vrs1 locus on chromosome 2H with the favorable alleles from Huadamai 6 was found to have the largest main effects on multiple seedling-related traits. Six QTL cluster regions associated with 16 seedling-related characteristics were observed on chromosome 2H, 3H and 7H. The majority of the 29 regions identified for five seedling characteristics were

Cancer-associated Isocitrate Dehydrogenase Mutations Inactivate NADPH-dependent Reductive Carboxylation*

PubMed Central

Leonardi, Roberta; Subramanian, Chitra; Jackowski, Suzanne; Rock, Charles O.

2012-01-01

Isocitrate dehydrogenase (IDH) is a reversible enzyme that catalyzes the NADP+-dependent oxidative decarboxylation of isocitrate (ICT) to α-ketoglutarate (αKG) and the NADPH/CO2-dependent reductive carboxylation of αKG to ICT. Reductive carboxylation by IDH1 was potently inhibited by NADP+ and, to a lesser extent, by ICT. IDH1 and IDH2 with cancer-associated mutations at the active site arginines were unable to carry out the reductive carboxylation of αKG. These mutants were also defective in ICT decarboxylation and converted αKG to 2-hydroxyglutarate using NADPH. These mutant proteins were thus defective in both of the normal reactions of IDH. Biochemical analysis of heterodimers between wild-type and mutant IDH1 subunits showed that the mutant subunit did not inactivate reductive carboxylation by the wild-type subunit. Cells expressing the mutant IDH are thus deficient in their capacity for reductive carboxylation and may be compromised in their ability to produce acetyl-CoA under hypoxia or when mitochondrial function is otherwise impaired. PMID:22442146

The influence of vernalization and daylength on expression of flowering-time genes in the shoot apex and leaves of barley (Hordeum vulgare).

PubMed Central

Sasani, Shahryar; Hemming, Megan N.; Oliver, Sandra N.; Greenup, Aaron; Tavakkol-Afshari, Reza; Mahfoozi, Siroos; Poustini, Kazem; Sharifi, Hamid-Reza; Dennis, Elizabeth S.; Peacock, W. James; Trevaskis, Ben

2009-01-01

Responses to prolonged low-temperature treatment of imbibed seeds (vernalization) were examined in barley (Hordeum vulgare). These occurred in two phases: the perception of prolonged cold, which occurred gradually at low temperatures, and the acceleration of reproductive development, which occurred after vernalization. Expression of the VERNALIZATION1 gene (HvVRN1) increased gradually in germinating seedlings during vernalization, both at the shoot apex and in the developing leaves. This occurred in darkness, independently of VERNALIZATION2 (HvVRN2), consistent with the hypothesis that expression of HvVRN1 is induced by prolonged cold independently of daylength flowering-response pathways. After vernalization, expression of HvVRN1 was maintained in the shoot apex and leaves. This was associated with accelerated inflorescence initiation and with down-regulation of HvVRN2 in the leaves. The largest determinant of HvVRN1 expression levels in vernalized plants was the length of seed vernalization treatment. Daylength did not influence HvVRN1 expression levels in shoot apices and typically did not affect expression in leaves. In the leaves of plants that had experienced a saturating seed vernalization treatment, expression of HvVRN1 was higher in long days, however. HvFT1 was expressed in the leaves of these plants in long days, which might account for the elevated HvVRN1 expression. Long-day up-regulation of HvVRN1 was not required for inflorescence initiation, but might accelerate subsequent stages of inflorescence development. Similar responses to seed vernalization were also observed in wheat (Triticum aestivum). These data support the hypothesis that VRN1 is induced by cold during winter to promote spring flowering in vernalization-responsive cereals. PMID:19357429

Comparative kinetics and reciprocal inhibition of nitrate and nitrite uptake in roots of uninduced and induced barley (Hordeum vulgare L.) seedlings

NASA Technical Reports Server (NTRS)

Aslam, M.; Travis, R. L.; Huffaker, R. C.

1992-01-01

Nitrate and NO2- transport by roots of 8-day-old uninduced and induced intact barley (Hordeum vulgare L. var CM 72) seedlings were compared to kinetic patterns, reciprocal inhibition of the transport systems, and the effect of the inhibitor, p-hydroxymercuribenzoate. Net uptake of NO3- and NO2- was measured by following the depletion of the ions from the uptake solutions. The roots of uninduced seedlings possessed a low concentration, saturable, low Km, possibly a constitutive uptake system, and a linear system for both NO3- and NO2-. The low Km system followed Michaelis-Menten kinetics and approached saturation between 40 and 100 micromolar, whereas the linear system was detected between 100 and 500 micromolar. In roots of induced seedlings, rates for both NO3- and NO2- uptake followed Michaelis-Menten kinetics and approached saturation at about 200 micromolar. In induced roots, two kinetically identifiable transport systems were resolved for each anion. At the lower substrate concentrations, less than 10 micromolar, the apparent low Kms of NO3- and NO2- uptake were 7 and 9 micromolar, respectively, and were similar to those of the low Km system in uninduced roots. At substrate concentrations between 10 and 200 micromolar, the apparent high Km values of NO3- uptake ranged from 34 to 36 micromolar and of NO2- uptake ranged from 41 to 49 micromolar. A linear system was also found in induced seedlings at concentrations above 500 micromolar. Double reciprocal plots indicated that NO3- and NO2- inhibited the uptake of each other competitively in both uninduced and induced seedlings; however, Ki values showed that NO3- was a more effective inhibitor than NO2-. Nitrate and NO2- transport by both the low and high Km systems were greatly inhibited by p-hydroxymercuribenzoate, whereas the linear system was only slightly inhibited.

Phylogenetic and comparative gene expression analysis of barley (Hordeum vulgare)WRKY transcription factor family reveals putatively retained functions betweenmonocots and dicots

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mangelsen, Elke; Kilian, Joachim; Berendzen, Kenneth W.

2008-02-01

WRKY proteins belong to the WRKY-GCM1 superfamily of zinc finger transcription factors that have been subject to a large plant-specific diversification. For the cereal crop barley (Hordeum vulgare), three different WRKY proteins have been characterized so far, as regulators in sucrose signaling, in pathogen defense, and in response to cold and drought, respectively. However, their phylogenetic relationship remained unresolved. In this study, we used the available sequence information to identify a minimum number of 45 barley WRKY transcription factor (HvWRKY) genes. According to their structural features the HvWRKY factors were classified into the previously defined polyphyletic WRKY subgroups 1 tomore » 3. Furthermore, we could assign putative orthologs of the HvWRKY proteins in Arabidopsis and rice. While in most cases clades of orthologous proteins were formed within each group or subgroup, other clades were composed of paralogous proteins for the grasses and Arabidopsis only, which is indicative of specific gene radiation events. To gain insight into their putative functions, we examined expression profiles of WRKY genes from publicly available microarray data resources and found group specific expression patterns. While putative orthologs of the HvWRKY transcription factors have been inferred from phylogenetic sequence analysis, we performed a comparative expression analysis of WRKY genes in Arabidopsis and barley. Indeed, highly correlative expression profiles were found between some of the putative orthologs. HvWRKY genes have not only undergone radiation in monocot or dicot species, but exhibit evolutionary traits specific to grasses. HvWRKY proteins exhibited not only sequence similarities between orthologs with Arabidopsis, but also relatedness in their expression patterns. This correlative expression is indicative for a putative conserved function of related WRKY proteins in mono- and dicot species.« less

An eceriferum locus, cer-zv, is associated with a defect in cutin responsible for water retention in barley (Hordeum vulgare) leaves.

PubMed

Li, Chao; Wang, Aidong; Ma, Xiaoying; Pourkheirandish, Mohammad; Sakuma, Shun; Wang, Ning; Ning, Shunzong; Nevo, Eviatar; Nawrath, Christiane; Komatsuda, Takao; Chen, Guoxiong

2013-03-01

Drought limits plant growth and threatens crop productivity. A barley (Hordeum vulgare) ethylene imine-induced monogenic recessive mutant cer-zv, which is sensitive to drought, was characterized and genetically mapped in the present study. Detached leaves of cer-zv lost 34.2 % of their initial weight after 1 h of dehydration. The transpiration was much higher in cer-zv leaves than in wild-type leaves under both light and dark conditions. The stomata of cer-zv leaves functioned normally, but the cuticle of cer-zv leaves showed increased permeability to ethanol and toluidine blue dye. There was a 50-90 % reduction in four major cutin monomers, but no reduction in wax loads was found in the cer-zv mutant as compared with the wild type. Two F(2) mapping populations were established by the crosses of 23-19 × cer-zv and cer-zv × OUH602. More polymorphisms were found in EST sequences between cer-zv and OUH602 than between cer-zv and 23-19. cer-zv was located in a pericentromeric region on chromosome 4H in a 10.8 cM interval in the 23-19 × cer-zv map based on 186 gametes tested and a 1.7 cM interval in the cer-zv × OUH602 map based on 176 gametes tested. It co-segregated with EST marker AK251484 in both maps. The results indicated that the cer-zv mutant is defective in cutin, which might be responsible for the increased transpiration rate and drought sensitivity, and that the F(2) of cer-zv × OUH602 might better facilitate high resolution mapping of cer-zv.

Contrasting Influence of NADPH and a NADPH-Regenerating System on the Metabolism of Carbonyl-Containing Compounds in Hepatic Microsomes

EPA Science Inventory

Carbonyl containing xenobiotics may be susceptible to NADPH-dependent cytochrome P450 (P450) and carbonyl-reduction reactions. In vitro hepatic microsome assays are routinely supplied NADPH either by direct addition of NADPH or via an NADPH-regenerating system (NRS). In contrast ...

The barley (Hordeum vulgare) cellulose synthase-like D2 gene (HvCslD2) mediates penetration resistance to host-adapted and nonhost isolates of the powdery mildew fungus.

PubMed

Douchkov, Dimitar; Lueck, Stefanie; Hensel, Goetz; Kumlehn, Jochen; Rajaraman, Jeyaraman; Johrde, Annika; Doblin, Monika S; Beahan, Cherie T; Kopischke, Michaela; Fuchs, René; Lipka, Volker; Niks, Rients E; Bulone, Vincent; Chowdhury, Jamil; Little, Alan; Burton, Rachel A; Bacic, Antony; Fincher, Geoffrey B; Schweizer, Patrick

2016-10-01

Cell walls and cellular turgor pressure shape and suspend the bodies of all vascular plants. In response to attack by fungal and oomycete pathogens, which usually breach their host's cell walls by mechanical force or by secreting lytic enzymes, plants often form local cell wall appositions (papillae) as an important first line of defence. The involvement of cell wall biosynthetic enzymes in the formation of these papillae is still poorly understood, especially in cereal crops. To investigate the role in plant defence of a candidate gene from barley (Hordeum vulgare) encoding cellulose synthase-like D2 (HvCslD2), we generated transgenic barley plants in which HvCslD2 was silenced through RNA interference (RNAi). The transgenic plants showed no growth defects but their papillae were more successfully penetrated by host-adapted, virulent as well as avirulent nonhost isolates of the powdery mildew fungus Blumeria graminis. Papilla penetration was associated with lower contents of cellulose in epidermal cell walls and increased digestion by fungal cell wall degrading enzymes. The results suggest that HvCslD2-mediated cell wall changes in the epidermal layer represent an important defence reaction both for nonhost and for quantitative host resistance against nonadapted wheat and host-adapted barley powdery mildew pathogens, respectively. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

Light Driven CO2 Fixation by Using Cyanobacterial Photosystem I and NADPH-Dependent Formate Dehydrogenase

PubMed Central

Ihara, Masaki; Kawano, Yusuke; Urano, Miho; Okabe, Ayako

2013-01-01

The ultimate goal of this research is to construct a new direct CO2 fixation system using photosystems in living algae. Here, we report light-driven formate production from CO2 by using cyanobacterial photosystem I (PS I). Formate, a chemical hydrogen carrier and important industrial material, can be produced from CO2 by using the reducing power and the catalytic function of formate dehydrogenase (FDH). We created a bacterial FDH mutant that experimentally switched the cofactor specificity from NADH to NADPH, and combined it with an in vitro-reconstituted cyanobacterial light-driven NADPH production system consisting of PS I, ferredoxin (Fd), and ferredoxin-NADP+-reductase (FNR). Consequently, light-dependent formate production under a CO2 atmosphere was successfully achieved. In addition, we introduced the NADPH-dependent FDH mutant into heterocysts of the cyanobacterium Anabaena sp. PCC 7120 and demonstrated an increased formate concentration in the cells. These results provide a new possibility for photo-biological CO2 fixation. PMID:23936519

Light driven CO2 fixation by using cyanobacterial photosystem I and NADPH-dependent formate dehydrogenase.

PubMed

Ihara, Masaki; Kawano, Yusuke; Urano, Miho; Okabe, Ayako

2013-01-01

The ultimate goal of this research is to construct a new direct CO2 fixation system using photosystems in living algae. Here, we report light-driven formate production from CO2 by using cyanobacterial photosystem I (PS I). Formate, a chemical hydrogen carrier and important industrial material, can be produced from CO2 by using the reducing power and the catalytic function of formate dehydrogenase (FDH). We created a bacterial FDH mutant that experimentally switched the cofactor specificity from NADH to NADPH, and combined it with an in vitro-reconstituted cyanobacterial light-driven NADPH production system consisting of PS I, ferredoxin (Fd), and ferredoxin-NADP(+)-reductase (FNR). Consequently, light-dependent formate production under a CO2 atmosphere was successfully achieved. In addition, we introduced the NADPH-dependent FDH mutant into heterocysts of the cyanobacterium Anabaena sp. PCC 7120 and demonstrated an increased formate concentration in the cells. These results provide a new possibility for photo-biological CO2 fixation.

A proteomics approach to study the molecular basis of enhanced salt tolerance in barley (Hordeum vulgare L.) conferred by the root mutualistic fungus Piriformospora indica.

PubMed

Alikhani, Mehdi; Khatabi, Behnam; Sepehri, Mozhgan; Nekouei, Mojtaba Khayam; Mardi, Mohsen; Salekdeh, Ghasem Hosseini

2013-06-01

Piriformospora indica is a root-interacting mutualistic fungus capable of enhancing plant growth, increasing plant resistance to a wide variety of pathogens, and improving plant stress tolerance under extreme environmental conditions. Understanding the molecular mechanisms by which P. indica can improve plant tolerance to stresses will pave the way to identifying the major mechanisms underlying plant adaptability to environmental stresses. We conducted greenhouse experiments at three different salt levels (0, 100 and 300 mM NaCl) on barley (Hordeum vulgare L.) cultivar "Pallas" inoculated with P. indica. Based on the analysis of variance, P. indica had a significant impact on the barley growth and shoot biomass under normal and salt stress conditions. P. indica modulated ion accumulation in colonized plants by increasing the foliar potassium (K(+))/sodium (Na(+)) ratio, as it is considered a reliable indicator of salt stress tolerance. P. indica induced calcium (Ca(2+)) accumulation and likely influenced the stress signal transduction. Subsequently, proteomic analysis of the barley leaf sheath using two-dimensional electrophoresis resulted in detection of 968 protein spots. Of these detected spots, the abundance of 72 protein spots changed significantly in response to salt treatment and P. indica-root colonization. Mass spectrometry analysis of responsive proteins led to the identification of 51 proteins. These proteins belonged to different functional categories including photosynthesis, cell antioxidant defense, protein translation and degradation, energy production, signal transduction and cell wall arrangement. Our results showed that P. indica induced a systemic response to salt stress by altering the physiological and proteome responses of the plant host.

Evaluation of phytotoxicity effect of olive mill wastewater treated by different technologies on seed germination of barley (Hordeum vulgare L.).

PubMed

Rusan, Munir J M; Albalasmeh, Ammar A; Zuraiqi, Said; Bashabsheh, Mohammad

2015-06-01

Olive-mill wastewater (OMW) is a by-product effluent of olive oil extraction process that is produced in large amount in the Mediterranean region. OMW is believed to induce phytotoxic effect on organisms including seed germination and plant growth. The objective of this study was to evaluate the impact of untreated and treated OMW with different techniques on seed germination of barley (Hordeum vulgare L.). The following treatments were investigated: (1) tap water (control); (2) OMW treated by aerobic biological technology in a Jacto Reactor (JR); (3) OMW treated by solar fenton oxidation (SFO); (4) OMW treated by microfiltration followed by nanofiltration (MF+NF); (5) OMW treated by microfiltration followed by reverse osmosis (MF+RO) process; (6) diluted OMW with tap water (25 % OMW); (7) diluted OMW with tap water (50 % OMW); (8) diluted OMW with tap water (75 % OMW); and (9) untreated OMW (100 % OMW). A germination test was conducted in an incubator at temperature of 23 (∘)C. In each petri dish, a filter paper was mounted and ten seeds of barley were placed on the filter paper. Five milliliter of water were added to each petri dish. The seed germination was determined by counting the number of germinated seeds to calculate the percentage of germination (G %). Germination rate index (GRI), seed vigor index (SVI), and phytotoxicity index (PI) were also calculated. Then, the dry weights and lengths of the shoots and the roots of the germinated seeds were measured. The results show that 100, 75, and 50 %OMW were very phytotoxic and completely prohibited seed germination. However, phytotoxicity decreased significantly following treatments of OMW with all techniques investigated and by the 25 % OMW dilution, as results of removing the phenols and other phytotoxic organic compounds from the OMW or by diluting it. This was evidenced by relative enhancement of the dry weights and lengths of shoot and root as well as the G %, GRI, SVG, and PI. It was concluded that if

Regiospecificity determinants of human heme oxygenase: differential NADPH- and ascorbate-dependent heme cleavage by the R183E mutant.

PubMed

Wang, Jinling; Lad, Latesh; Poulos, Thomas L; Ortiz de Montellano, Paul R

2005-01-28

The ability of the human heme oxygenase-1 (hHO-1) R183E mutant to oxidize heme in reactions supported by either NADPH-cytochrome P450 reductase or ascorbic acid has been compared. The NADPH-dependent reaction, like that of wild-type hHO-1, yields exclusively biliverdin IXalpha. In contrast, the R183E mutant with ascorbic acid as the reductant produces biliverdin IXalpha (79 +/- 4%), IXdelta (19 +/- 3%), and a trace of IXbeta. In the presence of superoxide dismutase and catalase, the yield of biliverdin IXdelta is decreased to 8 +/- 1% with a corresponding increase in biliverdin IXalpha. Spectroscopic analysis of the NADPH-dependent reaction shows that the R183E ferric biliverdin complex accumulates, because reduction of the iron, which is required for sequential iron and biliverdin release, is impaired. Reversal of the charge at position 183 makes reduction of the iron more difficult. The crystal structure of the R183E mutant, determined in the ferric and ferrous-NO bound forms, shows that the heme primarily adopts the same orientation as in wild-type hHO-1. The structure of the Fe(II).NO complex suggests that an altered active site hydrogen bonding network supports catalysis in the R183E mutant. Furthermore, Arg-183 contributes to the regiospecificity of the wild-type enzyme, but its contribution is not critical. The results indicate that the ascorbate-dependent reaction is subject to a lower degree of regiochemical control than the NADPH-dependent reaction. Ascorbate may be able to reduce the R183E ferric and ferrous dioxygen complexes in active site conformations that cannot be reduced by NADPH-cytochrome P450 reductase.

The voltage dependence of NADPH oxidase reveals why phagocytes need proton channels

NASA Astrophysics Data System (ADS)

DeCoursey, Thomas E.; Morgan, Deri; Cherny, Vladimir V.

2003-04-01

The enzyme NADPH oxidase in phagocytes is important in the body's defence against microbes: it produces superoxide anions (O2-, precursors to bactericidal reactive oxygen species). Electrons move from intracellular NADPH, across a chain comprising FAD (flavin adenine dinucleotide) and two haems, to reduce extracellular O2 to O2-. NADPH oxidase is electrogenic, generating electron current (Ie) that is measurable under voltage-clamp conditions. Here we report the complete current-voltage relationship of NADPH oxidase, the first such measurement of a plasma membrane electron transporter. We find that Ie is voltage-independent from -100mV to >0mV, but is steeply inhibited by further depolarization, and is abolished at about +190mV. It was proposed that H+ efflux mediated by voltage-gated proton channels compensates Ie, because Zn2+ and Cd2+ inhibit both H+ currents and O2- production. Here we show that COS-7 cells transfected with four NADPH oxidase components, but lacking H+ channels, produce O2- in the presence of Zn2+ concentrations that inhibit O2- production in neutrophils and eosinophils. Zn2+ does not inhibit NADPH oxidase directly, but through effects on H+ channels. H+ channels optimize NADPH oxidase function by preventing membrane depolarization to inhibitory voltages.

Contrasting influence of NADPH and a NADPH-regenerating system on the metabolism of carbonyl-containing compounds in hepatic microsomes.

PubMed

Mazur, Christopher S; Kenneke, John F; Goldsmith, Michael-Rock; Brown, Cather

2009-09-01

Carbonyl containing xenobiotics may be susceptible to NADPH-dependent cytochrome P450 (P450) and carbonyl-reduction reactions. In vitro hepatic microsome assays are routinely supplied NADPH either by direct addition of NADPH or via an NADPH-regenerating system (NRS). In contrast to oxidative P450 transformations, which occur on the periphery of a microsome vesicle, intraluminal carbonyl reduction depends on transport of cofactors across the endoplasmic reticulum (ER) membrane into the lumen. Glucose 6-phosphate, a natural cofactor and component of the NRS matrix, is readily transported across the ER membrane and facilitates intraluminal NADPH production, whereas direct addition of NADPH has limited access to the lumen. In this study, we compared the effects of direct addition of NADPH and use of an NRS on the P450-mediated transformation of propiconazole and 11 beta-hydroxysteroid dehydrogenase type 1 (HSD1) carbonyl reduction of cortisone and the xenobiotic triadimefon in hepatic microsomes. Our results demonstrate that the use of NADPH rather than NRS can underestimate the kinetic rates of intraluminal carbonyl reduction, whereas P450-mediated transformations were unaffected. Therefore, in vitro depletion rates measured for a carbonyl-containing xenobiotic susceptible to both intraluminal carbonyl reduction and P450 processes may not be properly assessed with direct addition of NADPH. In addition, we used in silico predictions as follows: 1) to show that 11 beta-HSD1 carbonyl reduction was energetically more favorable than oxidative P450 transformation; and 2) to calculate chemical binding score and the distance between the carbonyl group and the hydride to be transferred by NADPH to identify other 11 beta-HSD1 substrates for which reaction kinetics may be underestimated by direct addition of NADPH.

On the allopolyploid origin and genome structure of the closely related species Hordeum secalinum and Hordeum capense inferred by molecular karyotyping.

PubMed

Cuadrado, Ángeles; de Bustos, Alfredo; Jouve, Nicolás

2017-08-01

To provide additional information to the many phylogenetic analyses conducted within Hordeum , here the origin and interspecific affinities of the allotetraploids Hordeum secalinum and Hordeum capense were analysed by molecular karyotyping. Karyotypes were determined using genomic in situ hybridization (GISH) to distinguish the sub-genomes and , plus fluorescence in situ hybridization (FISH)/non-denaturing (ND)-FISH to determine the distribution of ten tandem repetitive DNA sequences and thus provide chromosome markers. Each chromosome pair in the six accessions analysed was identified, allowing the establishment of homologous and putative homeologous relationships. The low-level polymorphism observed among the H. secalinum accessions contrasted with the divergence recorded for the sub-genome of the H. capense accessions. Although accession H335 carries an intergenomic translocation, its chromosome structure was indistinguishable from that of H. secalinum . Hordeum secalinum and H. capense accession H335 share a hybrid origin involving Hordeum marinum subsp. gussoneanum as the genome donor and an unidentified genome progenitor. Hordeum capense accession BCC2062 either diverged, with remodelling of the sub-genome, or its genome was donated by a now extinct ancestor. A scheme of probable evolution shows the intricate pattern of relationships among the Hordeum species carrying the genome (including all H. marinum taxa and the hexaploid Hordeum brachyantherum ). © The Author 2017. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Vulgar Music and Technology

ERIC Educational Resources Information Center

Stivers, Richard

2007-01-01

Rock music, rap, and heavy metal are all forms of vulgar music. Vulgarity refers to actions and communication that are "common, noisy, and gross," and are "untranscendent." A technological society is a vulgar society in its base of materialism and exclusive concern with power. Its excessive rationality produces a need for escape, for ecstasy, for…

Transgenic barley (Hordeum vulgare L.) expressing the wheat aluminium resistance gene (TaALMT1) shows enhanced phosphorus nutrition and grain production when grown on an acid soil.

PubMed

Delhaize, Emmanuel; Taylor, Phillip; Hocking, Peter J; Simpson, Richard J; Ryan, Peter R; Richardson, Alan E

2009-06-01

Barley (Hordeum vulgare L.), genetically modified with the Al(3+) resistance gene of wheat (TaALMT1), was compared with a non-transformed sibling line when grown on an acidic and highly phosphate-fixing ferrosol supplied with a range of phosphorus concentrations. In short-term pot trials (26 days), transgenic barley expressing TaALMT1 (GP-ALMT1) was more efficient than a non-transformed sibling line (GP) at taking up phosphorus on acid soil, but the genotypes did not differ when the soil was limed. Differences in phosphorus uptake efficiency on acid soil could be attributed not only to the differential effects of aluminium toxicity on root growth between the genotypes, but also to differences in phosphorus uptake per unit root length. Although GP-ALMT1 out-performed GP on acid soil, it was still not as efficient at taking up phosphorus as plants grown on limed soil. GP-ALMT1 plants grown in acid soil possessed substantially smaller rhizosheaths than those grown in limed soil, suggesting that root hairs were shorter. This is a probable reason for the lower phosphorus uptake efficiency. When grown to maturity in large pots, GP-ALMT1 plants produced more than twice the grain as GP plants grown on acid soil and 80% of the grain produced by limed controls. Expression of TaALMT1 in barley was not associated with a penalty in either total shoot or grain production in the absence of Al(3+), with both genotypes showing equivalent yields in limed soil. These findings demonstrate that an important crop species can be genetically engineered to successfully increase grain production on an acid soil.

NADPH-dependent coenzyme Q reductase is the main enzyme responsible for the reduction of non-mitochondrial CoQ in cells.

PubMed

Takahashi, Takayuki; Okuno, Masaaki; Okamoto, Tadashi; Kishi, Takeo

2008-01-01

We purified an NADPH-dependent coenzyme Q reductase (NADPH-CoQ reductase) in rat liver cytosol and compared its enzymatic properties with those of the other CoQ10 reductases such as NADPH: quinone acceptor oxidoreductase 1 (NQO1), lipoamide dehydrogenase, thioredoxine reductase and glutathione reductase. NADPH-CoQ reductase was the only enzyme that preferred NADPH to NADH as an electron donor and was also different from the other CoQ10 reductases in the sensitivities to its inhibitors and stimulators. Especially, Zn2+ was the most powerful inhibitor for NADPH-CoQ reductase, but CoQ10 reduction by the other CoQ10 reductases could not be inhibited by Zn2+. Furthermore, the reduction of the CoQ9 incorporated into HeLa cells was also inhibited by Zn2+ in the presence of pyrithione, a zinc ionophore. Moreover, NQO1 gene silencing in HeLa cells by transfection of a small interfering RNA resulted in lowering of both the NQO1 protein level and the NQO1 activity by about 75%. However, this transfection did not affect the NADPH-CoQ reductase activity and the reduction of CoQ9 incorporated into the cells. These results suggest that the NADPH-CoQ reductase located in cytosol may be the main enzyme responsible for the reduction of non-mitochondrial CoQ in cells.

Method to Detect the Cellular Source of Over-Activated NADPH Oxidases Using NAD(P)H Fluorescence Lifetime Imaging.

PubMed

Bremer, Daniel; Leben, Ruth; Mothes, Ronja; Radbruch, Helena; Niesner, Raluca

2017-04-03

Fluorescence-lifetime imaging microscopy (FLIM) is a technique to generate images, in which the contrast is obtained by the excited-state lifetime of fluorescent molecules instead of their intensity and emission spectrum. The ubiquitous coenzymes NADH and NADPH, hereafter NAD(P)H, in cells show a short fluorescence lifetime ≈400 psec in the free-state and a longer fluorescence lifetime when bound to enzymes. The fluorescence lifetime of NAD(P)H in this state depends on the binding-site on the specific enzyme. In the case of NADPH bound to members of the NADPH oxidases family we measured a fluorescence lifetime of 3650 psec as compared to enzymes typically active in cells, in which case fluorescence lifetimes of ∼2000 psec are measured. Here we present a robust protocol based on NAD(P)H fluorescence lifetime imaging in isolated cells to distinguish between normally active enzymes and NADPH oxidases, mainly responsible for oxidative stress. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Red:far-red light conditions affect the emission of volatile organic compounds from barley (Hordeum vulgare), leading to altered biomass allocation in neighbouring plants

PubMed Central

Kegge, Wouter; Ninkovic, Velemir; Glinwood, Robert; Welschen, Rob A. M.; Voesenek, Laurentius A. C. J.; Pierik, Ronald

2015-01-01

Background and Aims Volatile organic compounds (VOCs) play various roles in plant–plant interactions, and constitutively produced VOCs might act as a cue to sense neighbouring plants. Previous studies have shown that VOCs emitted from the barley (Hordeum vulgare) cultivar ‘Alva’ cause changes in biomass allocation in plants of the cultivar ‘Kara’. Other studies have shown that shading and the low red:far-red (R:FR) conditions that prevail at high plant densities can reduce the quantity and alter the composition of the VOCs emitted by Arabidopsis thaliana, but whether this affects plant–plant signalling remains unknown. This study therefore examines the effects of far-red light enrichment on VOC emissions and plant–plant signalling between ‘Alva’ and ‘Kara’. Methods The proximity of neighbouring plants was mimicked by supplemental far-red light treatment of VOC emitter plants of barley grown in growth chambers. Volatiles emitted by ‘Alva’ under control and far-red light-enriched conditions were analysed using gas chromatography–mass spectrometry (GC-MS). ‘Kara’ plants were exposed to the VOC blend emitted by the ‘Alva’ plants that were subjected to either of the light treatments. Dry matter partitioning, leaf area, stem and total root length were determined for ‘Kara’ plants exposed to ‘Alva’ VOCs, and also for ‘Alva’ plants exposed to either control or far-red-enriched light treatments. Key Results Total VOC emissions by ‘Alva’ were reduced under low R:FR conditions compared with control light conditions, although individual volatile compounds were found to be either suppressed, induced or not affected by R:FR. The altered composition of the VOC blend emitted by ‘Alva’ plants exposed to low R:FR was found to affect carbon allocation in receiver plants of ‘Kara’. Conclusions The results indicate that changes in R:FR light conditions influence the emissions of VOCs in barley, and that these altered emissions

Red:far-red light conditions affect the emission of volatile organic compounds from barley (Hordeum vulgare), leading to altered biomass allocation in neighbouring plants.

PubMed

Kegge, Wouter; Ninkovic, Velemir; Glinwood, Robert; Welschen, Rob A M; Voesenek, Laurentius A C J; Pierik, Ronald

2015-05-01

Volatile organic compounds (VOCs) play various roles in plant-plant interactions, and constitutively produced VOCs might act as a cue to sense neighbouring plants. Previous studies have shown that VOCs emitted from the barley (Hordeum vulgare) cultivar 'Alva' cause changes in biomass allocation in plants of the cultivar 'Kara'. Other studies have shown that shading and the low red:far-red (R:FR) conditions that prevail at high plant densities can reduce the quantity and alter the composition of the VOCs emitted by Arabidopsis thaliana, but whether this affects plant-plant signalling remains unknown. This study therefore examines the effects of far-red light enrichment on VOC emissions and plant-plant signalling between 'Alva' and 'Kara'. The proximity of neighbouring plants was mimicked by supplemental far-red light treatment of VOC emitter plants of barley grown in growth chambers. Volatiles emitted by 'Alva' under control and far-red light-enriched conditions were analysed using gas chromatography-mass spectrometry (GC-MS). 'Kara' plants were exposed to the VOC blend emitted by the 'Alva' plants that were subjected to either of the light treatments. Dry matter partitioning, leaf area, stem and total root length were determined for 'Kara' plants exposed to 'Alva' VOCs, and also for 'Alva' plants exposed to either control or far-red-enriched light treatments. Total VOC emissions by 'Alva' were reduced under low R:FR conditions compared with control light conditions, although individual volatile compounds were found to be either suppressed, induced or not affected by R:FR. The altered composition of the VOC blend emitted by 'Alva' plants exposed to low R:FR was found to affect carbon allocation in receiver plants of 'Kara'. The results indicate that changes in R:FR light conditions influence the emissions of VOCs in barley, and that these altered emissions affect VOC-mediated plant-plant interactions. © The Author 2015. Published by Oxford University Press on

Exploiting algal NADPH oxidase for biophotovoltaic energy

DOE PAGES

Anderson, Alexander; Laohavisit, Anuphon; Blaby, Ian K.; ...

2015-01-29

Photosynthetic microbes exhibit light-dependent electron export across the cell membrane, which can generate electricity in biological photovoltaic (BPV) devices. How electrons are exported remains to be determined; the identification of mechanisms would help selection or generation of photosynthetic microbes capable of enhanced electrical output. We show that plasma membrane NADPH oxidase activity is a significant component of light-dependent generation of electricity by the unicellular green alga Chlamydomonas reinhardtii. NADPH oxidases export electrons across the plasma membrane to form superoxide anion from oxygen. The C. reinhardtii mutant lacking the NADPH oxidase encoded by RBO1 is impaired in both extracellular superoxide anionmore » production and current generation in a BPV device. Complementation with the wild-type gene restores both capacities, demonstrating the role of the enzyme in electron export. Monitoring light-dependent extracellular superoxide production with a colorimetric assay is shown to be an effective way of screening for electrogenic potential of candidate algal strains. Furthermore, the results show that algal NADPH oxidases are important for superoxide anion production and open avenues for optimizing the biological component of these devices.« less

NADPH Oxidase-Dependent Signaling in Endothelial Cells: Role in Physiology and Pathophysiology

PubMed Central

Ushio-Fukai, Masuko; Malik, Asrar B.

2009-01-01

Abstract Reactive oxygen species (ROS) including superoxide (O2·−) and hydrogen peroxide (H2O2) are produced endogenously in response to cytokines, growth factors; G-protein coupled receptors, and shear stress in endothelial cells (ECs). ROS function as signaling molecules to mediate various biological responses such as gene expression, cell proliferation, migration, angiogenesis, apoptosis, and senescence in ECs. Signal transduction activated by ROS, “oxidant signaling,” has received intense investigation. Excess amount of ROS contribute to various pathophysiologies, including endothelial dysfunction, atherosclerosis, hypertension, diabetes, and acute respiratory distress syndrome (ARDS). The major source of ROS in EC is a NADPH oxidase. The prototype phagaocytic NADPH oxidase is composed of membrane-bound gp91phox and p22hox, as well as cytosolic subunits such as p47phox, p67phox and small GTPase Rac. In ECs, in addition to all the components of phagocytic NADPH oxidases, homologues of gp91phox (Nox2) including Nox1, Nox4, and Nox5 are expressed. The aim of this review is to provide an overview of the emerging area of ROS derived from NADPH oxidase and oxidant signaling in ECs linked to physiological and pathophysiological functions. Understanding these mechanisms may provide insight into the NADPH oxidase and oxidant signaling components as potential therapeutic targets. Antioxid. Redox Signal. 11, 791–810. PMID:18783313

Antioxidant Protection of NADPH-Depleted Oligodendrocyte Precursor Cells Is Dependent on Supply of Reduced Glutathione.

PubMed

Kilanczyk, Ewa; Saraswat Ohri, Sujata; Whittemore, Scott R; Hetman, Michal

2016-08-01

The pentose phosphate pathway is the main source of NADPH, which by reducing oxidized glutathione, contributes to antioxidant defenses. Although oxidative stress plays a major role in white matter injury, significance of NADPH for oligodendrocyte survival has not been yet investigated. It is reported here that the NADPH antimetabolite 6-amino-NADP (6AN) was cytotoxic to cultured adult rat spinal cord oligodendrocyte precursor cells (OPCs) as well as OPC-derived oligodendrocytes. The 6AN-induced necrosis was preceded by increased production of superoxide, NADPH depletion, and lower supply of reduced glutathione. Moreover, survival of NADPH-depleted OPCs was improved by the antioxidant drug trolox. Such cells were also protected by physiological concentrations of the neurosteroid dehydroepiandrosterone (10(-8) M). The protection by dehydroepiandrosterone was associated with restoration of reduced glutathione, but not NADPH, and was sensitive to inhibition of glutathione synthesis. A similar protective mechanism was engaged by the cAMP activator forskolin or the G protein-coupled estrogen receptor (GPER/GPR30) ligand G1. Finally, treatment with the glutathione precursor N-acetyl cysteine reduced cytotoxicity of 6AN. Taken together, NADPH is critical for survival of OPCs by supporting their antioxidant defenses. Consequently, injury-associated inhibition of the pentose phosphate pathway may be detrimental for the myelination or remyelination potential of the white matter. Conversely, steroid hormones and cAMP activators may promote survival of NADPH-deprived OPCs by increasing a NADPH-independent supply of reduced glutathione. Therefore, maintenance of glutathione homeostasis appears as a critical effector mechanism for OPC protection against NADPH depletion and preservation of the regenerative potential of the injured white matter. © The Author(s) 2016.

Isolation and Characterization of a Soluble NADPH-Dependent Fe(III) Reductase from Geobacter sulfurreducens

PubMed Central

Kaufmann, Franz; Lovley, Derek R.

2001-01-01

NADPH is an intermediate in the oxidation of organic compounds coupled to Fe(III) reduction in Geobacter species, but Fe(III) reduction with NADPH as the electron donor has not been studied in these organisms. Crude extracts of Geobacter sulfurreducens catalyzed the NADPH-dependent reduction of Fe(III)-nitrilotriacetic acid (NTA). The responsible enzyme, which was recovered in the soluble protein fraction, was purified to apparent homogeneity in a four-step procedure. Its specific activity for Fe(III) reduction was 65 μmol · min−1 · mg−1. The soluble Fe(III) reductase was specific for NADPH and did not utilize NADH as an electron donor. Although the enzyme reduced several forms of Fe(III), Fe(III)-NTA was the preferred electron acceptor. The protein possessed methyl viologen:NADP+ oxidoreductase activity and catalyzed the reduction of NADP+ with reduced methyl viologen as electron donor at a rate of 385 U/mg. The enzyme consisted of two subunits with molecular masses of 87 and 78 kDa and had a native molecular mass of 320 kDa, as determined by gel filtration. The purified enzyme contained 28.9 mol of Fe, 17.4 mol of acid-labile sulfur, and 0.7 mol of flavin adenine dinucleotide per mol of protein. The genes encoding the two subunits were identified in the complete sequence of the G. sulfurreducens genome from the N-terminal amino acid sequences derived from the subunits of the purified protein. The sequences of the two subunits had about 30% amino acid identity to the respective subunits of the formate dehydrogenase from Moorella thermoacetica, but the soluble Fe(III) reductase did not possess formate dehydrogenase activity. This soluble Fe(III) reductase differs significantly from previously characterized dissimilatory and assimilatory Fe(III) reductases in its molecular composition and cofactor content. PMID:11443080

Genetic differentiation and geographical relationship of Asian barley landraces using SSRs

USDA-ARS?s Scientific Manuscript database

Genetic diversity in 403 morphologically distinctive landraces of barley (Hordeum vulgare L. subsp. vulgare) originating from seven geographical zones of Asia was studied using simple sequence repeat (SSR) markers. The seven polymorphic SSR markers representing each chromosome chosen for this study ...

Loss of centromeric histone H3 (CENH3) from centromeres precedes uniparental chromosome elimination in interspecific barley hybrids.

PubMed

Sanei, Maryam; Pickering, Richard; Kumke, Katrin; Nasuda, Shuhei; Houben, Andreas

2011-08-16

Uniparental chromosome elimination occurs in several interspecific hybrids of plants. We studied the mechanism underlying selective elimination of the paternal chromosomes during the early development of Hordeum vulgare × Hordeum bulbosum embryos. The following conclusions regarding the role of the centromere-specific histone H3 variant (CENH3) in the process of chromosome elimination were drawn: (i) centromere inactivity of H. bulbosum chromosomes triggers the mitosis-dependent process of uniparental chromosome elimination in unstable H. vulgare × H. bulbosum hybrids; (ii) centromeric loss of CENH3 protein rather than uniparental silencing of CENH3 genes causes centromere inactivity; (iii) in stable species combinations, cross-species incorporation of CENH3 occurs despite centromere-sequence differences, and not all CENH3 variants get incorporated into centromeres if multiple CENH3s are present in species combinations; and (iv) diploid barley species encode two CENH3 variants, the proteins of which are intermingled within centromeres throughout mitosis and meiosis.

The Sublime and the Vulgar.

ERIC Educational Resources Information Center

Swann, Karen

1990-01-01

Explores how Edmund Burke's discourse on the sublime helps illuminate attacks on the vulgarization of culture (as typified by Allan Bloom), both for the presumedly "vulgar" reader and for the champions of high culture. (MG)

Efficacy of Essential Oils of Thymus vulgaris and Origanum vulgare on Echinococcus granulosus

PubMed Central

Pensel, P. E.; Maggiore, M. A.; Gende, L. B.; Eguaras, M. J.; Denegri, M. G.; Elissondo, M. C.

2014-01-01

The aim of the present work was to determine the in vitro effect of T. vulgaris and O. vulgare essential oils against E. granulosus protoscoleces and cysts. Essential oils were added to the medium resulting in thymol final concentrations of 10 μg/mL. The essential oils had a time-dependent effect provoking the complete loss of protoscolex viability after 72 days of postincubation. The results were confirmed at the ultrastructure level. Loss of infectivity in protoscoleces incubated with O. vulgare after 60 days was observed. On the other hand, the weight of cysts recorded in mice inoculated with T. vulgaris treated protoscoleces was significantly lower than that obtained in control group. Gamma-glutamyl-transpeptidase activity was readily detected in the culture supernatant of protoscoleces treated either with the essential oils or thymol. T. vulgaris and O. vulgare essential oils and thymol can induce cell apoptosis of protoscoleces after short incubation times. The efficacy of T. vulgaris and O. vulgare essential oils was also demonstrated in vitro on E. granulosus murine cysts. Our data suggest that essential oils of T. vulgaris and O. vulgare have anthelmintic effect against protoscoleces and cysts of E. granulosus. PMID:25180033

Thioredoxin and NADPH-Dependent Thioredoxin Reductase C Regulation of Tetrapyrrole Biosynthesis.

PubMed

Da, Qingen; Wang, Peng; Wang, Menglong; Sun, Ting; Jin, Honglei; Liu, Bing; Wang, Jinfa; Grimm, Bernhard; Wang, Hong-Bin

2017-10-01

In chloroplasts, thioredoxin (TRX) isoforms and NADPH-dependent thioredoxin reductase C (NTRC) act as redox regulatory factors involved in multiple plastid biogenesis and metabolic processes. To date, less is known about the functional coordination between TRXs and NTRC in chlorophyll biosynthesis. In this study, we aimed to explore the potential functions of TRX m and NTRC in the regulation of the tetrapyrrole biosynthesis (TBS) pathway. Silencing of three genes, TRX m1 , TRX m2 , and TRX m4 ( TRX ms ), led to pale-green leaves, a significantly reduced 5-aminolevulinic acid (ALA)-synthesizing capacity, and reduced accumulation of chlorophyll and its metabolic intermediates in Arabidopsis ( Arabidopsis thaliana ). The contents of ALA dehydratase, protoporphyrinogen IX oxidase, the I subunit of Mg-chelatase, Mg-protoporphyrin IX methyltransferase (CHLM), and NADPH-protochlorophyllide oxidoreductase were decreased in triple TRX m- silenced seedlings compared with the wild type, although the transcript levels of the corresponding genes were not altered significantly. Protein-protein interaction analyses revealed a physical interaction between the TRX m isoforms and CHLM. 4-Acetoamido-4-maleimidylstilbene-2,2-disulfonate labeling showed the regulatory impact of TRX ms on the CHLM redox status. Since CHLM also is regulated by NTRC (Richter et al., 2013), we assessed the concurrent functions of TRX m and NTRC in the control of CHLM. Combined deficiencies of three TRX m isoforms and NTRC led to a cumulative decrease in leaf pigmentation, TBS intermediate contents, ALA synthesis rate, and CHLM activity. We discuss the coordinated roles of TRX m and NTRC in the redox control of CHLM stability with its corollary activity in the TBS pathway. © 2017 American Society of Plant Biologists. All Rights Reserved.

Role of Ser-257 in the sliding mechanism of NADP(H) in the reaction catalyzed by the Aspergillus fumigatus flavin-dependent ornithine N5-monooxygenase SidA.

PubMed

Shirey, Carolyn; Badieyan, Somayesadat; Sobrado, Pablo

2013-11-08

SidA (siderophore A) is a flavin-dependent N-hydroxylating monooxygenase that is essential for virulence in Aspergillus fumigatus. SidA catalyzes the NADPH- and oxygen-dependent formation of N(5)-hydroxyornithine. In this reaction, NADPH reduces the flavin, and the resulting NADP(+) is the last product to be released. The presence of NADP(+) is essential for activity, as it is required for stabilization of the C4a-hydroperoxyflavin, which is the hydroxylating species. As part of our efforts to determine the molecular details of the role of NADP(H) in catalysis, we targeted Ser-257 for site-directed mutagenesis and performed extensive characterization of the S257A enzyme. Using a combination of steady-state and stopped-flow kinetic experiments, substrate analogs, and primary kinetic isotope effects, we show that the interaction between Ser-257 and NADP(H) is essential for stabilization of the C4a-hydroperoxyflavin. Molecular dynamics simulation results suggest that Ser-257 functions as a pivot point, allowing the nicotinamide of NADP(+) to slide into position for stabilization of the C4a-hydroperoxyflavin.

Chlorella Induces Stomatal Closure via NADPH Oxidase-Dependent ROS Production and Its Effects on Instantaneous Water Use Efficiency in Vicia faba

PubMed Central

Li, Yan; Xu, Shan-Shan; Gao, Jing; Pan, Sha; Wang, Gen-Xuan

2014-01-01

Reactive oxygen species (ROS) have been established to participate in stomatal closure induced by live microbes and microbe-associated molecular patterns (MAMPs). Chlorella as a beneficial microorganism can be expected to trigger stomatal closure via ROS production. Here, we reported that Chlorella induced stomatal closure in a dose-and time-dependent manner in epidermal peels of Vicia faba. Using pharmacological methods in this work, we found that the Chlorella-induced stomatal closure was almost completely abolished by a hydrogen peroxide (H2O2) scavenger, catalase (CAT), significantly suppressed by an NADPH oxidase inhibitor, diphenylene iodonium chloride (DPI), and slightly affected by a peroxidase inhibitor, salicylhydroxamic acid (SHAM), suggesting that ROS production involved in Chlorella-induced stomatal closure is mainly mediated by DPI-sensitive NADPH oxidase. Additionally, Exogenous application of optimal concentrations of Chlorella suspension improved instantaneous water use efficiency (WUEi) in Vicia faba via a reduction in leaf transpiration rate (E) without a parallel reduction in net photosynthetic rate (Pn) assessed by gas-exchange measurements. The chlorophyll fluorescence and content analysis further demonstrated that short-term use of Chlorella did not influence plant photosynthetic reactions center. These results preliminarily reveal that Chlorella can trigger stomatal closure via NADPH oxidase-dependent ROS production in epidermal strips and improve WUEi in leave levels. PMID:24687099

Chlorella induces stomatal closure via NADPH oxidase-dependent ROS production and its effects on instantaneous water use efficiency in Vicia faba.

PubMed

Li, Yan; Xu, Shan-Shan; Gao, Jing; Pan, Sha; Wang, Gen-Xuan

2014-01-01

Reactive oxygen species (ROS) have been established to participate in stomatal closure induced by live microbes and microbe-associated molecular patterns (MAMPs). Chlorella as a beneficial microorganism can be expected to trigger stomatal closure via ROS production. Here, we reported that Chlorella induced stomatal closure in a dose-and time-dependent manner in epidermal peels of Vicia faba. Using pharmacological methods in this work, we found that the Chlorella-induced stomatal closure was almost completely abolished by a hydrogen peroxide (H2O2) scavenger, catalase (CAT), significantly suppressed by an NADPH oxidase inhibitor, diphenylene iodonium chloride (DPI), and slightly affected by a peroxidase inhibitor, salicylhydroxamic acid (SHAM), suggesting that ROS production involved in Chlorella-induced stomatal closure is mainly mediated by DPI-sensitive NADPH oxidase. Additionally, Exogenous application of optimal concentrations of Chlorella suspension improved instantaneous water use efficiency (WUEi) in Vicia faba via a reduction in leaf transpiration rate (E) without a parallel reduction in net photosynthetic rate (Pn) assessed by gas-exchange measurements. The chlorophyll fluorescence and content analysis further demonstrated that short-term use of Chlorella did not influence plant photosynthetic reactions center. These results preliminarily reveal that Chlorella can trigger stomatal closure via NADPH oxidase-dependent ROS production in epidermal strips and improve WUEi in leave levels.

NADPH Oxidases in Vascular Pathology

PubMed Central

Konior, Anna; Schramm, Agata; Czesnikiewicz-Guzik, Marta

2014-01-01

Abstract Significance: Reactive oxygen species (ROS) play a critical role in vascular disease. While there are many possible sources of ROS, nicotinamide adenine dinucleotide phosphate (NADPH) oxidases play a central role. They are a source of “kindling radicals,” which affect other enzymes, such as nitric oxide synthase endothelial nitric oxide synthase or xanthine oxidase. This is important, as risk factors for atherosclerosis (hypertension, diabetes, hypercholesterolemia, and smoking) regulate the expression and activity of NADPH oxidases in the vessel wall. Recent Advances: There are seven isoforms in mammals: Nox1, Nox2, Nox3, Nox4, Nox5, Duox1 and Duox2. Nox1, Nox2, Nox4, and Nox5 are expressed in endothelium, vascular smooth muscle cells, fibroblasts, or perivascular adipocytes. Other homologues have not been found or are expressed at very low levels; their roles have not been established. Nox1/Nox2 promote the development of endothelial dysfunction, hypertension, and inflammation. Nox4 may have a role in protecting the vasculature during stress; however, when its activity is increased, it may be detrimental. Calcium-dependent Nox5 has been implicated in oxidative damage in human atherosclerosis. Critical Issues: NADPH oxidase-derived ROS play a role in vascular pathology as well as in the maintenance of normal physiological vascular function. We also discuss recently elucidated mechanisms such as the role of NADPH oxidases in vascular protection, vascular inflammation, pulmonary hypertension, tumor angiogenesis, and central nervous system regulation of vascular function and hypertension. Future Directions: Understanding the role of individual oxidases and interactions between homologues in vascular disease is critical for efficient pharmacological regulation of vascular NADPH oxidases in both the laboratory and clinical practice. Antioxid. Redox Signal. 20, 2794–2814. PMID:24180474

Resistance of Saccharomyces cerevisiae to High Concentrations of Furfural Is Based on NADPH-Dependent Reduction by at Least Two Oxireductases ▿ †

PubMed Central

Heer, Dominik; Heine, Daniel; Sauer, Uwe

2009-01-01

Biofuels derived from lignocellulosic biomass hold promises for a sustainable fuel economy, but several problems hamper their economical feasibility. One important problem is the presence of toxic compounds in processed lignocellulosic hydrolysates, with furfural as a key toxin. While Saccharomyces cerevisiae has some intrinsic ability to reduce furfural to the less-toxic furfuryl alcohol, higher resistance is necessary for process conditions. By comparing an evolved, furfural-resistant strain and its parent in microaerobic, glucose-limited chemostats at increasing furfural challenge, we elucidate key mechanism and the molecular basis of both natural and high-level furfural resistance. At lower concentrations of furfural, NADH-dependent oxireductases are the main defense mechanism. At furfural concentrations above 15 mM, however, 13C-flux and global array-based transcript analysis demonstrated that the NADPH-generating flux through the pentose phosphate pathway increases and that NADPH-dependent oxireductases become the major resistance mechanism. The transcript analysis further revealed that iron transmembrane transport is upregulated in response to furfural. While these responses occur in both strains, high-level resistance in the evolved strain was based on strong induction of ADH7, the uncharacterized open reading frame (ORF) YKL071W, and four further, likely NADPH-dependent, oxireductases. By overexpressing the ADH7 gene and the ORF YKL071W, we inversely engineered significantly increased furfural resistance in the parent strain, thereby demonstrating that these two enzymes are key elements of the resistance phenotype. PMID:19854918

Thioredoxin f1 and NADPH-Dependent Thioredoxin Reductase C Have Overlapping Functions in Regulating Photosynthetic Metabolism and Plant Growth in Response to Varying Light Conditions.

PubMed

Thormählen, Ina; Meitzel, Tobias; Groysman, Julia; Öchsner, Alexandra Bianca; von Roepenack-Lahaye, Edda; Naranjo, Belén; Cejudo, Francisco J; Geigenberger, Peter

2015-11-01

Two different thiol redox systems exist in plant chloroplasts, the ferredoxin-thioredoxin (Trx) system, which depends on ferredoxin reduced by the photosynthetic electron transport chain and, thus, on light, and the NADPH-dependent Trx reductase C (NTRC) system, which relies on NADPH and thus may be linked to sugar metabolism in the dark. Previous studies suggested, therefore, that the two different systems may have different functions in plants. We now report that there is a previously unrecognized functional redundancy of Trx f1 and NTRC in regulating photosynthetic metabolism and growth. In Arabidopsis (Arabidopsis thaliana) mutants, combined, but not single, deficiencies of Trx f1 and NTRC led to severe growth inhibition and perturbed light acclimation, accompanied by strong impairments of Calvin-Benson cycle activity and starch accumulation. Light activation of key enzymes of these pathways, fructose-1,6-bisphosphatase and ADP-glucose pyrophosphorylase, was almost completely abolished. The subsequent increase in NADPH-NADP(+) and ATP-ADP ratios led to increased nitrogen assimilation, NADP-malate dehydrogenase activation, and light vulnerability of photosystem I core proteins. In an additional approach, reporter studies show that Trx f1 and NTRC proteins are both colocalized in the same chloroplast substructure. Results provide genetic evidence that light- and NADPH-dependent thiol redox systems interact at the level of Trx f1 and NTRC to coordinately participate in the regulation of the Calvin-Benson cycle, starch metabolism, and growth in response to varying light conditions. © 2015 American Society of Plant Biologists. All Rights Reserved.

Depletion of NADP(H) due to CD38 activation triggers endothelial dysfunction in the postischemic heart.

PubMed

Reyes, Levy A; Boslett, James; Varadharaj, Saradhadevi; De Pascali, Francesco; Hemann, Craig; Druhan, Lawrence J; Ambrosio, Giuseppe; El-Mahdy, Mohamed; Zweier, Jay L

2015-09-15

In the postischemic heart, coronary vasodilation is impaired due to loss of endothelial nitric oxide synthase (eNOS) function. Although the eNOS cofactor tetrahydrobiopterin (BH4) is depleted, its repletion only partially restores eNOS-mediated coronary vasodilation, indicating that other critical factors trigger endothelial dysfunction. Therefore, studies were performed to characterize the unidentified factor(s) that trigger endothelial dysfunction in the postischemic heart. We observed that depletion of the eNOS substrate NADPH occurs in the postischemic heart with near total depletion from the endothelium, triggering impaired eNOS function and limiting BH4 rescue through NADPH-dependent salvage pathways. In isolated rat hearts subjected to 30 min of ischemia and reperfusion (I/R), depletion of the NADP(H) pool occurred and was most marked in the endothelium, with >85% depletion. Repletion of NADPH after I/R increased NOS-dependent coronary flow well above that with BH4 alone. With combined NADPH and BH4 repletion, full restoration of NOS-dependent coronary flow occurred. Profound endothelial NADPH depletion was identified to be due to marked activation of the NAD(P)ase-activity of CD38 and could be prevented by inhibition or specific knockdown of this protein. Depletion of the NADPH precursor, NADP(+), coincided with formation of 2'-phospho-ADP ribose, a CD38-derived signaling molecule. Inhibition of CD38 prevented NADP(H) depletion and preserved endothelium-dependent relaxation and NO generation with increased recovery of contractile function and decreased infarction in the postischemic heart. Thus, CD38 activation is an important cause of postischemic endothelial dysfunction and presents a novel therapeutic target for prevention of this dysfunction in unstable coronary syndromes.

Advanced oxidation protein products induce inflammatory response in fibroblast-like synoviocytes through NADPH oxidase -dependent activation of NF-κB.

PubMed

Zheng, Shuai; Zhong, Zhao-Ming; Qin, Shuai; Chen, Guo-Xian; Wu, Qian; Zeng, Ji-Huan; Ye, Wen-Bin; Li, Wei; Yuan, Kai; Yao, Ling; Chen, Jian-Ting

2013-01-01

Advanced oxidation protein products (AOPPs), a marker of oxidative stress, are prevalent in many kinds of disorders. Rheumatoid arthritis (RA), mainly resulting from the dysfunction of fibroblast-like synoviocytes (FLSs), is related to oxidative stress. Although the increased levels of AOPPs in RA patients were reported, the effect of AOPPs on FLSs function still remains unclear. Therefore, our study aims to investigate whether AOPPs have an effect on the inflammatory response of FLSs in vitro. FLSs obtained from both knees of rats were treated with or without AOPPs-modified rat serum albumin (AOPPs-RSA) in vitro. The mRNA and protein expression of tumor necrosis factor (TNF)-α, interleukin(IL)-1β, matrix metalloproteinases(MMP)-3, MMP-13 and vascular endothelial growth factor (VEGF) were measured by real-time quantitative polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA), respectively. Reactive oxygen species (ROS) generation was detected by fluorescent microscope and fluorescence microplate reader. Immunoprecipitation, Co-Immunoprecipitation and western blot were performed to examine the activity of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and nuclear factor kappa B (NF-κB). Exposure of FLSs to AOPPs upregulated the mRNA and protein expression of TNF-α, IL-1β, MMP-3, MMP-13 and VEGF in a concentration dependent manner. AOPPs treatment triggered ROS production in FLSs, which was significantly abolished by ROS scavenger N-acetyl-L-cysteine (NAC), superoxide dismutase (SOD), NADPH oxidase inhibitors diphenyleneiodonium (DPI) and apocynin. Challenged AOPPs induced phosphorylation of p47(phox), triggered an interaction between p47(phox), p22(phox) and gp91(phox), and significantly upregulated expression of NADPH oxidase subunits p47(phox), p22(phox) and gp91(phox). IκB degradation and nuclear translocation of NF-κB p65 induced by AOPPs were significantly blocked by SOD, NAC, DPI and apocynin. These data indicate that

Structural and expressional analysis of the B-hordein genes in Tibetan hull-less barley

USDA-ARS?s Scientific Manuscript database

The B-hordein gene family was analyzed from two Tibetan hull-less barley cultivars, Z09 and Z26 (Hordeum vulgare subsp. vulgare). Fourteen B-hordein genes, designated BZ09-2 to BZ09-5 (from Z09) and BZ26-1 to BZ26-10 (from Z26), were sequenced. Seven of them similar to a previously reported BZ09-1...

Leucanthemum vulgare Lam. crude oil phytoremediation.

PubMed

Noori, Azam; Maivan, Hassan Zare; Alaie, Ebrahim; Newman, Lee A

2015-06-29

Sites with crude oil pollution have been successfully treated using phytoremediation, but expanding the range of plants that can be used and understanding how exposure impacts the plants are two areas of study that are important to continue. Leucanthemum vulgare has been shown to grow well under a variety of stressful conditions. To examine L. vulgare's ability to both survive crude oil exposure and to reduce crude oil concentrations in soil, plants were placed in soil containing 0, 2.5%, 5%, 7.5% or 10% w/w crude oil. Total Petroleum Hydrocarbons (TPH) concentration, peroxidase and catalase activity, proline and phenol content in roots and leaves were determined at the start of planting and every 2 months for six months. L. vulgare roots were successfully colonized with mycorrhizae under all conditions. Results showed positive correlation between antioxidant compound concentration and crude oil contamination. Also, a significant reduction occurred in TPH content of soil over time in planted pots as compared to controls. The lowest TPH content was recorded after 6 months under all treatments. Results showed L. vulgare could survive crude oil exposure and enhance reducing of crude oil from soil.

In Vivo and in Vitro Studies of Glucose-6-Phosphate Dehydrogenase from Barley Root Plastids in Relation to Reductant Supply for NO2- Assimilation.

PubMed Central

Wright, D. P.; Huppe, H. C.; Turpin, D. H.

1997-01-01

Pyridine nucleotide pools were measured in intact plastids from roots of barley (Hordeum vulgare L.) during the onset of NO2- assimilation and compared with the in vitro effect of the NADPH/NADP ratio on the activity of plastidic glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) from N-sufficient or N-starved roots. The NADPH/NADP ratio increased from 0.9 to 2.0 when 10 mM glucose-6-phosphate was supplied to intact plastids. The subsequent addition of 1 mM NaNO2 caused a rapid decline in this ratio to 1.5. In vitro, a ratio of 1.5 inactivated barley root plastid G6PDH by approximately 50%, suggesting that G6PDH could remain active during NO2- assimilation even at the high NADPH/NADP ratios that would favor a reduction of ferredoxin, the electron donor of NO2- reductase. Root plastid G6PDH was sensitive to reductive inhibition by dithiothreitol (DTT), but even at 50 mM DTT the enzyme remained more than 35% active. In root plastids from barley starved of N for 3 d, G6PDH had a substantially reduced specific activity, had a lower Km for NADP, and was less inhibited by DTT than the enzyme from N-sufficient root plastids, indicating that there was some effect of N starvation on the G6PDH activity in barley root plastids. PMID:12223780

Hydrolysis of (1,4)-β-D-mannans in barley (Hordeum vulgare L.) is mediated by the concerted action of (1,4)-β-D-mannan endohydrolase and β-D-mannosidase

PubMed Central

Hrmova, Maria; Burton, Rachel A.; Biely, Peter; Lahnstein, Jelle; Fincher, Geoffrey B.

2006-01-01

A family GH5 (family 5 glycoside hydrolase) (1,4)-β-D-mannan endohydrolase or β-D-mannanase (EC 3.2.1.78), designated HvMAN1, has been purified 300-fold from extracts of 10-day-old barley (Hordeum vulgare L.) seedlings using ammonium sulfate fractional precipitation, followed by ion exchange, hydrophobic interaction and size-exclusion chromatography. The purified HvMAN1 is a relatively unstable enzyme with an apparent molecular mass of 43 kDa, a pI of 7.8 and a pH optimum of 4.75. The HvMAN1 releases Man (mannose or D-mannopyranose)-containing oligosaccharides of degree of polymerization 2–6 from mannans, galactomannans and glucomannans. With locust-bean galactomannan and mannopentaitol as substrates, the enzyme has Km constants of 0.16 mg·ml−1 and 5.3 mM and kcat constants of 12.9 and 3.9 s−1 respectively. Product analyses indicate that transglycosylation reactions occur during hydrolysis of (1,4)-β-D-manno-oligosaccharides. The complete sequence of 374 amino acid residues of the mature enzyme has been deduced from the nucleotide sequence of a near full-length cDNA, and has allowed a three-dimensional model of the HvMAN1 to be constructed. The barley HvMAN1 gene is a member of a small (1,4)-β-D-mannan endohydrolase family of at least six genes, and is transcribed at low levels in a number of organs, including the developing endosperm, but also in the basal region of young roots and in leaf tips. A second barley enzyme that participates in mannan depolymerization through its ability to hydrolyse (1,4)-β-D-manno-oligosaccharides to Man is a family GH1 β-D-mannosidase, now designated HvβMANNOS1, but previously identified as a β-D-glucosidase [Hrmova, MacGregor, Biely, Stewart and Fincher (1998) J. Biol. Chem. 273, 11134–11143], which hydrolyses 4NP (4-nitrophenyl) β-D-mannoside three times faster than 4NP β-D-glucoside, and has an action pattern typical of a (1,4)-β-D-mannan exohydrolase. PMID:16771710

NADPH oxidase/ROS-dependent PYK2 activation is involved in TNF-α-induced matrix metalloproteinase-9 expression in rat heart-derived H9c2 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Chuen-Mao, E-mail: chuenmao@mail.cgu.edu.tw; Heart Failure Center, Division of Cardiology, Department of Internal Medicine, Chang Gung Memorial Hospital at Keelung, Keelung, Taiwan; Lee, I-Ta

TNF-α plays a mediator role in the pathogenesis of chronic heart failure contributing to cardiac remodeling and peripheral vascular disturbances. The implication of TNF-α in inflammatory responses has been shown to be mediated through up-regulation of matrix metalloproteinase-9 (MMP-9). However, the detailed mechanisms of TNF-α-induced MMP-9 expression in rat embryonic-heart derived H9c2 cells are largely not defined. We demonstrated that in H9c2 cells, TNF-α induced MMP-9 mRNA and protein expression associated with an increase in the secretion of pro-MMP-9. TNF-α-mediated responses were attenuated by pretreatment with the inhibitor of ROS (N-acetyl-L-cysteine, NAC), NADPH oxidase [apocynin (APO) or diphenyleneiodonium chloride (DPI)],more » MEK1/2 (U0126), p38 MAPK (SB202190), JNK1/2 (SP600125), NF-κB (Bay11-7082), or PYK2 (PF-431396) and transfection with siRNA of TNFR1, p47{sup phox}, p42, p38, JNK1, p65, or PYK2. Moreover, TNF-α markedly induced NADPH oxidase-derived ROS generation in these cells. TNF-α-enhanced p42/p44 MAPK, p38 MAPK, JNK1/2, and NF-κB (p65) phosphorylation and in vivo binding of p65 to the MMP-9 promoter were inhibited by U0126, SB202190, SP600125, NAC, DPI, or APO. In addition, TNF-α-mediated PYK2 phosphorylation was inhibited by NAC, DPI, or APO. PYK2 inhibition could reduce TNF-α-stimulated MAPKs and NF-κB activation. Thus, in H9c2 cells, we are the first to show that TNF-α-induced MMP-9 expression is mediated through a TNFR1/NADPH oxidase/ROS/PYK2/MAPKs/NF-κB cascade. We demonstrated that NADPH oxidase-derived ROS generation is involved in TNF-α-induced PYK2 activation in these cells. Understanding the regulation of MMP-9 expression and NADPH oxidase activation by TNF-α on H9c2 cells may provide potential therapeutic targets of chronic heart failure. - Highlights: • TNF-α induces MMP-9 secretion and expression via a TNFR1-dependent pathway. • TNF-α induces ROS/PYK2-dependent MMP-9 expression in H9c2 cells. • TNF

Clavicipitaceous anamorphic endophytes in Hordeum germplasm

Treesearch

A. Dan Wilson

2007-01-01

The incidence of clavicipitaceous anamorphic endophytes, non-choke inducing endosymbiotic fungi of the genus Neotyphodium that systemically infect grasses, in eighteen Hordeum species from the U.S. National Plant Germplasm System was examined using light and Scanning Electron Microscopy (SEM). Seventeen plant inventory accessions...

Stress inducible expression of the DREB1A transcription factor from xeric, Hordeum spontaneum L. in turf and forage grass (Paspalum notatum Flugge) enhances abiotic stress tolerance.

PubMed

James, Victoria A; Neibaur, Isaac; Altpeter, Fredy

2008-02-01

The dehydration-responsive element binding proteins (DREB1)/C-repeat (CRT) binding factors (CBF) function as transcription activators and bind to the DRE/CRT cis-acting element commonly present in the promoters of abiotic stress-regulated genes. A DREB1A transcription factor ortholog was isolated from a xeric, wild barley (Hordeum spontaneum L.) accession, originating from the Negev desert. Sequence comparison revealed a very high degree of sequence conservation of HsDREB1A to the published barley (Hordeum vulgare L.) DREB1A. Constitutive expression of the HsDREB1A gene was able to trans-activate a reporter gene under transcriptional control of the stress-inducible HVA1s and Dhn8 promoters. HsDREB1A was subcloned under transcriptional control of the stress-inducible barley HVA1s promoter and introduced into the apomictic bahiagrass (Paspalum notatum Flugge) cultivar 'Argentine'. HsDREB1A integration and stress inducible expression was detected in primary transgenic bahiagrass plants and apomictic seed progeny by Southern blot, RT-PCR and northern blot analysis respectively. Transgenic bahiagrass plants with stress-inducible expression of HsDREB1A survived severe salt stress and repeated cycles of severe dehydration stress under controlled environment conditions, in contrast to non-transgenic plants. The observed abiotic stress tolerance is very desirable in turf and forage grasses like bahiagrass, where seasonal droughts and irrigation restrictions affect establishment, persistence or productivity of this perennial crop.

NADPH oxidase inhibitors: a patent review.

PubMed

Kim, Jung-Ae; Neupane, Ganesh Prasad; Lee, Eung Seok; Jeong, Byeong-Seon; Park, Byung Chul; Thapa, Pritam

2011-08-01

NADPH oxidases, a family of multi-subunit enzyme complexes, catalyze the production of reactive oxygen species (ROS), which may contribute to the pathogenesis of a variety of diseases. In addition to the first NADPH oxidase found in phagocytes, four non-phagocytic NADPH oxidase isoforms have been identified, which all differ in their catalytic subunit (Nox1-5) and tissue distribution. This paper provides a comprehensive review of the patent literature on NADPH oxidase inhibitors, small molecule Nox inhibitors, peptides and siRNAs. Since each member of the NADPH oxidase family has great potential as a therapeutic target, several different compounds have been registered as NADPH oxidase inhibitors in the patent literature. As yet, none have gone through clinical trials, and some have not completed preclinical trials, including safety and specificity evaluation. Recently, small molecule pyrazolopyridine and triazolopyrimidine derivatives have been submitted as potent NADPH oxidase inhibitors and reported as first-in-class inhibitors for idiopathic pulmonary fibrosis and acute stroke, respectively. Further clinical efficacy and safety data are warranted to prove their actual clinical utility.

Enhancing biomass and ethanol production by increasing NADPH production in Synechocystis sp. PCC 6803.

PubMed

Choi, Yun-Nam; Park, Jong Moon

2016-08-01

This study demonstrates that increased NADPH production can improve biomass and ethanol production in cyanobacteria. We over-expressed the endogenous zwf gene, which encodes glucose-6-phosphate dehydrogenase of pentose phosphate pathway, in the model cyanobacterium Synechocystis sp. PCC 6803. zwf over-expression resulted in increased NADPH production, and promoted biomass production compared to the wild type in both autotrophic and mixotrophic conditions. Ethanol production pathway including NADPH-dependent alcohol dehydrogenase was also integrated with and without zwf over-expression. Excessive NADPH production by zwf over-expression could improve both biomass and ethanol production in the autotrophic conditions. Copyright © 2016 Elsevier Ltd. All rights reserved.

Silencing of NADPH-Dependent Oxidoreductase Genes (yqhD and dkgA) in Furfural-Resistant Ethanologenic Escherichia coli▿

PubMed Central

Miller, E. N.; Jarboe, L. R.; Yomano, L. P.; York, S. W.; Shanmugam, K. T.; Ingram, L. O.

2009-01-01

Low concentrations of furfural are formed as a side product during the dilute acid hydrolysis of hemicellulose. Growth is inhibited by exposure to furfural but resumes after the complete reduction of furfural to the less toxic furfuryl alcohol. Growth-based selection was used to isolate a furfural-resistant mutant of ethanologenic Escherichia coli LY180, designated strain EMFR9. Based on mRNA expression levels in the parent and mutant in response to furfural challenge, genes encoding 12 oxidoreductases were found to vary by more than twofold (eight were higher in EMFR9; four were higher in the parent). All 12 genes were cloned. When expressed from plasmids, none of the eight genes in the first group increased furfural tolerance in the parent (LY180). Expression of three of the silenced genes (yqhD, dkgA, and yqfA) in EMFR9 was found to decrease furfural tolerance compared to that in the parent. Purified enzymes encoded by yqhD and dkgA were shown to have NADPH-dependent furfural reductase activity. Both exhibited low Km values for NADPH (8 μM and 23 μM, respectively), similar to those of biosynthetic reactions. Furfural reductase activity was not associated with yqfA. Deleting yqhD and dkgA in the parent (LY180) increased furfural tolerance, but not to the same extent observed in the mutant EMFR9. Together, these results suggest that the process of reducing furfural by using an enzyme with a low Km for NADPH rather than a direct inhibitory action is the primary cause for growth inhibition by low concentrations of furfural. PMID:19429550

Dual utilization of NADPH and NADH cofactors enhances xylitol production in engineered Saccharomyces cerevisiae.

PubMed

Jo, Jung-Hyun; Oh, Sun-Young; Lee, Hyeun-Soo; Park, Yong-Cheol; Seo, Jin-Ho

2015-12-01

Xylitol, a natural sweetener, can be produced by hydrogenation of xylose in hemicelluloses. In microbial processes, utilization of only NADPH cofactor limited commercialization of xylitol biosynthesis. To overcome this drawback, Saccharomyces cerevisiae D452-2 was engineered to express two types of xylose reductase (XR) with either NADPH-dependence or NADH-preference. Engineered S. cerevisiae DWM expressing both the XRs exhibited higher xylitol productivity than the yeast strain expressing NADPH-dependent XR only (DWW) in both batch and glucose-limited fed-batch cultures. Furthermore, the coexpression of S. cerevisiae ZWF1 and ACS1 genes in the DWM strain increased intracellular concentrations of NADPH and NADH and improved maximum xylitol productivity by 17%, relative to that for the DWM strain. Finally, the optimized fed-batch fermentation of S. cerevisiae DWM-ZWF1-ACS1 resulted in 196.2 g/L xylitol concentration, 4.27 g/L h productivity and almost the theoretical yield. Expression of the two types of XR utilizing both NADPH and NADH is a promising strategy to meet the industrial demands for microbial xylitol production. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Thioredoxin and NADPH-Dependent Thioredoxin Reductase C Regulation of Tetrapyrrole Biosynthesis1[OPEN

PubMed Central

Sun, Ting; Jin, Honglei; Wang, Jinfa

2017-01-01

In chloroplasts, thioredoxin (TRX) isoforms and NADPH-dependent thioredoxin reductase C (NTRC) act as redox regulatory factors involved in multiple plastid biogenesis and metabolic processes. To date, less is known about the functional coordination between TRXs and NTRC in chlorophyll biosynthesis. In this study, we aimed to explore the potential functions of TRX m and NTRC in the regulation of the tetrapyrrole biosynthesis (TBS) pathway. Silencing of three genes, TRX m1, TRX m2, and TRX m4 (TRX ms), led to pale-green leaves, a significantly reduced 5-aminolevulinic acid (ALA)-synthesizing capacity, and reduced accumulation of chlorophyll and its metabolic intermediates in Arabidopsis (Arabidopsis thaliana). The contents of ALA dehydratase, protoporphyrinogen IX oxidase, the I subunit of Mg-chelatase, Mg-protoporphyrin IX methyltransferase (CHLM), and NADPH-protochlorophyllide oxidoreductase were decreased in triple TRX m-silenced seedlings compared with the wild type, although the transcript levels of the corresponding genes were not altered significantly. Protein-protein interaction analyses revealed a physical interaction between the TRX m isoforms and CHLM. 4-Acetoamido-4-maleimidylstilbene-2,2-disulfonate labeling showed the regulatory impact of TRX ms on the CHLM redox status. Since CHLM also is regulated by NTRC (Richter et al., 2013), we assessed the concurrent functions of TRX m and NTRC in the control of CHLM. Combined deficiencies of three TRX m isoforms and NTRC led to a cumulative decrease in leaf pigmentation, TBS intermediate contents, ALA synthesis rate, and CHLM activity. We discuss the coordinated roles of TRX m and NTRC in the redox control of CHLM stability with its corollary activity in the TBS pathway. PMID:28827456

The Contribution of Nicotinamide Nucleotide Transhydrogenase to Peroxide Detoxification Is Dependent on the Respiratory State and Counterbalanced by Other Sources of NADPH in Liver Mitochondria*

PubMed Central

Ronchi, Juliana Aparecida; Francisco, Annelise; Passos, Luiz Augusto Correa; Figueira, Tiago Rezende; Castilho, Roger Frigério

2016-01-01

The forward reaction of nicotinamide nucleotide transhydrogenase (NNT) reduces NADP+ at the expense of NADH oxidation and H+ movement down the electrochemical potential across the inner mitochondrial membrane, establishing an NADPH/NADP+ ratio severalfold higher than the NADH/NAD+ ratio in the matrix. In turn, NADPH drives processes, such as peroxide detoxification and reductive biosynthesis. In this study, we generated a congenic mouse model carrying a mutated NntC57BL/6J allele from the C57BL/6J substrain. Suspensions of isolated mitochondria from Nnt+/+, Nnt+/−, and Nnt−/− mouse liver were biochemically evaluated and challenged with exogenous peroxide under different respiratory states. The respiratory substrates were also varied, and the participation of concurrent NADPH sources (i.e. isocitrate dehydrogenase-2, malic enzymes, and glutamate dehydrogenase) was assessed. The principal findings include the following: Nnt+/− and Nnt−/− exhibit ∼50% and absent NNT activity, respectively, but the activities of concurrent NADPH sources are unchanged. The lack of NNT activity in Nnt−/− mice impairs peroxide metabolism in intact mitochondria. The contribution of NNT to peroxide metabolism is decreased during ADP phosphorylation compared with the non-phosphorylating state; however, it is accompanied by increased contributions of concurrent NADPH sources, especially glutamate dehydrogenase. NNT makes a major contribution to peroxide metabolism during the blockage of mitochondrial electron transport. Interestingly, peroxide metabolism in the Nnt+/− mitochondria matched that in the Nnt+/+ mitochondria. Overall, this study demonstrates that the respiratory state and/or substrates that sustain energy metabolism markedly influence the relative contribution of NNT (i.e. varies between nearly 0 and 100%) to NADPH-dependent mitochondrial peroxide metabolism. PMID:27474736

The Contribution of Nicotinamide Nucleotide Transhydrogenase to Peroxide Detoxification Is Dependent on the Respiratory State and Counterbalanced by Other Sources of NADPH in Liver Mitochondria.

PubMed

Ronchi, Juliana Aparecida; Francisco, Annelise; Passos, Luiz Augusto Correa; Figueira, Tiago Rezende; Castilho, Roger Frigério

2016-09-16

The forward reaction of nicotinamide nucleotide transhydrogenase (NNT) reduces NADP(+) at the expense of NADH oxidation and H(+) movement down the electrochemical potential across the inner mitochondrial membrane, establishing an NADPH/NADP(+) ratio severalfold higher than the NADH/NAD(+) ratio in the matrix. In turn, NADPH drives processes, such as peroxide detoxification and reductive biosynthesis. In this study, we generated a congenic mouse model carrying a mutated Nnt(C57BL/6J) allele from the C57BL/6J substrain. Suspensions of isolated mitochondria from Nnt(+/+), Nnt(+/-), and Nnt(-/-) mouse liver were biochemically evaluated and challenged with exogenous peroxide under different respiratory states. The respiratory substrates were also varied, and the participation of concurrent NADPH sources (i.e. isocitrate dehydrogenase-2, malic enzymes, and glutamate dehydrogenase) was assessed. The principal findings include the following: Nnt(+/-) and Nnt(-/-) exhibit ∼50% and absent NNT activity, respectively, but the activities of concurrent NADPH sources are unchanged. The lack of NNT activity in Nnt(-/-) mice impairs peroxide metabolism in intact mitochondria. The contribution of NNT to peroxide metabolism is decreased during ADP phosphorylation compared with the non-phosphorylating state; however, it is accompanied by increased contributions of concurrent NADPH sources, especially glutamate dehydrogenase. NNT makes a major contribution to peroxide metabolism during the blockage of mitochondrial electron transport. Interestingly, peroxide metabolism in the Nnt(+/-) mitochondria matched that in the Nnt(+/+) mitochondria. Overall, this study demonstrates that the respiratory state and/or substrates that sustain energy metabolism markedly influence the relative contribution of NNT (i.e. varies between nearly 0 and 100%) to NADPH-dependent mitochondrial peroxide metabolism. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Pigment epithelium-derived factor stimulates skeletal muscle glycolytic activity through NADPH oxidase-dependent reactive oxygen species production.

PubMed

Carnagarin, Revathy; Carlessi, Rodrigo; Newsholme, Philip; Dharmarajan, Arun M; Dass, Crispin R

2016-09-01

Pigment epithelium-derived factor is a multifunctional serpin implicated in insulin resistance in metabolic disorders. Recent evidence suggests that exposure of peripheral tissues such as skeletal muscle to PEDF has profound metabolic consequences with predisposition towards chronic conditions such as obesity, type 2 diabetes, metabolic syndrome and polycystic ovarian syndrome. Chronic inflammation shifts muscle metabolism towards increased glycolysis and decreased oxidative metabolism. In the present study, we demonstrate a novel effect of PEDF on cellular metabolism in mouse cell line (C2C12) and human primary skeletal muscle cells. PEDF addition to skeletal muscle cells induced enhanced phospholipase A2 activity. This was accompanied with increased production of reactive oxygen species in a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-dependent manner that triggered a shift towards a more glycolytic phenotype. Extracellular flux analysis and glucose consumption assays demonstrated that PEDF treatment resulted in enhanced glycolysis but did not change mitochondrial respiration. Our results demonstrate that skeletal muscle cells express a PEDF-inducible oxidant generating system that enhances glycolysis but is sensitive to antioxidants and NADPH oxidase inhibition. Copyright © 2016 Elsevier Ltd. All rights reserved.

Distribution and Properties of a Potassium-dependent Asparaginase Isolated from Developing Seeds of Pisum sativum and Other Plants 1

PubMed Central

Sodek, Ladaslav; Lea, Peter J.; Miflin, Benjamin J.

1980-01-01

Asparaginase (EC 3.5.1.1) was isolated from the developing seed of Pisum sativum. The enzyme is dependent upon the presence of K+ for activity, although Na+ and Rb+ may substitute to a lesser extent. Maximum activity was obtained at K+ concentrations above 20 millimolar. Potassium ions protected the enzyme against heat denaturation. The enzyme has a molecular weight of 68,300. Asparaginase activity developed initially in the testa, with maximum activity (3.6 micromoles per hour per seed) being present 13 days after flowering. Maximum activity (1.2 micromoles per hour per seed) did not develop in the cotyledon until 21 days after flowering. Glutamine synthetase and glutamate dehydrogenase were also present in the testae and cotyledons but maximum activity developed later than that of asparaginase. Potassium-dependent asparaginase activity was also detected in the developing seeds of Vicia faba, Phaseolus multiflorus, Zea mays, Hordeum vulgare, and two Lupinus varieties. No stimulation of activity was detected with the enzyme isolated from Lupinus polyphyllus, which has previously been shown to contain a K+-independent enzyme. PMID:16661136

Genetic Diversity and Ecological Niche Modelling of Wild Barley: Refugia, Large-Scale Post-LGM Range Expansion and Limited Mid-Future Climate Threats?

PubMed Central

Russell, Joanne; van Zonneveld, Maarten; Dawson, Ian K.; Booth, Allan; Waugh, Robbie; Steffenson, Brian

2014-01-01

Describing genetic diversity in wild barley (Hordeum vulgare ssp. spontaneum) in geographic and environmental space in the context of current, past and potential future climates is important for conservation and for breeding the domesticated crop (Hordeum vulgare ssp. vulgare). Spatial genetic diversity in wild barley was revealed by both nuclear- (2,505 SNP, 24 nSSR) and chloroplast-derived (5 cpSSR) markers in 256 widely-sampled geo-referenced accessions. Results were compared with MaxEnt-modelled geographic distributions under current, past (Last Glacial Maximum, LGM) and mid-term future (anthropogenic scenario A2, the 2080s) climates. Comparisons suggest large-scale post-LGM range expansion in Central Asia and relatively small, but statistically significant, reductions in range-wide genetic diversity under future climate. Our analyses support the utility of ecological niche modelling for locating genetic diversity hotspots and determine priority geographic areas for wild barley conservation under anthropogenic climate change. Similar research on other cereal crop progenitors could play an important role in tailoring conservation and crop improvement strategies to support future human food security. PMID:24505252

Barley whole exome capture: a tool for genomic research in the genus Hordeum and beyond

PubMed Central

Mascher, Martin; Richmond, Todd A; Gerhardt, Daniel J; Himmelbach, Axel; Clissold, Leah; Sampath, Dharanya; Ayling, Sarah; Steuernagel, Burkhard; Pfeifer, Matthias; D'Ascenzo, Mark; Akhunov, Eduard D; Hedley, Pete E; Gonzales, Ana M; Morrell, Peter L; Kilian, Benjamin; Blattner, Frank R; Scholz, Uwe; Mayer, Klaus FX; Flavell, Andrew J; Muehlbauer, Gary J; Waugh, Robbie; Jeddeloh, Jeffrey A; Stein, Nils

2013-01-01

Advanced resources for genome-assisted research in barley (Hordeum vulgare) including a whole-genome shotgun assembly and an integrated physical map have recently become available. These have made possible studies that aim to assess genetic diversity or to isolate single genes by whole-genome resequencing and in silico variant detection. However such an approach remains expensive given the 5 Gb size of the barley genome. Targeted sequencing of the mRNA-coding exome reduces barley genomic complexity more than 50-fold, thus dramatically reducing this heavy sequencing and analysis load. We have developed and employed an in-solution hybridization-based sequence capture platform to selectively enrich for a 61.6 megabase coding sequence target that includes predicted genes from the genome assembly of the cultivar Morex as well as publicly available full-length cDNAs and de novo assembled RNA-Seq consensus sequence contigs. The platform provides a highly specific capture with substantial and reproducible enrichment of targeted exons, both for cultivated barley and related species. We show that this exome capture platform provides a clear path towards a broader and deeper understanding of the natural variation residing in the mRNA-coding part of the barley genome and will thus constitute a valuable resource for applications such as mapping-by-sequencing and genetic diversity analyzes. PMID:23889683

NADPH-generating systems in bacteria and archaea

PubMed Central

Spaans, Sebastiaan K.; Weusthuis, Ruud A.; van der Oost, John; Kengen, Servé W. M.

2015-01-01

Reduced nicotinamide adenine dinucleotide phosphate (NADPH) is an essential electron donor in all organisms. It provides the reducing power that drives numerous anabolic reactions, including those responsible for the biosynthesis of all major cell components and many products in biotechnology. The efficient synthesis of many of these products, however, is limited by the rate of NADPH regeneration. Hence, a thorough understanding of the reactions involved in the generation of NADPH is required to increase its turnover through rational strain improvement. Traditionally, the main engineering targets for increasing NADPH availability have included the dehydrogenase reactions of the oxidative pentose phosphate pathway and the isocitrate dehydrogenase step of the tricarboxylic acid (TCA) cycle. However, the importance of alternative NADPH-generating reactions has recently become evident. In the current review, the major canonical and non-canonical reactions involved in the production and regeneration of NADPH in prokaryotes are described, and their key enzymes are discussed. In addition, an overview of how different enzymes have been applied to increase NADPH availability and thereby enhance productivity is provided. PMID:26284036

Endophytic Fungi in a Hordeum Germplasm Collection

Treesearch

A. Dan Wilson; S.L. Clement; W.J. Kaiser

1991-01-01

The incidence of clavicipitaceous anamorphic endophytes in a Hordeum spp. germplasm collection is reported. The potential application of endophytes as biocontrol agents against pests of cereal crops is recognized. Suggestions are proposed to modify existing germplasm maintenance procedures to ensure that both seed viability and endophyte viability...

Regulating NETosis: Increasing pH Promotes NADPH Oxidase-Dependent NETosis

PubMed Central

Khan, Meraj A.; Philip, Lijy M.; Cheung, Guillaume; Vadakepeedika, Shawn; Grasemann, Hartmut; Sweezey, Neil; Palaniyar, Nades

2018-01-01

Neutrophils migrating from the blood (pH 7.35–7.45) into the surrounding tissues encounter changes in extracellular pH (pHe) conditions. Upon activation of NADPH oxidase 2 (Nox), neutrophils generate large amounts of H+ ions reducing the intracellular pH (pHi). Nevertheless, how extracellular pH regulates neutrophil extracellular trap (NET) formation (NETosis) is not clearly established. We hypothesized that increasing pH increases Nox-mediated production of reactive oxygen species (ROS) and neutrophil protease activity, stimulating NETosis. Here, we found that raising pHe (ranging from 6.6 to 7.8; every 0.2 units) increased pHi of both activated and resting neutrophils within 10–20 min (Seminaphtharhodafluor dual fluorescence measurements). Since Nox activity generates H+ ions, pHi is lower in neutrophils that are activated compared to resting. We also found that higher pH stimulated Nox-dependent ROS production (R123 generation; flow cytometry, plate reader assay, and imaging) during spontaneous and phorbol myristate acetate-induced NETosis (Sytox Green assays, immunoconfocal microscopy, and quantifying NETs). In neutrophils that are activated and not resting, higher pH stimulated histone H4 cleavage (Western blots) and NETosis. Raising pH increased Escherichia coli lipopolysaccharide-, Pseudomonas aeruginosa (Gram-negative)-, and Staphylococcus aureus (Gram-positive)-induced NETosis. Thus, higher pHe promoted Nox-dependent ROS production, protease activity, and NETosis; lower pH has the opposite effect. These studies provided mechanistic steps of pHe-mediated regulation of Nox-dependent NETosis. Raising pH either by sodium bicarbonate or Tris base (clinically known as Tris hydroxymethyl aminomethane, tromethamine, or THAM) increases NETosis. Each Tris molecule can bind 3H+ ions, whereas each bicarbonate HCO3− ion binds 1H+ ion. Therefore, the amount of Tris solution required to cause the same increase in pH level is less than that of equimolar

Regulating NETosis: Increasing pH Promotes NADPH Oxidase-Dependent NETosis.

PubMed

Khan, Meraj A; Philip, Lijy M; Cheung, Guillaume; Vadakepeedika, Shawn; Grasemann, Hartmut; Sweezey, Neil; Palaniyar, Nades

2018-01-01

Neutrophils migrating from the blood (pH 7.35-7.45) into the surrounding tissues encounter changes in extracellular pH (pH e ) conditions. Upon activation of NADPH oxidase 2 (Nox), neutrophils generate large amounts of H + ions reducing the intracellular pH (pH i ). Nevertheless, how extracellular pH regulates neutrophil extracellular trap (NET) formation (NETosis) is not clearly established. We hypothesized that increasing pH increases Nox-mediated production of reactive oxygen species (ROS) and neutrophil protease activity, stimulating NETosis. Here, we found that raising pH e (ranging from 6.6 to 7.8; every 0.2 units) increased pH i of both activated and resting neutrophils within 10-20 min (Seminaphtharhodafluor dual fluorescence measurements). Since Nox activity generates H + ions, pH i is lower in neutrophils that are activated compared to resting. We also found that higher pH stimulated Nox-dependent ROS production (R123 generation; flow cytometry, plate reader assay, and imaging) during spontaneous and phorbol myristate acetate-induced NETosis (Sytox Green assays, immunoconfocal microscopy, and quantifying NETs). In neutrophils that are activated and not resting, higher pH stimulated histone H4 cleavage (Western blots) and NETosis. Raising pH increased Escherichia coli lipopolysaccharide-, Pseudomonas aeruginosa (Gram-negative)-, and Staphylococcus aureus (Gram-positive)-induced NETosis. Thus, higher pH e promoted Nox-dependent ROS production, protease activity, and NETosis; lower pH has the opposite effect. These studies provided mechanistic steps of pH e -mediated regulation of Nox-dependent NETosis. Raising pH either by sodium bicarbonate or Tris base (clinically known as Tris hydroxymethyl aminomethane, tromethamine, or THAM) increases NETosis. Each Tris molecule can bind 3H + ions, whereas each bicarbonate HCO3 - ion binds 1H + ion. Therefore, the amount of Tris solution required to cause the same increase in pH level is less than that of equimolar

A redox-mediated modulation of stem bolting in transgenic Nicotiana sylvestris differentially expressing the external mitochondrial NADPH dehydrogenase.

PubMed

Liu, Yun-Jun; Nunes-Nesi, Adriano; Wallström, Sabá V; Lager, Ida; Michalecka, Agnieszka M; Norberg, Fredrik E B; Widell, Susanne; Fredlund, Kenneth M; Fernie, Alisdair R; Rasmusson, Allan G

2009-07-01

Cytosolic NADPH can be directly oxidized by a calcium-dependent NADPH dehydrogenase, NDB1, present in the plant mitochondrial electron transport chain. However, little is known regarding the impact of modified cytosolic NADPH reduction levels on growth and metabolism. Nicotiana sylvestris plants overexpressing potato (Solanum tuberosum) NDB1 displayed early bolting, whereas sense suppression of the same gene led to delayed bolting, with consequential changes in flowering time. The phenotype was dependent on light irradiance but not linked to any change in biomass accumulation. Whereas the leaf NADPH/NADP(+) ratio was unaffected, the stem NADPH/NADP(+) ratio was altered following the genetic modification and strongly correlated with the bolting phenotype. Metabolic profiling of the stem showed that the NADP(H) change affected relatively few, albeit central, metabolites, including 2-oxoglutarate, glutamate, ascorbate, sugars, and hexose-phosphates. Consistent with the phenotype, the modified NDB1 level also affected the expression of putative floral meristem identity genes of the SQUAMOSA and LEAFY types. Further evidence for involvement of the NADPH redox in stem development was seen in the distinct decrease in the stem apex NADPH/NADP(+) ratio during bolting. Additionally, the potato NDB1 protein was specifically detected in mitochondria, and a survey of its abundance in major organs revealed that the highest levels are found in green stems. These results thus strongly suggest that NDB1 in the mitochondrial electron transport chain can, by modifying cell redox levels, specifically affect developmental processes.

Molecular cloning and functional characterization of NADPH-dependent cytochrome P450 reductase from the green microalga Botryococcus braunii, B race.

PubMed

Tsou, Chung-Yau; Matsunaga, Shigeki; Okada, Shigeru

2018-01-01

The green microalga Botryococcus braunii of the B race accumulates various lipophilic compounds containing a 10,11-oxidosqualene epoxide moiety in addition to large amounts of triterpene hydrocarbons. While 2,3-squalene epoxidases have already been isolated and characterized from the alga, the enzyme that catalyzes the 10,11-epoxidation of squalene has remained elusive. In order to obtain a molecular tool to explore a 10,11-squalene epoxidase, cDNA cloning of an NADPH-dependent cytochrome P450 reductase (CPR) that is required by both squalene epoxidases and cytochrome P450 enzymes was carried out. The isolated cDNA contained an open reading frame (1998 bp) that encoded for a protein with 665 amino acid residues with a predicted molecular weight of 71.46 kDa and a theoretical pI of 5.49. Analysis of the deduced amino acid sequence revealed the presence of conserved motifs, including FMN, FAD, and NADPH binding domains, which are typical of other CPRs and necessary for enzyme activity. By truncation of the N-terminal transmembrane anchor and addition of a 6× His-tag, BbCPR was heterologously produced in Escherichia coli and purified by Ni-NTA affinity chromatography. The purified recombinant enzyme showed optimal reducing activity of cytochrome c at around a neutral pH at a temperature range of 30-37°C. For steady state kinetic parameters, the recombinant enzyme had a k m for cytochrome c and NADPH of 11.7±1.6 and 9.4±1.4 μM, and a k cat for cytochrome c and NADPH of 2.78±0.09 and 3.66±0.11 μmol/min/mg protein, respectively. This is the first study to perform the functional characterization of a CPR from eukaryotic microalgae. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Leaf rust of cultivated barley: pathology and control.

PubMed

Park, Robert F; Golegaonkar, Prashant G; Derevnina, Lida; Sandhu, Karanjeet S; Karaoglu, Haydar; Elmansour, Huda M; Dracatos, Peter M; Singh, Davinder

2015-01-01

Leaf rust of barley is caused by the macrocyclic, heteroecious rust pathogen Puccinia hordei, with aecia reported from selected species of the genera Ornithogalum, Leopoldia, and Dipcadi, and uredinia and telia occurring on Hordeum vulgare, H. vulgare ssp. spontaneum, Hordeum bulbosum, and Hordeum murinum, on which distinct parasitic specialization occurs. Although Puccinia hordei is sporadic in its occurrence, it is probably the most common and widely distributed rust disease of barley. Leaf rust has increased in importance in recent decades in temperate barley-growing regions, presumably because of more intensive agricultural practices. Although total crop loss does not occur, under epidemic conditions yield reductions of up to 62% have been reported in susceptible varieties. Leaf rust is primarily controlled by the use of resistant cultivars, and, to date, 21 seedling resistance genes and two adult plant resistance (APR) genes have been identified. Virulence has been detected for most seedling resistance genes but is unknown for the APR genes Rph20 and Rph23. Other potentially new sources of APR have been reported, and additivity has been described for some of these resistances. Approaches to achieving durable resistance to leaf rust in barley are discussed.

Wheat and barley exposure to nanoceria: Implications for agricultural productivity

EPA Science Inventory

The impacts of man-made nanomaterials on agricultural productivity are not yet well understood. A soil microcosm study was performed to assess the physiological, phenological, and yield responses of wheat (Triticum aestivum) and barley (Hordeum vulgare L.) exposed to nanoceria (n...

Correlation of enteric NADPH-d positive cell counts with the duration of incubation period in NADPH-d histochemistry.

PubMed

Cserni, Tamas; O' Donnel, Annemarie; Paran, Sri; Puri, Prem

2009-03-01

Nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) staining can be used in the enteric nervous system to determine nitrergic neuronal counts, critical in motility disorders such as intestinal neuronal dysplasia and hypoganglionosis. The reported incubation periods of specimens with NADPH-d staining solution has varied from 2 to 24 h. The aim of this study is to investigate the impact of the incubation period on the overall NADPH-d positive cell counts in porcine rectal submucosal plexus. The submucosal plexus of rectal specimens from 12-week-old pigs (n = 5) were studied. Conventional frozen sections were used to identify nitrergic neurons while whole-mount preparations were used to quantify the effect of prolonged duration of incubation on positively identified ganglion cells with NADPH-d histochemistry. The same submucosal ganglia on the conventional sections, and a minimum of 12 ganglia per whole-mount preparation specimen were photographed sequentially at 2, 6, and 24 h and used to count the number of nitrergic cells per ganglion. The same staining solution was used throughout the experiment. Results were analysed using a one-way ANOVA test. Prolonged incubation with the staining solution revealed new NADPH-d positive cells in the ganglia on the conventional sections. The total number of neurons counted in the 12 adjacent ganglia in the whole-mount specimens was 180 +/- 55, the mean neuronal cell per ganglion was 15 +/- 8 after 2 h of incubation. This increased to 357 +/- 17, and to 29 +/- 12 after 6 h (p < 0.05). A further increase was observed of 515 +/- 19 and 43 +/- 17 after 24 h (p < 0.05). When the photomicrographs were retrospectively analysed, not even the outline of the neuronal cells that stained with prolonged incubation was evident at the earlier time points. NADPH-d positive cell counts increase in proportion to the duration of incubation in NADPH-d histochemistry. Comparative studies attempting to quantify nitrergic cell counts in

Crystal structure of conjugated polyketone reductase (CPR-C1) from Candida parapsilosis IFO 0708 complexed with NADPH.

PubMed

Qin, Hui-Min; Yamamura, Akihiro; Miyakawa, Takuya; Kataoka, Michihiko; Maruoka, Shintaro; Ohtsuka, Jun; Nagata, Koji; Shimizu, Sakayu; Tanokura, Masaru

2013-11-01

Conjugated polyketone reductase (CPR-C1) from Candida parapsilosis IFO 0708 is a member of the aldo-keto reductase (AKR) superfamily and reduces ketopantoyl lactone to d-pantoyl lactone in a NADPH-dependent and stereospecific manner. We determined the crystal structure of CPR-C1.NADPH complex at 2.20 Å resolution. CPR-C1 adopted a triose-phosphate isomerase (TIM) barrel fold at the core of the structure in which Thr25 and Lys26 of the GXGTX motif bind uniquely to the adenosine 2'-phosphate group of NADPH. This finding provides a novel structural basis for NADPH binding of the AKR superfamily. Copyright © 2013 Wiley Periodicals, Inc.

Improvement of NADPH bioavailability in Escherichia coli through the use of phosphofructokinase deficient strains.

PubMed

Wang, Yipeng; San, Ka-Yiu; Bennett, George N

2013-08-01

NADPH-dependent reactions play important roles in production of industrially valuable compounds. In this study, we used phosphofructokinase (PFK)-deficient strains to direct fructose-6-phosphate to be oxidized through the pentose phosphate pathway (PPP) to increase NADPH generation. pfkA or pfkB single deletion and double-deletion strains were tested for their ability to produce lycopene. Since lycopene biosynthesis requires many NADPH, levels of lycopene were compared in a set of isogenic strains, with the pfkA single deletion strain showing the highest lycopene yield. Using another NADPH-requiring process, a one-step reduction reaction of 2-chloroacrylate to 2-chloropropionic acid by 2-haloacrylate reductase, the pfkA pfkB double-deletion strain showed the highest yield of 2-chloropropionic acid product. The combined effect of glucose-6-phosphate dehydrogenase overexpression or lactate dehydrogenase deletion with PFK deficiency on NADPH bioavailability was also studied. The results indicated that the flux distribution of fructose-6-phosphate between glycolysis and the pentose phosphate pathway determines the amount of NAPDH available for reductive biosynthesis.

Spectroscopic Analysis of Desiccation-Induced Alterations of the Chlorophyllide Transformation Pathway in Etiolated Barley Leaves1

PubMed Central

Le Lay, Pascaline; Böddi, Béla; Kovacevic, Dragan; Juneau, Philippe; Dewez, David; Popovic, Radovan

2001-01-01

Effects of water deficit on the chlorophyllide (Chlide) transformation pathway were studied in etiolated barley (Hordeum vulgare) leaves by analyzing absorption spectra and 77-K fluorescence spectra deconvoluted in components. Chlide transformations were examined in dehydrated leaves exposed to a 35-ms saturating flash triggering protochlorophyllide (Pchlide) and Chlide transformation processes. During the 90 min following the flash, we found that dehydration induced modifications of Chlide transformations, but no effect on Pchlide phototransformation into Chlide was observed. During this time, content of NADPH-Pchlide oxydoreductase in leaves did not change. Chlide transformation process in dehydrated leaves was characterized by the alteration of the Shibata shift process, by the appearance of a new Chlide species emitting at 692 nm, and by the favored formation of Chl(ide) A668F676. The formation of Chl(ide) A668F676, so-called “free Chlide,” was probably induced by disaggregation of highly aggregated Chlide complexes. Here, we offer evidence for the alteration of photoactive Pchlide regeneration process, which may be caused by the desiccation-induced inhibition of Pchlide synthesis. PMID:11553748

Changes in life history parameters of corn leaf aphid, Rhopalosiphum maidus (Homoptera: Aphididae), under four different elevated temperature and CO2 combinations

USDA-ARS?s Scientific Manuscript database

Biological characteristics of corn leaf aphid, Rhopalosiphum maidis (Fitch), on barley, Hordeum vulgare L., were examined for two generations under four different elevated temperature and CO2 combinations. The developmental duration for each life stage was significantly reduced under the elevated te...

Phenotypic Variation for Diastatic power, ß-Amylase Activity, and ß-Amylase Thermostability vs. Allelic Variation at the Bmy1 Locus in a Sample of North American Barley Germplasm

USDA-ARS?s Scientific Manuscript database

Malting quality data including diastatic power, ß-amylase activity, and ß-amylase thermostability, were collected on malts from three barley (Hordeum vulgare L.) breeding program trials containing two growth habits and 165 lines grown in multiple environments. We attempted to identify causal polymor...

Fungitoxicity of some higher plants and synergistic activity of their essential oils against Sclerotium rolfsii sacc. causing foot-rot disease of barley.

PubMed

Singh, R K

Twenty five plant species were screened for their volatile components against hyphal growth and sclerotia formation of Sclerotium rolfsii causing foot rot disease of barley (Hordeum vulgare). Leaves of Chenopodium ambrosioides (CA), Lippia alba (LA), Azadirachta indica (AI) and Eucalyptus globulus (EG) were found to be strongly toxic. Their volatile active factors were isolated in the form of essential oils which were tested for toxicity individually and in six combinations (1:1 v/v) viz. CA-LA, LA-AI, CA-AI, CA-EG, and EG-AI. The oil combinations were found to be more fungitoxic than the individual oils. The CA-LA, LA-AI, EG-AI, and CA-EG combinations exhibited a broad fnngitoxic spectrum while CA-AI, LA-EG combinations possessed a narrow range of toxicity. None of the six oil combinations showed phytotoxic behaviour on seed germination, seedling growth and general morphology of Hordeum vulgare.

Nitrate-Dependent O2 Evolution in Intact Leaves 1

PubMed Central

de la Torre, Angel; Delgado, Begoña; Lara, Catalina

1991-01-01

Evolution of O2 by illuminated intact detached leaves from barley (Hordeum vulgare L. cv Athos) and pea (Pisum sativum L. cv Lincoln) in a CO2-saturating atmosphere was enhanced when KNO3 (1-2.5 millimolar) had been previously supplied through the transpiration stream. The extra O2 evolution observed after feeding KNO3 increased with the light intensity, being maximal at near saturating photon flux densities and resulting in no changes in the initial slope of the O2 versus light-intensity curve. No stimulation of O2 evolution was otherwise observed after feeding KCl or NH4Cl. The data indicate that nitrate assimilation uses photosynthetically generated reductant and stimulates the rate of non-cyclic electron flow by acting as a second electron-accepting assimilatory process in addition to CO2 fixation. PMID:16668272

Regulation of NADPH-dependent Nitric Oxide and reactive oxygen species signalling in endothelial and melanoma cells by a photoactive NADPH analogue

PubMed Central

Rouaud, Florian; Romero-Perez, Miguel; Wang, Huan; Lobysheva, Irina; Ramassamy, Booma; Henry, Etienne; Tauc, Patrick; Giacchero, Damien; Boucher, Jean-Luc; Deprez, Eric; Rocchi, Stéphane; Slama-Schwok, Anny

2014-01-01

Nitric Oxide (NO) and Reactive oxygen species (ROS) are endogenous regulators of angiogenesis-related events as endothelial cell proliferation and survival, but NO/ROS defect or unbalance contribute to cancers. We recently designed a novel photoactive inhibitor of NO-Synthases (NOS) called NS1, which binds their NADPH site in vitro. Here, we show that NS1 inhibited NO formed in aortic rings. NS1-induced NO decrease led to an inhibition of angiogenesis in a model of VEGF-induced endothelial tubes formation. Beside this effect, NS1 reduced ROS levels in endothelial and melanoma A375 cells and in aorta. In metastatic melanoma cells, NS1 first induced a strong decrease of VEGF and blocked melanoma cell cycle at G2/M. NS1 decreased NOX4 and ROS levels that could lead to a specific proliferation arrest and cell death. In contrast, NS1 did not perturb melanocytes growth. Altogether, NS1 revealed a possible cross-talk between eNOS- and NOX4 –associated pathways in melanoma cells via VEGF, Erk and Akt modulation by NS1 that could be targeted to stop proliferation. NS1 thus constitutes a promising tool that modulates NO and redox stresses by targeting and directly inhibiting eNOS and, at least indirectly, NADPH oxidase(s), with great potential to control angiogenesis. PMID:25296975

Antiurolithic activity of Origanum vulgare is mediated through multiple pathways

PubMed Central

2011-01-01

Background Origanum vulgare Linn has traditionally been used in the treatment of urolithiasis. Therefore, we investigated the crude extract of Origanum vulgare for possible antiurolithic effect, to rationalize its medicinal use. Methods The crude aqueous-methanolic extract of Origanum vulgare (Ov.Cr) was studied using the in vitro and in vivo methods. In the in vitro experiments, supersaturated solution of calcium and oxalate, kidney epithelial cell lines (MDCK) and urinary bladder of rabbits were used, whereas, in the in vivo studies, rat model of urolithiasis was used for the study of preventive and curative effect. Results In the in vitro experiments, Ov.Cr exhibited a concentration-dependent (0.25-4 mg/ml) inhibitory effect on the slope of nucleation and aggregation and also decreased the number of calcium oxalate monohydrate crystals (COM) produced in calcium oxalate metastable solutions. It also showed concentration-dependent antioxidant effect against DPPH free radical and lipid peroxidation induced in rat kidney tissue homogenate. Ov.Cr reduced the cell toxicity using MTT assay and LDH release in renal epithelial cells (MDCK) exposed to oxalate (0.5 mM) and COM (66 μg/cm2) crystals. Ov.Cr relaxed high K+ (80 mM) induced contraction in rabbit urinary bladder strips, and shifted the calcium concentration-response curves (CRCs) towards right with suppression of the maximum response similar to that of verapamil, a standard calcium channel blocker. In male Wistar rats receiving lithogenic treatment comprising of 0.75% ethylene glycol in drinking water given for 3 weeks along with ammonium chloride (NH4Cl) for the first 5 days, Ov.Cr treatment (10-30 mg/kg) prevented as well as reversed toxic changes including loss of body weight, polyurea, crystalluria, oxaluria, raised serum urea and creatinine levels and crystal deposition in kidneys compared to their respective controls. Conclusion These data indicating the antiurolithic activity in Ov.Cr, possibly mediated

Mitochondrial Impairment May Increase Cellular NAD(P)H: Resazurin Oxidoreductase Activity, Perturbing the NAD(P)H-Based Viability Assays.

PubMed

Aleshin, Vasily A; Artiukhov, Artem V; Oppermann, Henry; Kazantsev, Alexey V; Lukashev, Nikolay V; Bunik, Victoria I

2015-08-21

Cellular NAD(P)H-dependent oxidoreductase activity with artificial dyes (NAD(P)H-OR) is an indicator of viability, as the cellular redox state is important for biosynthesis and antioxidant defense. However, high NAD(P)H due to impaired mitochondrial oxidation, known as reductive stress, should increase NAD(P)H-OR yet perturb viability. To better understand this complex behavior, we assayed NAD(P)H-OR with resazurin (Alamar Blue) in glioblastoma cell lines U87 and T98G, treated with inhibitors of central metabolism, oxythiamin, and phosphonate analogs of 2-oxo acids. Targeting the thiamin diphosphate (ThDP)-dependent enzymes, the inhibitors are known to decrease the NAD(P)H production in the pentose phosphate shuttle and/or upon mitochondrial oxidation of 2-oxo acids. Nevertheless, the inhibitors elevated NAD(P)H-OR with resazurin in a time- and concentration-dependent manner, suggesting impaired NAD(P)H oxidation rather than increased viability. In particular, inhibition of the ThDP-dependent enzymes affects metabolism of malate, which mediates mitochondrial oxidation of cytosolic NAD(P)H. We showed that oxythiamin not only inhibited mitochondrial 2-oxo acid dehydrogenases, but also induced cell-specific changes in glutamate and malate dehydrogenases and/or malic enzyme. As a result, inhibition of the 2-oxo acid dehydrogenases compromises mitochondrial metabolism, with the dysregulated electron fluxes leading to increases in cellular NAD(P)H-OR. Perturbed mitochondrial oxidation of NAD(P)H may thus complicate the NAD(P)H-based viability assay.

Enhanced production of GDP-L-fucose by overexpression of NADPH regenerator in recombinant Escherichia coli.

PubMed

Lee, Won-Heong; Chin, Young-Wook; Han, Nam Soo; Kim, Myoung-Dong; Seo, Jin-Ho

2011-08-01

Biosynthesis of guanosine 5'-diphosphate-L-fucose (GDP-L-fucose) requires NADPH as a reducing cofactor. In this study, endogenous NADPH regenerating enzymes such as glucose-6-phosphate dehydrogenase (G6PDH), isocitrate dehydrogenase (Icd), and NADP(+)-dependent malate dehydrogenase (MaeB) were overexpressed to increase GDP-L-fucose production in recombinant Escherichia coli. The effects of overexpression of each NADPH regenerating enzyme on GDP-L-fucose production were investigated in a series of batch and fed-batch fermentations. Batch fermentations showed that overexpression of G6PDH was the most effective for GDP-L-fucose production. However, GDP-L-fucose production was not enhanced by overexpression of G6PDH in the glucose-limited fed-batch fermentation. Hence, a glucose feeding strategy was optimized to enhance GDP-L-fucose production. Fed-batch fermentation with a pH-stat feeding mode for sufficient supply of glucose significantly enhanced GDP-L-fucose production compared with glucose-limited fed-batch fermentation. A maximum GDP-L-fucose concentration of 235.2 ± 3.3 mg l(-1), corresponding to a 21% enhancement in the GDP-L-fucose production compared with the control strain overexpressing GDP-L-fucose biosynthetic enzymes only, was achieved in the pH-stat fed-batch fermentation of the recombinant E. coli overexpressing G6PDH. It was concluded that sufficient glucose supply and efficient NADPH regeneration are crucial for NADPH-dependent GDP-L-fucose production in recombinant E. coli.

Selective Rac1 inhibition protects renal tubular epithelial cells from oxalate-induced NADPH oxidase-mediated oxidative cell injury

PubMed Central

Thamilselvan, Vijayalakshmi; Menon, Mani

2013-01-01

Oxalate-induced oxidative cell injury is one of the major mechanisms implicated in calcium oxalate nucleation, aggregation and growth of kidney stones. We previously demonstrated that oxalate-induced NADPH oxidase-derived free radicals play a significant role in renal injury. Since NADPH oxidase activation requires several regulatory proteins, the primary goal of this study was to characterize the role of Rac GTPase in oxalate-induced NADPH oxidase-mediated oxidative injury in renal epithelial cells. Our results show that oxalate significantly increased membrane translocation of Rac1 and NADPH oxidase activity of renal epithelial cells in a time-dependent manner. We found that NSC23766, a selective inhibitor of Rac1, blocked oxalate-induced membrane translocation of Rac1 and NADPH oxidase activity. In the absence of Rac1 inhibitor, oxalate exposure significantly increased hydrogen peroxide formation and LDH release in renal epithelial cells. In contrast, Rac1 inhibitor pretreatment, significantly decreased oxalate-induced hydrogen peroxide production and LDH release. Furthermore, PKC α and δ inhibitor, oxalate exposure did not increase Rac1 protein translocation, suggesting that PKC resides upstream from Rac1 in the pathway that regulates NADPH oxidase. In conclusion, our data demonstrate for the first time that Rac1-dependent activation of NADPH oxidase might be a crucial mechanism responsible for oxalate-induced oxidative renal cell injury. These findings suggest that Rac1 signaling plays a key role in oxalate-induced renal injury, and may serve as a potential therapeutic target to prevent calcium oxalate crystal deposition in stone formers and reduce recurrence. PMID:21814770

Evaluation of a barley core collection for spot form net blotch reaction reveals distinct genotype specific pathogen virulence and host susceptibility

USDA-ARS?s Scientific Manuscript database

Spot form net blotch (SFNB) caused by Pyrenophora teres Drechs. f. maculata Smedeg., (anamorph Drechslera teres [Sacc.] Shoem.) is a major foliar disease of barley (Hordeum vulgare L.) worldwide. SFNB epidemics have recently been observed in major barley producing countries, suggesting that the loca...

Sequencing of 15,622 gene-bearing BACs clarifies the gene-dense regions of the barley genome

USDA-ARS?s Scientific Manuscript database

Barley (Hordeum vulgare L.) possesses a large and highly repetitive genome of 5.1 Gb that has hindered the development of a complete sequence. In 2012, the International Barley Sequencing Consortium released a resource integrating whole-genome shotgun sequences with a physical and genetic framework....

Granzyme B of cytotoxic T cells induces extramitochondrial reactive oxygen species production via caspase-dependent NADPH oxidase activation.

PubMed

Aguiló, Juan I; Anel, Alberto; Catalán, Elena; Sebastián, Alvaro; Acín-Pérez, Rebeca; Naval, Javier; Wallich, Reinhard; Simon, Markus M; Pardo, Julián

2010-07-01

Induction of reactive oxygen species (ROS) is a hallmark of granzyme B (gzmB)-mediated pro-apoptotic processes and target cell death. However, it is unclear to what extent the generated ROS derive from mitochondrial and/or extra-mitochondrial sources. To clarify this point, we have produced a mutant EL4 cell line, termed EL4-rho(0), which lacks mitochondrial DNA, associated with a decreased mitochondrial membrane potential and a defective ROS production through the electron transport chain of oxidative phosphorylation. When incubated with either recombinant gzmB plus streptolysin or ex vivo gzmB(+) cytotoxic T cells, EL4-rho(0) cells showed phosphatydylserine translocation, caspase 3 activation, Bak conformational change, cytochrome c release and apoptotic morphology comparable to EL4 cells. Moreover, EL4-rho(0) cells produced ROS at levels similar to EL4 under these conditions. GzmB-mediated ROS production was almost totally abolished in both cell lines by the pan-caspase inhibitor, Z-VAD-fmk. However, addition of apocynin, a specific inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, led to a significant reduction of ROS production and cell death only in EL4-rho(0) but not EL4 cells. These data suggest that gzmB-induced cell death is accompanied by a caspase-dependent pathway of extra-mitochondrial ROS production, most probably through activation of NADPH oxidase.

Targeting NADPH oxidases in vascular pharmacology

PubMed Central

Schramm, Agata; Matusik, Paweł; Osmenda, Grzegorz; Guzik, Tomasz J

2012-01-01

Oxidative stress is a molecular dysregulation in reactive oxygen species (ROS) metabolism, which plays a key role in the pathogenesis of atherosclerosis, vascular inflammation and endothelial dysfunction. It is characterized by a loss of nitric oxide (NO) bioavailability. Large clinical trials such as HOPE and HPS have not shown a clinical benefit of antioxidant vitamin C or vitamin E treatment, putting into question the role of oxidative stress in cardiovascular disease. A change in the understanding of the molecular nature of oxidative stress has been driven by the results of these trials. Oxidative stress is no longer perceived as a simple imbalance between the production and scavenging of ROS, but as a dysfunction of enzymes involved in ROS production. NADPH oxidases are at the center of these events, underlying the dysfunction of other oxidases including eNOS uncoupling, xanthine oxidase and mitochondrial dysfunction. Thus NADPH oxidases are important therapeutic targets. Indeed, HMG-CoA reductase inhibitors (statins) as well as drugs interfering with the renin-angiotensin-aldosterone system inhibit NADPH oxidase activation and expression. Angiotensin-converting enzyme (ACE) inhibitors, AT1 receptor antagonists (sartans) and aliskiren, as well as spironolactone or eplerenone, have been discussed. Molecular aspects of NADPH oxidase regulation must be considered, while thinking about novel pharmacological targeting of this family of enzymes consisting of several homologs Nox1, Nox2, Nox3, Nox4 and Nox5 in humans. In order to properly design trials of antioxidant therapies, we must develop reliable techniques for the assessment of local and systemic oxidative stress. Classical antioxidants could be combined with novel oxidase inhibitors. In this review, we discuss NADPH oxidase inhibitors such as VAS2870, VAS3947, GK-136901, S17834 or plumbagin. Therefore, our efforts must focus on generating small molecular weight inhibitors of NADPH oxidases, allowing the

Neovascularization in an arterio-venous loop-containing tissue engineering chamber: role of NADPH oxidase

PubMed Central

Jiang, F; Zhang, G; Hashimoto, I; Kumar, B S; Bortolotto, S; Morrison, W A; Dusting, G J

2008-01-01

Using an in vivo arterio-venous loop-containing tissue-engineering chamber, we have created a variety of vascularized tissue blocks, including functional myocardium. The viability of the transplanted cells is limited by the rate of neovascularization in the chamber. A Nox2-containing nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is thought to have a critical role in ischaemic angiogenesis. In this study we investigated whether NADPH oxidase is involved in the neovascularization process in the tissue-engineering chamber. New blood vessels originating from the venous and the arterial ends of the loop could be identified after 3 days, and the vessel density (by lectin staining) peaked after 7 days and was maintained for at least 14 days. This was accompanied by granulation tissue formation and concomitant increase in the mRNA level of Nox4 NADPH oxidase. Although the total level of Nox2 mRNA in the chamber tissue decreased from day 3 to day 7, immunohistochemistry identified a strong expression of Nox2 in the endothelial cells of the new vessels. In human microvascular endothelial cells, the NADPH oxidase inhibitor apocynin reduced NADPH oxidase activity and inhibited the angiogenic responses in vitro. Local treatment with the NADPH oxidase inhibitors apocynin or gp91ds-tat peptide significantly suppressed the vessel growth in the chamber. In conclusion, NADPH oxidase-dependent redox signalling is important for neovascularization in this novel tissue-engineering chamber in vivo, and boosting this signalling might be a new approach to extending vascularization and tissue growth. PMID:19012731

Biochemical and molecular characterization of an azoreductase from Staphylococcus aureus, a tetrameric NADPH-dependent flavoprotein

PubMed Central

Chen, Huizhong; Hopper, Sherryll L.; Cerniglia, Carl E.

2018-01-01

Azo dyes are a predominant class of colourants used in tattooing, cosmetics, foods and consumer products. A gene encoding NADPH-flavin azoreductase (Azo1) from the skin bacterium Staphylococcus aureus ATCC 25923 was identified and overexpressed in Escherichia coli. RT-PCR results demonstrated that the azo1 gene was constitutively expressed at the mRNA level in S. aureus. Azo1 was found to be a tetramer with a native molecular mass of 85 kDa containing four non-covalently bound FMN. Azo1 requires NADPH, but not NADH, as an electron donor for its activity. The enzyme was resolved to dimeric apoprotein by removing the flavin prosthetic groups using hydrophobic-interaction chromatography. The dimeric apoprotein was reconstituted on-column and in free stage with FMN, resulting in the formation of a fully functional native-like tetrameric enzyme. The enzyme cleaved the model azo dye 2-[4-(dimethylamino)phenylazo]benzoic acid (Methyl Red) into N,N-dimethyl-p-phenylenediamine and 2-aminobenzoic acid. The apparent Km values for NADPH and Methyl Red substrates were 0·;074 and 0·057 mM, respectively. The apparent Vmax was 0·4 µM min−1 (mg protein)−1. Azo1 was also able to metabolize Orange II, Amaranth, Ponceau BS and Ponceau S azo dyes. Azo1 represents the first azoreductase to be identified and characterized from human skin microflora. PMID:15870453

Improved NADPH supply for xylitol production by engineered Escherichia coli with glycolytic mutations.

PubMed

Chin, Jonathan W; Cirino, Patrick C

2011-01-01

Escherichia coli engineered to uptake xylose while metabolizing glucose was previously shown to produce high levels of xylitol from a mixture of glucose and xylose when expressing NADPH-dependent xylose reductase from Candida boidinii (CbXR) (Cirino et al., Biotechnol Bioeng. 2006;95:1167-1176). We then described the effects of deletions of key metabolic pathways (e.g., Embden-Meyerhof-Parnas and pentose phosphate pathway) and reactions (e.g., transhydrogenase and NADH dehydrogenase) on resting-cell xylitol yield (Y RPG: moles of xylitol produced per mole of glucose consumed) (Chin et al., Biotechnol Bioeng. 2009;102:209-220). These prior results demonstrated the importance of direct NADPH supply by NADP+-utilizing enzymes in central metabolism for driving heterologous NADPH-dependent reactions. This study describes strain modifications that improve coupling between glucose catabolism (oxidation) and xylose reduction using two fundamentally different strategies. We first examined the effects of deleting the phosphofructokinase (pfk) gene(s) on growth-uncoupled xylitol production and found that deleting both pfkA and sthA (encoding the E. coli-soluble transhydrogenase) improved the xylitol Y RPG from 3.4 ± 0.6 to 5.4 ± 0.4. The second strategy focused on coupling aerobic growth on glucose to xylitol production by deleting pgi (encoding phosphoglucose isomerase) and sthA. Impaired growth due to imbalanced NADPH metabolism (Sauer et al., J Biol Chem. 2004;279:6613-6619) was alleviated upon expressing CbXR, resulting in xylitol production similar to that of the growth-uncoupled precursor strains but with much less acetate secretion and more efficient utilization of glucose. Intracellular nicotinamide cofactor levels were also quantified, and the magnitude of the change in the NADPH/NADP+ ratio measured from cells consuming glucose in the absence vs. presence of xylose showed a strong correlation to the resulting Y RPG. Copyright © 2011 American Institute of Chemical

Phosphatidic acid as a second messenger in human polymorphonuclear leukocytes. Effects on activation of NADPH oxidase.

PubMed Central

Agwu, D E; McPhail, L C; Sozzani, S; Bass, D A; McCall, C E

1991-01-01

Receptor-mediated agonists, such as FMLP, induce an early, phospholipase D (PLD)-mediated accumulation of phosphatidic acid (PA) which may play a role in the activation of NADPH oxidase in human PMN. We have determined the effect of changes in PA production on O2 consumption in intact PMN and the level of NADPH oxidase activity measured in a cell-free assay. Pretreatment of cells with various concentrations of propranolol enhanced (less than or equal to 200 microM) or inhibited (greater than 300 microM) PLD-induced production of PA (mass and radiolabel) in a manner that correlated with enhancement or inhibition of O2 consumption in PMN stimulated with 1 microM FMLP in the absence of cytochalasin B. The concentration-dependent effects of propranolol on FMLP-induced NADPH oxidase activation was confirmed by direct assay of the enzyme in subcellular fractions. In PA extracted from cells pretreated with 200 microM propranolol before stimulation with 1 microM FMLP, phospholipase A1 (PLA1)-digestion for 90 min, followed by quantitation of residual PA, showed that a minimum of 44% of PA in control (undigested) sample was diacyl-PA; alkylacyl-PA remained undigested by PLA1. Propranolol was also observed to have a concentration-dependent enhancement of mass of 1,2-DG formed in PMN stimulated with FMLP. DG levels reached a maximum at 300 microM propranolol and remained unchanged up to 500 microM propranolol. However, in contrast to PA levels, the level of DG produced did not correlate with NADPH oxidase activation. Exogenously added didecanoyl-PA activated NADPH oxidase in a concentration-dependent manner (1-300 microM) in a reconstitution assay using membrane and cytosolic fractions from unstimulated PMN. In addition, PA synergized with SDS for oxidase activation. Taken together, these results indicate that PA plays a second messenger role in the activation of NADPH oxidase in human PMN and that regulation of phospholipase D is a key step in the activation pathway. Images

Greenhouse gas emissions from an irrigated dairy forage rotation as influenced by fertilizer and manure applications

USDA-ARS?s Scientific Manuscript database

Information is needed regarding the effect of nitrogen source on greenhouse gas (GHG) emissions from irrigated semiarid agricultural soils. We report nitrous oxide, carbon dioxide, and methane emissions from a silage corn (Zea mays L.) (2013)–barley (Hordeum vulgare L.) (2014)–alfalfa (Medicago sati...

Genetic mapping of race-specific stem rust resistance in the synthetic hexaploid W7984 x Opata M85 mapping population

USDA-ARS?s Scientific Manuscript database

Stem rust (caused by Puccinia graminis f. sp. tritici) has historically caused severe yield losses of wheat (Triticum aestivum) worldwide and has been one of the most feared diseases of wheat and barley (Hordeum vulgare). Stem rust has been controlled successfully through the use of resistant varie...

Registration of Sawtooth low-phytate, hulled spring barley

USDA-ARS?s Scientific Manuscript database

The Agricultural Research Service, U.S. Department of Agriculture (USDA-ARS), has released 'Sawtooth', (Hordeum vulgare L.) (Reg. No. xxxxxx, P.I. xxxxxx). Sawtooth is a hulless, low-phytate, spring barley, the second to be developed and released by the USDA-ARS. Compared to the previously released ...

Bioreduction with Efficient Recycling of NADPH by Coupled Permeabilized Microorganisms▿

PubMed Central

Zhang, Wei; O'Connor, Kevin; Wang, Daniel I. C.; Li, Zhi

2009-01-01

The glucose dehydrogenase (GDH) from Bacillus subtilis BGSC 1A1 was cloned and functionally expressed in Escherichia coli BL21(pGDH1) and XL-1 Blue(pGDH1). Controlled permeabilization of recombinant E. coli BL21 and XL-1 Blue with EDTA-toluene under optimized conditions resulted in permeabilized cells with specific activities of 61 and 14 U/g (dry weight) of cells, respectively, for the conversion of NADP+ to NADPH upon oxidation of glucose. The permeabilized recombinant strains were more active than permeabilized B. subtilis BGSC 1A1, did not exhibit NADPH/NADH oxidase activity, and were useful for regeneration of both NADH and NADPH. Coupling of permeabilized cells of Bacillus pumilus Phe-C3 containing an NADPH-dependent ketoreductase and an E. coli recombinant expressing GDH as a novel biocatalytic system allowed enantioselective reduction of ethyl 3-keto-4,4,4-trifluorobutyrate with efficient recycling of NADPH; a total turnover number (TTN) of 4,200 mol/mol was obtained by using E. coli BL21(pGDH1) as the cofactor-regenerating microorganism with initial addition of 0.005 mM NADP+. The high TTN obtained is in the practical range for producing fine chemicals. Long-term stability of the permeabilized cell couple and a higher product concentration were demonstrated by 68 h of bioreduction of ethyl 3-keto-4,4,4-trifluorobutyrate with addition of 0.005 mM NADP+ three times; 50.5 mM (R)-ethyl 3-hydroxy-4,4,4-trifluorobutyrate was obtained with 95% enantiomeric excess, 84% conversion, and an overall TTN of 3,400 mol/mol. Our method results in practical synthesis of (R)-ethyl 3-hydroxy-4,4,4-trifluorobutyrate, and the principle described here is generally applicable to other microbial reductions with cofactor recycling. PMID:19047388

Mitochondrial Impairment May Increase Cellular NAD(P)H: Resazurin Oxidoreductase Activity, Perturbing the NAD(P)H-Based Viability Assays

PubMed Central

Aleshin, Vasily A.; Artiukhov, Artem V.; Oppermann, Henry; Kazantsev, Alexey V.; Lukashev, Nikolay V.; Bunik, Victoria I.

2015-01-01

Cellular NAD(P)H-dependent oxidoreductase activity with artificial dyes (NAD(P)H-OR) is an indicator of viability, as the cellular redox state is important for biosynthesis and antioxidant defense. However, high NAD(P)H due to impaired mitochondrial oxidation, known as reductive stress, should increase NAD(P)H-OR yet perturb viability. To better understand this complex behavior, we assayed NAD(P)H-OR with resazurin (Alamar Blue) in glioblastoma cell lines U87 and T98G, treated with inhibitors of central metabolism, oxythiamin, and phosphonate analogs of 2-oxo acids. Targeting the thiamin diphosphate (ThDP)-dependent enzymes, the inhibitors are known to decrease the NAD(P)H production in the pentose phosphate shuttle and/or upon mitochondrial oxidation of 2-oxo acids. Nevertheless, the inhibitors elevated NAD(P)H-OR with resazurin in a time- and concentration-dependent manner, suggesting impaired NAD(P)H oxidation rather than increased viability. In particular, inhibition of the ThDP-dependent enzymes affects metabolism of malate, which mediates mitochondrial oxidation of cytosolic NAD(P)H. We showed that oxythiamin not only inhibited mitochondrial 2-oxo acid dehydrogenases, but also induced cell-specific changes in glutamate and malate dehydrogenases and/or malic enzyme. As a result, inhibition of the 2-oxo acid dehydrogenases compromises mitochondrial metabolism, with the dysregulated electron fluxes leading to increases in cellular NAD(P)H-OR. Perturbed mitochondrial oxidation of NAD(P)H may thus complicate the NAD(P)H-based viability assay. PMID:26308058

Crystallization and preliminary X-ray analysis of the NADPH-dependent 3-quinuclidinone reductase from Rhodotorula rubra

PubMed Central

Takeshita, Daijiro; Kataoka, Michihiko; Miyakawa, Takuya; Miyazono, Ken-ichi; Uzura, Atsuko; Nagata, Koji; Shimizu, Sakayu; Tanokura, Masaru

2009-01-01

(R)-3-Quinuclidinol is a useful compound that is applicable to the synthesis of various pharmaceuticals. The NADPH-dependent carbonyl reductase 3-­quinuclidinone reductase from Rhodotorula rubra catalyzes the stereospecific reduction of 3-quinuclidinone to (R)-3-quinuclidinol and is expected to be utilized in industrial production of this alcohol. 3-Quinuclidinone reductase from R. rubra was expressed in Escherichia coli and purified using Ni-affinity and ion-exchange column chromatography. Crystals of the protein were obtained by the sitting-drop vapour-diffusion method using PEG 8000 as the precipitant. The crystals belonged to space group P41212, with unit-cell parameters a = b = 91.3, c = 265.4 Å, and diffracted X-rays to 2.2 Å resolution. The asymmetric unit contained four molecules of the protein and the solvent content was 48.4%. PMID:19478454

SIRT1 inhibits NADPH oxidase activation and protects endothelial function in the rat aorta: implications for vascular aging.

PubMed

Zarzuelo, María José; López-Sepúlveda, Rocío; Sánchez, Manuel; Romero, Miguel; Gómez-Guzmán, Manuel; Ungvary, Zoltan; Pérez-Vizcaíno, Francisco; Jiménez, Rosario; Duarte, Juan

2013-05-01

Vascular aging is characterized by up-regulation of NADPH oxidase, oxidative stress and endothelial dysfunction. Previous studies demonstrate that the activity of the evolutionarily conserved NAD(+)-dependent deacetylase SIRT1 declines with age and that pharmacological activators of SIRT1 confer significant anti-aging cardiovascular effects. To determine whether dysregulation of SIRT1 promotes NADPH oxidase-dependent production of reactive oxygen species (ROS) and impairs endothelial function we assessed the effects of three structurally different inhibitors of SIRT1 (nicotinamide, sirtinol, EX527) in aorta segments isolated from young Wistar rats. Inhibition of SIRT1 induced endothelial dysfunction, as shown by the significantly reduced relaxation to the endothelium-dependent vasodilators acetylcholine and the calcium ionophore A23187. Endothelial dysfunction induced by SIRT1 inhibition was prevented by treatment of the vessels with the NADPH oxidase inhibitor apocynin or superoxide dismutase. Inhibition of SIRT1 significantly increased vascular superoxide production, enhanced NADPH oxidase activity, and mRNA expression of its subunits p22(phox) and NOX4, which were prevented by resveratrol. Peroxisome proliferator-activated receptor-α (PPARα) activation mimicked the effects of resveratrol while PPARα inhibition prevented the effects of this SIRT1 activator. SIRT1 co-precipitated with PPARα and nicotinamide increased the acetylation of the PPARα coactivator PGC-1α, which was suppressed by resveratrol. In conclusion, impaired activity of SIRT1 induces endothelial dysfunction and up-regulates NADPH oxidase-derived ROS production in the vascular wall, mimicking the vascular aging phenotype. Moreover, a new mechanism for controlling endothelial function after SIRT1 activation involves a decreased PGC-1α acetylation and the subsequent PPARα activation, resulting in both decreased NADPH oxidase-driven ROS production and NO inactivation. Copyright © 2013

Biological invasion of oxeye daisy (Leucanthemum vulgare) in North America: Pre-adaptation, post-introduction evolution, or both?

PubMed

Stutz, Sonja; Mráz, Patrik; Hinz, Hariet L; Müller-Schärer, Heinz; Schaffner, Urs

2018-01-01

Species may become invasive after introduction to a new range because phenotypic traits pre-adapt them to spread and become dominant. In addition, adaptation to novel selection pressures in the introduced range may further increase their potential to become invasive. The diploid Leucanthemum vulgare and the tetraploid L. ircutianum are native to Eurasia and have been introduced to North America, but only L. vulgare has become invasive. To investigate whether phenotypic differences between the two species in Eurasia could explain the higher abundance of L. vulgare in North America and whether rapid evolution in the introduced range may have contributed to its invasion success, we grew 20 L. vulgare and 21 L. ircutianum populations from Eurasia and 21 L. vulgare populations from North America under standardized conditions and recorded performance and functional traits. In addition, we recorded morphological traits to investigate whether the two closely related species can be clearly distinguished by morphological means and to what extent morphological traits have changed in L. vulgare post-introduction. We found pronounced phenotypic differences between L. vulgare and L. ircutianum from the native range as well as between L. vulgare from the native and introduced ranges. The two species differed significantly in morphology but only moderately in functional or performance traits that could have explained the higher invasion success of L. vulgare in North America. In contrast, leaf morphology was similar between L. vulgare from the native and introduced range, but plants from North America flowered later, were larger and had more and larger flower heads than those from Eurasia. In summary, we found litte evidence that specific traits of L. vulgare may have pre-adapted this species to become more invasive than L. ircutianum, but our results indicate that rapid evolution in the introduced range likely contributed to the invasion success of L. vulgare.

BYK191023 (2-[2-(4-methoxy-pyridin-2-yl)-ethyl]-3h-imidazo[4,5-b]pyridine) is an NADPH- and time-dependent irreversible inhibitor of inducible nitric-oxide synthase.

PubMed

Tiso, Mauro; Strub, Andreas; Hesslinger, Christian; Kenney, Claire T; Boer, Rainer; Stuehr, Dennis J

2008-04-01

Imidazopyridine derivates were recently shown to be a novel class of selective and arginine-competitive inhibitors of inducible nitric-oxide synthase (iNOS), and 2-[2-(4-methoxypyridin-2-yl)-ethyl]-3H-imidazo[4,5-b]pyridine (BYK191023) was found to have very high selectivity in enzymatic and cellular models ( Mol Pharmacol 69: 328-337, 2006 ). Here, we show that BYK191023 irreversibly inactivates murine iNOS in an NADPH- and time-dependent manner, whereas it acts only as a reversible l-arginine-competitive inhibitor in the absence of NADPH or during anaerobic preincubation. Time-dependent irreversible inhibition by BYK191023 could also be demonstrated in intact cells using the RAW macrophage or iNOS-overexpressing human embryonic kidney 293 cell lines. The mechanism of BYK191023 inhibition in the presence of NADPH was studied using spectral, kinetic, chromatographic, and radioligand binding methods. BYK191023-bound iNOS was spectrally indistinguishable from l-arginine-bound iNOS, pointing to an interaction of BYK191023 with the catalytic center of the enzyme. [(3)H]BYK191023 was recovered quantitatively from irreversibly inactivated iNOS, and no inhibitor metabolite was detected by high-performance liquid chromatography (HPLC). Size exclusion chromatography revealed only about 20% iNOS dissociation into monomers. Furthermore, HPLC and spectrophotometric analysis showed that the irreversible inhibition was associated with loss of heme from iNOS and a reduced ability to form the distinctive ferrous heme-CO complex (cytochrome P450). Thus, enzyme inactivation is mainly caused by heme loss, and it occurs in the inhibitor-bound enzyme in the presence of electron flux from NADPH.

New Advances in Marker Assisted Selection for Winter Hardiness in Oats.

USDA-ARS?s Scientific Manuscript database

Oat (Avena sativa L.) breeding and genetics research has lagged behind other small grains, such as wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.), in the development of PCR based markers and map construction due to fewer oat researchers and reduced research funding. As a result, marke...

Registration of Harriman low-phytate, hulled spring barley

USDA-ARS?s Scientific Manuscript database

The Agricultural Research Service, U.S. Department of Agriculture (USDA-ARS), has released 'Harriman', (Hordeum vulgare L.) (Reg. No. xxxxxx, P.I. xxxxxx). Harriman is a hulled, low-phytate barley, the second to be developed and released by the USDA-ARS. Compared to the previously released hulled, l...

NADPH oxidases of the brain: distribution, regulation, and function.

PubMed

Infanger, David W; Sharma, Ram V; Davisson, Robin L

2006-01-01

The NADPH oxidase is a multi-subunit enzyme that catalyzes the reduction of molecular oxygen to form superoxide (O(2)(-)). While classically linked to the respiratory burst in neutrophils, recent evidence now shows that O(2)(-) (and associated reactive oxygen species, ROS) generated by NADPH oxidase in nonphagocytic cells serves myriad functions in health and disease. An entire new family of NADPH Oxidase (Nox) homologues has emerged, which vary widely in cell and tissue distribution, as well as in function and regulation. A major concept in redox signaling is that while NADPH oxidase-derived ROS are necessary for normal cellular function, excessive oxidative stress can contribute to pathological disease. This certainly is true in the central nervous system (CNS), where normal NADPH oxidase function appears to be required for processes such as neuronal signaling, memory, and central cardiovascular homeostasis, but overproduction of ROS contributes to neurotoxicity, neurodegeneration, and cardiovascular diseases. Despite implications of NADPH oxidase in normal and pathological CNS processes, still relatively little is known about the mechanisms involved. This paper summarizes the evidence for NADPH oxidase distribution, regulation, and function in the CNS, emphasizing the diversity of Nox isoforms and their new and emerging role in neuro-cardiovascular function. In addition, perspectives for future research and novel therapeutic targets are offered.

Interrupted reperfusion reduces the activation of NADPH oxidase after cerebral I/R injury.

PubMed

Shen, Jia; Bai, Xiao-Yin; Qin, Yuan; Jin, Wei-Wei; Zhou, Jing-Yin; Zhou, Ji-Ping; Yan, Ying-Gang; Wang, Qiong; Bruce, Iain C; Chen, Jiang-Hua; Xia, Qiang

2011-06-15

Interrupted reperfusion reduces ischemia/reperfusion (I/R) injury. This study was designed to determine whether NADPH oxidase participates in the neural protection against global I/R injury after interrupted reperfusion. Mice were randomly divided into five groups: sham (sham-operated), I/R (20-min global I/R), RR (I/R+interrupted reperfusion), Apo (I/R+apocynin administration), and RR+Apo. Behavioral tests (pole test, beam walking, and Morris water maze) and Nissl staining were undertaken in all five groups; superoxide levels, expression of gp91(phox) and p47(phox), p47(phox) translocation, and Rac1 activation were measured in the sham, I/R, and RR groups. The motor coordination, bradykinesia, and spatial learning and memory, as well as the neuron survival rates, were better in the RR, Apo, and RR+Apo groups than in the I/R group. The NADPH oxidase-dependent superoxide levels, p47(phox) and gp91(phox) expression, p47(phox) translocation, and Rac1 activation were lower in the RR group than in the I/R group. In conclusion, the neural protective effect of interrupted reperfusion is at least partly mediated by decreasing the expression and assembly of NADPH oxidase and the levels of NADPH oxidase-derived superoxide. The most striking reduction Rac1-GTP in the RR group suggests that interrupted reperfusion also acts on the activation of assembled NADPH oxidase by reducing the availability of Rac1-GTP. Copyright © 2011 Elsevier Inc. All rights reserved.

Development of a method using multispectral imagery and spatial pattern metrics to quantify stress to wheat fields caused by Diuraphis noxia

USDA-ARS?s Scientific Manuscript database

The Russian wheat aphid, Diuraphis noxia, is an important pest of winter wheat, Triticum aestivum, and barley, Hordeum vulgare that has caused an annual economic loss estimated at over 1 billion dollars since it first appeared in the United States. The objective of this study was to determine the p...

Biochemistry, Structure and Function of Non-Wheat Proteins: Case Study of Barley ß-Amylase

USDA-ARS?s Scientific Manuscript database

The importance of a protein is not always evident and may be due to its multifunctional nature. ß-Amylase in seeds of barley (Hordeum vulgare L.) constitutes approximately 2% of the total protein in mature seeds and is assumed to be important when storage proteins are mobilized to support protein s...

Registration of 'Mesa' Russian wheat aphid-resistant winter feed barley

USDA-ARS?s Scientific Manuscript database

'Mesa' (Reg. No., PI 659768), Russian wheat aphid [RWA, Diruaphis noxia (Kurdjumov)]-resistant six-rowed winter feed barley Hordeum vulgare L.) tested as 97BX 43-99A and STARS 1401B, was developed and released by USDA-ARS, Stillwater, OK. Although all crossing, selection, and evaluation was done by...

Registration of STARS 1501B and STARS 1502B barley germplasm with Rsg2 resistance to greenbug

USDA-ARS?s Scientific Manuscript database

STARS 1501B (Reg. No. GP- , PI 675335) greenbug-resistant, six-rowed, spring, feed barley (Hordeum vulgare L.) germplasm and STARS 1502B (Reg. No. GP-, PI 675336) greenbug-resistant, six-rowed, winter, feed barley germplasm, tested as GR 001 and GR 010 respectively, were developed and released by US...

Responses of growth and primary metabolism of water-stressed barley roots to rehydration

USDA-ARS?s Scientific Manuscript database

Barley seedlings [Hordeum vulgare L. Brant] were grown in pots in controlled environment chambers and drought treatments were imposed 11 days after sowing. Soil water content decreased from 92% to 10% after an additional 14 days of water stress. Shoot and root growth ceased after 4 and 9 days of wat...

Cytochemical and Cytofluorometric Evidence for Guard Cell Photosystems 1

PubMed Central

Vaughn, Kevin C.; Outlaw, William H.

1983-01-01

Evidence for photosynthetic linear electron transport in guard cells was obtained with two sensitive methods of high spacial resolution. Light-dependent diaminobenzidine oxidation (an indicator of PSI) and DCMU-sensitive, light-dependent thiocarbamyl nitroblue tetrazolium reduction (an indicator of PSII) were observed in guard cell plastids of Hordeum vulgare L. cv Himalaya using electron microscopic cytochemical procedures. DCMU-sensitive Chl a fluorescence induction (an indicator of PSII) was detected in individual guard cell pairs of Vicia faba L. cv Longpod using an ultramicrofluorometer. At least for these species, we conclude these results are proof for the presence of PSII in guard cell chloroplasts, which until now has been somewhat controversial. Images Fig. 2 Fig. 1 PMID:16662840

Oscillatory shear stress stimulates endothelial production of O2- from p47phox-dependent NAD(P)H oxidases, leading to monocyte adhesion

NASA Technical Reports Server (NTRS)

Hwang, Jinah; Saha, Aniket; Boo, Yong Chool; Sorescu, George P.; McNally, J. Scott; Holland, Steven M.; Dikalov, Sergei; Giddens, Don P.; Griendling, Kathy K.; Harrison, David G.;

Anthelmintic Activity of Crude Extract and Essential Oil of Tanacetum vulgare (Asteraceae) against Adult Worms of Schistosoma mansoni

PubMed Central

Godinho, Loyana Silva; Aleixo de Carvalho, Lara Soares; Barbosa de Castro, Clarissa Campos; Dias, Mirna Meana; Pinto, Priscila de Faria; Crotti, Antônio Eduardo Miller; Pinto, Pedro Luiz Silva; de Moraes, Josué; Da Silva Filho, Ademar A.

2014-01-01

Schistosomiasis, a parasitic disease caused by trematode flatworms of the genus Schistosoma, affects more than 200 million people worldwide, and its control is dependent on a single drug, praziquantel. Tanacetum vulgare (Asteraceae) is used in folk medicine as a vermifuge. This study aimed to investigate the in vitro schistosomicidal activity of the crude extract (TV) and the essential oil (TV-EO) from the aerial parts of T. vulgare. TV-EO was obtained by hydrodistillation and analyzed by GC/MS, which allowed the identification of β-thujone (84.13%) as the major constituent. TV and TV-EO, at 200 μg/mL, decreased motor activity and caused 100% mortality of all adult worms. At 100 and 50 μg/mL, only TV caused death of all adult worms, while TV-EO was inactive. TV (200 μg/mL) was also able to reduce viability and decrease production of developed eggs. Confocal laser scanning microscopy showed morphological alterations in the tegument of the S. mansoni surface after incubation with TV (50 and 100 μg/mL). Quantitative analysis on the schistosomes tegument showed that TV caused changes in the numbers of tubercles of S. mansoni male worms in a dose-dependent manner. The findings suggest that T. vulgare is a potential source of schistosomicidal compounds. PMID:24672320

Hordeum chilense genome, a useful tool to investigate the endosperm yellow pigment content in the Triticeae

PubMed Central

2012-01-01

Background The wild barley Hordeum chilense fulfills some requirements for being a useful tool to investigate the endosperm yellow pigment content (YPC) in the Triticeae including its diploid constitution, the availability of genetic resources (addition and deletion stocks and a high density genetic map) and, especially, its high seed YPC not silenced in tritordeums (amphiploids derived from H. chilense and wheat). Thus, the aim of this work was to test the utility of the H. chilense genome for investigating the YPC in the Triticeae. Results Twelve genes related to endosperm carotenoid content and/or YPC in grasses (Dxr, Hdr [synonym ispH], Ggpps1, Psy2, Psy3, Pds, Zds, e-Lcy, b-Lcy, Hyd3, Ccd1 and Ppo1) were identified, and mapped in H. chilense using rice genes to identify orthologs from barley, wheat, sorghum and maize. Macrocolinearity studies revealed that gene positions were in agreement in H. vulgare and H. chilense. Additionally, three main regions associated with YPC were identified in chromosomes 2Hch, 3Hch and 7Hch in H. chilense, the former being the most significant one. Conclusions The results obtained are consistent with previous findings in wheat and suggest that Ggpps1, Zds and Hyd3 on chromosome 2Hch may be considered candidate genes in wheat for further studies in YPC improvement. Considering the syntenic location of carotenoid genes in H. chilense, we have concluded that the Hch genome may constitute a valuable tool for YPC studies in the Triticeae. PMID:23122232

Myeloperoxidase amplified high glucose-induced endothelial dysfunction in vasculature: Role of NADPH oxidase and hypochlorous acid.

PubMed

Tian, Rong; Ding, Yun; Peng, Yi-Yuan; Lu, Naihao

2017-03-11

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-derived reactive oxygen species (ROS) such as superoxide and hydrogen peroxide (H 2 O 2 ), have emerged as important molecules in the pathogenesis of diabetic endothelial dysfunction. Additionally, neutrophils-derived myeloperoxidase (MPO) and MPO-catalyzed hypochlorous acid (HOCl) play important roles in the vascular injury. However, it is unknown whether MPO can use vascular-derived ROS to induce diabetic endothelial dysfunction. In the present study, we demonstrated that NADPH oxidase was the main source of ROS formation in high glucose-cultured human umbilical vein endothelial cells (HUVECs), and played a critical role in high glucose-induced endothelial dysfunction such as cell apoptosis, loss of cell viability and reduction of nitric oxide (NO). However, the addition of MPO could amplify the high glucose-induced endothelial dysfunction which was inhibited by the presence of apocynin (NADPH oxidase inhibitor), catalase (H 2 O 2 scavenger), or methionine (HOCl scavenger), demonstrating the contribution of NADPH oxidase-H 2 O 2 -MPO-HOCl pathway in the MPO/high glucose-induced vascular injury. In high glucose-incubated rat aortas, MPO also exacerbated the NADPH oxidase-induced impairment of endothelium-dependent relaxation. Consistent with these in vitro data, in diabetic rat aortas, both MPO expresion and NADPH oxidase activity were increased while the endothelial function was simultaneously impaired. The results suggested that vascular-bound MPO could amplify high glucose-induced vascular injury in diabetes. MPO-NADPH oxidase-HOCl may represent an important pathogenic pathway in diabetic vascular diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

Ethanol increases matrix metalloproteinase-12 expression via NADPH oxidase-dependent ROS production in macrophages

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Mi Jin; Nepal, Saroj; Lee, Eung-Seok

2013-11-15

Matrix metalloproteinase-12 (MMP-12), an enzyme responsible for degradation of extracellular matrix, plays an important role in the progression of various diseases, including inflammation and fibrosis. Although most of those are pathogenic conditions induced by ethanol ingestion, the effect of ethanol on MMP-12 has not been explored. In the present study, we investigated the effect of ethanol on MMP-12 expression and its potential mechanisms in macrophages. Here, we demonstrated that ethanol treatment increased MMP-12 expression in primary murine peritoneal macrophages and RAW 264.7 macrophages at both mRNA and protein levels. Ethanol treatment also significantly increased the activity of nicotinamide adenine dinucleotidemore » (NADPH) oxidase and the expression of NADPH oxidase-2 (Nox2). Pretreatment with an anti-oxidant (N-acetyl cysteine) or a selective inhibitor of NADPH oxidase (diphenyleneiodonium chloride (DPI)) prevented ethanol-induced MMP-12 expression. Furthermore, knockdown of Nox2 by small interfering RNA (siRNA) prevented ethanol-induced ROS production and MMP-12 expression in RAW 264.7 macrophages, indicating a critical role for Nox2 in ethanol-induced intracellular ROS production and MMP-12 expression in macrophages. We also showed that ethanol-induced Nox2 expression was suppressed by transient transfection with dominant negative IκB-α plasmid or pretreatment with Bay 11-7082, a selective inhibitor of NF-κB, in RAW 264.7 macrophages. In addition, ethanol-induced Nox2 expression was also attenuated by treatment with a selective inhibitor of p38 MAPK, suggesting involvement of p38 MAPK/NF-κB pathway in ethanol-induced Nox2 expression. Taken together, these results demonstrate that ethanol treatment elicited increase in MMP-12 expression via increase in ROS production derived from Nox2 in macrophages. - Highlights: • Ethanol increases ROS production through up-regulation of Nox2 in macrophages. • Enhanced oxidative stress contributes to

Chloroplast NADPH-Dependent Thioredoxin Reductase from Chlorella vulgaris Alleviates Environmental Stresses in Yeast Together with 2-Cys Peroxiredoxin

PubMed Central

Machida, Takeshi; Ishibashi, Akiko; Kirino, Ai; Sato, Jun-ichi; Kawasaki, Shinji; Niimura, Youichi; Honjoh, Ken-ichi; Miyamoto, Takahisa

2012-01-01

Chloroplast NADPH-dependent thioredoxin reductase (NTRC) catalyzes the reduction of 2-Cys peroxiredoxin (2-Cys Prx) and, thus, probably functions as an antioxidant system. The functions of the enzyme in oxidative and salt stresses have been reported previously. We have previously identified and characterized NTRC in Chlorella vulgaris. In the present study, we isolated a full-length cDNA clone encoding 2-Cys Prx from C. vulgaris and investigated the involvement of Chlorella NTRC/2-Cys Prx system in several environmental stress tolerances by using yeast as a eukaryotic model. Deduced Chlorella 2-Cys Prx was homologous to those of chloroplast 2-Cys Prxs from plants, and two conserved cysteine residues were found in the deduced sequence. Enzyme assay showed that recombinant mature C. vulgaris NTRC (mCvNTRC) transferred electrons from NADPH to recombinant mature C. vulgaris 2-Cys Prx (mCvPrx), and mCvPrx decomposed hydrogen peroxide, tert-butyl hydroperoxide, and peroxynitrite by cooperating with mCvNTRC. Based on the results, the mCvNTRC/mCvPrx antioxidant system was identified in Chlorella. The antioxidant system genes were expressed in yeast separately or coordinately. Stress tolerances of yeast against freezing, heat, and menadione-induced oxidative stresses were significantly improved by expression of mCvNTRC, and the elevated tolerances were more significant when both mCvNTRC and mCvPrx were co-expressed. Our results reveal a novel feature of NTRC: it functions as an antioxidant system with 2-Cys Prx in freezing and heat stress tolerances. PMID:23029353

Current status of NADPH oxidase research in cardiovascular pharmacology.

PubMed

Rodiño-Janeiro, Bruno K; Paradela-Dobarro, Beatriz; Castiñeiras-Landeira, María Isabel; Raposeiras-Roubín, Sergio; González-Juanatey, José R; Alvarez, Ezequiel

2013-01-01

The implications of reactive oxygen species in cardiovascular disease have been known for some decades. Rationally, therapeutic antioxidant strategies combating oxidative stress have been developed, but the results of clinical trials have not been as good as expected. Therefore, to move forward in the design of new therapeutic strategies for cardiovascular disease based on prevention of production of reactive oxygen species, steps must be taken on two fronts, ie, comprehension of reduction-oxidation signaling pathways and the pathophysiologic roles of reactive oxygen species, and development of new, less toxic, and more selective nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitors, to clarify both the role of each NADPH oxidase isoform and their utility in clinical practice. In this review, we analyze the value of NADPH oxidase as a therapeutic target for cardiovascular disease and the old and new pharmacologic agents or strategies to prevent NADPH oxidase activity. Some inhibitors and different direct or indirect approaches are available. Regarding direct NADPH oxidase inhibition, the specificity of NADPH oxidase is the focus of current investigations, whereas the chemical structure-activity relationship studies of known inhibitors have provided pharmacophore models with which to search for new molecules. From a general point of view, small-molecule inhibitors are preferred because of their hydrosolubility and oral bioavailability. However, other possibilities are not closed, with peptide inhibitors or monoclonal antibodies against NADPH oxidase isoforms continuing to be under investigation as well as the ongoing search for naturally occurring compounds. Likewise, some different approaches include inhibition of assembly of the NADPH oxidase complex, subcellular translocation, post-transductional modifications, calcium entry/release, electron transfer, and genetic expression. High-throughput screens for any of these activities could provide new

Current status of NADPH oxidase research in cardiovascular pharmacology

PubMed Central

Rodiño-Janeiro, Bruno K; Paradela-Dobarro, Beatriz; Castiñeiras-Landeira, María Isabel; Raposeiras-Roubín, Sergio; González-Juanatey, José R; Álvarez, Ezequiel

2013-01-01

The implications of reactive oxygen species in cardiovascular disease have been known for some decades. Rationally, therapeutic antioxidant strategies combating oxidative stress have been developed, but the results of clinical trials have not been as good as expected. Therefore, to move forward in the design of new therapeutic strategies for cardiovascular disease based on prevention of production of reactive oxygen species, steps must be taken on two fronts, ie, comprehension of reduction-oxidation signaling pathways and the pathophysiologic roles of reactive oxygen species, and development of new, less toxic, and more selective nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitors, to clarify both the role of each NADPH oxidase isoform and their utility in clinical practice. In this review, we analyze the value of NADPH oxidase as a therapeutic target for cardiovascular disease and the old and new pharmacologic agents or strategies to prevent NADPH oxidase activity. Some inhibitors and different direct or indirect approaches are available. Regarding direct NADPH oxidase inhibition, the specificity of NADPH oxidase is the focus of current investigations, whereas the chemical structure-activity relationship studies of known inhibitors have provided pharmacophore models with which to search for new molecules. From a general point of view, small-molecule inhibitors are preferred because of their hydrosolubility and oral bioavailability. However, other possibilities are not closed, with peptide inhibitors or monoclonal antibodies against NADPH oxidase isoforms continuing to be under investigation as well as the ongoing search for naturally occurring compounds. Likewise, some different approaches include inhibition of assembly of the NADPH oxidase complex, subcellular translocation, post-transductional modifications, calcium entry/release, electron transfer, and genetic expression. High-throughput screens for any of these activities could provide new

Inhibition of Human Vascular NADPH Oxidase by Apocynin Derived Oligophenols

PubMed Central

Mora-Pale, Mauricio; Weïwer, Michel; Yu, Jingjing; Linhardt, Robert J.; Dordick, Jonathan S.

2009-01-01

Enzymatic oxidation of apocynin, which may mimic in vivo metabolism, affords a large number of oligomers (apocynin oxidation products, AOP) that inhibit vascular NADPH oxidase. In vitro studies of NADPH oxidase activity were performed to identify active inhibitors, resulting in a trimer hydroxylated quinone (IIIHyQ) that inhibited NADPH oxidase with an IC50 = 31 nM. Apocynin itself possessed minimal inhibitory activity. NADPH oxidase is believed to be inhibited through prevention of the interaction between two NADPH oxidase subunits, p47phox and p22phox. To that end, while apocynin was unable to block the interaction of his-tagged p47phox with a surface immobilized biotinalyted p22phox peptide, the IIIHyQ product strongly interfered with this interaction (apparent IC50 = 1.6 μM). These results provide evidence that peroxidase-catalyzed AOP, which consist of oligomeric phenols and quinones, inhibit critical interactions that are involved in the assembly and activation of human vascular NADPH oxidase. PMID:19523836

The Impacts of Phosphorus Deficiency on the Photosynthetic Electron Transport Chain.

PubMed

Carstensen, Andreas; Herdean, Andrei; Schmidt, Sidsel Birkelund; Sharma, Anurag; Spetea, Cornelia; Pribil, Mathias; Husted, Søren

2018-05-01

Phosphorus (P) is an essential macronutrient, and P deficiency limits plant productivity. Recent work showed that P deficiency affects electron transport to photosystem I (PSI), but the underlying mechanisms are unknown. Here, we present a comprehensive biological model describing how P deficiency disrupts the photosynthetic machinery and the electron transport chain through a series of sequential events in barley ( Hordeum vulgare ). P deficiency reduces the orthophosphate concentration in the chloroplast stroma to levels that inhibit ATP synthase activity. Consequently, protons accumulate in the thylakoids and cause lumen acidification, which inhibits linear electron flow. Limited plastoquinol oxidation retards electron transport to the cytochrome b 6 f complex, yet the electron transfer rate of PSI is increased under steady-state growth light and is limited under high-light conditions. Under P deficiency, the enhanced electron flow through PSI increases the levels of NADPH, whereas ATP production remains restricted and, hence, reduces CO 2 fixation. In parallel, lumen acidification activates the energy-dependent quenching component of the nonphotochemical quenching mechanism and prevents the overexcitation of photosystem II and damage to the leaf tissue. Consequently, plants can be severely affected by P deficiency for weeks without displaying any visual leaf symptoms. All of the processes in the photosynthetic machinery influenced by P deficiency appear to be fully reversible and can be restored in less than 60 min after resupply of orthophosphate to the leaf tissue. © 2018 American Society of Plant Biologists. All Rights Reserved.

The Impacts of Phosphorus Deficiency on the Photosynthetic Electron Transport Chain1[OPEN

PubMed Central

2018-01-01

Phosphorus (P) is an essential macronutrient, and P deficiency limits plant productivity. Recent work showed that P deficiency affects electron transport to photosystem I (PSI), but the underlying mechanisms are unknown. Here, we present a comprehensive biological model describing how P deficiency disrupts the photosynthetic machinery and the electron transport chain through a series of sequential events in barley (Hordeum vulgare). P deficiency reduces the orthophosphate concentration in the chloroplast stroma to levels that inhibit ATP synthase activity. Consequently, protons accumulate in the thylakoids and cause lumen acidification, which inhibits linear electron flow. Limited plastoquinol oxidation retards electron transport to the cytochrome b6f complex, yet the electron transfer rate of PSI is increased under steady-state growth light and is limited under high-light conditions. Under P deficiency, the enhanced electron flow through PSI increases the levels of NADPH, whereas ATP production remains restricted and, hence, reduces CO2 fixation. In parallel, lumen acidification activates the energy-dependent quenching component of the nonphotochemical quenching mechanism and prevents the overexcitation of photosystem II and damage to the leaf tissue. Consequently, plants can be severely affected by P deficiency for weeks without displaying any visual leaf symptoms. All of the processes in the photosynthetic machinery influenced by P deficiency appear to be fully reversible and can be restored in less than 60 min after resupply of orthophosphate to the leaf tissue. PMID:29540590

Hypertonic Saline Suppresses NADPH Oxidase-Dependent Neutrophil Extracellular Trap Formation and Promotes Apoptosis.

PubMed

Nadesalingam, Ajantha; Chen, Jacky H K; Farahvash, Armin; Khan, Meraj A

2018-01-01

Tonicity of saline (NaCl) is important in regulating cellular functions and homeostasis. Hypertonic saline is administered to treat many inflammatory diseases, including cystic fibrosis. Excess neutrophil extracellular trap (NET) formation, or NETosis, is associated with many pathological conditions including chronic inflammation. Despite the known therapeutic benefits of hypertonic saline, its underlying mechanisms are not clearly understood. Therefore, we aimed to elucidate the effects of hypertonic saline in modulating NETosis. For this purpose, we purified human neutrophils and induced NETosis using agonists such as diacylglycerol mimetic phorbol myristate acetate (PMA), Gram-negative bacterial cell wall component lipopolysaccharide (LPS), calcium ionophores (A23187 and ionomycin from Streptomyces conglobatus ), and bacteria ( Pseudomonas aeruginosa and Staphylococcus aureus ). We then analyzed neutrophils and NETs using Sytox green assay, immunostaining of NET components and apoptosis markers, confocal microscopy, and pH sensing reagents. This study found that hypertonic NaCl suppresses nicotinamide adenine dinucleotide phosphate oxidase (NADPH2 or NOX2)-dependent NETosis induced by agonists PMA, Escherichia coli LPS (0111:B4 and O128:B12), and P. aeruginosa . Hypertonic saline also suppresses LPS- and PMA- induced reactive oxygen species production. It was determined that supplementing H 2 O 2 reverses the suppressive effect of hypertonic saline on NOX2-dependent NETosis. Many of the aforementioned suppressive effects were observed in the presence of equimolar concentrations of choline chloride and osmolytes (d-mannitol and d-sorbitol). This suggests that the mechanism by which hypertonic saline suppresses NOX2-dependent NETosis is via neutrophil dehydration. Hypertonic NaCl does not significantly alter the intracellular pH of neutrophils. We found that hypertonic NaCl induces apoptosis while suppressing NOX2-dependent NETosis. In contrast, hypertonic

Registration of 'Dan' winter hulless barley

USDA-ARS?s Scientific Manuscript database

Dan’ (Reg. No. CV- , PI 659066) six-rowed winter hulless barley (Hordeum vulgare L.) was developed and released by the Virginia Agricultural Experiment Station in March 2009. Dan was derived from the cross VA96-41-17 / SC872143. It was released for production in the eastern United States, as a poten...

Registration of ‘Secretariat’ winter barley

USDA-ARS?s Scientific Manuscript database

Secretariat’ (PI 673931) is a six-row hulled winter feed barley (Hordeum vulgare L.) cultivar developed by the Virginia Agricultural Experiment Station and released in May 2014. Secretariat, formerly designated VA08B-85, was derived from the cross VA00B-199 / VA00B-259 and was developed using a mod...

Anti-Inflammatory, Antioxidant, Antibiotic, and Cytotoxic Activities of Tanacetum vulgare L. Essential Oil and Its Constituents.

PubMed

Coté, Héloïse; Boucher, Marie-Anne; Pichette, André; Legault, Jean

2017-05-25

Background: Tanacetum vulgare L. (Asteraceae) is a perennial herb that has been used to treat multiple ailments. Regional variability of the chemical composition of T. vulgare essential oils is well-known. Despite these regional chemotypes, most relevant studies did not analyze the complete chemical composition of the T. vulgare essential oil and its constituents in relation to their biological activities. Here, we assess the anti-inflammatory, antioxidant, antibacterial, and cytotoxic activities of T. vulgare collected from northern Quebec (Saguenay-Lac-St-Jean), Canada. Methods: Essential oil was extracted from plants by steam distillation and analyzed using GC-FID. Biological activities of essential oil and its main constituents were evaluated in vitro. Results: We identified the major compounds as camphor, borneol, and 1,8-cineole. The oil possesses anti-inflammatory activity inhibiting NO production. It also inhibits intracellular DCFH oxidation induced by tert-butylhydroperoxide. Anti-inflammatory activity of essential oil appears driven mainly by α-humulene while antioxidant activity is provided by α-pinene and caryophyllene oxide. Essential oil from T vulgare was active against both Escherichia coli and Staphylococcus aureus with camphor and caryophyllene oxide responsible for antibacterial activity. Finally, T. vulgare essential oil was slightly cytotoxic against the human healthy cell line WS1 while α-humulene and caryophyllene oxide were moderately cytotoxic against A-549, DLD-1, and WS1. Conclusion: We report, for the first time, links between the specific compounds found in T. vulgare essential oil and anti-inflammatory, antioxidant, antibacterial, and cytotoxic activities. T. vulgare essential oil possesses interesting biological properties.

Chemical composition and biological activity of essential oils of Origanum vulgare L. subsp. vulgare L. under different growth conditions.

PubMed

De Falco, Enrica; Mancini, Emilia; Roscigno, Graziana; Mignola, Enrico; Taglialatela-Scafati, Orazio; Senatore, Felice

2013-12-04

This research was aimed at investigating the essential oil production, chemical composition and biological activity of a crop of pink flowered oregano (Origanum vulgare L. subsp. vulgare L.) under different spatial distribution of the plants (single and binate rows). This plant factor was shown to affect its growth, soil covering, fresh biomass, essential oil amount and composition. In particular, the essential oil percentage was higher for the binate row treatment at the full bloom. The chemical composition of the oils obtained by hydrodistillation was fully characterized by GC and GC-MS. The oil from plants grown in single rows was rich in sabinene, while plants grown in double rows were richer in ocimenes. The essential oils showed antimicrobial action, mainly against Gram-positive pathogens and particularly Bacillus cereus and B. subtilis.

ROS mediated selection for increased NADPH availability in Escherichia coli.

PubMed

Reynolds, Thomas S; Courtney, Colleen M; Erickson, Keesha E; Wolfe, Lisa M; Chatterjee, Anushree; Nagpal, Prashant; Gill, Ryan T

2017-11-01

The economical production of chemicals and fuels by microbial processes remains an intense area of interest in biotechnology. A key limitation in such efforts concerns the availability of key co-factors, in this case NADPH, required for target pathways. Many of the strategies pursued for increasing NADPH availability in Escherichia coli involve manipulations to the central metabolism, which can create redox imbalances and overall growth defects. In this study we used a reactive oxygen species based selection to search for novel methods of increasing NADPH availability. We report a loss of function mutation in the gene hdfR appears to increase NADPH availability in E. coli. Additionally, we show this excess NADPH can be used to improve the production of 3HP in E. coli. © 2017 Wiley Periodicals, Inc.

Calcium mobilization and Rac1 activation are required for VCAM-1 (vascular cell adhesion molecule-1) stimulation of NADPH oxidase activity.

PubMed Central

Cook-Mills, Joan M; Johnson, Jacob D; Deem, Tracy L; Ochi, Atsuo; Wang, Lei; Zheng, Yi

2004-01-01

VCAM-1 (vascular cell adhesion molecule-1) plays an important role in the regulation of inflammation in atherosclerosis, asthma, inflammatory bowel disease and transplantation. VCAM-1 activates endothelial cell NADPH oxidase, and this oxidase activity is required for VCAM-1-dependent lymphocyte migration. We reported previously that a mouse microvascular endothelial cell line promotes lymphocyte migration that is dependent on VCAM-1, but not on other known adhesion molecules. Here we have investigated the signalling mechanisms underlying VCAM-1 function. Lymphocyte binding to VCAM-1 on the endothelial cell surface activated an endothelial cell calcium flux that could be inhibited with anti-alpha4-integrin and mimicked by anti-VCAM-1-coated beads. VCAM-1 stimulation of calcium responses could be blocked by an inhibitor of intracellular calcium mobilization, a calcium channel inhibitor or a calcium chelator, resulting in the inhibition of NADPH oxidase activity. Addition of ionomycin overcame the calcium channel blocker suppression of VCAM-1-stimulated NADPH oxidase activity, but could not reverse the inhibitory effect imposed by intracellular calcium blockage, indicating that both intracellular and extracellular calcium mobilization are required for VCAM-1-mediated activation of NADPH oxidase. Furthermore, VCAM-1 specifically activated the Rho-family GTPase Rac1, and VCAM-1 activation of NADPH oxidase was blocked by a dominant negative Rac1. Thus VCAM-1 stimulates the mobilization of intracellular and extracellular calcium and Rac1 activity that are required for the activation of NADPH oxidase. PMID:14594451

Intraspecific differences in drought tolerance and acclimation in hydraulics of Ligustrum vulgare and Viburnum lantana.

PubMed

Beikircher, Barbara; Mayr, Stefan

2009-06-01

An adequate general drought tolerance and the ability to acclimate to changing hydraulic conditions are important features for long-lived woody plants. In this study, we compared hydraulic safety (water potential at 50% loss of conductivity, Psi(50)), hydraulic efficiency (specific conductivity, k(s)), xylem anatomy (mean tracheid diameter, d(mean), mean hydraulic diameter, d(h), conduit wall thickness, t, conduit wall reinforcement, (t/b)(h)(2)) and stomatal conductance, g(s), of forest plants as well as irrigated and drought-treated garden plants of Ligustrum vulgare L. and Viburnum lantana L. Forest plants of L. vulgare and V. lantana were significantly less resistant to drought-induced cavitation (Psi(50) at -2.82 +/- 0.13 MPa and -2.79 +/- 0.17 MPa) than drought-treated garden plants (- 4.58 +/- 0.26 MPa and -3.57 +/- 0.15 MPa). When previously irrigated garden plants were subjected to drought, a significant decrease in d(mean) and d(h) and an increase in t and (t/b)(h)(2) were observed in L. vulgare. In contrast, in V. lantana conduit diameters increased significantly but no change in t and (t/b)(h)(2) was found. Stomatal closure occurred at similar water potentials (Psi(sc)) in forest plants and drought-treated garden plants, leading to higher safety margins (Psi(sc) - Psi(50)) of the latter (L. vulgare 1.63 MPa and V. lantana 0.43 MPa). These plants also showed higher g(s) at moderate Psi, more abrupt stomatal closure and lower cuticular conductivity. Data indicate that the development of drought-tolerant xylem as well as stomatal regulation play an important role in drought acclimation, whereby structural and physiological responses to drought are species-specific and depend on the plant's hydraulic strategy.

Relative importance of redox buffers GSH and NAD(P)H in age-related neurodegeneration and Alzheimer disease-like mouse neurons.

PubMed

Ghosh, Debolina; Levault, Kelsey R; Brewer, Gregory J

2014-08-01

Aging, a major risk factor in Alzheimer's disease (AD), is associated with an oxidative redox shift, decreased redox buffer protection, and increased free radical reactive oxygen species (ROS) generation, probably linked to mitochondrial dysfunction. While NADH is the ultimate electron donor for many redox reactions, including oxidative phosphorylation, glutathione (GSH) is the major ROS detoxifying redox buffer in the cell. Here, we explored the relative importance of NADH and GSH to neurodegeneration in aging and AD neurons from nontransgenic and 3xTg-AD mice by inhibiting their synthesis to determine whether NADH can compensate for the GSH loss to maintain redox balance. Neurons stressed by either depleting NAD(P)H or GSH indicated that NADH redox control is upstream of GSH levels. Further, although depletion of NAD(P)H or GSH correlated linearly with neuron death, compared with GSH depletion, higher neurodegeneration was observed when NAD(P)H was extrapolated to zero, especially in old age, and in the 3xTg-AD neurons. We also observed an age-dependent loss of gene expression of key redox-dependent biosynthetic enzymes, NAMPT (nicotinamide phosphoribosyltransferase), and NNT (nicotinamide nucleotide transhydrogenase). Moreover, age-related correlations between brain NNT or NAMPT gene expression and NADPH levels suggest that these genes contribute to the age-related declines in NAD(P)H. Our data indicate that in aging and more so in AD-like neurons, NAD(P)H redox control is upstream of GSH and an oxidative redox shift that promotes neurodegeneration. Thus, NAD(P)H generation may be a more efficacious therapeutic target upstream of GSH and ROS. © 2014 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

Registration of ‘Kardia’, a Two-Rowed Spring Food Barley

USDA-ARS?s Scientific Manuscript database

‘Kardia’ (Reg. No. XXXX, XXXX), a two-rowed spring food barley (Hordeum vulgare L.) developed by the USDA-ARS, Aberdeen, ID, in cooperation with the University of Idaho Agricultural Experiment Station, was released in 2015. Kardia is derived from the cross of ‘03AH3054 / 98Ab12019’ and was advanced...

NADPH Oxidase-Driven Phagocyte Recruitment Controls Candida albicans Filamentous Growth and Prevents Mortality

PubMed Central

Brothers, Kimberly M.; Gratacap, Remi L.; Barker, Sarah E.; Newman, Zachary R.; Norum, Ashley; Wheeler, Robert T.

2013-01-01

Candida albicans is a human commensal and clinically important fungal pathogen that grows as both yeast and hyphal forms during human, mouse and zebrafish infection. Reactive oxygen species (ROS) produced by NADPH oxidases play diverse roles in immunity, including their long-appreciated function as microbicidal oxidants. Here we demonstrate a non-traditional mechanistic role of NADPH oxidase in promoting phagocyte chemotaxis and intracellular containment of fungi to limit filamentous growth. We exploit the transparent zebrafish model to show that failed NADPH oxidase-dependent phagocyte recruitment to C. albicans in the first four hours post-infection permits fungi to germinate extracellularly and kill the host. We combine chemical and genetic tools with high-resolution time-lapse microscopy to implicate both phagocyte oxidase and dual-specific oxidase in recruitment, suggesting that both myeloid and non-myeloid cells promote chemotaxis. We show that early non-invasive imaging provides a robust tool for prognosis, strongly connecting effective early immune response with survival. Finally, we demonstrate a new role of a key regulator of the yeast-to-hyphal switching program in phagocyte-mediated containment, suggesting that there are species-specific methods for modulation of NADPH oxidase-independent immune responses. These novel links between ROS-driven chemotaxis and fungal dimorphism expand our view of a key host defense mechanism and have important implications for pathogenesis. PMID:24098114

NADPH oxidases: novel therapeutic targets for neurodegenerative diseases.

PubMed

Gao, Hui-Ming; Zhou, Hui; Hong, Jau-Shyong

2012-06-01

Oxidative stress is a key pathologic factor in neurodegenerative diseases such as Alzheimer and Parkinson diseases (AD, PD). The failure of free-radical-scavenging antioxidants in clinical trials pinpoints an urgent need to identify and to block major sources of oxidative stress in neurodegenerative diseases. As a major superoxide-producing enzyme complex in activated phagocytes, phagocyte NADPH oxidase (PHOX) is essential for host defense. However, recent preclinical evidence has underscored a pivotal role of overactivated PHOX in chronic neuroinflammation and progressive neurodegeneration. Deficiency in PHOX subunits mitigates neuronal damage induced by diverse insults/stresses relevant to neurodegenerative diseases. More importantly, suppression of PHOX activity correlates with reduced neuronal impairment in models of neurodegenerative diseases. The discovery of PHOX and non-phagocyte NADPH oxidases in astroglia and neurons further reinforces the crucial role of NADPH oxidases in oxidative stress-mediated chronic neurodegeneration. Thus, proper modulation of NADPH oxidase activity might hold therapeutic potential for currently incurable neurodegenerative diseases. Published by Elsevier Ltd.

Essential oil diversity of European Origanum vulgare L. (Lamiaceae).

PubMed

Lukas, Brigitte; Schmiderer, Corinna; Novak, Johannes

2015-11-01

This investigation focused on the qualitative and quantitative composition of essential oil compounds of European Origanum vulgare. Extracts of 502 individual O. vulgare plants from 17 countries and 51 populations were analyzed via GC. Extracts of 49 plants of 5 populations of Israeli Origanum syriacum and 30 plants from 3 populations of Turkish Origanum onites were included to exemplify essential oil characteristics of 'high-quality' oregano. The content of essential oil compounds of European O. vulgare ranged between 0.03% and 4.6%. The monoterpenes were primarily made up of sabinene, myrcene, p-cymene, 1,8-cineole, β-ocimene, γ-terpinene, sabinene hydrate, linalool, α-terpineol, carvacrol methyl ether, linalyl acetate, thymol and carvacrol. Among the sesquiterpenes β-caryophyllene, germacrene D, germacrene D-4-ol, spathulenol, caryophyllene oxide and oplopanone were often present in higher amounts. According to the proportions of cymyl-compounds, sabinyl-compounds and the acyclic linalool/linalyl acetate three different main monoterpene chemotypes were defined. The cymyl- and the acyclic pathway were usually active in plants from the Mediterranean climate whereas an active sabinyl-pathway was a characteristic of plants from the Continental climate. Copyright © 2015 Elsevier Ltd. All rights reserved.

A Barley Efflux Transporter Operates in a Na+-Dependent Manner, as Revealed by a Multidisciplinary Platform[OPEN

PubMed Central

Nagarajan, Yagnesh; Rongala, Jay; Luang, Sukanya; Shadiac, Nadim; Sutton, Tim; Tyerman, Stephen D.; McPhee, Gordon; Voelcker, Nicolas H.; Lee, Jung-Goo

2016-01-01

Plant growth and survival depend upon the activity of membrane transporters that control the movement and distribution of solutes into, around, and out of plants. Although many plant transporters are known, their intrinsic properties make them difficult to study. In barley (Hordeum vulgare), the root anion-permeable transporter Bot1 plays a key role in tolerance to high soil boron, facilitating the efflux of borate from cells. However, its three-dimensional structure is unavailable and the molecular basis of its permeation function is unknown. Using an integrative platform of computational, biophysical, and biochemical tools as well as molecular biology, electrophysiology, and bioinformatics, we provide insight into the origin of transport function of Bot1. An atomistic model, supported by atomic force microscopy measurements, reveals that the protein folds into 13 transmembrane-spanning and five cytoplasmic α-helices. We predict a trimeric assembly of Bot1 and the presence of a Na+ ion binding site, located in the proximity of a pore that conducts anions. Patch-clamp electrophysiology of Bot1 detects Na+-dependent polyvalent anion transport in a Nernstian manner with channel-like characteristics. Using alanine scanning, molecular dynamics simulations, and transport measurements, we show that conductance by Bot1 is abolished by removal of the Na+ ion binding site. Our data enhance the understanding of the permeation functions of Bot1. PMID:26672067

NO nerves in a tapeworm. NADPH-diaphorase histochemistry in adult Hymenolepis diminuta.

PubMed

Gustafsson, M K; Lindholm, A M; Terenina, N B; Reuter, M

1996-12-01

The free radical nitric oxide (NO), which is synthesized by nitric oxide synthase (NOS), has recently been discovered to function as a neuronal messenger. The presence of NOS was detected in the nervous system of adult Hymenolepis diminuta with NADPH-diaphorase (NADPH-d) histochemistry. The NADPH-d histochemical reaction is regarded as a selective marker for NOS in neuronal tissue. NADPH-d staining was observed in nerve fibres in the main and minor nerve cords and the transverse ring commissures, and in cell bodies in the brain commissure, along the main nerve cords, in the suckers and the rostellar sac. NADPH-d staining was also observed in the wall of the internal seminal vesicle and the genital atrium. The pattern of NADPH-d staining was compared with that of the 5-HT immunoreactive nervous elements. The NADPH-d staining reaction and the 5-HT immunoreactivity occur in separate sets of neurons. This is the first time the NADPH-d reaction has been demonstrated in the nervous system of a flatworm, indicating that NOS is present and that NO can be produced at this level of evolution.

Coupling Drosophila melanogaster Cryptochrome Light Activation and Oxidation of the Kvβ Subunit Hyperkinetic NADPH Cofactor.

PubMed

Hong, Gongyi; Pachter, Ruth; Ritz, Thorsten

2018-06-28

Motivated by the observations on the involvement of light-induced processes in the Drosophila melanogaster cryptochrome (DmCry) in regulation of the neuronal firing rate, which is achieved by a redox-state change of its voltage-dependent K + channel Kvβ subunit hyperkinetic (Hk) reduced nicotinamide adenine dinucleotide phosphate (NADPH) cofactor, we propose in this work two hypothetical pathways that may potentially enable such coupling. In the first pathway, triggered by blue-light-induced formation of a radical pair [FAD •- TRP •+ ] in DmCry, the hole (TRP •+ ) may hop to Hk, for example, through a tryptophan chain and oxidize NADPH, possibly leading to inhibition of the N-terminus inactivation in the K + channel. In a second possible pathway, DmCry's FAD •- is reoxidized by molecular oxygen, producing H 2 O 2 , which then diffuses to Hk and oxidizes NADPH. In this work, by applying a combination of quantum and empirical-based methods for free-energy calculations, we find that the oxidation of NADPH by TRP •+ or H 2 O 2 and the reoxidation of FAD •- by O 2 are thermodynamically feasible. Our results may have an implication in identifying a magnetic sensing signal transduction pathway, specifically upon Drosophila's Hk NADPH cofactor oxidation, with a subsequent inhibition of the K + channel N-terminus inactivation gate, permitting K + flux.

Crystal structures and atomic model of NADPH oxidase.

PubMed

Magnani, Francesca; Nenci, Simone; Millana Fananas, Elisa; Ceccon, Marta; Romero, Elvira; Fraaije, Marco W; Mattevi, Andrea

2017-06-27

NADPH oxidases (NOXs) are the only enzymes exclusively dedicated to reactive oxygen species (ROS) generation. Dysregulation of these polytopic membrane proteins impacts the redox signaling cascades that control cell proliferation and death. We describe the atomic crystal structures of the catalytic flavin adenine dinucleotide (FAD)- and heme-binding domains of Cylindrospermum stagnale NOX5. The two domains form the core subunit that is common to all seven members of the NOX family. The domain structures were then docked in silico to provide a generic model for the NOX family. A linear arrangement of cofactors (NADPH, FAD, and two membrane-embedded heme moieties) injects electrons from the intracellular side across the membrane to a specific oxygen-binding cavity on the extracytoplasmic side. The overall spatial organization of critical interactions is revealed between the intracellular loops on the transmembrane domain and the NADPH-oxidizing dehydrogenase domain. In particular, the C terminus functions as a toggle switch, which affects access of the NADPH substrate to the enzyme. The essence of this mechanistic model is that the regulatory cues conformationally gate NADPH-binding, implicitly providing a handle for activating/deactivating the very first step in the redox chain. Such insight provides a framework to the discovery of much needed drugs that selectively target the distinct members of the NOX family and interfere with ROS signaling.

Ebselen and congeners inhibit NADPH oxidase 2-dependent superoxide generation by interrupting the binding of regulatory subunits.

PubMed

Smith, Susan M E; Min, Jaeki; Ganesh, Thota; Diebold, Becky; Kawahara, Tsukasa; Zhu, Yerun; McCoy, James; Sun, Aiming; Snyder, James P; Fu, Haian; Du, Yuhong; Lewis, Iestyn; Lambeth, J David

2012-06-22

NADPH oxidases (Nox) are a primary source of reactive oxygen species (ROS), which function in normal physiology and, when overproduced, in pathophysiology. Recent studies using mice deficient in Nox2 identify this isoform as a novel target against Nox2-implicated inflammatory diseases. Nox2 activation depends on the binding of the proline-rich domain of its heterodimeric partner p22phox to p47phox. A high-throughput screen that monitored this interaction via fluorescence polarization identified ebselen and several of its analogs as inhibitors. Medicinal chemistry was performed to explore structure-activity relationships and to optimize potency. Ebselen and analogs potently inhibited Nox1 and Nox2 activity but were less effective against other isoforms. Ebselen also blocked translocation of p47phox to neutrophil membranes. Thus, ebselen and its analogs represent a class of compounds that inhibit ROS generation by interrupting the assembly of Nox2-activating regulatory subunits. Copyright © 2012 Elsevier Ltd. All rights reserved.

NADPH OXIDASE: STRUCTURE AND ACTIVATION MECHANISMS (REVIEW). NOTE I.

PubMed

Filip-Ciubotaru, Florina; Manciuc, Carmen; Stoleriu, Gabriela; Foia, Liliana

2016-01-01

NADPH oxidase (nicotinamide adenine dinucleotide phosphate-oxidase), with its generically termed NOX isoforms, is the major source of ROS (reactive oxigen species) in biological systems. ROS are small oxygen-derived molecules with an important role in various biological processes (physiological or pathological). If under physiological conditions some processes are beneficial and necessary for life, under pathophysiological conditions they are noxious, harmful. NADPH oxidases are present in phagocytes and in a wide variety of nonphagocytic cells. The enzyme generates superoxide by transferring electrons from NADPH inside the cell across the membrane and coupling them to molecular oxygen to produce superoxide anion, a reactive free-radical. Structurally, NADPH oxidase is a multicomponent enzyme which includes two integral membrane proteins, glycoprotein gp9 1 Phox and adaptor protein p22(phox), which together form the heterodimeric flavocytochrome b558 that constitutes the core of the enzyme. During the resting state, the multidomain regulatory subunits p40P(phox), p47(phox), p67(Phox) are located in the cytosol organized as a complex. The activation of phagocytic NADPH oxidase occurs through a complex series of protein interactions.

Molecular Insights of p47phox Phosphorylation Dynamics in the Regulation of NADPH Oxidase Activation and Superoxide Production*

PubMed Central

Meijles, Daniel N.; Fan, Lampson M.; Howlin, Brendan J.; Li, Jian-Mei

2014-01-01

Phagocyte superoxide production by a multicomponent NADPH oxidase is important in host defense against microbial invasion. However inappropriate NADPH oxidase activation causes inflammation. Endothelial cells express NADPH oxidase and endothelial oxidative stress due to prolonged NADPH oxidase activation predisposes many diseases. Discovering the mechanism of NADPH oxidase activation is essential for developing novel treatment of these diseases. The p47phox is a key regulatory subunit of NADPH oxidase; however, due to the lack of full protein structural information, the mechanistic insight of p47phox phosphorylation in NADPH oxidase activation remains incomplete. Based on crystal structures of three functional domains, we generated a computational structural model of the full p47phox protein. Using a combination of in silico phosphorylation, molecular dynamics simulation and protein/protein docking, we discovered that the C-terminal tail of p47phox is critical for stabilizing its autoinhibited structure. Ser-379 phosphorylation disrupts H-bonds that link the C-terminal tail to the autoinhibitory region (AIR) and the tandem Src homology 3 (SH3) domains, allowing the AIR to undergo phosphorylation to expose the SH3 pocket for p22phox binding. These findings were confirmed by site-directed mutagenesis and gene transfection of p47phox−/− coronary microvascular cells. Compared with wild-type p47phox cDNA transfected cells, the single mutation of S379A completely blocked p47phox membrane translocation, binding to p22phox and endothelial O2⨪ production in response to acute stimulation of PKC. p47phox C-terminal tail plays a key role in stabilizing intramolecular interactions at rest. Ser-379 phosphorylation is a molecular switch which initiates p47phox conformational changes and NADPH oxidase-dependent superoxide production by cells. PMID:24970888

Ultrafine carbon particles promote rotenone-induced dopamine neuronal loss through activating microglial NADPH oxidase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Yinxi; Liu, Dan; Zhang, Huifeng

Background: Atmospheric ultrafine particles (UFPs) and pesticide rotenone were considered as potential environmental risk factors for Parkinson's disease (PD). However, whether and how UFPs alone and in combination with rotenone affect the pathogenesis of PD remains largely unknown. Methods: Ultrafine carbon black (ufCB, a surrogate of UFPs) and rotenone were used individually or in combination to determine their roles in chronic dopaminergic (DA) loss in neuron-glia, and neuron-enriched, mix-glia cultures. Immunochemistry using antibody against tyrosine hydroxylase was performed to detect DA neuronal loss. Measurement of extracellular superoxide and intracellular reactive oxygen species (ROS) were performed to examine activation of NADPHmore » oxidase. Genetic deletion and pharmacological inhibition of NADPH oxidase and MAC-1 receptor in microglia were employed to examine their role in DA neuronal loss triggered by ufCB and rotenone. Results: In rodent midbrain neuron-glia cultures, ufCB and rotenone alone caused neuronal death in a dose-dependent manner. In particularly, ufCB at doses of 50 and 100 μg/cm{sup 2} induced significant loss of DA neurons. More importantly, nontoxic doses of ufCB (10 μg/cm{sup 2}) and rotenone (2 nM) induced synergistic toxicity to DA neurons. Microglial activation was essential in this process. Furthermore, superoxide production from microglial NADPH oxidase was critical in ufCB/rotenone-induced neurotoxicity. Studies in mix-glia cultures showed that ufCB treatment activated microglial NADPH oxidase to induce superoxide production. Firstly, ufCB enhanced the expression of NADPH oxidase subunits (gp91{sup phox}, p47{sup phox} and p40{sup phox}); secondly, ufCB was recognized by microglial surface MAC-1 receptor and consequently promoted rotenone-induced p47{sup phox} and p67{sup phox} translocation assembling active NADPH oxidase. Conclusion: ufCB and rotenone worked in synergy to activate NADPH oxidase in microglia, leading to

A possible role of NADPH-dependent cytochrome P450nor isozyme in glycolysis under denitrifying conditions.

PubMed

Watsuji, Tomo-o; Takaya, Naoki; Nakamura, Akira; Shoun, Hirofumi

2003-05-01

The denitrifying fungus Cylindrocarpon tonkinense contains two isozymes of cytochrome P450nor. One isozyme, P450nor1, uses NADH specifically as its electron donor whereas the other isozyme P450nor2 prefers NADPH to NADH. Here we show that P450nor1 is localized in both cytosol and mitochondria, like P450nor of Fusarium oxysporum, while P450nor2 is exclusively in cytosol. We also found that the addition of glucose as a carbon source to the culture media leads to the production of much more P450nor2 in the fungal cells than a non-fermentable substrate (glycerol or acetate) does. These results suggest that the NADP-dependent pentose phosphate cycle acts predominantly in C. tonkinense as the glycolysis pathway under the denitrifying conditions, which was confirmed by the observation that glucose induced enzyme activities involved in the cycle. These results showed that P450nor2 should act as the electron sink under anaerobic, denitrifying conditions to regenerate NADP+ for the pentose phosphate cycle.

Attenuation of NADPH oxidase activation and glomerular filtration barrier remodeling with statin treatment.

PubMed

Whaley-Connell, Adam; Habibi, Javad; Nistala, Ravi; Cooper, Shawna A; Karuparthi, Poorna R; Hayden, Melvin R; Rehmer, Nathan; DeMarco, Vincent G; Andresen, Bradley T; Wei, Yongzhong; Ferrario, Carlos; Sowers, James R

2008-02-01

Activation of reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase by angiotensin II is integral to the formation of oxidative stress in the vasculature and the kidney. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibition is associated with reductions of oxidative stress in the vasculature and kidney and associated decreases in albuminuria. Effects of 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibition on oxidative stress in the kidney and filtration barrier integrity are poorly understood. To investigate, we used transgenic TG(mRen2)27 (Ren2) rats, which harbor the mouse renin transgene and renin-angiotensin system activation, and an immortalized murine podocyte cell line. We treated young, male Ren2 and Sprague-Dawley rats with rosuvastatin (20 mg/kg IP) or placebo for 21 days. Compared with controls, we observed increases in systolic blood pressure, albuminuria, renal NADPH oxidase activity, and 3-nitrotryosine staining, with reductions in the rosuvastatin-treated Ren2. Structural changes on light and transmission electron microscopy, consistent with periarteriolar fibrosis and podocyte foot-process effacement, were attenuated with statin treatment. Nephrin expression was diminished in the Ren2 kidney and trended to normalize with statin treatment. Angiotensin II-dependent increases in podocyte NADPH oxidase activity and subunit expression (NOX2, NOX4, Rac, and p22(phox)) and reactive oxygen species generation were decreased after in vitro statin treatment. These data support a role for increased NADPH oxidase activity and subunit expression with resultant reactive oxygen species formation in the kidney and podocyte. Furthermore, statin attenuation of NADPH oxidase activation and reactive oxygen species formation in the kidney/podocyte seems to play roles in the abrogation of oxidative stress-induced filtration barrier injury and consequent albuminuria.

Structure of conjugated polyketone reductase from Candida parapsilosis IFO 0708 reveals conformational changes for substrate recognition upon NADPH binding.

PubMed

Qin, Hui-Min; Yamamura, Akihiro; Miyakawa, Takuya; Kataoka, Michihiko; Nagai, Takahiro; Kitamura, Nahoko; Urano, Nobuyuki; Maruoka, Shintaro; Ohtsuka, Jun; Nagata, Koji; Shimizu, Sakayu; Tanokura, Masaru

2014-01-01

Conjugated polyketone reductase C2 (CPR-C2) from Candida parapsilosis IFO 0708, identified as a nicotinamide adenine dinucleotide phosphate (NADPH)-dependent ketopantoyl lactone reductase, belongs to the aldo-keto reductase superfamily. This enzyme reduces ketopantoyl lactone to D-pantoyl lactone in a strictly stereospecific manner. To elucidate the structural basis of the substrate specificity, we determined the crystal structures of the apo CPR-C2 and CPR-C2/NADPH complex at 1.70 and 1.80 Å resolutions, respectively. CPR-C2 adopted a triose-phosphate isomerase barrel fold at the core of the structure. Binding with the cofactor NADPH induced conformational changes in which Thr27 and Lys28 moved 15 and 5.0 Å, respectively, in the close vicinity of the adenosine 2'-phosphate group of NADPH to form hydrogen bonds. Based on the comparison of the CPR-C2/NADPH structure with 3-α-hydroxysteroid dehydrogenase and mutation analyses, we constructed substrate binding models with ketopantoyl lactone, which provided insight into the substrate specificity by the cofactor-induced structure. The results will be useful for the rational design of CPR-C2 mutants targeted for use in the industrial manufacture of ketopantoyl lactone.

Enhanced Purification of Recombinant Rat NADPH-P450 Reductase by Using a Hexahistidine-Tag.

PubMed

Park, Hyoung-Goo; Lim, Young-Ran; Han, Songhee; Jeong, Dabin; Kim, Donghak

2017-05-28

NADPH-P450 reductase (NPR) transfers electrons from NADPH to cytochrome P450 and heme oxygenase enzymes to support their catalytic activities. This protein is localized within the endoplasmic reticulum membrane and utilizes FMN, FAD, and NADPH as cofactors. Although NPR is essential toward enabling the biochemical and pharmacological analyses of P450 enzymes, its production as a recombinant purified protein requires a series of tedious efforts and a high cost due to the use of NADP + in the affinity chromatography process. In the present study, the rat NPR clone containing a 6× Histidine-tag (NPR-His) was constructed and heterologously expressed. The NPR-His protein was purified using Ni 2+ -affinity chromatography, and its functional features were characterized. A single band at 78 kDa was observed from SDS-PAGE and the purified protein displayed a maximum absorbance at 455 nm, indicating the presence of an oxidized flavin cofactor. Cytochrome c and nitroblue tetrazolium were reduced by purified NPR-His in an NADPH-dependent manner. The purified NPR-His successfully supported the catalytic activities of human P450 1A2 and 2A6 and fungal CYP52A21, yielding results similar to those obtained using conventional purified rat reductase. This study will facilitate the use of recombinant NPR-His protein in the various fields of P450 research.

Antibacterial and antioxidant activities of essential oils isolated from Thymbra capitata L. (Cav.) andOriganum vulgare L.

PubMed

Faleiro, Leonor; Miguel, Graça; Gomes, Sónia; Costa, Ludmila; Venâncio, Florencia; Teixeira, Adriano; Figueiredo, A Cristina; Barroso, José G; Pedro, Luis G

2005-10-19

Antilisterial activities of Thymbra capitata and Origanum vulgare essential oils were tested against 41 strains of Listeria monocytogenes. The oil of T. capitata was mainly constituted by one component, carvacrol (79%), whereas for O. vulgare three components constituted 70% of the oil, namely, thymol (33%), gamma-terpinene (26%), and p-cymene (11%). T. capitata essential oil had a significantly higher antilisterial activity in comparison to O. vulgare oil and chloramphenicol. No significant differences in L. monocytogenes susceptibilities to the essential oils tested were registered. The minimum inhibitory concentration values of T. capitata essential oil and of carvacrol were quite similar, ranging between 0.05 and 0.2 microL/mL. Antioxidant activity was also tested, the essential oil of T. capitata showing significantly higher antioxidant activity than that of O. vulgare. Use of T. capitata and O. vulgare essential oils can constitute a powerful tool in the control of L. monocytogenes in food and other industries.

Thermodynamic and NMR analyses of NADPH binding to lipocalin-type prostaglandin D synthase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qin, Shubin; Shimamoto, Shigeru; Maruno, Takahiro

2015-12-04

Lipocalin-type prostaglandin D synthase (L-PGDS) is one of the most abundant proteins in human cerebrospinal fluid (CSF) with dual functions as a prostaglandin D{sub 2} (PGD{sub 2}) synthase and a transporter of lipophilic ligands. Recent studies revealed that L-PGDS plays important roles in protecting against various neuronal diseases induced by reactive oxygen species (ROS). However, the molecular mechanisms of such protective actions of L-PGDS remain unknown. In this study, we conducted thermodynamic and nuclear magnetic resonance (NMR) analyses, and demonstrated that L-PGDS binds to nicotinamide coenzymes, including NADPH, NADP{sup +}, and NADH. Although a hydrophilic ligand is not common formore » L-PGDS, these ligands, especially NADPH showed specific interaction with L-PGDS at the upper pocket of its ligand-binding cavity with an unusually bifurcated shape. The binding affinity of L-PGDS for NADPH was comparable to that previously reported for NADPH oxidases and NADPH in vitro. These results suggested that L-PGDS potentially attenuates the activities of NADPH oxidases through interaction with NADPH. Given that NADPH is the substrate for NADPH oxidases that play key roles in neuronal cell death by generating excessive ROS, these results imply a novel linkage between L-PGDS and ROS. - Highlights: • Interactions of L-PGDS with nicotinamide coenzymes were studied by ITC and NMR. • The binding affinity of L-PGDS was strongest to NADPH among nicotinamide coenzymes. • NADPH binds to the upper part of L-PGDS ligand-binding cavity. • L-PGDS binds to both lipophilic and hydrophilic ligands. • This study implies a novel linkage between L-PGDS and reactive oxygen species.« less

Intracellular NADPH Levels Affect the Oligomeric State of the Glucose 6-Phosphate Dehydrogenase

PubMed Central

Tramonti, Angela; Lanini, Claudio; Cialfi, Samantha; De Biase, Daniela; Falcone, Claudio

2012-01-01

In the yeast Kluyveromyces lactis, glucose 6-phosphate dehydrogenase (G6PDH) is detected as two differently migrating forms on native polyacrylamide gels. The pivotal metabolic role of G6PDH in K. lactis led us to investigate the mechanism controlling the two activities in respiratory and fermentative mutant strains. An extensive analysis of these mutants showed that the NAD+(H)/NADP+(H)-dependent cytosolic alcohol (ADH) and aldehyde (ALD) dehydrogenase balance affects the expression of the G6PDH activity pattern. Under fermentative/ethanol growth conditions, the concomitant activation of ADH and ALD activities led to cytosolic accumulation of NADPH, triggering an alteration in the oligomeric state of the G6PDH caused by displacement/release of the structural NADP+ bound to each subunit of the enzyme. The new oligomeric G6PDH form with faster-migrating properties increases as a consequence of intracellular redox unbalance/NADPH accumulation, which inhibits G6PDH activity in vivo. The appearance of a new G6PDH-specific activity band, following incubation of Saccharomyces cerevisiae and human cellular extracts with NADP+, also suggests that a regulatory mechanism of this activity through NADPH accumulation is highly conserved among eukaryotes. PMID:23064253

Oleic, Linoleic and Linolenic Acids Increase ROS Production by Fibroblasts via NADPH Oxidase Activation

PubMed Central

Hatanaka, Elaine; Dermargos, Alexandre; Hirata, Aparecida Emiko; Vinolo, Marco Aurélio Ramirez; Carpinelli, Angelo Rafael; Newsholme, Philip; Armelin, Hugo Aguirre; Curi, Rui

2013-01-01

The effect of oleic, linoleic and γ-linolenic acids on ROS production by 3T3 Swiss and Rat 1 fibroblasts was investigated. Using lucigenin-amplified chemiluminescence, a dose-dependent increase in extracellular superoxide levels was observed during the treatment of fibroblasts with oleic, linoleic and γ-linolenic acids. ROS production was dependent on the addition of β-NADH or NADPH to the medium. Diphenyleneiodonium inhibited the effect of oleic, linoleic and γ-linolenic acids on fibroblast superoxide release by 79%, 92% and 82%, respectively. Increased levels of p47phox phosphorylation due to fatty acid treatment were detected by Western blotting analyses of fibroblast proteins. Increased p47phox mRNA expression was observed using real-time PCR. The rank order for the fatty acid stimulation of the fibroblast oxidative burst was as follows: γ-linolenic > linoleic > oleic. In conclusion, oleic, linoleic and γ-linolenic acids stimulated ROS production via activation of the NADPH oxidase enzyme complex in fibroblasts. PMID:23579616

DNA replication after mutagenic treatment in Hordeum vulgare.

PubMed

Kwasniewska, Jolanta; Kus, Arita; Swoboda, Monika; Braszewska-Zalewska, Agnieszka

2016-12-01

The temporal and spatial properties of DNA replication in plants related to DNA damage and mutagenesis is poorly understood. Experiments were carried out to explore the relationships between DNA replication, chromatin structure and DNA damage in nuclei from barley root tips. We quantitavely analysed the topological organisation of replication foci using pulse EdU labelling during the S phase and its relationship with the DNA damage induced by mutagenic treatment with maleic hydrazide (MH), nitroso-N-methyl-urea (MNU) and gamma ray. Treatment with mutagens did not change the characteristic S-phase patterns in the nuclei; however, the frequencies of the S-phase-labelled cells after treatment differed from those observed in the control cells. The analyses of DNA replication in barley nuclei were extended to the micronuclei induced by mutagens. Replication in the chromatin of the micronuclei was rare. The results of simultanous TUNEL reaction to identify cells with DNA strand breaks and the labelling of the S-phase cells with EdU revealed the possibility of DNA replication occurring in damaged nuclei. For the first time, the intensity of EdU fluorescence to study the rate of DNA replication was analysed. Copyright © 2016 Elsevier B.V. All rights reserved.

Peroxiredoxins and NADPH-dependent thioredoxin systems in the model legume Lotus japonicus.

PubMed

Tovar-Méndez, Alejandro; Matamoros, Manuel A; Bustos-Sanmamed, Pilar; Dietz, Karl-Josef; Cejudo, Francisco Javier; Rouhier, Nicolas; Sato, Shusei; Tabata, Satoshi; Becana, Manuel

2011-07-01

Peroxiredoxins (Prxs), thioredoxins (Trxs), and NADPH-thioredoxin reductases (NTRs) constitute central elements of the thiol-disulfide redox regulatory network of plant cells. This study provides a comprehensive survey of this network in the model legume Lotus japonicus. The aims were to identify and characterize these gene families and to assess whether the NTR-Trx systems are operative in nodules. Quantitative reverse transcription-polymerase chain reaction and immunological and proteomic approaches were used for expression profiling. We identified seven Prx, 14 Trx, and three NTR functional genes. The PrxQ1 gene was found to be transcribed in two alternative spliced variants and to be expressed at high levels in leaves, stems, petals, pods, and seeds and at low levels in roots and nodules. The 1CPrx gene showed very high expression in the seed embryos and low expression in vegetative tissues and was induced by nitric oxide and cytokinins. In sharp contrast, cytokinins down-regulated all other Prx genes, except PrxQ1, in roots and nodules, but only 2CPrxA and PrxQ1 in leaves. Gene-specific changes in Prx expression were also observed in response to ethylene, abscisic acid, and auxins. Nodules contain significant mRNA and protein amounts of cytosolic PrxIIB, Trxh1, and NTRA and of plastidic NTRC. Likewise, they express cytosolic Trxh3, Trxh4, Trxh8, and Trxh9, mitochondrial PrxIIF and Trxo, and plastidic Trxm2, Trxm4, and ferredoxin-Trx reductase. These findings reveal a complex regulation of Prxs that is dependent on the isoform, tissue, and signaling molecule and support that redox NTR-Trx systems are functional in the cytosol, mitochondria, and plastids of nodules.

[Oxygen and the superoxide anion. Modulation of NADPH oxidase?].

PubMed

Delbosc, S; Cristol, J P; Descomps, B; Chénard, J; Sirois, P

2001-01-01

Oxidative stress which results from an imbalance between oxidant production and antioxidant defense mechanisms can promote modifications of lipids, proteins and nucleic acids. This review focuses on the different pathways leading to Reactive Oxygen Species (ROS) production in particular on NADPH oxidase activation. This enzyme is localized in numerous cells including phagocytes and vascular cells and composed of membrane and cytosolic sub-units. The activation of the NADPH oxidase is largely involved in inflammation associated diseases such as asthma, Systemic Inflammatory Response Syndrome and aging associated diseases such as atherosclerosis and neurodeneratives diseases. The modulation of NADPH oxidase could be a way to limit or prevent the development of these diseases.

Oxygen and Carbon Dioxide Fluxes from Barley Shoots Depend on Nitrate Assimilation 1

PubMed Central

Bloom, Arnold J.; Caldwell, Richard M.; Finazzo, John; Warner, Robert L.; Weissbart, Joseph

1989-01-01

A custom oxygen analyzer in conjunction with an infrared carbon dioxide analyzer and humidity sensors permitted simultaneous measurements of oxygen, carbon dioxide, and water vapor fluxes from the shoots of intact barley plants (Hordeum vulgare L. cv Steptoe). The oxygen analyzer is based on a calciazirconium sensor and can resolve concentration differences to within 2 microliters per liter against the normal background of 210,000 microliters per liter. In wild-type plants receiving ammonium as their sole nitrogen source or in nitrate reductase-deficient mutants, photosynthetic and respiratory fluxes of oxygen equaled those of carbon dioxide. By contrast, wild-type plants exposed to nitrate had unequal oxygen and carbon dioxide fluxes: oxygen evolution at high light exceeded carbon dioxide consumption by 26% and carbon dioxide evolution in the dark exceeded oxygen consumption by 25%. These results indicate that a substantial portion of photosynthetic electron transport or respiration generates reductant for nitrate assimilation rather than for carbon fixation or mitochondrial electron transport. PMID:16667024

NADPH Oxidase Activation Contributes to Heavy Ion Irradiation–Induced Cell Death

PubMed Central

Wang, Yupei; Liu, Qing; Zhao, Weiping; Zhou, Xin; Miao, Guoying; Sun, Chao

2017-01-01

Increased oxidative stress plays an important role in heavy ion radiation–induced cell death. The mechanism involved in the generation of elevated reactive oxygen species (ROS) is not fully illustrated. Here we show that NADPH oxidase activation is closely related to heavy ion radiation–induced cell death via excessive ROS generation. Cell death and cellular ROS can be greatly reduced in irradiated cancer cells with the preincubation of diphenyleneiodium, an inhibitor of NADPH oxidase. Most of the NADPH oxidase (NOX) family proteins (NOX1, NOX2, NOX3, NOX4, and NOX5) showed increased expression after heavy ion irradiation. Meanwhile, the cytoplasmic subunit p47phox was translocated to the cell membrane and localized with NOX2 to form reactive NADPH oxidase. Our data suggest for the first time that ROS generation, as mediated by NADPH oxidase activation, could be an important contributor to heavy ion irradiation–induced cell death. PMID:28473742

NADPH oxidase activity and reactive oxygen species production in brain and kidney of adult male hypertensive Ren-2 transgenic rats.

PubMed

Vokurková, M; Rauchová, H; Řezáčová, L; Vaněčková, I; Zicha, J

2015-01-01

Hypothalamic paraventricular nucleus (PVN) and rostral ventrolateral medulla (RVLM) play an important role in brain control of blood pressure (BP). One of the important mechanisms involved in the pathogenesis of hypertension is the elevation of reactive oxygen species (ROS) production by nicotine adenine dinucleotide phosphate (NADPH) oxidase. The aim of our present study was to investigate NADPH oxidase-mediated superoxide (O(2)(-)) production and to search for the signs of lipid peroxidation in hypothalamus and medulla oblongata as well as in renal medulla and cortex of hypertensive male rats transgenic for the murine Ren-2 renin gene (Ren-2 TGR) and their age-matched normotensive controls - Hannover Sprague Dawley rats (HanSD). We found no difference in the activity of NADPH oxidase measured as a lucigenin-mediated O(2)(-) production in the hypothalamus and medulla oblongata. However, we observed significantly elevated NADPH oxidase in both renal cortex and medulla of Ren-2 TGR compared with HanSD. Losartan (LOS) treatment (10 mg/kg body weight/day) for 2 months (Ren-2 TGR+LOS) did not change NADPH oxidase-dependent O(2)(-) production in the kidney. We detected significantly elevated indirect markers of lipid peroxidation measured as thiobarbituric acid-reactive substances (TBARS) in Ren-2 TGR, while they were significantly decreased in Ren-2 TGR+LOS. In conclusion, the present study shows increased NADPH oxidase activities in renal cortex and medulla with significantly increased TBARS in renal cortex. No significant changes of NADPH oxidase and markers of lipid peroxidation were detected in the studied brain regions.

The use of the ph1b mutant to induce recombination between the chromosomes of wheat and barley

PubMed Central

Rey, María-Dolores; Calderón, María C.; Prieto, Pilar

2015-01-01

Intensive breeding has led to a narrowing in the genetic base of our major crops. In wheat, access to the extensive gene pool residing in its many and varied relatives (some cultivated, others wild) is hampered by the block on recombination imposed by the Ph1 (Pairing homoeologous 1) gene. Here, the ph1b mutant has been exploited to induced allosyndesis between wheat chromosomes and those of both Hordeum vulgare (cultivated barley) and H. chilense (a wild barley). A number of single chromosome Hordeum sp. substitution and addition lines in wheat were crossed and backcrossed to the ph1b mutant to produce plants in which pairing between the wheat and the non-wheat chromosomes was not suppressed by the presence of Ph1. Genomic in situ hybridization was applied to almost 500 BC1F2 progeny as a screen for allosyndetic recombinants. Chromosome rearrangements were detected affecting H. chilense chromosomes 4Hch, 5Hch, 6Hch, and 7Hch and H. vulgare chromosomes 4Hv, 6Hv, and 7Hv. Two of these were clearly the product of a recombination event involving chromosome 4Hch and a wheat chromosome. PMID:25852713

Cytosolic NADPH Homeostasis in Glucose-starved Procyclic Trypanosoma brucei Relies on Malic Enzyme and the Pentose Phosphate Pathway Fed by Gluconeogenic Flux*

PubMed Central

Allmann, Stefan; Morand, Pauline; Ebikeme, Charles; Gales, Lara; Biran, Marc; Hubert, Jane; Brennand, Ana; Mazet, Muriel; Franconi, Jean-Michel; Michels, Paul A. M.; Portais, Jean-Charles; Boshart, Michael; Bringaud, Frédéric

2013-01-01

All living organisms depend on NADPH production to feed essential biosyntheses and for oxidative stress defense. Protozoan parasites such as the sleeping sickness pathogen Trypanosoma brucei adapt to different host environments, carbon sources, and oxidative stresses during their infectious life cycle. The procyclic stage develops in the midgut of the tsetse insect vector, where they rely on proline as carbon source, although they prefer glucose when grown in rich media. Here, we investigate the flexible and carbon source-dependent use of NADPH synthesis pathways in the cytosol of the procyclic stage. The T. brucei genome encodes two cytosolic NADPH-producing pathways, the pentose phosphate pathway (PPP) and the NADP-dependent malic enzyme (MEc). Reverse genetic blocking of those pathways and a specific inhibitor (dehydroepiandrosterone) of glucose-6-phosphate dehydrogenase together established redundancy with respect to H2O2 stress management and parasite growth. Blocking both pathways resulted in ∼10-fold increase of susceptibility to H2O2 stress and cell death. Unexpectedly, the same pathway redundancy was observed in glucose-rich and glucose-depleted conditions, suggesting that gluconeogenesis can feed the PPP to provide NADPH. This was confirmed by (i) a lethal phenotype of RNAi-mediated depletion of glucose-6-phosphate isomerase (PGI) in the glucose-depleted Δmec/Δmec null background, (ii) an ∼10-fold increase of susceptibility to H2O2 stress observed for the Δmec/Δmec/RNAiPGI double mutant when compared with the single mutants, and (iii) the 13C enrichment of glycolytic and PPP intermediates from cells incubated with [U-13C]proline, in the absence of glucose. Gluconeogenesis-supported NADPH supply may also be important for nucleotide and glycoconjugate syntheses in the insect host. PMID:23665470

Protein-bound NAD(P)H Lifetime is Sensitive to Multiple Fates of Glucose Carbon.

PubMed

Sharick, Joe T; Favreau, Peter F; Gillette, Amani A; Sdao, Sophia M; Merrins, Matthew J; Skala, Melissa C

2018-04-03

While NAD(P)H fluorescence lifetime imaging (FLIM) can detect changes in flux through the TCA cycle and electron transport chain (ETC), it remains unclear whether NAD(P)H FLIM is sensitive to other potential fates of glucose. Glucose carbon can be diverted from mitochondria by the pentose phosphate pathway (via glucose 6-phosphate dehydrogenase, G6PDH), lactate production (via lactate dehydrogenase, LDH), and rejection of carbon from the TCA cycle (via pyruvate dehydrogenase kinase, PDK), all of which can be upregulated in cancer cells. Here, we demonstrate that multiphoton NAD(P)H FLIM can be used to quantify the relative concentrations of recombinant LDH and malate dehydrogenase (MDH) in solution. In multiple epithelial cell lines, NAD(P)H FLIM was also sensitive to inhibition of LDH and PDK, as well as the directionality of LDH in cells forced to use pyruvate versus lactate as fuel sources. Among the parameters measurable by FLIM, only the lifetime of protein-bound NAD(P)H (τ 2 ) was sensitive to these changes, in contrast to the optical redox ratio, mean NAD(P)H lifetime, free NAD(P)H lifetime, or the relative amount of free and protein-bound NAD(P)H. NAD(P)H τ 2 offers the ability to non-invasively quantify diversions of carbon away from the TCA cycle/ETC, which may support mechanisms of drug resistance.

Rosuvastatin prevents angiotensin II-induced vascular changes by inhibition of NAD(P)H oxidase and COX-1

PubMed Central

Colucci, Rocchina; Fornai, Matteo; Duranti, Emiliano; Antonioli, Luca; Rugani, Ilaria; Aydinoglu, Fatma; Ippolito, Chiara; Segnani, Cristina; Bernardini, Nunzia; Taddei, Stefano; Blandizzi, Corrado; Virdis, Agostino

2013-01-01

Background and Purpose NAD(P)H oxidase and COX-1 participate in vascular damage induced by angiotensin II. We investigated the effect of rosuvastatin on endothelial dysfunction, vascular remodelling, changes in extracellular matrix components and mechanical properties of small mesenteric arteries from angiotensin II-infused rats. Experimental Approach Male rats received angiotensin II (120 ng·kg−1·min−1, subcutaneously) for 14 days with or without rosuvastatin (10 mg·kg−1·day−1, oral gavage) or vehicle. Vascular functions and morphological parameters were assessed by pressurized myography. Key Results In angiotensin II-infused rats, ACh-induced relaxation was attenuated compared with controls, less sensitive to L-NAME, enhanced by SC-560 (COX-1 inhibitor) or SQ-29548 (prostanoid TP receptor antagonist), and normalized by the antioxidant ascorbic acid or NAD(P)H oxidase inhibitors. After rosuvastatin, relaxations to ACh were normalized, fully sensitive to L-NAME, and no longer affected by SC-560, SQ-29548 or NAD(P)H oxidase inhibitors. Angiotensin II enhanced intravascular superoxide generation, eutrophic remodelling, collagen and fibronectin depositions, and decreased elastin content, resulting in increased vessel stiffness. All these changes were prevented by rosuvastatin. Angiotensin II increased phosphorylation of NAD(P)H oxidase subunit p47phox and its binding to subunit p67phox, effects inhibited by rosuvastatin. Rosuvastatin down-regulated vascular Nox4/NAD(P)H isoform and COX-1 expression, attenuated the vascular release of 6-keto-PGF1α, and enhanced copper/zinc-superoxide dismutase expression. Conclusion and Implications Rosuvastatin prevents angiotensin II-induced alterations in resistance arteries in terms of function, structure, mechanics and composition. These effects depend on restoration of NO availability, prevention of NAD(P)H oxidase-derived oxidant excess, reversal of COX-1 induction and its prostanoid production, and stimulation of

A LED-based method for monitoring NAD(P)H and FAD fluorescence in cell cultures and brain slices.

PubMed

Rösner, Jörg; Liotta, Agustin; Schmitz, Dietmar; Heinemann, Uwe; Kovács, Richard

2013-01-30

Nicotinamide- and flavine-adenine-dinucleotides (NAD(P)H and FADH₂) are electron carriers involved in cellular energy metabolism and in a multitude of enzymatic processes. As reduced NAD(P)H and oxidised FAD molecules are fluorescent, changes in tissue auto-fluorescence provide valuable information on the cellular redox state and energy metabolism. Since fluorescence excitation, by mercury arc lamps (HBO) is inherently coupled to photo-bleaching and photo-toxicity, microfluorimetric monitoring of energy metabolism might benefit from the replacement of HBO lamps by light emitting diodes (LEDs). Here we describe a LED-based custom-built setup for monitoring NAD(P)H and FAD fluorescence at the level of single cells (HEK293) and of brain slices. We compared NAD(P)H bleaching characteristics with two light sources (HBO lamp and LED) as well as sensitivity and signal to noise ratio of three different detector types (multi-pixel photon counter (MPPC), photomultiplier tube (PMT) and photodiode). LED excitation resulted in reduced photo-bleaching at the same fluorescence output in comparison to excitation with the HBO lamp. Transiently increasing LED power resulted in reversible bleaching of NAD(P)H fluorescence. Recovery kinetics were dependent on metabolic substrates indicating coupling of NAD(P)H fluorescence to metabolism. Electrical stimulation of brain slices induced biphasic redox changes, as indicated by NAD(P)H/FAD fluorescence transients. Increasing the gain of PMT and decreasing the LED power resulted in similar sensitivity as obtained with the MPPC and the photodiode, without worsening the signal to noise ratio. In conclusion, replacement of HBO lamp with LED might improve conventional PMT based microfluorimetry of tissue auto-fluorescence. Copyright © 2012 Elsevier B.V. All rights reserved.

Regulation of the NADPH Oxidase RBOHD During Plant Immunity.

PubMed

Kadota, Yasuhiro; Shirasu, Ken; Zipfel, Cyril

2015-08-01

Pathogen recognition induces the production of reactive oxygen species (ROS) by NADPH oxidases in both plants and animals. ROS have direct antimicrobial properties, but also serve as signaling molecules to activate further immune outputs. However, ROS production has to be tightly controlled to avoid detrimental effects on host cells, but yet must be produced in the right amount, at the right place and at the right time upon pathogen perception. Plant NADPH oxidases belong to the respiratory burst oxidase homolog (RBOH) family, which contains 10 members in the model plant Arabidopsis thaliana. The perception of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs) leads to a rapid, specific and strong production of ROS, which is dependent on RBOHD. RBOHD is mainly controlled by Ca(2+) via direct binding to EF-hand motifs and phosphorylation by Ca(2+)-dependent protein kinases. Recent studies have, however, revealed a critical role for a Ca(2+)-independent regulation of RBOHD. The plasma membrane-associated cytoplasmic kinase BIK1 (BOTRYTIS-INDUCED KINASE1), which is a direct substrate of the PRR complex, directly interacts with and phosphorylates RBOHD upon PAMP perception. Impairment of these phosphorylation events completely abolishes the function of RBOHD in immunity. These results suggest that RBOHD activity is tightly controlled by multilayered regulations. In this review, we summarize recent advances in our understanding of the regulatory mechanisms controlling RBOHD activation. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Nitric Oxide Synthase and Neuronal NADPH Diaphorase are Identical in Brain and Peripheral Tissues

NASA Astrophysics Data System (ADS)

Dawson, Ted M.; Bredt, David S.; Fotuhi, Majid; Hwang, Paul M.; Snyder, Solomon H.

1991-09-01

NADPH diaphorase staining neurons, uniquely resistant to toxic insults and neurodegenerative disorders, have been colocalized with neurons in the brain and peripheral tissue containing nitric oxide synthase (EC 1.14.23.-), which generates nitric oxide (NO), a recently identified neuronal messenger molecule. In the corpus striatum and cerebral cortex, NO synthase immunoreactivity and NADPH diaphorase staining are colocalized in medium to large aspiny neurons. These same neurons colocalize with somatostatin and neuropeptide Y immunoreactivity. NO synthase immunoreactivity and NADPH diaphorase staining are colocalized in the pedunculopontine nucleus with choline acetyltransferase-containing cells and are also colocalized in amacrine cells of the inner nuclear layer and ganglion cells of the retina, myenteric plexus neurons of the intestine, and ganglion cells of the adrenal medulla. Transfection of human kidney cells with NO synthase cDNA elicits NADPH diaphorase staining. The ratio of NO synthase to NADPH diaphorase staining in the transfected cells is the same as in neurons, indicating that NO synthase fully accounts for observed NADPH staining. The identity of neuronal NO synthase and NADPH diaphorase suggests a role for NO in modulating neurotoxicity.

Foeniculum vulgare Mill: A Review of Its Botany, Phytochemistry, Pharmacology, Contemporary Application, and Toxicology

PubMed Central

Patel, Vainav V.; Bandivdekar, Atmaram H.

2014-01-01

Foeniculum vulgare Mill commonly called fennel has been used in traditional medicine for a wide range of ailments related to digestive, endocrine, reproductive, and respiratory systems. Additionally, it is also used as a galactagogue agent for lactating mothers. The review aims to gather the fragmented information available in the literature regarding morphology, ethnomedicinal applications, phytochemistry, pharmacology, and toxicology of Foeniculum vulgare. It also compiles available scientific evidence for the ethnobotanical claims and to identify gaps required to be filled by future research. Findings based on their traditional uses and scientific evaluation indicates that Foeniculum vulgare remains to be the most widely used herbal plant. It has been used for more than forty types of disorders. Phytochemical studies have shown the presence of numerous valuable compounds, such as volatile compounds, flavonoids, phenolic compounds, fatty acids, and amino acids. Compiled data indicate their efficacy in several in vitro and in vivo pharmacological properties such as antimicrobial, antiviral, anti-inflammatory, antimutagenic, antinociceptive, antipyretic, antispasmodic, antithrombotic, apoptotic, cardiovascular, chemomodulatory, antitumor, hepatoprotective, hypoglycemic, hypolipidemic, and memory enhancing property. Foeniculum vulgare has emerged as a good source of traditional medicine and it provides a noteworthy basis in pharmaceutical biology for the development/formulation of new drugs and future clinical uses. PMID:25162032

Inhibition of arsenic induced-rat liver injury by grape seed exact through suppression of NADPH oxidase and TGF-{beta}/Smad activation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pan Xinjuan; Dai Yujie; Li Xing

2011-08-01

Chronic arsenic exposure induces oxidative damage to liver leading to liver fibrosis. We aimed to define the effect of grape seed extract (GSE), an antioxidant dietary supplement, on arsenic-induced liver injury. First, Male Sprague-Dawley rats were exposed to a low level of arsenic in drinking water (30 ppm) with or without GSE (100 mg/kg, every other day by oral gavage) for 12 months and the effect of GSE on arsenic-induced hepatotoxicity was examined. The results from this study revealed that GSE co-treatment significantly attenuated arsenic-induced low antioxidant defense, oxidative damage, proinflammatory cytokines and fibrogenic genes. Moreover, GSE reduced arsenic-stimulated Smad2/3more » phosphorylation and protein levels of NADPH oxidase subunits (Nox2, Nox4 and p47phox). Next, we explored the molecular mechanisms underlying GSE inhibition of arsenic toxicity using cultured rat hepatic stellate cells (HSCs). From the in vitro study, we found that GSE dose-dependently reduced arsenic-stimulated ROS production and NADPH oxidase activities. Both NADPH oxidases flavoprotein inhibitor DPI and Nox4 siRNA blocked arsenic-induced ROS production, whereas Nox4 overexpression suppressed the inhibitory effects of GSE on arsenic-induced ROS production and NADPH oxidase activities, as well as expression of TGF-{beta}1, type I procollagen (Coll-I) and {alpha}-smooth muscle actin ({alpha}-SMA) mRNA. We also observed that GSE dose-dependently inhibited TGF-{beta}1-induced transactivation of the TGF-{beta}-induced smad response element p3TP-Lux, and that forced expression of Smad3 attenuated the inhibitory effects of GSE on TGF-{beta}1-induced mRNA expression of Coll-I and {alpha}-SMA. Collectively, GSE could be a potential dietary therapeutic agent for arsenic-induced liver injury through suppression of NADPH oxidase and TGF-{beta}/Smad activation. - Research Highlights: > GSE attenuated arsenic-induced low antioxidant defense, oxidative damage, proinflammatory cytokines

NADPH oxidase mediates depressive behavior induced by chronic stress in mice.

PubMed

Seo, Ji-Seon; Park, Jin-Young; Choi, Juli; Kim, Tae-Kyung; Shin, Joo-Hyun; Lee, Ja-Kyeong; Han, Pyung-Lim

2012-07-11

Stress is a potent risk factor for depression, yet the underlying mechanism is not clearly understood. In the present study, we explored the mechanism of development and maintenance of depression in a stress-induced animal model. Mice restrained for 2 h daily for 14 d showed distinct depressive behavior, and the altered behavior persisted for >3 months in the absence of intervention. Acute restraint induced a surge of oxidative stress in the brain, and stress-induced oxidative stress progressively increased with repetition of stress. In vitro, the stress hormone glucocorticoid generated superoxide via upregulation of NADPH oxidase. Consistently, repeated restraints increased the expression of the key subunits of NADPH oxidase, p47phox and p67phox, in the brain. Moreover, stressed brains markedly upregulated the expression of p47phox to weak restress evoked in the poststress period, and this molecular response was reminiscent of amplified ROS surge to restress. Pharmacological inhibition of NADPH oxidase by the NADPH oxidase inhibitor apocynin during the stress or poststress period completely blocked depressive behavior. Consistently, heterozygous p47phox knock-out mice (p47phox(+/-)) or molecular inhibition of p47phox with Lenti shRNA-p47phox in the hippocampus suppressed depressive behavior. These results suggest that repeated stress promotes depressive behavior through the upregulation of NADPH oxidase and the resultant metabolic oxidative stress, and that the inhibition of NADPH oxidase provides beneficial antidepression effects.

P2x7 Receptor-NADPH Oxidase-Axis Mediates Protein radical Formation And Kupffer Cell Activation in Carbon Tetrachloride-Mediated Steatohepatitis in Obese Mice

PubMed Central

Chatterjee, Saurabh; Rana, Ritu; Corbett, Jean; Kadiiska, Maria B.; Goldstein, Joyce; Mason, Ronald P.

2012-01-01

While some studies show that carbon tetrachloride-mediated metabolic oxidative stress exacerbates steatohepatitic-like lesions in obese mice, the redox mechanisms that trigger the innate immune system and accentuate the inflammatory cascade remain unclear. Here we have explored the role of the purinergic receptor P2X7-NADPH oxidase axis as a primary event in recognizing the heightened release of extracellular ATP from CCl4-treated hepatocytes and generating redoxmediated Kupffer cell activation in obese mice. We found that an underlying condition of obesity led to the formation of protein radicals and post-translational nitration, primarily in Kupffer cells, at 24 h post-CCl4 administration. The free radical-mediated oxidation of cellular macromolecules, which was NADPH oxidase- and P2X7 receptor-dependent, correlated well with the release of TNF- α and MCP-2 from Kupffer cells. The Kupffer cells in CCl4-treated mice exhibited increased expression of MHC Class II proteins and showed an activated phenotype. Increased expression of MHC Class II was inhibited by the NADPH oxidase inhibitor apocynin , P2X7 receptor antagonist A438709 hydrochloride, and genetic deletions of the NADPH oxidase p47 phox subunit or the P2X7 receptor. The P2X7 receptor acted upstream of NADPH oxidase activation by up-regulating the expression of the p47 phox subunit and p47 phox binding to the membrane subunit, gp91 phox. We conclude that the P2X7 receptor is a primary mediator of oxidative stress-induced exacerbation of inflammatory liver injury in obese mice via NADPH oxidase-dependent mechanisms. PMID:22343416

Ultra-fast HPM detectors improve NAD(P)H FLIM

NASA Astrophysics Data System (ADS)

Becker, Wolfgang; Wetzker, Cornelia; Benda, Aleš

2018-02-01

Metabolic imaging by NAD(P)H FLIM requires the decay functions in the individual pixels to be resolved into the decay components of bound and unbound NAD(P)H. Metabolic information is contained in the lifetime and relative amplitudes of the components. The separation of the decay components and the accuracy of the amplitudes and lifetimes improves substantially by using ultra-fast HPM-100-06 and HPM-100-07 hybrid detectors. The IRF width in combination with the Becker & Hickl SPC-150N and SPC-150NX TCSPC modules is less than 20 ps. An IRF this fast does not interfere with the fluorescence decay. The usual deconvolution process in the data analysis then virtually becomes a simple curve fitting, and the parameters of the NAD(P)H decay components are obtained at unprecedented accuracy.

Electron spin resonance characterization of vascular xanthine and NAD(P)H oxidase activity in patients with coronary artery disease: relation to endothelium-dependent vasodilation.

PubMed

Spiekermann, Stephan; Landmesser, Ulf; Dikalov, Sergey; Bredt, Martin; Gamez, Graciela; Tatge, Helma; Reepschläger, Nina; Hornig, Burkhard; Drexler, Helmut; Harrison, David G

2003-03-18

Increased inactivation of nitric oxide by superoxide (O2*-) contributes to endothelial dysfunction in patients with coronary disease (CAD). We therefore characterized the vascular activities of xanthine oxidase and NAD(P)H oxidase, 2 major O2*--producing enzyme systems, and their relationship with flow-dependent, endothelium-mediated vasodilation (FDD) in patients with CAD. Xanthine- and NAD(P)H-mediated O*.- formation was determined in coronary arteries from 10 patients with CAD and 10 controls by using electron spin resonance spectroscopy. Furthermore, activity of endothelium-bound xanthine oxidase in vivo and FDD of the radial artery were determined in 21 patients with CAD and 10 controls. FDD was measured before and after infusion of the antioxidant vitamin C (25 mg/min i.a.) to determine the portion of FDD inhibited by radicals. In coronary arteries from patients with CAD, xanthine- and NAD(P)H-mediated O2*- formation was increased compared with controls (xanthine: 12+/-2 versus 7+/-1 nmol O2*-/ microg protein; NADH: 11+/-1 versus 7+/-1 nmol O2*-/ microg protein; and NADPH: 12+/-2 versus 9+/-1 nmol O2*-/ microg protein; each P<0.05). Endothelium-bound xanthine oxidase activity was increased by >200% in patients with CAD (25+/-4 versus 9+/-1 nmol O2*-/ microL plasma per min; P<0.05) and correlated inversely with FDD (r=-0.55; P<0.05) and positively with the effect of vitamin C on FDD (r=0.54; P<0.05). The present study represents the first electron spin resonance measurements of xanthine and NAD(P)H oxidase activity in human coronary arteries and supports the concept that increased activities of both enzymes contribute to increased vascular oxidant stress in patients with CAD. Furthermore, the present study suggests that increased xanthine oxidase activity contributes to endothelial dysfunction in patients with CAD and may thereby promote the atherosclerotic process.

NADPH oxidase-mediated generation of reactive oxygen species is critically required for survival of undifferentiated human promyelocytic leukemia cell line HL-60.

PubMed

Dong, Jing-Mei; Zhao, Sheng-Guo; Huang, Guo-Yin; Liu, Qing

2004-06-01

Nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) mediated generation of reactive oxygen species (ROS) was originally identified as the powerful host defense machinery against microorganism in phagocytes. But recent reports indicated that some non-phagocytic cells also have the NADPH oxidase activity, and the ROS produced by it may act as cell signal molecule. But as far as today, whether the NADPH oxidase also plays similar role in phagocyte has not been paid much attention. Utilizing the undifferentiated HL-60 promyelocytic leukemia cells as a model, the aim of the present study was to determine whether NADPH oxidase plays a role on ROS generation in undifferentiated HL-60, and the ROS mediated by it was essential for cell's survival. For the first time, we verified that the release of ROS in undifferentiated HL-60 was significantly increased by the stimulation with Calcium ionophore or opsonized zymosan, which are known to trigger respiration burst in phagocytes by NADPH oxidase pathway. Diphenylene iodonium (DPI) or apocynin (APO), two inhibitors of NADPH oxidase, significantly suppressed the increasing of ROS caused by opsonized zymosan. Cell survival assay and fluorescence double dyeing with acridine orange and ethidium bromide showed that DPI and APO, as well as superoxide dismutase (SOD) and catalase (CAT) concentration-dependently decreased the viability of undifferentiated HL-60 cells, whereas exogenous H2O2 can rescue the cells from death obviously. Our results suggested that the ROS, generated by NADPH oxidase play an essential role in the survival of undifferentiated HL-60 cells.

NADH/NADPH bi-cofactor-utilizing and thermoactive ketol-acid reductoisomerase from Sulfolobus acidocaldarius.

PubMed

Chen, Chin-Yu; Ko, Tzu-Ping; Lin, Kuan-Fu; Lin, Bo-Lin; Huang, Chun-Hsiang; Chiang, Cheng-Hung; Horng, Jia-Cherng

2018-05-08

Ketol-acid reductoisomerase (KARI) is a bifunctional enzyme in the second step of branched-chain amino acids biosynthetic pathway. Most KARIs prefer NADPH as a cofactor. However, KARI with a preference for NADH is desirable in industrial applications including anaerobic fermentation for the production of branched-chain amino acids or biofuels. Here, we characterize a thermoacidophilic archaeal Sac-KARI from Sulfolobus acidocaldarius and present its crystal structure at a 1.75-Å resolution. By comparison with other holo-KARI structures, one sulphate ion is observed in each binding site for the 2'-phosphate of NADPH, implicating its NADPH preference. Sac-KARI has very high affinity for NADPH and NADH, with K M values of 0.4 μM for NADPH and 6.0 μM for NADH, suggesting that both are good cofactors at low concentrations although NADPH is favoured over NADH. Furthermore, Sac-KARI can catalyze 2(S)-acetolactate (2S-AL) with either cofactor from 25 to 60 °C, but the enzyme has higher activity by using NADPH. In addition, the catalytic activity of Sac-KARI increases significantly with elevated temperatures and reaches an optimum at 60 °C. Bi-cofactor utilization and the thermoactivity of Sac-KARI make it a potential candidate for use in metabolic engineering or industrial applications under anaerobic or harsh conditions.

NADPH as a potential intrinsic probe for tumour margin estimation

NASA Astrophysics Data System (ADS)

Stewart, Hazel; Hupp, Ted R.; Birch, David J. S.

2018-03-01

The fluorescent properties of the reduced coenzyme NADH and its phosphorylated derivative (NADPH) have been explored in order to assess their potential as an intrinsic probe for cancer surgery. NADPH production is increased in cancer cells to quench reactive oxygen species and meet higher demands for biosynthesis, and has attractive fluorescent properties such as emission towards the visible part of the spectrum and a relatively long fluorescence lifetime upon binding to enzymes ( 1 - 6.5 ns) that helps discriminate against other endogenous species. Different environmental effects on NAD(P)H fluorescence are reported here, including an increase in lifetime upon oxygen removal, an ability to retain its fluorescent properties in a complex medium (a silica phantom) and its fluorescence lifetime also being distinguishable in a cell environment. In addition, the development of a miniaturized liquid light guide filter-based timecorrelated single photon counting fluorescence lifetime system is reported as a step towards time-resolved visual imaging in cancer surgery. This system has been demonstrated as being capable of accurately measuring NAD(P)H fluorescence lifetimes in both simple solvent and cellular environments.

Improved strategies for electrochemical 1,4-NAD(P)H2 regeneration: A new era of bioreactors for industrial biocatalysis.

PubMed

Morrison, Clifford S; Armiger, William B; Dodds, David R; Dordick, Jonathan S; Koffas, Mattheos A G

Industrial enzymatic reactions requiring 1,4-NAD(P)H 2 to perform redox transformations often require convoluted coupled enzyme regeneration systems to regenerate 1,4-NAD(P)H 2 from NAD(P) and recycle the cofactor for as many turnovers as possible. Renewed interest in recycling the cofactor via electrochemical means is motivated by the low cost of performing electrochemical reactions, easy monitoring of the reaction progress, and straightforward product recovery. However, electrochemical cofactor regeneration methods invariably produce adventitious reduced cofactor side products which result in unproductive loss of input NAD(P). We review various literature strategies for mitigating adventitious product formation by electrochemical cofactor regeneration systems, and offer insight as to how a successful electrochemical bioreactor system could be constructed to engineer efficient 1,4-NAD(P)H 2 -dependent enzyme reactions of interest to the industrial biocatalysis community. Copyright © 2017 Elsevier Inc. All rights reserved.

NAD(P)H oxidase mediates the endothelial barrier dysfunction induced by TNF-alpha.

PubMed

Gertzberg, Nancy; Neumann, Paul; Rizzo, Victor; Johnson, Arnold

2004-01-01

We tested the hypothesis that the NAD(P)H oxidase-dependent generation of superoxide anion (O2-*) mediates tumor necrosis factor-alpha (TNF)-induced alterations in the permeability of pulmonary microvessel endothelial monolayers (PMEM). The permeability of PMEM was assessed by the clearance rate of Evans blue-labeled albumin. The NAD(P)H oxidase subcomponents p47phox and p22phox were assessed by immunofluorescent microscopy and Western blot. The reactive oxygen species O2-* was measured by the fluorescence of 6-carboxy-2',7'-dichlorodihydrofluorescein diacetatedi(acetoxymethyl ester), 5 (and 6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate-acetyl ester, and dihydroethidium. TNF treatment (50 ng/ml for 4.0 h) induced 1) p47phox translocation, 2) an increase in p22phox protein, 3) increased localization of p47phox with p22phox, 4) O2-* generation, and 5) increased permeability to albumin. p22phox antisense oligonucleotide prevented the TNF-induced effect on p22phox, p47phox, O2-*, and permeability. The scrambled nonsense oligonucleotide had no effect. The TNF-induced increase in O2-* and permeability to albumin was also prevented by the O2-* scavenger Cu-Zn superoxide dismutase (100 U/ml). The results indicate that the activation of NAD(P)H oxidase, via the generation of O2-*, mediates TNF-induced barrier dysfunction in PMEM.

The Phytotoxicity of Designated Pollutants

DTIC Science & Technology

1981-12-01

necrotic injury on tomato and barley leaves .* . . .* . . . . . . . .e .o . . . . o . . . . .o . 74 A4 Pictorial key for necrotic injury on lettuce and...water flushed with water. ................ 58 43 Growth of barley seedlings from soil previously treated with jet fuel .................. 60 44 Growth of...Variety or Cultivar Alfalfa Medicago sativa L. Cu F101, Bonanza Barley Hordeum vulgare L. CM67 Bean Phaseolus vulgaris L. Pinto U.I. III Carrot Daucus

NADPH Oxidase as a Therapeutic Target for Oxalate Induced Injury in Kidneys

PubMed Central

Peck, Ammon B.; Khan, Saeed R.

2013-01-01

A major role of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase family of enzymes is to catalyze the production of superoxides and other reactive oxygen species (ROS). These ROS, in turn, play a key role as messengers in cell signal transduction and cell cycling, but when they are produced in excess they can lead to oxidative stress (OS). Oxidative stress in the kidneys is now considered a major cause of renal injury and inflammation, giving rise to a variety of pathological disorders. In this review, we discuss the putative role of oxalate in producing oxidative stress via the production of reactive oxygen species by isoforms of NADPH oxidases expressed in different cellular locations of the kidneys. Most renal cells produce ROS, and recent data indicate a direct correlation between upregulated gene expressions of NADPH oxidase, ROS, and inflammation. Renal tissue expression of multiple NADPH oxidase isoforms most likely will impact the future use of different antioxidants and NADPH oxidase inhibitors to minimize OS and renal tissue injury in hyperoxaluria-induced kidney stone disease. PMID:23840917

Composition and antiproliferative effect of essential oil of Origanum vulgare against tumor cell lines.

PubMed

Begnini, Karine Rech; Nedel, Fernanda; Lund, Rafael Guerra; Carvalho, Pedro Henrique de Azambuja; Rodrigues, Maria Regina Alves; Beira, Fátima Tereza Alves; Del-Pino, Francisco Augusto Burkert

2014-10-01

Cancer is a leading cause of death and is responsible for one in eight deaths worldwide. The use of herbs as complementary medicine for cancer, especially advanced cancer, has recently increased. The aim of this study was to evaluate in vitro, the antiproliferative effect of Origanum vulgare against human breast adenocarcinoma (MCF-7), and human colon adenocarcinoma (HT-29). The essential oil (EO) was extracted from a bought amount of O. vulgare dried leaves and analyzed in a gas chromatograph interfaced with a mass selective detector. The cytotoxicity test was performed by sulforhodamine B assay. The results show that the EO is composed mostly of 4-terpineol and induces a high cytotoxicity effect in HT-29. In the MCF-7 cell line the EO was less effective. In conclusion, this study showed that O. vulgare main component is 4-terpineol and was effective in inducing cancer cell growth inhibition.

Fructose increases corticosterone production in association with NADPH metabolism alterations in rat epididymal white adipose tissue.

PubMed

Prince, Paula D; Santander, Yanina A; Gerez, Estefania M; Höcht, Christian; Polizio, Ariel H; Mayer, Marcos A; Taira, Carlos A; Fraga, Cesar G; Galleano, Monica; Carranza, Andrea

2017-08-01

Metabolic syndrome is an array of closely metabolic disorders that includes glucose intolerance/insulin resistance, central obesity, dyslipidemia, and hypertension. Fructose, a highly lipogenic sugar, has profound metabolic effects in adipose tissue, and has been associated with the etiopathology of many components of the metabolic syndrome. In adipocytes, the enzyme 11 β-HSD1 amplifies local glucocorticoid production, being a key player in the pathogenesis of central obesity and metabolic syndrome. 11 β-HSD1 reductase activity is dependent on NADPH, a cofactor generated by H6PD inside the endoplasmic reticulum. Our focus was to explore the effect of fructose overload on epididymal white adipose tissue (EWAT) machinery involved in glucocorticoid production and NADPH and oxidants metabolism. Male Sprague-Dawley rats fed with a fructose solution (10% (w/v) in tap water) during 9 weeks developed some characteristic features of metabolic syndrome, such as hypertriglyceridemia, and hypertension. In addition, high levels of plasma and EWAT corticosterone were detected. Activities and expressions of H6PD and 11 β-HSD1, NAPDH content, superoxide anion production, expression of NADPH oxidase 2 subunits, and indicators of oxidative metabolism were measured. Fructose overloaded rats showed an increased potential in oxidant production respect to control rats. In parallel, in EWAT from fructose overloaded rats we found higher expression/activity of H6PD and 11 β-HSD1, and NADPH/NADP + ratio. Our in vivo results support that fructose overload installs in EWAT conditions favoring glucocorticoid production through higher H6PD expression/activity supplying NADPH for enhanced 11 β-HSD1 expression/activity, becoming this tissue a potential extra-adrenal source of corticosterone under these experimental conditions. Copyright © 2017 Elsevier Inc. All rights reserved.

Arginase Inhibition Suppresses Native Low-Density Lipoprotein-Stimulated Vascular Smooth Muscle Cell Proliferation by NADPH Oxidase Inactivation.

PubMed

Koo, Bon Hyeock; Yi, Bong Gu; Wang, Wi Kwang; Ko, In Young; Hoe, Kwang Lae; Kwon, Young Guen; Won, Moo Ho; Kim, Young Myeong; Lim, Hyun Kyo; Ryoo, Sungwoo

2018-05-01

Vascular smooth muscle cell (VSMC) proliferation induced by native low-density lipoprotein (nLDL) stimulation is dependent on superoxide production from activated NADPH oxidase. The present study aimed to investigate whether the novel arginase inhibitor limonin could suppress nLDL-induced VSMC proliferation and to examine related mechanisms. Isolated VSMCs from rat aortas were treated with nLDL, and cell proliferation was measured by WST-1 and BrdU assays. NADPH oxidase activation was evaluated by lucigenin-induced chemiluminescence, and phosphorylation of protein kinase C (PKC) βII and extracellular signal-regulated kinase (ERK) 1/2 was determined by western blot analysis. Mitochondrial reactive oxygen species (ROS) generation was assessed using MitoSOX-red, and intracellular L-arginine concentrations were determined by high-performance liquid chromatography (HPLC) in the presence or absence of limonin. Limonin inhibited arginase I and II activity in the uncompetitive mode, and prevented nLDL-induced VSMC proliferation in a p21Waf1/Cip1-dependent manner without affecting arginase protein levels. Limonin blocked PKCβII phosphorylation, but not ERK1/2 phosphorylation, and translocation of p47phox to the membrane was decreased, as was superoxide production in nLDL-stimulated VSMCs. Moreover, mitochondrial ROS generation was increased by nLDL stimulation and blocked by preincubation with limonin. Mitochondrial ROS production was responsible for the phosphorylation of PKCβII. HPLC analysis showed that arginase inhibition with limonin increases intracellular L-arginine concentrations, but decreases polyamine concentrations. L-Arginine treatment prevented PKCβII phosphorylation without affecting ERK1/2 phosphorylation. Increased L-arginine levels following limonin-dependent arginase inhibition prohibited NADPH oxidase activation in a PKCβII-dependent manner, and blocked nLDL-stimulated VSMC proliferation. © Copyright: Yonsei University College of Medicine 2018.

Arginase Inhibition Suppresses Native Low-Density Lipoprotein-Stimulated Vascular Smooth Muscle Cell Proliferation by NADPH Oxidase Inactivation

PubMed Central

Wang, Wi-Kwang; Ko, In-Young; Hoe, Kwang-Lae; Kwon, Young-Guen; Won, Moo-Ho; Kim, Young-Myeong

2018-01-01

Purpose Vascular smooth muscle cell (VSMC) proliferation induced by native low-density lipoprotein (nLDL) stimulation is dependent on superoxide production from activated NADPH oxidase. The present study aimed to investigate whether the novel arginase inhibitor limonin could suppress nLDL-induced VSMC proliferation and to examine related mechanisms. Materials and Methods Isolated VSMCs from rat aortas were treated with nLDL, and cell proliferation was measured by WST-1 and BrdU assays. NADPH oxidase activation was evaluated by lucigenin-induced chemiluminescence, and phosphorylation of protein kinase C (PKC) βII and extracellular signal-regulated kinase (ERK) 1/2 was determined by western blot analysis. Mitochondrial reactive oxygen species (ROS) generation was assessed using MitoSOX-red, and intracellular L-arginine concentrations were determined by high-performance liquid chromatography (HPLC) in the presence or absence of limonin. Results Limonin inhibited arginase I and II activity in the uncompetitive mode, and prevented nLDL-induced VSMC proliferation in a p21Waf1/Cip1-dependent manner without affecting arginase protein levels. Limonin blocked PKCβII phosphorylation, but not ERK1/2 phosphorylation, and translocation of p47phox to the membrane was decreased, as was superoxide production in nLDL-stimulated VSMCs. Moreover, mitochondrial ROS generation was increased by nLDL stimulation and blocked by preincubation with limonin. Mitochondrial ROS production was responsible for the phosphorylation of PKCβII. HPLC analysis showed that arginase inhibition with limonin increases intracellular L-arginine concentrations, but decreases polyamine concentrations. L-Arginine treatment prevented PKCβII phosphorylation without affecting ERK1/2 phosphorylation. Conclusion Increased L-arginine levels following limonin-dependent arginase inhibition prohibited NADPH oxidase activation in a PKCβII-dependent manner, and blocked nLDL-stimulated VSMC proliferation. PMID

Functional expression and characterization of recombinant NADPH-P450 reductase from Malassezia globosa.

PubMed

Lee, Hwayoun; Park, Hyoung-Goo; Lim, Young-Ran; Lee, Im-Soon; Kim, Beom Joon; Seong, Cheul-Hun; Chun, Young-Jin; Kim, Donghak

2012-01-01

Malassezia globosa is a common pathogenic fungus that causes skin diseases including dandruff and seborrheic dermatitis in humans. Analysis of its genome identified a gene (MGL_1677) coding for a putative NADPH-P450 reductase (NPR) to support the fungal cytochrome P450 enzymes. The heterologously expressed recombinant M. globosa NPR protein was purified, and its functional features were characterized. The purified protein generated a single band on SDS-PAGE at 80.74 kDa and had an absorption maximum at 452 nm, indicating its possible function as an oxidized flavin cofactor. It evidenced NADPH-dependent reducing activity for cytochrome c or nitroblue tetrazolium. Human P450 1A2 and 2A6 were able to successfully catalyze the O-deethylation of 7- ethoxyresorufin and the 7-hydroxylation of coumarin, respectively, with the support of the purified NPR. These results demonstrate that purified NPR is an orthologous reductase protein that supports cytochrome P450 enzymes in M. globosa.

Cell-free NADPH oxidase activation assays: "in vitro veritas".

PubMed

Pick, Edgar

2014-01-01

The superoxide (O2 (∙-))-generating NADPH oxidase complex of phagocytes comprises a membrane-imbedded heterodimeric flavocytochrome, known as cytochrome b 558 (consisting of Nox2 and p22 (phox) ) and four cytosolic regulatory proteins, p47 (phox) , p67 (phox) , p40 (phox) , and the small GTPase Rac. Under physiological conditions, in the resting phagocyte, O2 (∙-) generation is initiated by engagement of membrane receptors by a variety of stimuli, followed by specific signal transduction sequences leading to the translocation of the cytosolic components to the membrane and their association with the cytochrome. A consequent conformational change in Nox2 initiates the electron "flow" along a redox gradient, from NADPH to oxygen, leading to the one-electron reduction of molecular oxygen to O2 (∙-). Methodological difficulties in the dissection of this complex mechanism led to the design "cell-free" systems (also known as "broken cells" or in vitro systems). In these, membrane receptor stimulation and all or part of the signal transduction sequence are missing, the accent being placed on the actual process of "NADPH oxidase assembly," thus on the formation of the complex between cytochrome b 558 and the cytosolic components and the resulting O2 (∙-) generation. Cell-free assays consist of a mixture of the individual components of the NADPH oxidase complex, derived from resting phagocytes or in the form of purified recombinant proteins, exposed in vitro to an activating agent (distinct from and unrelated to whole cell stimulants), in the presence of NADPH and oxygen. Activation is commonly quantified by measuring the primary product of the reaction, O2 (∙-), trapped immediately after its generation by an appropriate acceptor in a kinetic assay, permitting the calculation of the linear rate of O2 (∙-) production, but numerous variations exist, based on the assessment of reaction products or the consumption of substrates. Cell-free assays played a paramount

NADPH oxidases: new kids on the block.

PubMed

Geiszt, Miklós

2006-07-15

Reactive oxygen species (ROS) play a pivotal role in many physiological processes including host defense, hormone biosynthesis, fertilization and cellular signaling. Altered production of ROS has been implicated in the development of immunodeficiency, hypothyroidism and cardiovascular pathologies. In the last few years, several enzymes were identified at the molecular level, which are now thought to be responsible for ROS production observed in diverse tissues. These enzymes show a high degree of homology to the phagocytic NADPH oxidase and are now designated the Nox family of NADPH oxidases. This review updates our knowledge on six new members of the Nox family: Nox1, Nox3, Nox4, Nox5, Duox1 and Duox2.

The response of Hordeum spontaneum desert ecotype to drought and excessive light intensity is characterized by induction of O2 dependent photochemical activity and anthocyanin accumulation.

PubMed

Eppel, Amir; Keren, Nir; Salomon, Eitan; Volis, Sergei; Rachmilevitch, Shimon

2013-03-01

The goal of the current research was to study the role of anthocyanin accumulation, O(2)-related photochemical processes and non-photochemical quenching (NPQ) in the response of desert and Mediterranean plants to drought and excessive light. Plants of Hordeum spontaneum were collected from Mediterranean and desert environments and were subjected to terminal drought for 25 days and then measured for PSII yield at 2 and 21% O(2), NPQ, net carbon assimilation, stomatal conductance, leaf relative water content (LRWC), anthocyanin concentration and leaf absorbance. Under terminal drought, LRWC, carbon assimilation and stomatal conductance decreased similarly and significantly in both the Mediterranean and the desert ecotypes. Anthocyanin accumulated more in the desert ecotype than in the Mediterranean ecotype. NPQ increased more in the Mediterranean ecotype as compared with the desert ecotype. PSII yield decreased significantly in the Mediterranean ecotype under drought and was much lower than in the desert ecotype under drought. The relatively high PSII yield under drought in the desert ecotype was O(2) dependent. The response of the H. spontaneum ecotype from a desert environment to drought stress was characterized by anthocyanin accumulation and induction of O(2) dependent photochemical activity, while the response of the Mediterranean ecotype was based on a higher induction of NPQ. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Monocyte and macrophage-targeted NADPH oxidase mediates antifungal host defense and regulation of acute inflammation in mice

PubMed Central

Grimm, Melissa J.; Vethanayagam, R. Robert; Almyroudis, Nikolaos G.; Dennis, Carly G.; Khan, A. Nazmul H.; D’Auria, Anthony; Singel, Kelly L.; Davidson, Bruce A.; Knight, Paul R.; Blackwell, Timothy S.; Hohl, Tobias M.; Mansour, Michael K.; Vyas, Jatin M.; Röhm, Marc; Urban, Constantin F.; Kelkka, Tiina; Holmdahl, Rikard; Segal, Brahm H.

2013-01-01

Chronic granulomatous disease, an inherited disorder of the NADPH oxidase in which phagocytes are defective in the generation of superoxide anion and downstream reactive oxidant species, is characterized by severe bacterial and fungal infections and excessive inflammation. Although NADPH oxidase isoforms exist in several lineages, reactive oxidant generation is greatest in neutrophils, where NADPH oxidase has been deemed vital for pathogen killing. In contrast, the function and importance of NADPH oxidase in macrophages are less clear. Therefore, we evaluated susceptibility to pulmonary aspergillosis in globally NADPH oxidase-deficient mice versus transgenic mice with monocyte/macrophage-targeted NADPH oxidase activity. We found that the lethal inoculum was more than 100-fold greater in transgenic versus globally NADPH oxidase-deficient mice. Consistent with these in vivo results, NADPH oxidase in mouse alveolar macrophages limited germination of phagocytosed Aspergillus fumigatus spores. Finally, globally NADPH oxidase-deficient mice developed exuberant neutrophilic lung inflammation and pro-inflammatory cytokine responses to zymosan, a fungal cell wall-derived product composed principally of particulate beta-glucans, whereas inflammation in transgenic and wildtype mice was mild and transient. Together, our studies identify a central role for monocyte/macrophage NADPH oxidase in controlling fungal infection and in limiting acute lung inflammation. PMID:23509361

NADPH oxidase activation in neutrophils: Role of the Phosphorylation of its subunits.

PubMed

Belambri, Sahra A; Rolas, Loïc; Raad, Houssam; Hurtado-Nedelec, Margarita; Dang, Pham My-Chan; El-Benna, Jamel

2018-05-14

Neutrophils are key cells of innate immunity and during inflammation. Upon activation, they produce large amounts of superoxide anion (O 2 -. ) and ensuing reactive oxygen species (ROS) to kill phagocytized microbes. The enzyme responsible for O 2 -. production is called the phagocyte NADPH oxidase. This is a multicomponent enzyme system that becomes active after assembly of four cytosolic proteins (p47 phox , p67 phox , p40 phox and Rac2) with the transmembrane proteins (p22 phox and gp91 phox , which form the cytochrome b 558 ). gp91 phox represents the catalytic subunit of the NADPH oxidase and is also called NOX2. NADPH oxidase-derived ROS are essential for microbial killing and innate immunity; however, excessive ROS production induces tissue injury and prolonged inflammatory reactions that contribute to inflammatory diseases. Thus, NADPH oxidase activation must be tightly regulated in time and space in order to limit ROS production. NADPH oxidase activation is regulated by several processes such as phosphorylation of its components, exchange of GDP/GTP on Rac2 and binding of p47 phox and p40 phox to phospholipids. This review aims to provide new insights into the role of the phosphorylation of the NADPH oxidase components, i.e., gp91 phox , p22 phox , p47 phox , p67 phox and p40 phox , in the activation of this enzyme. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Structural analysis of NADPH depleted bovine liver catalase and its inhibitor complexes

PubMed Central

Sugadev, Ragumani; Ponnuswamy, M.N.; Sekar, K.

2011-01-01

To study the functional role of NADPH during mammalian catalase inhibition, the X-ray crystal structures of NADPH-depleted bovine liver catalase and its inhibitor complexes, cyanide and azide, determined at 2.8Å resolution. From the complex structures it is observed that subunits with and without an inhibitor/catalytic water molecule are linked by N-terminal domain swapping. Comparing mammalian- and fungal- catalases, we speculate that NADPH-depleted mammalian catalases may function as a domain-swapped dimer of dimers, especially during inactivation by inhibitors like cyanide and azide. We further speculate that in mammalian catalases the N-terminal hinge-loop region and α-helix is the structural element that senses NADPH binding. Although the above arguments are speculative and need further verification, as a whole our studies have opened up a new possibility, viz. that mammalian catalase acts as a domain-swapped dimer of dimers, especially during inhibitor binding. To generalize this concept to the formation of the inactive state in mammalian catalases in the absence of tightly bound NADPH molecules needs further exploration. The present study adds one more intriguing fact to the existing mysteries of mammalian catalases. PMID:21968615

Latent manganese deficiency increases transpiration in barley (Hordeum vulgare).

PubMed

Hebbern, Christopher A; Laursen, Kristian Holst; Ladegaard, Anne H; Schmidt, Sidsel B; Pedas, Pai; Bruhn, Dan; Schjoerring, Jan K; Wulfsohn, Dvoralai; Husted, Søren

2009-03-01

To investigate if latent manganese (Mn) deficiency leads to increased transpiration, barley plants were grown for 10 weeks in hydroponics with daily additions of Mn in the low nM range. The Mn-starved plants did not exhibit visual leaf symptoms of Mn deficiency, but Chl a fluorescence measurements revealed that the quantum yield efficiency of PSII (F(v)/F(m)) was reduced from 0.83 in Mn-sufficient control plants to below 0.5 in Mn-starved plants. Leaf Mn concentrations declined from 30 to 7 microg Mn g(-1) dry weight in control and Mn-starved plants, respectively. Mn-starved plants had up to four-fold higher transpiration than control plants. Stomatal closure and opening upon light/dark transitions took place at the same rate in both Mn treatments, but the nocturnal leaf conductance for water vapour was still twice as high in Mn-starved plants compared with the control. The observed increase in transpiration was substantiated by (13)C-isotope discrimination analysis and gravimetric measurement of the water consumption, showing significantly lower water use efficiency in Mn-starved plants. The extractable wax content of leaves of Mn-starved plants was approximately 40% lower than that in control plants, and it is concluded that the increased leaf conductance and higher transpirational water loss are correlated with a reduction in the epicuticular wax layer under Mn deficiency.

Trimethyltin-Induced Microglial Activation via NADPH Oxidase and MAPKs Pathway in BV-2 Microglial Cells.

PubMed

Kim, Da Jung; Kim, Yong Sik

2015-01-01

Trimethyltin (TMT) is known as a potent neurotoxicant that causes neuronal cell death and neuroinflammation, particularly in the hippocampus. Microglial activation is one of the prominent pathological features of TMT neurotoxicity. Nevertheless, it remains unclear how microglial activation occurs in TMT intoxication. In this study, we aimed to investigate the signaling pathways in TMT-induced microglial activation using BV-2 murine microglial cells. Our results revealed that TMT generates reactive oxygen species (ROS) and increases the expression of CD11b and nuclear factor-κB- (NF-κB-) mediated nitric oxide (NO) and tumor necrosis factor- (TNF-) α in BV-2 cells. We also observed that NF-κB activation was controlled by p38 and JNK phosphorylation. Moreover, TMT-induced ROS generation occurred via nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in BV-2 cells. Interestingly, treatment with the NADPH oxidase inhibitor apocynin significantly suppressed p38 and JNK phosphorylation and NF-κB activation and ultimately the production of proinflammatory mediators upon TMT exposure. These findings indicate that NADPH oxidase-dependent ROS generation activated p38 and JNK mitogen-activated protein kinases (MAPKs), which then stimulated NF-κB to release proinflammatory mediators in the TMT-treated BV-2 cells.

Trimethyltin-Induced Microglial Activation via NADPH Oxidase and MAPKs Pathway in BV-2 Microglial Cells

PubMed Central

Kim, Da Jung; Kim, Yong Sik

2015-01-01

Trimethyltin (TMT) is known as a potent neurotoxicant that causes neuronal cell death and neuroinflammation, particularly in the hippocampus. Microglial activation is one of the prominent pathological features of TMT neurotoxicity. Nevertheless, it remains unclear how microglial activation occurs in TMT intoxication. In this study, we aimed to investigate the signaling pathways in TMT-induced microglial activation using BV-2 murine microglial cells. Our results revealed that TMT generates reactive oxygen species (ROS) and increases the expression of CD11b and nuclear factor-κB- (NF-κB-) mediated nitric oxide (NO) and tumor necrosis factor- (TNF-) α in BV-2 cells. We also observed that NF-κB activation was controlled by p38 and JNK phosphorylation. Moreover, TMT-induced ROS generation occurred via nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in BV-2 cells. Interestingly, treatment with the NADPH oxidase inhibitor apocynin significantly suppressed p38 and JNK phosphorylation and NF-κB activation and ultimately the production of proinflammatory mediators upon TMT exposure. These findings indicate that NADPH oxidase-dependent ROS generation activated p38 and JNK mitogen-activated protein kinases (MAPKs), which then stimulated NF-κB to release proinflammatory mediators in the TMT-treated BV-2 cells. PMID:26221064

In vitro cytotoxicity of allelopathic plants Adonis vernalis L. Origanum vulgare ssp. vulgare L. and Nepeta nuda subsp. nuda

NASA Astrophysics Data System (ADS)

Koleva, Vanya; Dragoeva, Asya; Stoyanova, Zheni; Yordanova, Zhenia; Ali, Selime; Uzunov, Nikolay M.; Melendez-Alafort, Laura; Rosato, Antonio; Enchev, Dobromir D.

2018-03-01

Medicinal plants produce various secondary metabolites as a part of their chemical defence and survival in nature. These compounds have a wide range of biological activities. Nowadays, medicinal plants are used as source of allelochemicals and new effective anticancer agents. Our previous studies revealed allelopathic potential of water extracts of Adonis vernalis L. (Ranunculaceae), Origanum vulgare ssp. vulgare L. and Nepeta nuda subsp. nuda (Lamiaceae). Present study aimed to evaluate the effect of the same extracts in vitro on human hepatoma cell line SK-HEP-1. Cell proliferation/viability was assessed using Premixed WST-1 Cell Proliferation Reagent. Adonis water extract (1.83mg/ml) had notable negative influence on cancer cell line tested. Oregano (3.5 mg/ml) also exerted negative effect, but to a lesser degree. On the contrary, nepeta water extract (6.59 mg/ml) had an opposite effect, stimulating cell proliferation. One possible explanation could be the type of extraction: after treatment with nepeta methanol extract (6.59 mg/ml) cell viability was significantly reduced. In conclusion, Adonis vernalis and Nepeta nuda subsp. nuda possess metabolites with growth inhibitory effect on human hepatoma cell line SK-HEP-1. Further research is needed to clarify biological activity of lower concentrations which are appropriate to enable the design of new anticancer drugs.

Indonesian herbal medicine prevents hypertension-induced left ventricular hypertrophy by diminishing NADPH oxidase-dependent oxidative stress.

PubMed

Sulistyowati, Erna; Hsu, Jong-Hau; Cheng, Yuan-Bin; Chang, Fang-Rong; Chen, Ying-Fu; Yeh, Jwu-Lai

2017-10-17

Indonesian herbal medicine Centella asiatica , Justicia gendarussa and Imperata cylindrica decoction (CJID) are known to be efficacious for hypertension. Oxidative stress plays an important role in hypertension-induced left ventricular hypertrophy (H-LVH). This study evaluated whether CJID inhibit cardiac remodeling in spontaneously hypertensive rats (SHRs) through mechanism of oxidative stress-related cardiac-NADPH oxidase (NOXs) pathway: NOX1, NOX2 and NOX4. Forty-weeks-old SHRs and normotensive-WKY rats, were both randomly divided into 2 groups: CJID and control. All rats were treated for 5 weeks. Systolic blood pressure (SBP) and heart rate (HR) were measured. LV morphology, function and performance were assessed by histological staining and echocardiography. Serum and cardiac superoxide dismutase (SOD) activity and malondialdehyde (MDA) content were assessed. Cardiac superoxide and hydrogen peroxide (H 2 O 2 ) productions, protein expressions of SOD2, SOD3, NOX1, NOX2 and NOX4 were also determined. We found that SBP and HR were significantly decreased in SHRs-treated group. Echocardiography showed that CJID significantly improved LV morphometry and function. CJID decreased MDA level, but increased SOD activity. Cardiac superoxide and H 2 O 2 generation were decreased in SHRs-treated group. CJID caused cardiac SODs expressions to be increased but NOXs expressions to be suppressed. In conclusion, CJID prevents H-LVH by reducing reactive oxygen species production via the NOXs-dependent pathway.

Indonesian herbal medicine prevents hypertension-induced left ventricular hypertrophy by diminishing NADPH oxidase-dependent oxidative stress

PubMed Central

Sulistyowati, Erna; Hsu, Jong-Hau; Cheng, Yuan-Bin; Chang, Fang-Rong; Chen, Ying-Fu; Yeh, Jwu-Lai

2017-01-01

Indonesian herbal medicine Centella asiatica, Justicia gendarussa and Imperata cylindrica decoction (CJID) are known to be efficacious for hypertension. Oxidative stress plays an important role in hypertension-induced left ventricular hypertrophy (H-LVH). This study evaluated whether CJID inhibit cardiac remodeling in spontaneously hypertensive rats (SHRs) through mechanism of oxidative stress-related cardiac-NADPH oxidase (NOXs) pathway: NOX1, NOX2 and NOX4. Forty-weeks-old SHRs and normotensive-WKY rats, were both randomly divided into 2 groups: CJID and control. All rats were treated for 5 weeks. Systolic blood pressure (SBP) and heart rate (HR) were measured. LV morphology, function and performance were assessed by histological staining and echocardiography. Serum and cardiac superoxide dismutase (SOD) activity and malondialdehyde (MDA) content were assessed. Cardiac superoxide and hydrogen peroxide (H2O2) productions, protein expressions of SOD2, SOD3, NOX1, NOX2 and NOX4 were also determined. We found that SBP and HR were significantly decreased in SHRs-treated group. Echocardiography showed that CJID significantly improved LV morphometry and function. CJID decreased MDA level, but increased SOD activity. Cardiac superoxide and H2O2 generation were decreased in SHRs-treated group. CJID caused cardiac SODs expressions to be increased but NOXs expressions to be suppressed. In conclusion, CJID prevents H-LVH by reducing reactive oxygen species production via the NOXs-dependent pathway. PMID:29156835

Changes in pH and NADPH regulate the DNA binding activity of neuronal PAS domain protein 2, a mammalian circadian transcription factor.

PubMed

Yoshii, Katsuhiro; Tajima, Fumihisa; Ishijima, Sumio; Sagami, Ikuko

2015-01-20

Neuronal PAS domain protein 2 (NPAS2) is a core clock transcription factor that forms a heterodimer with BMAL1 to bind the E-box in the promoter of clock genes and is regulated by various environmental stimuli such as heme, carbon monoxide, and NAD(P)H. In this study, we investigated the effects of pH and NADPH on the DNA binding activity of NPAS2. In an electrophoretic mobility shift (EMS) assay, the pH of the reaction mixture affected the DNA binding activity of the NPAS2/BMAL1 heterodimer but not that of the BMAL1/BMAL1 homodimer. A change in pH from 7.0 to 7.5 resulted in a 1.7-fold increase in activity in the absence of NADPH, and NADPH additively enhanced the activity up to 2.7-fold at pH 7.5. The experiments using truncated mutants revealed that N-terminal amino acids 1-61 of NPAS2 were sufficient to sense the change in both pH and NADPH. We further analyzed the kinetics of formation and DNA binding of the NPAS2/BMAL1 heterodimer at various pH values. In the absence of NADPH, a change in pH from 6.5 to 8.0 decreased the KD(app) value of the E-box from 125 to 22 nM, with an 8-fold increase in the maximal level of DNA binding for the NPAS2/BMAL1 heterodimer. The addition of NADPH resulted in a further decrease in KD(app) to 9 nM at pH 8.0. Furthermore, NPAS2-dependent transcriptional activity in a luciferase assay using NIH3T3 cells also increased with the pH of the culture medium. These results suggest that NPAS2 has a role as a pH and metabolite sensor in regulating circadian rhythms.

The effect of drought on photosynthetic plasticity in Marrubium vulgare plants growing at low and high altitudes.

PubMed

Habibi, Ghader; Ajory, Neda

2015-11-01

Photosynthesis is a biological process most affected by water deficit. Plants have various photosynthetic mechanisms that are matched to specific climatic zones. We studied the photosynthetic plasticity of C3 plants at water deficit using ecotypes of Marrubium vulgare L. from high (2,200 m) and low (1,100 m) elevation sites in the Mishou-Dagh Mountains of Iran. Under experimental drought, high-altitude plants showed more tolerance to water stress based on most of the parameters studied as compared to the low-altitude plants. Increased tolerance in high-altitude plants was achieved by lower levels of daytime stomatal conductance (g s) and reduced damaging effect on maximal quantum yield of photosystem II (PSII) (F v /F m ) coupled with higher levels of carotenoids and non-photochemical quenching (NPQ). High-altitude plants exhibited higher water use efficiency (WUE) than that in low-altitude plants depending on the presence of thick leaves and the reduced daytime stomatal conductance. Additionally, we have studied the oscillation in H(+) content and diel gas exchange patterns to determine the occurrence of C3 or weak CAM (Crassulacean acid metabolism) in M. vulgare through 15 days drought stress. Under water-stressed conditions, low-altitude plants exhibited stomatal conductance and acid fluctuations characteristic of C3 photosynthesis, though high-altitude plants exhibited more pronounced increases in nocturnal acidity and phosphoenolpyruvate carboxylase (PEPC) activity, suggesting photosynthetic flexibility. These results indicated that the regulation of carotenoids, NPQ, stomatal conductance and diel patterns of CO2 exchange presented the larger differences among studied plants at different altitudes and seem to be the protecting mechanisms controlling the photosynthetic performance of M. vulgare plants under drought conditions.

Functional Assembly of Soluble and Membrane Recombinant Proteins of Mammalian NADPH Oxidase Complex.

PubMed

Souabni, Hajer; Ezzine, Aymen; Bizouarn, Tania; Baciou, Laura

2017-01-01

Activation of phagocyte cells from an innate immune system is associated with a massive consumption of molecular oxygen to generate highly reactive oxygen species (ROS) as microbial weapons. This is achieved by a multiprotein complex, the so-called NADPH oxidase. The activity of phagocyte NADPH oxidase relies on an assembly of more than five proteins, among them the membrane heterodimer named flavocytochrome b 558 (Cytb 558 ), constituted by the tight association of the gp91 phox (also named Nox2) and p22 phox proteins. The Cytb 558 is the membrane catalytic core of the NADPH oxidase complex, through which the reducing equivalent provided by NADPH is transferred via the associated prosthetic groups (one flavin and two hemes) to reduce dioxygen into superoxide anion. The other major proteins (p47 phox , p67 phox , p40 phox , Rac) requisite for the complex activity are cytosolic proteins. Thus, the NADPH oxidase functioning relies on a synergic multi-partner assembly that in vivo can be hardly studied at the molecular level due to the cell complexity. Thus, a cell-free assay method has been developed to study the NADPH oxidase activity that allows measuring and eventually quantifying the ROS generation based on optical techniques following reduction of cytochrome c. This setup is a valuable tool for the identification of protein interactions, of crucial components and additives for a functional enzyme. Recently, this method was improved by the engineering and the production of a complete recombinant NADPH oxidase complex using the combination of purified proteins expressed in bacterial and yeast host cells. The reconstitution into artificial membrane leads to a fully controllable system that permits fine functional studies.

Targeting NADPH oxidase decreases oxidative stress in the transgenic sickle cell mouse penis.

PubMed

Musicki, Biljana; Liu, Tongyun; Sezen, Sena F; Burnett, Arthur L

2012-08-01

Sickle cell disease (SCD) is a state of chronic vasculopathy characterized by endothelial dysfunction and increased oxidative stress, but the sources and mechanisms responsible for reactive oxygen species (ROS) production in the penis are unknown. We evaluated whether SCD activates NADPH oxidase, induces endothelial nitric oxide synthase (eNOS) uncoupling, and decreases antioxidants in the SCD mouse penis. We further tested the hypothesis that targeting NADPH oxidase decreases oxidative stress in the SCD mouse penis. SCD transgenic (sickle) mice were used as an animal model of SCD. Hemizygous (hemi) mice served as controls. Mice received an NADPH oxidase inhibitor apocynin (10 mM in drinking water) or vehicle. Penes were excised at baseline for molecular studies. Markers of oxidative stress (4-hydroxy-2-nonenal [HNE]), sources of ROS (eNOS uncoupling and NADPH oxidase subunits p67(phox) , p47(phox) , and gp91(phox) ), and enzymatic antioxidants (superoxide dismutase [SOD]1, SOD2, catalase, and glutathione peroxidase-1 [GPx1]) were measured by Western blot in penes. Sources of ROS, oxidative stress, and enzymatic antioxidants in the SCD penis. Relative to hemi mice, SCD increased (P<0.05) protein expression of NADPH oxidase subunits p67(phox) , p47(phox) , and gp91(phox) , 4-HNE-modified proteins, induced eNOS uncoupling, and reduced Gpx1 expression in the penis. Apocynin treatment of sickle mice reversed (P<0.05) the abnormalities in protein expressions of p47(phox) , gp91(phox) (but not p67(phox) ) and 4-HNE, but only slightly (P>0.05) prevented eNOS uncoupling in the penis. Apocynin treatment of hemi mice did not affect any of these parameters. NADPH oxidase and eNOS uncoupling are sources of oxidative stress in the SCD penis; decreased GPx1 further contributes to oxidative stress. Inhibition of NADPH oxidase upregulation decreases oxidative stress, implying a major role for NADPH oxidase as a ROS source and a potential target for improving vascular function in

Targeting NADPH Oxidase Decreases Oxidative Stress in the Transgenic Sickle Cell Mouse Penis

PubMed Central

Musicki, Biljana; Liu, Tongyun; Sezen, Sena F.; Burnett, Arthur L.

2012-01-01

Introduction Sickle cell disease (SCD) is a state of chronic vasculopathy characterized by endothelial dysfunction and increased oxidative stress, but the sources and mechanisms responsible for reactive oxygen species (ROS) production in the penis are unknown. Aims We evaluated whether SCD activates NADPH oxidase, induces endothelial nitric oxide synthase (eNOS) uncoupling, and decreases antioxidants in the SCD mouse penis. We further tested the hypothesis that targeting NADPH oxidase decreases oxidative stress in the SCD mouse penis. Methods SCD transgenic (sickle) mice were used as an animal model of SCD. Hemizygous (hemi) mice served as controls. Mice received an NADPH oxidase inhibitor apocynin (10 mM in drinking water) or vehicle. Penes were excised at baseline for molecular studies. Markers of oxidative stress (4-hydroxy-2-nonenal [HNE]), sources of ROS (eNOS uncoupling and NADPH oxidase subunits p67phox, p47phox, and gp91phox), and enzymatic antioxidants (superoxide dismutase [SOD]1, SOD2, catalase, and glutathione peroxidase-1 [GPx1]) were measured by Western blot in penes. Main Outcome Measures Sources of ROS, oxidative stress, and enzymatic antioxidants in the SCD penis. Results Relative to hemi mice, SCD increased (P < 0.05) protein expression of NADPH oxidase subunits p67phox, p47phox, and gp91phox, 4-HNE-modified proteins, induced eNOS uncoupling, and reduced Gpx1 expression in the penis. Apocynin treatment of sickle mice reversed (P < 0.05) the abnormalities in protein expressions of p47phox, gp91phox (but not p67phox) and 4-HNE, but only slightly (P > 0.05) prevented eNOS uncoupling in the penis. Apocynin treatment of hemi mice did not affect any of these parameters. Conclusion NADPH oxidase and eNOS uncoupling are sources of oxidative stress in the SCD penis; decreased GPx1 further contributes to oxidative stress. Inhibition of NADPH oxidase upregulation decreases oxidative stress, implying a major role for NADPH oxidase as a ROS source and a

NADPH-diaphorase activity and neurovascular coupling in the rat cerebral cortex.

PubMed

Vlasenko, O V; Maisky, V A; Maznychenko, A V; Pilyavskii, A I

2008-01-01

The distribution of NADPH-diaphorase-reactive (NADPH-dr) neurons and neuronal processes in the cerebral cortex and basal forebrain and their association with parenchymal vessels were studied in normal adult rats using NADPH-d histochemical protocol. The intensely stained cortical interneurons and reactive subcortically originating afferents, and stained microvessels were examined through a light microscope at law (x250) and high (x630) magnifications. NADPH-dr interneurons were concentrated in layers 2-6 of the M1 and M2 areas. However, clear predominance in their concentration (14 +/- 0.8 P < 0.05 per section) was found in layer 6. A mean number of labeled neurons in auditory (AuV), granular and agranular (GI, AIP) areas of the insular cortex was calculated to reach 12.3 +/- 0.7, 18.5 +/- 1.0 and 23.3 +/- 1.7 units per section, respectively (P < 0.05). The distinct apposition of labelled neurons to intracortical vessels was found in the M1, M2. The order of frequency of neurovascular coupling in different zones of the cerebral cortex was as following sequence: AuV (31.2%, n = 1040) > GI (18.0%, n = 640) > S1 (13.3%, n = 720) > M1 (6.3%, n = 1360). A large number of structural associations between labeled cells and vessels in the temporal and insular cortex indicate that NADPH-d-reactive interneurons can contribute to regulation of the cerebral regional blood flow in these areas.

NADPH oxidase contributes to coronary endothelial dysfunction in the failing heart.

PubMed

Zhang, Ping; Hou, Mingxiao; Li, Yunfang; Xu, Xin; Barsoum, Michel; Chen, Yingjie; Bache, Robert J

2009-03-01

Increased reactive oxygen species (ROS) produced by the failing heart can react with nitric oxide (NO), thereby decreasing NO bioavailability. This study tested the hypothesis that increased ROS generation contributes to coronary endothelial dysfunction in the failing heart. Congestive heart failure (CHF) was produced in six dogs by ventricular pacing at 240 beats/min for 4 wk. Studies were performed at rest and during treadmill exercise under control conditions and after treatment with the NADPH oxidase inhibitor and antioxidant apocynin (4 mg/kg iv). Apocynin caused no significant changes in heart rate, aortic pressure, left ventricular (LV) systolic pressure, LV end-diastolic pressure, or maximum rate of LV pressure increase at rest or during exercise in normal or CHF dogs. Apocynin caused no change in coronary blood flow (CBF) in normal dogs but increased CBF at rest and during exercise in animals with CHF (P < 0.05). Intracoronary ACh caused dose-dependent increases of CBF that were blunted in CHF. Apocynin had no effect on the response to ACh in normal dogs but augmented the response to ACh in CHF dogs (P < 0.05). The oxidative stress markers nitrotyrosine and 4-hydroxy-2-nonenal were significantly greater in failing than in normal myocardium. Furthermore, coelenterazine chemiluminescence for O(2)(-) was more than twice normal in failing myocardium, and this difference was abolished by apocynin. Western blot analysis of myocardial lysates demonstrated that the p47(phox) and p22(phox) subunits of NADPH were significantly increased in the failing hearts, while real-time PCR demonstrated that Nox2 mRNA was significantly increased. The data indicate that increased ROS generation in the failing heart is associated with coronary endothelial dysfunction and suggest that NADPH oxidase may contribute to this abnormality.

Dynamic single-cell NAD(P)H measurement reveals oscillatory metabolism throughout the E. coli cell division cycle.

PubMed

Zhang, Zheng; Milias-Argeitis, Andreas; Heinemann, Matthias

2018-02-01

Recent work has shown that metabolism between individual bacterial cells in an otherwise isogenetic population can be different. To investigate such heterogeneity, experimental methods to zoom into the metabolism of individual cells are required. To this end, the autofluoresence of the redox cofactors NADH and NADPH offers great potential for single-cell dynamic NAD(P)H measurements. However, NAD(P)H excitation requires UV light, which can cause cell damage. In this work, we developed a method for time-lapse NAD(P)H imaging in single E. coli cells. Our method combines a setup with reduced background emission, UV-enhanced microscopy equipment and optimized exposure settings, overall generating acceptable NAD(P)H signals from single cells, with minimal negative effect on cell growth. Through different experiments, in which we perturb E. coli's redox metabolism, we demonstrated that the acquired fluorescence signal indeed corresponds to NAD(P)H. Using this new method, for the first time, we report that intracellular NAD(P)H levels oscillate along the bacterial cell division cycle. The developed method for dynamic measurement of NAD(P)H in single bacterial cells will be an important tool to zoom into metabolism of individual cells.

Khz-cp (crude polysaccharide extract obtained from the fusion of Ganoderma lucidum and Polyporus umbellatus mycelia) induces apoptosis by increasing intracellular calcium levels and activating P38 and NADPH oxidase-dependent generation of reactive oxygen species in SNU-1 cells.

PubMed

Kim, Tae Hwan; Kim, Ju Sung; Kim, Zoo Haye; Huang, Ren Bin; Chae, Young Lye; Wang, Ren Sheng

2014-07-10

Khz-cp is a crude polysaccharide extract that is obtained after nuclear fusion in Ganoderma lucidum and Polyporus umbellatus mycelia (Khz). It inhibits the growth of cancer cells. Khz-cp was extracted by solvent extraction. The anti-proliferative activity of Khz-cp was confirmed by using Annexin-V/PI-flow cytometry analysis. Intracellular calcium increase and measurement of intracellular reactive oxygen species (ROS) were performed by using flow cytometry and inverted microscope. SNU-1 cells were treated with p38, Bcl-2 and Nox family siRNA. siRNA transfected cells was employed to investigate the expression of apoptotic, growth and survival genes in SNU-1 cells. Western blot analysis was performed to confirm the expression of the genes. In the present study, Khz-cp induced apoptosis preferentially in transformed cells and had only minimal effects on non-transformed cells. Furthermore, Khz-cp was found to induce apoptosis by increasing the intracellular Ca2+ concentration ([Ca2+]i) and activating P38 to generate reactive oxygen species (ROS) via NADPH oxidase and the mitochondria. Khz-cp-induced apoptosis was caspase dependent and occurred via a mitochondrial pathway. ROS generation by NADPH oxidase was critical for Khz-cp-induced apoptosis, and although mitochondrial ROS production was also required, it appeared to occur secondary to ROS generation by NADPH oxidase. Activation of NADPH oxidase was shown by the translocation of the regulatory subunits p47phox and p67phox to the cell membrane and was necessary for ROS generation by Khz-cp. Khz-cp triggered a rapid and sustained increase in [Ca2+]i that activated P38. P38 was considered to play a key role in the activation of NADPH oxidase because inhibition of its expression or activity abrogated membrane translocation of the p47phox and p67phox subunits and ROS generation. In summary, these data indicate that Khz-cp preferentially induces apoptosis in cancer cells and that the signaling mechanisms involve an

Hypoglycemic neuronal death is triggered by glucose reperfusion and activation of neuronal NADPH oxidase

PubMed Central

Suh, Sang Won; Gum, Elizabeth T.; Hamby, Aaron M.; Chan, Pak H.; Swanson, Raymond A.

2007-01-01

Hypoglycemic coma and brain injury are potential complications of insulin therapy. Certain neurons in the hippocampus and cerebral cortex are uniquely vulnerable to hypoglycemic cell death, and oxidative stress is a key event in this cell death process. Here we show that hypoglycemia-induced oxidative stress and neuronal death are attributable primarily to the activation of neuronal NADPH oxidase during glucose reperfusion. Superoxide production and neuronal death were blocked by the NADPH oxidase inhibitor apocynin in both cell culture and in vivo models of insulin-induced hypoglycemia. Superoxide production and neuronal death were also blocked in studies using mice or cultured neurons deficient in the p47phox subunit of NADPH oxidase. Chelation of zinc with calcium disodium EDTA blocked both the assembly of the neuronal NADPH oxidase complex and superoxide production. Inhibition of the hexose monophosphate shunt, which utilizes glucose to regenerate NADPH, also prevented superoxide formation and neuronal death, suggesting a mechanism linking glucose reperfusion to superoxide formation. Moreover, the degree of superoxide production and neuronal death increased with increasing glucose concentrations during the reperfusion period. These results suggest that high blood glucose concentrations following hypoglycemic coma can initiate neuronal death by a mechanism involving extracellular zinc release and activation of neuronal NADPH oxidase. PMID:17404617

The Importance of NADPH Oxidases and Redox Signaling in Angiogenesis

PubMed Central

Prieto-Bermejo, Rodrigo; Hernández-Hernández, Angel

2017-01-01

Eukaryotic cells have to cope with the constant generation of reactive oxygen species (ROS). Although the excessive production of ROS might be deleterious for cell biology, there is a plethora of evidence showing that moderate levels of ROS are important for the control of cell signaling and gene expression. The family of the nicotinamide adenine dinucleotide phosphate oxidases (NADPH oxidases or Nox) has evolved to produce ROS in response to different signals; therefore, they fulfil a central role in the control of redox signaling. The role of NADPH oxidases in vascular physiology has been a field of intense study over the last two decades. In this review we will briefly analyze how ROS can regulate signaling and gene expression. We will address the implication of NADPH oxidases and redox signaling in angiogenesis, and finally, the therapeutic possibilities derived from this knowledge will be discussed. PMID:28505091

Extra virgin olive oil rich in polyphenols modulates VEGF-induced angiogenic responses by preventing NADPH oxidase activity and expression.

PubMed

Calabriso, Nadia; Massaro, Marika; Scoditti, Egeria; D'Amore, Simona; Gnoni, Antonio; Pellegrino, Mariangela; Storelli, Carlo; De Caterina, Raffaele; Palasciano, Giuseppe; Carluccio, Maria Annunziata

2016-02-01

Previous studies have shown the antiinflammatory, antioxidant and antiangiogenic properties by pure olive oil polyphenols; however, the effects of olive oil phenolic fraction on the inflammatory angiogenesis are unknown. In this study, we investigated the effects of the phenolic fraction (olive oil polyphenolic extract, OOPE) from extra virgin olive oil and related circulating metabolites on the VEGF-induced angiogenic responses and NADPH oxidase activity and expression in human cultured endothelial cells. We found that OOPE (1-10 μg/ml), at concentrations achievable nutritionally, significantly reduced, in a concentration-dependent manner, the VEGF-induced cell migration, invasiveness and tube-like structure formation through the inhibition of MMP-2 and MMP-9. OOPE significantly (P<0.05) reduced VEGF-induced intracellular reactive oxygen species by modulating NADPH oxidase activity, p47phox membrane translocation and the expression of Nox2 and Nox4. Moreover, the treatment of endothelial cells with serum obtained 4 h after acute intake of extra virgin olive oil, with high polyphenol content, decreased VEGF-induced NADPH oxidase activity and Nox4 expression, as well as, MMP-9 expression, as compared with fasting control serum. Overall, native polyphenols and serum metabolites of extra virgin olive oil rich in polyphenols are able to lower the VEGF-induced angiogenic responses by preventing endothelial NADPH oxidase activity and decreasing the expression of selective NADPH oxidase subunits. Our results provide an alternative mechanism by which the consumption of olive oil rich in polyphenols may account for a reduction of oxidative stress inflammatory-related sequelae associated with chronic degenerative diseases. Copyright © 2015 Elsevier Inc. All rights reserved.

The effect of abandoned mining ponds on trace elements dynamics in the soil-plant system

NASA Astrophysics Data System (ADS)

Gabarrón, María; Faz, Ángel; Zornoza, Raúl; Acosta, Jose A.

2017-04-01

In semiarid climate regions lack of vegetation and dryer climate contribute to erosion of abandoned mining surface areas making them up important potential sources of metal pollution into the environment. The objectives of this study were to determine the influence of mine ponds in agriculture and forest soils, and identify the dynamic of metals in the soil-plant system for native plant species (Ballota hirsuta) and crop species (Hordeum vulgare) in two ancient mining districts: La Unión and Mazarrón. To achieve these objectives, wastes samples from mine ponds and soil samples (rhizosphere and non-rhizosphere soils) from natural and agricultural lands were collected. In addition, six plants (Ballota hirsuta) from natural area and 3 plants (Hordeum vulgare) from crops were collected. Physicochemical properties and total, water soluble and bioavailable metals (Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb, and Zn) and arsenic were measured in waste/soil samples. The chemical speciation of metals in soil was estimated by a sequential extraction procedure. For plants analyses, each plant were divided in roots, stem and leaves and metal content measured by ICP-MS. Results indicated that mine, natural and agricultural soils were contaminated by As, Cd, Cu, Pb, and Zn. Chemical partitioning revealed higher mobility of metals in mine ponds than natural and agriculture soils while only Fe and As are completely bound to the soil matrix due to the mineralogical compositions of soils. The accumulation of metals in Ballota hirsuta in La Union decrease as Fe>As>Cr>Ni>Cu>Zn>Cd>Mn>Co>Pb while in Mazarrón did as As>Fe>Cr>Pb>Cu>Ni>Co>Mn>Zn>Cd. Ballota hirsuta showed high ability to bio-accumulate Cu, Cr, Fe, Ni, and As, transferring a large amount to edible parts without exceeding the toxicity limits for animals. Results for barley plants (Hordeum vulgare) showed the ability to absorb and accumulate As, Fe, Mn, Pb and Zn, although the transfer ability of As, Cd and Pb was lower. Although the

Identification and cloning of an NADPH-dependent hydroxycinnamoyl-CoA double bond reductase involved in dihydrochalcone formation in Malus×domestica Borkh.

PubMed

Ibdah, Mwafaq; Berim, Anna; Martens, Stefan; Valderrama, Andrea Lorena Herrera; Palmieri, Luisa; Lewinsohn, Efraim; Gang, David R

2014-11-01

The apple tree (Malus sp.) is an agriculturally and economically important source of food and beverages. Many of the health beneficial properties of apples are due to (poly)phenolic metabolites that they contain, including various dihydrochalcones. Although many of the genes and enzymes involved in polyphenol biosynthesis are known in many plant species, the specific reactions that lead to the biosynthesis of the dihydrochalcone precursor, p-dihydrocoumaroyl-CoA (3), are unknown. To identify genes involved in the synthesis of these metabolites, existing genome databases of the Rosaceae were screened for apple genes with significant sequence similarity to Arabidopsis alkenal double bond reductases. Herein described are the isolation and characterization of a Malus hydroxycinnamoyl-CoA double bond reductase, which catalyzed the NADPH-dependent reduction of p-coumaroyl-CoA and feruloyl-CoA to p-dihydrocoumaroyl-CoA and dihydroferuloyl-CoA, respectively. Its apparent Km values for p-coumaroyl-CoA, feruloyl-CoA and NADPH were 96.6, 92.9 and 101.3μM, respectively. The Malus double bond reductase preferred feruloyl-CoA to p-coumaroyl-CoA as a substrate by a factor of 2.1 when comparing catalytic efficiencies in vitro. Expression analysis of the hydroxycinnamoyl-CoA double bond reductase gene revealed that its transcript levels showed significant variation in tissues of different developmental stages, but was expressed when expected for involvement in dihydrochalcone formation. Thus, the hydroxycinnamoyl-CoA double bond reductase appears to be responsible for the reduction of the α,β-unsaturated double bond of p-coumaroyl-CoA, the first step of dihydrochalcone biosynthesis in apple tissues, and may be involved in the production of these compounds. Copyright © 2014 Elsevier Ltd. All rights reserved.

Two Atypical l-Cysteine-regulated NADPH-dependent Oxidoreductases Involved in Redox Maintenance, l-Cystine and Iron Reduction, and Metronidazole Activation in the Enteric Protozoan Entamoeba histolytica*

PubMed Central

Jeelani, Ghulam; Husain, Afzal; Sato, Dan; Ali, Vahab; Suematsu, Makoto; Soga, Tomoyoshi; Nozaki, Tomoyoshi

2010-01-01

We discovered novel catalytic activities of two atypical NADPH-dependent oxidoreductases (EhNO1/2) from the enteric protozoan parasite Entamoeba histolytica. EhNO1/2 were previously annotated as the small subunit of glutamate synthase (glutamine:2-oxoglutarate amidotransferase) based on similarity to authentic bacterial homologs. As E. histolytica lacks the large subunit of glutamate synthase, EhNO1/2 were presumed to play an unknown role other than glutamine/glutamate conversion. Transcriptomic and quantitative reverse PCR analyses revealed that supplementation or deprivation of extracellular l-cysteine caused dramatic up- or down-regulation, respectively, of EhNO2, but not EhNO1 expression. Biochemical analysis showed that these FAD- and 2[4Fe-4S]-containing enzymes do not act as glutamate synthases, a conclusion which was supported by phylogenetic analyses. Rather, they catalyze the NADPH-dependent reduction of oxygen to hydrogen peroxide and l-cystine to l-cysteine and also function as ferric and ferredoxin-NADP+ reductases. EhNO1/2 showed notable differences in substrate specificity and catalytic efficiency; EhNO1 had lower Km and higher kcat/Km values for ferric ion and ferredoxin than EhNO2, whereas EhNO2 preferred l-cystine as a substrate. In accordance with these properties, only EhNO1 was observed to physically interact with intrinsic ferredoxin. Interestingly, EhNO1/2 also reduced metronidazole, and E. histolytica transformants overexpressing either of these proteins were more sensitive to metronidazole, suggesting that EhNO1/2 are targets of this anti-amebic drug. To date, this is the first report to demonstrate that small subunit-like proteins of glutamate synthase could play an important role in redox maintenance, l-cysteine/l-cystine homeostasis, iron reduction, and the activation of metronidazole. PMID:20592025

Antimalarial NADPH-Consuming Redox-Cyclers As Superior Glucose-6-Phosphate Dehydrogenase Deficiency Copycats.

PubMed

Bielitza, Max; Belorgey, Didier; Ehrhardt, Katharina; Johann, Laure; Lanfranchi, Don Antoine; Gallo, Valentina; Schwarzer, Evelin; Mohring, Franziska; Jortzik, Esther; Williams, David L; Becker, Katja; Arese, Paolo; Elhabiri, Mourad; Davioud-Charvet, Elisabeth

2015-05-20

Early phagocytosis of glucose-6-phosphate dehydrogenase (G6PD)-deficient erythrocytes parasitized by Plasmodium falciparum were shown to protect G6PD-deficient populations from severe malaria. Here, we investigated the mechanism of a novel antimalarial series, namely 3-[substituted-benzyl]-menadiones, to understand whether these NADPH-consuming redox-cyclers, which induce oxidative stress, mimic the natural protection of G6PD deficiency. We demonstrated that the key benzoylmenadione metabolite of the lead compound acts as an efficient redox-cycler in NADPH-dependent methaemoglobin reduction, leading to the continuous formation of reactive oxygen species, ferrylhaemoglobin, and subsequent haemichrome precipitation. Structure-activity relationships evidenced that both drug metabolites and haemoglobin catabolites contribute to potentiate drug effects and inhibit parasite development. Disruption of redox homeostasis by the lead benzylmenadione was specifically induced in Plasmodium falciparum parasitized erythrocytes and not in non-infected cells, and was visualized via changes in the glutathione redox potential of living parasite cytosols. Furthermore, the redox-cycler shows additive and synergistic effects in combination with compounds affecting the NADPH flux in vivo. The lead benzylmenadione 1c is the first example of a novel redox-active agent that mimics the behavior of a falciparum parasite developing inside a G6PD-deficient red blood cell (RBC) giving rise to malaria protection, and it exerts specific additive effects that are inhibitory to parasite development, without harm for non-infected G6PD-sufficient or -deficient RBCs. This strategy offers an innovative perspective for the development of future antimalarial drugs for G6PD-sufficient and -deficient populations.

New insights into the roles of NADPH oxidases in sexual development and ascospore germination in Sordaria macrospora.

PubMed

Dirschnabel, Daniela Elisabeth; Nowrousian, Minou; Cano-Domínguez, Nallely; Aguirre, Jesus; Teichert, Ines; Kück, Ulrich

2014-03-01

NADPH oxidase (NOX)-derived reactive oxygen species (ROS) act as signaling determinants that induce different cellular processes. To characterize NOX function during fungal development, we utilized the genetically tractable ascomycete Sordaria macrospora. Genome sequencing of a sterile mutant led us to identify the NADPH oxidase encoding nox1 as a gene required for fruiting body formation, regular hyphal growth, and hyphal fusion. These phenotypes are shared by nor1, lacking the NOX regulator NOR1. Further phenotypic analyses revealed a high correlation between increased ROS production and hyphal fusion deficiencies in nox1 and other sterile mutants. A genome-wide transcriptional profiling analysis of mycelia and isolated protoperithecia from wild type and nox1 revealed that nox1 inactivation affects the expression of genes related to cytoskeleton remodeling, hyphal fusion, metabolism, and mitochondrial respiration. Genetic analysis of nox2, lacking the NADPH oxidase 2 gene, nor1, and transcription factor deletion mutant ste12, revealed a strict melanin-dependent ascospore germination defect, indicating a common genetic pathway for these three genes. We report that gsa3, encoding a G-protein α-subunit, and sac1, encoding cAMP-generating adenylate cyclase, act in a separate pathway during the germination process. The finding that cAMP inhibits ascospore germination in a melanin-dependent manner supports a model in which cAMP inhibits NOX2 activity, thus suggesting a link between both pathways. Our results expand the current knowledge on the role of NOX enzymes in fungal development and provide a frame to define upstream and downstream components of the NOX signaling pathways in fungi.

Human sperm NADH and NADPH diaphorase cytochemistry: correlation with sperm motility.

PubMed

Zini, A; O'Bryan, M K; Israel, L; Schlegel, P N

1998-03-01

We have examined the correlation between the retention of residual sperm cytoplasm and sperm motility in semen from men presenting for infertility evaluation. Semen samples (n = 12) were obtained from nonazoospermic men presenting for infertility evaluation at our institution. Samples were fractionated into high-, intermediate-, and low-density subpopulations by Percoll gradients in order to examine the correlation between the retention of residual sperm cytoplasm and sperm motility. Residual sperm cytoplasm retention was detected by cytochemical staining of sperm for nicotinamide adenine dinucleotide (NADH)- or nicotinamide adenine dinucleotide phosphate (NADPH)-dependent diaphorase activity. The different sperm subpopulations (low, intermediate, and high density) had significantly different percentages of sperm with droplet retention (analysis of variance, P < 0.05). Using either NADH or NADPH diaphorase staining as a marker of the cytoplasmic space, a significant negative correlation was observed between the percentage of sperm with residual cytoplasmic droplets and the percentage of motile sperm (r = -0.58 and -0.61, respectively, P < 0.05). Assessment of residual sperm cytoplasm retention is a simple diagnostic test. Although this test is of unproven value in the management of infertile men, this and other studies suggest that it may provide useful data on sperm function.

Reconstituted high-density lipoprotein suppresses leukocyte NADPH oxidase activation by disrupting lipid rafts.

PubMed

Peshavariya, Hitesh; Dusting, Gregory J; Di Bartolo, Belinda; Rye, Kerry-Anne; Barter, Philip J; Jiang, Fan

2009-08-01

Reconstituted discoidal high-density lipoprotein (rHDL) has potent vascular protective actions. Native HDL suppresses cellular generation of reactive oxygen species, whereas this antioxidant effect of rHDL is less clear. This study examined the effects of rHDL on NADPH oxidase, a major source of cellular superoxide generation, in both leukocytes and human umbilical vein endothelial cells. Superoxide was measured with lucigenin-enhanced chemiluminescence. Expression of NADPH oxidase sub-units was determined by real-time PCR. Pre-treatment of HL-60 cells with rHDL (10 and 25 microM) for 1 h significantly reduced phorbol 12-myristate 13-acetate-stimulated superoxide production. Treatment with rHDL for up to 24 h did not change the mRNA expression of NADPH oxidase sub-units. In HL-60 cells, depletion of cholesterol from the plasma membrane by methyl-beta-cyclodextrin mimicked the effect of rHDL, whereas cholesterol repletion blunted the effects of rHDL. Treatment with rHDL induced disruption of the lipid raft structures and blunted PMA-induced redistribution of p47phox into lipid rafts. In contrast, treatment of endothelial cells with rHDL for up to 18 h had no effect on either basal or tumour necrosis factor-alpha-stimulated NADPH oxidase activity, but markedly suppressed the cytokine-induced expression of proinflammatory adhesion molecules. The results suggest that rHDL inhibits NADPH oxidase activation in leukocytes, probably by interrupting the assembly of NADPH oxidase sub-units at the lipid rafts. This effect may contribute to the vascular protective actions of rHDL against inflammation-mediated oxidative damage.

Localization of nitric oxide synthase and NADPH-diaphorase in guinea pig and human cochleae.

PubMed

Ruan, R S; Leong, S K; Yeoh, K H

1997-01-01

The distributions of nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) and nitric oxide synthase (NOS) in mammalian cochlea were studied at light and electron microscope levels by NADPH-d histochemistry and brain NOS (bNOS) immunohistochemistry. The cochleae from 15 albino guinea pigs were perilymphatically fixed with 2% periodate-lysine-paraformaldehyde, decalcified in 10% EDTA and processed for light and electron microscopy after NADPH-d or NOS staining in frozen and vibratome sections respectively. One human cochlea was available for light microscope examination of NADPH-d or bNOS stained sections. Light microscope results revealed that type I neurons and nerve fibers of the spiral ganglion cells were labeled by bNOS immunohistochemistry as well as NADPH-d histochemistry in both guinea pig and human cochleae. At subcellular level, NADPH-d reaction product was localized in the mitochondria of the neuronal cytoplasm and axoplasm and in the cytoplasm of the vascular endothelium. The immunoreaction products of bNOS were evenly distributed in the neuronal cytoplasm and axoplasm. Myelinated and unmyelinated fibers in the intraganglionic spiral bundle and the inner spiral and inner radial fibers below the inner hair cells were labeled for bNOS. The nerve endings below the outer hair cells were not stained. NOS immunoreaction product was also found in the outer hair cells, Schwann cells of myelinated nerve fibers, Deiter's cells, pillar cells and the tympanic lamina cells. No difference was found in the staining pattern of both NADPH-d and NOS reaction products between human and guinea pig cochleae at the light microscope level. The results suggest that NO plays an important role in the maintenance of auditory function in the mammal.

NADPH Oxidase versus Mitochondria-Derived ROS in Glucose-Induced Apoptosis of Pericytes in Early Diabetic Retinopathy

PubMed Central

Mustapha, Nik M.; Tarr, Joanna M.; Kohner, Eva M.; Chibber, Rakesh

2010-01-01

Objectives. Using apocynin (inhibitor of NADPH oxidase), and Mitoquinol 10 nitrate (MitoQ; mitochondrial-targeted antioxidant), we addressed the importance of mitochondria versus NADPH oxidase-derived ROS in glucose-induced apoptosis of pericytes. Methods. NADPH oxidase was localised using Western blot analysis and cytochrome C reduction assay. Apoptosis was detected by measuring caspase-3 activity. Intracellular glucose concentration, ROS formation and Nε-(carboxymethyl) lysine (CML) content were measured using Amplex Red assay kit, dihydroethidium (DHE), and competitive immunoabsorbant enzyme-linked assay (ELISA), respectively. Results. NADPH oxidase was localised in the cytoplasm of pericytes suggesting ROS production within intracellular compartments. High glucose (25 mM) significantly increased apoptosis, intracellular glucose concentration, and CML content. Apoptosis was associated with increased gp91phox expression, activity of NADPH oxidase, and intracellular ROS production. Apocynin and not MitoQ significantly blunted the generation of ROS, formation of intracellular CML and apoptosis. Conclusions. NADPH oxidase and not mitochondria-derived ROS is responsible for the accelerated apoptosis of pericytes in diabetic retinopathy. PMID:20652059

Nox4 NADPH oxidase-derived reactive oxygen species, via endogenous carbon monoxide, promote survival of brain endothelial cells during TNF-α-induced apoptosis

PubMed Central

Basuroy, Shyamali; Tcheranova, Dilyara; Bhattacharya, Sujoy; Leffler, Charles W.

2011-01-01

We investigated the role of reactive oxygen species (ROS) in promoting cell survival during oxidative stress induced by the inflammatory mediator tumor necrosis factor-α (TNF-α) in cerebral microvascular endothelial cells (CMVEC) from newborn piglets. Nox4 is the major isoform of NADPH oxidase responsible for TNF-α-induced oxidative stress and apoptosis in CMVEC. We present novel data that Nox4 NADPH oxidase-derived ROS also initiate a cell survival mechanism by increasing production of a gaseous antioxidant mediator carbon monoxide (CO) by constitutive heme oxygenase-2 (HO-2). TNF-α rapidly enhanced endogenous CO production in a superoxide- and NADPH oxidase-dependent manner in CMVEC with innate, but not with small interfering RNA (siRNA)-downregulated Nox4 activity. CORM-A1, a CO-releasing compound, inhibited Nox4-mediated ROS production and enhanced cell survival in TNF-α-challenged CMVEC. The ROS-induced CO-mediated survival mechanism requires functional interactions between the protein kinase B/Akt and extracellular signal-related kinase (ERK)/p38 MAPK signaling pathways activated by TNF-α. In Akt siRNA-transfected CMVEC and in cells with pharmacologically inhibited Akt, Erk1/2, and p38 mitogen-activated protein kinase (MAPK) activities, CORM-A1 was no longer capable of blocking Nox4 activation and apoptosis caused by TNF-α. Overall, Nox4 NADPH oxidase-derived ROS initiate both death and survival pathways in TNF-α-challenged CMVEC. The ROS-dependent cell survival pathway is mediated by an endogenous antioxidant CO, which inhibits Nox4 activation via a mechanism that includes Akt, ERK1/2, and p38 MAPK signaling pathways. The ability of CO to inhibit TNF-α-induced ERK1/2 and p38 MAPK activities in an Akt-dependent manner appears to be the key element in ROS-dependent survival of endothelial cells during TNF-α-mediated brain inflammatory disease. PMID:21123734

Anthelmintic effects of the essential oil of fennel (Foeniculum vulgare Mill., Apiaceae) against Schistosoma mansoni.

PubMed

Wakabayashi, Kamila A L; de Melo, Nathalya I; Aguiar, Daniela P; de Oliveira, Pollyanna F; Groppo, Milton; da Silva Filho, Ademar A; Rodrigues, Vanderlei; Cunha, Wilson R; Tavares, Denise C; Magalhães, Lizandra G; Crotti, Antônio E M

2015-07-01

Foeniculum vulgare Mill. (Apiaceae), known as fennel, is a widespread aromatic herbaceous plant, and its essential oil is used as additive in the food, pharmaceutical, cosmetic, and perfume industries. The in vitro antischistosomal activity and cytotoxic effects against V79 cells of the essential oil of F. vulgare cultivated in southeastern Brazil (FV-EO) was investigated. The FV-EO was obtained by hydrodistillation and characterized by GC-FID and GC/MS analyses. (E)-Anethole (69.8%) and limonene (22.5%) were identified as the major constituents. Its anthelmintic activity against Schistosoma mansoni was evaluated at concentrations of 10, 50, and 100 μg/ml, and it was found to be active against adult S. mansoni worms, although it was less effective than the positive control praziquantel (PZQ) in terms of separation of the coupled pairs, mortality, and decreased motor activity. However, FV-EO elicited an interesting dose-dependent reduction in the number of S. mansoni eggs. On their own, (E)-anethole and the limonene enantiomers were much less effective than FV-EO and PZQ. An XTT-cytotoxicity-based assay evidenced no FV-EO cytotoxicity against V79 cells. In summary, FV-EO displayed moderate in vitro schistosomicidal activity against adult S. mansoni worms, exerted remarkable inhibitory effects on the egg development, and was of low toxicity. Copyright © 2015 Verlag Helvetica Chimica Acta AG, Zürich.

PKC delta and NADPH oxidase in retinoic acid-induced neuroblastoma cell differentiation.

PubMed

Nitti, Mariapaola; Furfaro, Anna Lisa; Cevasco, Claudia; Traverso, Nicola; Marinari, Umberto Maria; Pronzato, Maria Adelaide; Domenicotti, Cinzia

2010-05-01

The role of reactive oxygen species (ROS) in the regulation of signal transduction processes has been well established in many cell types and recently the fine tuning of redox signalling in neurons received increasing attention. With regard to this, the involvement of NADPH oxidase (NOX) in neuronal pathophysiology has been proposed but deserves more investigation. In the present study, we used SH-SY5Y neuroblastoma cells to analyse the role of NADPH oxidase in retinoic acid (RA)-induced differentiation, pointing out the involvement of protein kinase C (PKC) delta in the activation of NOX. Retinoic acid induces neuronal differentiation as revealed by the increased expression of MAP2, the decreased cell doubling rate, and the gain in neuronal morphological features and these events are accompanied by the increased expression level of PKC delta and p67(phox), one of the components of NADPH oxidase. Using DPI to inhibit NOX activity we show that retinoic acid acts through this enzyme to induce morphological changes linked to the differentiation. Moreover, using rottlerin to inhibit PKC delta or transfection experiments to overexpress it, we show that retinoic acid acts through this enzyme to induce MAP2 expression and to increase p67(phox) membrane translocation leading to NADPH oxidase activation. These findings identify the activation of PKC delta and NADPH oxidase as crucial steps in RA-induced neuroblastoma cell differentiation. 2010 Elsevier Inc. All rights reserved.

NADPH oxidase activation contributes to native low-density lipoprotein-induced proliferation of human aortic smooth muscle cells.

PubMed

Park, Il Hwan; Hwang, Hye Mi; Jeon, Byeong Hwa; Kwon, Hyung-Joo; Hoe, Kwang Lae; Kim, Young Myeong; Ryoo, Sungwoo

2015-06-12

Elevated plasma concentration of native low-density lipoprotein (nLDL) is associated with vascular smooth muscle cell (VSMC) activation and cardiovascular disease. We investigated the mechanisms of superoxide generation and its contribution to pathophysiological cell proliferation in response to nLDL stimulation. Lucigenin-induced chemiluminescence was used to measure nLDL-induced superoxide production in human aortic smooth muscle cells (hAoSMCs). Superoxide production was increased by nicotinamide adenine dinucleotide phosphate (NADPH) and decreased by NADPH oxidase inhibitors in nLDL-stimulated hAoSMC and hAoSMC homogenates, as well as in prepared membrane fractions. Extracellular signal-regulated kinase 1/2 (Erk1/2), protein kinase C-θ (PKCθ) and protein kinase C-β (PKCβ) were phosphorylated and maximally activated within 3 min of nLDL stimulation. Phosphorylated Erk1/2 mitogen-activated protein kinase, PKCθ and PKCβ stimulated interactions between p47phox and p22phox; these interactions were prevented by MEK and PKC inhibitors (PD98059 and calphostin C, respectively). These inhibitors decreased nLDL-dependent superoxide production and blocked translocation of p47phox to the membrane, as shown by epifluorescence imaging and cellular fractionation experiments. Proliferation assays showed that a small interfering RNA against p47phox, as well as superoxide scavenger and NADPH oxidase inhibitors, blocked nLDL-induced hAoSMC proliferation. The nLDL stimulation in deendothelialized aortic rings from C57BL/6J mice increased dihydroethidine fluorescence and induced p47phox translocation that was blocked by PD98059 or calphostin C. Isolated aortic SMCs from p47phox(-/-) mice (mAoSMCs) did not respond to nLDL stimulation. Furthermore, NADPH oxidase 1 (Nox1) was responsible for superoxide generation and cell proliferation in nLDL-stimulated hAoSMCs. These data demonstrated that NADPH oxidase activation contributed to cell proliferation in nLDL-stimulated hAoSMCs.

NADPH oxidases in the arbuscular mycorrhizal symbiosis.

PubMed

Belmondo, Simone; Calcagno, Cristina; Genre, Andrea; Puppo, Alain; Pauly, Nicolas; Lanfranco, Luisa

2016-01-01

Plant NADPH oxidases are the major source of reactive oxygen species (ROS) that plays key roles as both signal and stressor in several plant processes, including defense responses against pathogens. ROS accumulation in root cells during arbuscular mycorrhiza (AM) development has raised the interest in understanding how ROS-mediated defense programs are modulated during the establishment of this mutualistic interaction. We have recently analyzed the expression pattern of 5 NADPH oxidase (also called RBOH) encoding genes in Medicago truncatula, showing that only one of them (MtRbohE) is specifically upregulated in arbuscule-containing cells. In line with this result, RNAi silencing of MtRbohE generated a strong alteration in root colonization, with a significant reduction in the number of arbusculated cells. On this basis, we propose that MtRBOHE-mediated ROS production plays a crucial role in the intracellular accommodation of arbuscules.

NADPH oxidases in the arbuscular mycorrhizal symbiosis

PubMed Central

Belmondo, Simone; Calcagno, Cristina; Genre, Andrea; Puppo, Alain; Pauly, Nicolas; Lanfranco, Luisa

2016-01-01

ABSTRACT Plant NADPH oxidases are the major source of reactive oxygen species (ROS) that plays key roles as both signal and stressor in several plant processes, including defense responses against pathogens. ROS accumulation in root cells during arbuscular mycorrhiza (AM) development has raised the interest in understanding how ROS-mediated defense programs are modulated during the establishment of this mutualistic interaction. We have recently analyzed the expression pattern of 5 NADPH oxidase (also called RBOH) encoding genes in Medicago truncatula, showing that only one of them (MtRbohE) is specifically upregulated in arbuscule-containing cells. In line with this result, RNAi silencing of MtRbohE generated a strong alteration in root colonization, with a significant reduction in the number of arbusculated cells. On this basis, we propose that MtRBOHE-mediated ROS production plays a crucial role in the intracellular accommodation of arbuscules. PMID:27018627

Inhibition of the NADPH oxidase regulates HO-1 expression in chronic myeloid leukemia

PubMed Central

Singh, Melissa M.; Irwin, Mary E.; Gao, Yin; Ban, Kechen; Shi, Ping; Arlinghaus, Ralph B.; Amin, Hesham M.; Chandra, Joya

2011-01-01

Background Patients with blast crisis phase chronic myelogeneous leukemia (CML) have poor response to tyrosine kinase inhibitors designed to inhibit the BCR-ABL1 oncogene. Recent work has shown that heme oxygenase 1 (HO-1) expression is increased in BCR-ABL1 expressing cells and that inhibition of HO-1 in CML leads to reduced cellular growth suggesting HO-1 may be a plausible target for therapy. Here we sought to clarify the mechanism of HO-1 overexpression and the role of the NADPH oxidase as a contributor to this mechanism in CML. Methods HO-1 expression was evaluated in CML bone marrow specimens from patients in various stages of disease, in a transplant based model for CML and in CML cell lines. Chemical and genetic inhibition of the NADPH oxidase was carried out in CML cells. Results Blast crisis CML patient specimens displayed higher levels of HO-1 staining than chronic or accelerated phase. HO-1 upregulation in BCR-ABL1 expressing cells was suppressed by diphenyliodonium (DPI), a chemical inhibitor of the NADPH oxidase. Targeting the NADPH oxidase through RNAi to Rac1, a dominant negative Rac1 construct or an inhibitor of Rac1 activity also blunted HO-1 protein expression. Moreover, inhibition of the NADPH oxidase by RNAi directed towards p47phox similarly abrogated HO-1 levels. Conclusion BCR-ABL1 expression upregulates HO-1, a survival factor for CML cells. This upregulation is more pronounced in blast crisis CML relative to early stage disease and is mediated by the NADPH oxidase components Rac1 and p47phox. Expression of p47phox is increased in BCR-ABL1 expressing cells. PMID:22139798

New Insights Into the Roles of NADPH Oxidases in Sexual Development and Ascospore Germination in Sordaria macrospora

PubMed Central

Dirschnabel, Daniela Elisabeth; Nowrousian, Minou; Cano-Domínguez, Nallely; Aguirre, Jesus; Teichert, Ines; Kück, Ulrich

2014-01-01

NADPH oxidase (NOX)-derived reactive oxygen species (ROS) act as signaling determinants that induce different cellular processes. To characterize NOX function during fungal development, we utilized the genetically tractable ascomycete Sordaria macrospora. Genome sequencing of a sterile mutant led us to identify the NADPH oxidase encoding nox1 as a gene required for fruiting body formation, regular hyphal growth, and hyphal fusion. These phenotypes are shared by ∆nor1, lacking the NOX regulator NOR1. Further phenotypic analyses revealed a high correlation between increased ROS production and hyphal fusion deficiencies in ∆nox1 and other sterile mutants. A genome-wide transcriptional profiling analysis of mycelia and isolated protoperithecia from wild type and ∆nox1 revealed that nox1 inactivation affects the expression of genes related to cytoskeleton remodeling, hyphal fusion, metabolism, and mitochondrial respiration. Genetic analysis of ∆nox2, lacking the NADPH oxidase 2 gene, ∆nor1, and transcription factor deletion mutant ∆ste12, revealed a strict melanin-dependent ascospore germination defect, indicating a common genetic pathway for these three genes. We report that gsa3, encoding a G-protein α-subunit, and sac1, encoding cAMP-generating adenylate cyclase, act in a separate pathway during the germination process. The finding that cAMP inhibits ascospore germination in a melanin-dependent manner supports a model in which cAMP inhibits NOX2 activity, thus suggesting a link between both pathways. Our results expand the current knowledge on the role of NOX enzymes in fungal development and provide a frame to define upstream and downstream components of the NOX signaling pathways in fungi. PMID:24407906

NADPH Oxidase Inhibition Improves Neurological Outcomes in Surgically-Induced Brain Injury

PubMed Central

Lo, Wendy; Bravo, Thomas; Jadhav, Vikram; Zhang, John H.; Tang, Jiping

2007-01-01

Neurosurgical procedures can result in brain injury by various means including direct trauma, hemorrhage, retractor stretch, and electrocautery. This surgically-induced brain injury (SBI) can cause post-operative complications such as brain edema. By creating a mouse model of SBI, we tested whether NADPH oxidase, an important reactive oxygen species producing enzyme, is involved in SBI using transgenic mice lacking gp91phox subunit of NADPH oxidase (gp91phox KO) and apocynin, a specific inhibitor of NADPH oxidase. Neurological function and brain edema were evaluated at 24 hours post-SBI in gp91phox KO and wild-type littermates grouped into SBI and sham-surgery groups. Alternatively, mice were grouped into vehicle- and apocynin-treated (5mg/kg, i.p. 30 minutes before SBI) groups. Oxidative stress indicated by lipid peroxidation (LPO) was measured at 3 and 24 hours post SBI. The gp91phox KO mice, but not the apocynin-treated mice showed significantly improved neurological scores. Brain edema was observed in both gp91phox KO and wild-type groups after SBI; however, there was no significant difference between these two groups. Brain edema was also not affected by apocynin-pretreatment. LPO levels were significantly higher in SBI group in both gp91phox KO and wild-type groups as compared to sham group. A trend, although without statistical significance, was noted towards attenuation of LPO in the gp91phox KO animals as compared to wild-type group. LPO levels were significantly attenuated at 3 hours post-SBI by apocynin pretreatment but not at 24 hours post-SBI. These results suggest that chronic and acute inhibition of NADPH oxidase activity does not reduce brain edema after SBI. Long-term inhibition of NADPH oxidase, however improves neurological functions after SBI. PMID:17317004

Activation of neuronal nitric oxide synthase in cerebellum of chronic hepatic encephalopathy rats is associated with up-regulation of NADPH-producing pathway.

PubMed

Singh, Santosh; Trigun, Surendra K

2010-09-01

Cerebellum-associated functions get affected during mild hepatic encephalopathy (MHE) in patients with chronic liver failure (CLF). Involvement of nitrosative and antioxidant factors in the pathogenesis of chronic hepatic encephalopathy is an evolving concept and needs to be defined in a true CLF animal model. This article describes profiles of NADPH-dependent neuronal nitric oxide synthase (nNOS) and those of glutathione peroxidase and glutathione reductase (GR) vis-a-vis regulation of NADPH-producing pathway in the cerebellum of CLF rats induced by administration of thioacetamide (100 mg kg⁻¹ b.w., i.p.) up to 10 days and confirming MHE on Morris water maze tests. Significant increases in the expression of nNOS protein and nitric oxide (NOx) level coincided with a similar increment in NADPH-diaphorase activity in the cerebellum of CLF rats. Glutathione peroxidase and GR utilize NADPH to regenerate reduced glutathione (GSH) in the cells. Both these enzymes and GSH level were found to be static and thus suggested efficient turnover of GSH in the cerebellum of MHE rats. Relative levels of glucose-6-phosphate dehydrogenase (G6PD) vs. phosphofructokinase 2 (PFK2) determine the rate of pentose phosphate pathway (PPP) responsible to synthesize NADPH. The cerebellum of CLF rats showed overactivation of G6PD with a significant decline in the expression of PFK2 and thus suggested activation of PPP in the cerebellum during MHE. It is concluded that concordant activations of PPP and nNOS in cerebellum of MHE rats could be associated with the implication of NOx in the pathogenesis of MHE.

NADPH oxidases as novel pharmacologic targets against influenza A virus infection.

PubMed

Vlahos, Ross; Selemidis, Stavros

2014-12-01

Influenza A viruses represent a major global health care challenge, with imminent pandemics, emerging antiviral resistance, and long lag times for vaccine development, raising a pressing need for novel pharmacologic strategies that ideally target the pathology irrespective of the infecting strain. Reactive oxygen species (ROS) pervade all facets of cell biology with both detrimental and protective properties. Indeed, there is compelling evidence that activation of the NADPH oxidase 2 (NOX2) isoform of the NADPH oxidase family of ROS-producing enzymes promotes lung oxidative stress, inflammation, injury, and dysfunction resulting from influenza A viruses of low to high pathogenicity, as well as impeding virus clearance. By contrast, the dual oxidase isoforms produce ROS that provide vital protective antiviral effects for the host. In this review, we propose that inhibitors of NOX2 are better alternatives than broad-spectrum antioxidant approaches for treatment of influenza pathologies, for which clinical efficacy may have been limited owing to poor bioavailability and inadvertent removal of beneficial ROS. Finally, we briefly describe the current suite of NADPH oxidase inhibitors and the molecular features of the NADPH oxidase enzymes that could be exploited by drug discovery for development of more specific and novel inhibitors to prevent or treat disease caused by influenza. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

Resveratrol protects vascular endothelial cells from high glucose-induced apoptosis through inhibition of NADPH oxidase activation-driven oxidative stress.

PubMed

Chen, Feng; Qian, Li-Hua; Deng, Bo; Liu, Zhi-Min; Zhao, Ying; Le, Ying-Ying

2013-09-01

Hyperglycemia-induced oxidative stress has been implicated in diabetic vascular complications in which NADPH oxidase is a major source of reactive oxygen species (ROS) generation. Resveratrol is a naturally occurring polyphenol, which has vasoprotective effects in diabetic animal models and inhibits high glucose (HG)-induced oxidative stress in endothelial cells. We aimed to examine whether HG-induced NADPH oxidase activation and ROS production contribute to glucotoxicity to endothelial cells and the effect of resveratrol on glucotoxicity. Using a murine brain microvascular endothelial cell line bEnd3, we found that NADPH oxidase inhibitor (apocynin) and resveratrol both inhibited HG-induced endothelial cell apoptosis. HG-induced elevation of NADPH oxidase activity and production of ROS were inhibited by apocynin, suggesting that HG induces endothelial cell apoptosis through NADPH oxidase-mediated ROS production. Mechanistic studies revealed that HG upregulated NADPH oxidase subunit Nox1 but not Nox2, Nox4, and p22(phox) expression through NF-κB activation, which resulted in elevation of NADPH oxidase activity and consequent ROS production. Resveratrol prevented HG-induced endothelial cell apoptosis through inhibiting HG-induced NF-κB activation, NADPH oxidase activity elevation, and ROS production. HG induces endothelial cell apoptosis through NF-κB/NADPH oxidase/ROS pathway, which was inhibited by resveratrol. Our findings provide new potential therapeutic targets against brain vascular complications of diabetes. © 2013 John Wiley & Sons Ltd.

Identification of a Phytase Gene in Barley (Hordeum vulgare L.)

PubMed Central

Dai, Fei; Qiu, Long; Ye, Lingzhen; Wu, Dezhi; Zhou, Meixue; Zhang, Guoping

2011-01-01

Background Endogenous phytase plays a crucial role in phytate degradation and is thus closely related to nutrient efficiency in barley products. The understanding of genetic information of phytase in barley can provide a useful tool for breeding new barley varieties with high phytase activity. Methodology/Principal Findings Quantitative trait loci (QTL) analysis for phytase activity was conducted using a doubled haploid population. Phytase protein was purified and identified by the LC-ESI MS/MS Shotgun method. Purple acid phosphatase (PAP) gene was sequenced and the position was compared with the QTL controlling phytase activity. A major QTL for phytase activity was mapped to chromosome 5 H in barley. The gene controlling phytase activity in the region was named as mqPhy. The gene HvPAP a was mapped to the same position as mqPhy, supporting the colinearity between HvPAP a and mqPhy. Conclusions/Significance It is the first report on QTLs for phytase activity and the results showed that HvPAP a, which shares a same position with the QTL, is a major phytase gene in barley grains. PMID:21533044

New greenbug resistant sources in winter barley, Hordeum vulgare (L.)

USDA-ARS?s Scientific Manuscript database

Greenbug, Schizaphis graminum (Rondonai), is a chronic problem for small grains in the Southern Plains causing significant economic losses in outbreak years. Central to the pest status of greenbug is the occurrence of resistance-breaking biotypes. Rsg1 and Rsg2 are the only 2 genes for greenbug re...

Adverse effects of the classic antioxidant uric acid in adipocytes: NADPH oxidase-mediated oxidative/nitrosative stress.

PubMed

Sautin, Yuri Y; Nakagawa, Takahiko; Zharikov, Sergey; Johnson, Richard J

2007-08-01

Uric acid is considered a major antioxidant in human blood that may protect against aging and oxidative stress. Despite its proposed protective properties, elevated levels of uric acid are commonly associated with increased risk for cardiovascular disease and mortality. Furthermore, recent experimental studies suggest that uric acid may have a causal role in hypertension and metabolic syndrome. All these conditions are thought to be mediated by oxidative stress. In this study we demonstrate that differentiation of cultured mouse adipocytes is associated with increased production of reactive oxygen species (ROS) and uptake of uric acid. Soluble uric acid stimulated an increase in NADPH oxidase activity and ROS production in mature adipocytes but not in preadipocytes. The stimulation of NADPH oxidase-dependent ROS by uric acid resulted in activation of MAP kinases p38 and ERK1/2, a decrease in nitric oxide bioavailability, and an increase in protein nitrosylation and lipid oxidation. Collectively, our results suggest that hyperuricemia induces redox-dependent signaling and oxidative stress in adipocytes. Since oxidative stress in the adipose tissue has recently been recognized as a major cause of insulin resistance and cardiovascular disease, hyperuricemia-induced alterations in oxidative homeostasis in the adipose tissue might play an important role in these derangements.

Regulation of superoxide anion production by NADPH oxidase in monocytes/macrophages: contributions to atherosclerosis.

PubMed

Cathcart, Martha K

2004-01-01

Monocyte extravasation into the vessel wall has been shown to be a critical step in the development of atherosclerosis. Upon activation, monocytes produce a burst of superoxide anion due to activation of the NADPH oxidase enzyme complex. Monocyte-derived superoxide anion contributes to oxidant stress in inflammatory sites, is required for monocyte-mediated LDL oxidation, and alters basic cell functions such as adhesion and proliferation. We hypothesize that monocyte-derived superoxide anion production contributes to atherosclerotic lesion formation. In this brief review, we summarize our current understanding of the signal transduction pathways regulating NADPH oxidase activation and related superoxide anion production in activated human monocytes. Novel pathways are identified that may serve as future targets for therapeutic intervention in this pathogenic process. The contributions of superoxide anion and NADPH oxidase to atherogenesis are discussed. Future experiments are needed to clarify the exact role of NADPH oxidase-derived superoxide anion in atherogenesis, particularly that derived from monocytes.

A Novel F420-dependent Thioredoxin Reductase Gated by Low Potential FAD

PubMed Central

Susanti, Dwi; Loganathan, Usha; Mukhopadhyay, Biswarup

2016-01-01

A recent report suggested that the thioredoxin-dependent metabolic regulation, which is widespread in all domains of life, existed in methanogenic archaea about 3.5 billion years ago. We now show that the respective electron delivery enzyme (thioredoxin reductase, TrxR), although structurally similar to flavin-containing NADPH-dependent TrxRs (NTR), lacked an NADPH-binding site and was dependent on reduced coenzyme F420 (F420H2), a stronger reductant with a mid-point redox potential (E′0) of −360 mV; E′0 of NAD(P)H is −320 mV. Because F420 is a deazaflavin, this enzyme was named deazaflavin-dependent flavin-containing thioredoxin reductase (DFTR). It transferred electrons from F420H2 to thioredoxin via protein-bound flavin; Km values for thioredoxin and F420H2 were 6.3 and 28.6 μm, respectively. The E′0 of DFTR-bound flavin was approximately −389 mV, making electron transfer from NAD(P)H or F420H2 to flavin endergonic. However, under high partial pressures of hydrogen prevailing on early Earth and present day deep-sea volcanoes, the potential for the F420/F420H2 pair could be as low as −425 mV, making DFTR efficient. The presence of DFTR exclusively in ancient methanogens and mostly in the early Earth environment of deep-sea volcanoes and DFTR's characteristics suggest that the enzyme developed on early Earth and gave rise to NTR. A phylogenetic analysis revealed six more novel-type TrxR groups and suggested that the broader flavin-containing disulfide oxidoreductase family is more diverse than previously considered. The unprecedented structural similarities between an F420-dependent enzyme (DFTR) and an NADPH-dependent enzyme (NTR) brought new thoughts to investigations on F420 systems involved in microbial pathogenesis and antibiotic production. PMID:27590343

Restructuring of the dinucleotide-binding fold in an NADP(H) sensor protein

PubMed Central

Zheng, Xiaofeng; Dai, Xueyu; Zhao, Yanmei; Chen, Qiang; Lu, Fei; Yao, Deqiang; Yu, Quan; Liu, Xinping; Zhang, Chuanmao; Gu, Xiaocheng; Luo, Ming

2007-01-01

NAD(P) has long been known as an essential energy-carrying molecule in cells. Recent data, however, indicate that NAD(P) also plays critical signaling roles in regulating cellular functions. The crystal structure of a human protein, HSCARG, with functions previously unknown, has been determined to 2.4-Å resolution. The structure reveals that HSCARG can form an asymmetrical dimer with one subunit occupied by one NADP molecule and the other empty. Restructuring of its NAD(P)-binding Rossmann fold upon NADP binding changes an extended loop to an α-helix to restore the integrity of the Rossmann fold. The previously unobserved restructuring suggests that HSCARG may assume a resting state when the level of NADP(H) is normal within the cell. When the NADP(H) level passes a threshold, an extensive restructuring of HSCARG would result in the activation of its regulatory functions. Immunofluorescent imaging shows that HSCARG redistributes from being associated with intermediate filaments in the resting state to being dispersed in the nucleus and the cytoplasm. The structural change of HSCARG upon NADP(H) binding could be a new regulatory mechanism that responds only to a significant change of NADP(H) levels. One of the functions regulated by HSCARG may be argininosuccinate synthetase that is involved in NO synthesis. PMID:17496144

Peroxiredoxins and NADPH-Dependent Thioredoxin Systems in the Model Legume Lotus japonicus1[W][OA

PubMed Central

Tovar-Méndez, Alejandro; Matamoros, Manuel A.; Bustos-Sanmamed, Pilar; Dietz, Karl-Josef; Cejudo, Francisco Javier; Rouhier, Nicolas; Sato, Shusei; Tabata, Satoshi; Becana, Manuel

2011-01-01

Peroxiredoxins (Prxs), thioredoxins (Trxs), and NADPH-thioredoxin reductases (NTRs) constitute central elements of the thiol-disulfide redox regulatory network of plant cells. This study provides a comprehensive survey of this network in the model legume Lotus japonicus. The aims were to identify and characterize these gene families and to assess whether the NTR-Trx systems are operative in nodules. Quantitative reverse transcription-polymerase chain reaction and immunological and proteomic approaches were used for expression profiling. We identified seven Prx, 14 Trx, and three NTR functional genes. The PrxQ1 gene was found to be transcribed in two alternative spliced variants and to be expressed at high levels in leaves, stems, petals, pods, and seeds and at low levels in roots and nodules. The 1CPrx gene showed very high expression in the seed embryos and low expression in vegetative tissues and was induced by nitric oxide and cytokinins. In sharp contrast, cytokinins down-regulated all other Prx genes, except PrxQ1, in roots and nodules, but only 2CPrxA and PrxQ1 in leaves. Gene-specific changes in Prx expression were also observed in response to ethylene, abscisic acid, and auxins. Nodules contain significant mRNA and protein amounts of cytosolic PrxIIB, Trxh1, and NTRA and of plastidic NTRC. Likewise, they express cytosolic Trxh3, Trxh4, Trxh8, and Trxh9, mitochondrial PrxIIF and Trxo, and plastidic Trxm2, Trxm4, and ferredoxin-Trx reductase. These findings reveal a complex regulation of Prxs that is dependent on the isoform, tissue, and signaling molecule and support that redox NTR-Trx systems are functional in the cytosol, mitochondria, and plastids of nodules. PMID:21562331

The Intimate and Controversial Relationship between Voltage Gated Proton Channels and the Phagocyte NADPH Oxidase

PubMed Central

DeCoursey, Thomas E.

2016-01-01

Summary One of the most fascinating and exciting periods in my scientific career entailed dissecting the symbiotic relationship between two membrane transporters, the NADPH oxidase complex and voltage gated proton channels (HV1). By the time I entered this field, there had already been substantial progress toward understanding NADPH oxidase, but HV1 were known only to a tiny handful of cognoscenti around the world. Having identified the first proton currents in mammalian cells in 1991, I needed to find a clear function for these molecules if the work was to become fundable. The then-recent discoveries of Henderson, Chappell, and colleagues in 1987–1988 that led them to hypothesize interactions of both molecules during the respiratory burst of phagocytes provided an excellent opportunity. In a nutshell, both transporters function by moving electrical charge across the membrane: NADPH oxidase moves electrons and HV1 moves protons. The consequences of electrogenic NADPH oxidase activity on both membrane potential and pH strongly self-limit this enzyme. Fortunately, both consequences specifically activate HV1, and HV1 activity counteracts both consequences, a kind of yin-yang relationship. Notwithstanding a decade starting in 1995 when many believed the opposite, these are two separate molecules that function independently despite their being functionally interdependent in phagocytes. The relationship between NADPH oxidase and HV1 has become a paradigm that somewhat surprisingly has now extended well beyond the phagocyte NADPH oxidase -- an industrial strength producer of reactive oxygen species (ROS) -- to myriad other cells that produce orders of magnitude less ROS for signaling purposes. These cells with their seven NADPH oxidase (NOX) isoforms provide a vast realm of mechanistic obscurity that will occupy future studies for years to come. PMID:27558336

The intimate and controversial relationship between voltage-gated proton channels and the phagocyte NADPH oxidase.

PubMed

DeCoursey, Thomas E

2016-09-01

One of the most fascinating and exciting periods in my scientific career entailed dissecting the symbiotic relationship between two membrane transporters, the Nicotinamide adenine dinucleotide phosphate reduced form (NADPH) oxidase complex and voltage-gated proton channels (HV 1). By the time I entered this field, there had already been substantial progress toward understanding NADPH oxidase, but HV 1 were known only to a tiny handful of cognoscenti around the world. Having identified the first proton currents in mammalian cells in 1991, I needed to find a clear function for these molecules if the work was to become fundable. The then-recent discoveries of Henderson, Chappell, and colleagues in 1987-1988 that led them to hypothesize interactions of both molecules during the respiratory burst of phagocytes provided an excellent opportunity. In a nutshell, both transporters function by moving electrical charge across the membrane: NADPH oxidase moves electrons and HV 1 moves protons. The consequences of electrogenic NADPH oxidase activity on both membrane potential and pH strongly self-limit this enzyme. Fortunately, both consequences specifically activate HV 1, and HV 1 activity counteracts both consequences, a kind of yin-yang relationship. Notwithstanding a decade starting in 1995 when many believed the opposite, these are two separate molecules that function independently despite their being functionally interdependent in phagocytes. The relationship between NADPH oxidase and HV 1 has become a paradigm that somewhat surprisingly has now extended well beyond the phagocyte NADPH oxidase - an industrial strength producer of reactive oxygen species (ROS) - to myriad other cells that produce orders of magnitude less ROS for signaling purposes. These cells with their seven NADPH oxidase (NOX) isoforms provide a vast realm of mechanistic obscurity that will occupy future studies for years to come. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Triticum vulgare extract exerts an anti-inflammatory action in two in vitro models of inflammation in microglial cells

PubMed Central

Sanguigno, Luca; Casamassa, Antonella; Funel, Niccola; Minale, Massimiliano; Riccio, Rodolfo; Riccio, Salvatore; Boscia, Francesca; Brancaccio, Paola; Pollina, Luca Emanuele; Anzilotti, Serenella; Di Renzo, Gianfranco

2018-01-01

Triticum vulgare has been extensively used in traditional medicine thanks to its properties of accelerating tissue repair. The specific extract of Triticum vulgare manufactured by Farmaceutici Damor (TVE-DAMOR) is already present in some pharmaceutical formulations used in the treatment of decubitus ulcers, skin lesions and burns. It has been recently suggested that this Triticum vulgare extract may possess potential anti-inflammatory properties. In the light of these premises the aim of the present paper was to verify the anti-inflammatory role of TVE, using the LPS-stimulated microglia model of inflammation. In particular the effect of different concentrations of TVE on the release of several mediators of inflammation such as nitric oxide, IL-6, PGE2 and TNF alpha was evaluated. More important, the anti-inflammatory effect of TVE was confirmed also in primary rat microglia cultures. The results of the present study show that TVE exerts anti-inflammatory properties since it reduces the release of all the evaluated markers of inflammation, such as NO, IL6, TNF alpha and PGE2 in LPS-activated BV2 microglial cells. Intriguingly, TVE reduced microglia activation and NO release also in primary microglia. Indeed, to verify the pathway of modulation of the inflammatory markers reported above, we found that TVE restores the cytoplasmic expression of p65 protein, kwown as specific marker associated with activation of inflammatory response. The evidence for an inhibitory activity on inflammation of this specific extract of Triticum vulgare may open the way to the possibility of a therapeutical use of the Triticum vulgare extract as an anti-inflammatory compound in certain pathological states such as burns, decubitus ulcers, folliculitis and inflammation of peripheral nerve. PMID:29902182

Toxicity assessment of untreated/treated electroplating sludge using human and plant bioassay.

PubMed

Orescanin, Visnja; Durgo, Ksenija; Mikelic, Ivanka Lovrencic; Halkijevic, Ivan; Kuspilic, Marin

2018-04-30

The purpose of this work was to assess the risk to the environment arising from the electroplating sludge from both chemical and toxicological point of view. Both approaches were used for the assessment of the treatment efficiency which consisted of CaO based solidification followed by thermal treatment at 400°C. The elemental composition was determined in the bulk samples and the leachates of untreated sludge. The toxicity of the leachate was determined using two human colorectal adenocarcinoma cell lines (Caco-2 and SW 480) and Hordeum vulgare L. based plant bioassay. The same toxicity tests were employed to the leachate of the treated sludge. Untreated sludge showed extremely high cytotoxic effect to both human and plant bio-system in dose-dependent manner. The percentages higher than 0.5% and 0.05% of the leachate caused significant cytotoxic effect on Caco-2 and SW 480 cells, respectively. The percentages of the leachate higher than 0.05% also showed significant toxic effect to H. vulgare L. bio-system with complete arrest of seed germination following the treatment with 100% to 5% of the leachate. The leachate of the treated sludge showed no toxicity to any of the test systems confirming the efficiency and justification of the employed procedures for the detoxification of electroplating sludge.

Crystal Structure of Perakine Reductase, Founding Member of a Novel Aldo-Keto Reductase (AKR) Subfamily That Undergoes Unique Conformational Changes during NADPH Binding*

PubMed Central

Sun, Lianli; Chen, Yixin; Rajendran, Chitra; Mueller, Uwe; Panjikar, Santosh; Wang, Meitian; Mindnich, Rebekka; Rosenthal, Cindy; Penning, Trevor M.; Stöckigt, Joachim

2012-01-01

Perakine reductase (PR) catalyzes the NADPH-dependent reduction of the aldehyde perakine to yield the alcohol raucaffrinoline in the biosynthetic pathway of ajmaline in Rauvolfia, a key step in indole alkaloid biosynthesis. Sequence alignment shows that PR is the founder of the new AKR13D subfamily and is designated AKR13D1. The x-ray structure of methylated His6-PR was solved to 2.31 Å. However, the active site of PR was blocked by the connected parts of the neighbor symmetric molecule in the crystal. To break the interactions and obtain the enzyme-ligand complexes, the A213W mutant was generated. The atomic structure of His6-PR-A213W complex with NADPH was determined at 1.77 Å. Overall, PR folds in an unusual α8/β6 barrel that has not been observed in any other AKR protein to date. NADPH binds in an extended pocket, but the nicotinamide riboside moiety is disordered. Upon NADPH binding, dramatic conformational changes and movements were observed: two additional β-strands in the C terminus become ordered to form one α-helix, and a movement of up to 24 Å occurs. This conformational change creates a large space that allows the binding of substrates of variable size for PR and enhances the enzyme activity; as a result cooperative kinetics are observed as NADPH is varied. As the founding member of the new AKR13D subfamily, PR also provides a structural template and model of cofactor binding for the AKR13 family. PMID:22334702

The transcriptional regulator NtrC controls glucose-6-phosphate dehydrogenase expression and polyhydroxybutyrate synthesis through NADPH availability in Herbaspirillum seropedicae.

PubMed

Sacomboio, Euclides Nenga Manuel; Kim, Edson Yu Sin; Correa, Henrique Leonardo Ruchaud; Bonato, Paloma; Pedrosa, Fabio de Oliveira; de Souza, Emanuel Maltempi; Chubatsu, Leda Satie; Müller-Santos, Marcelo

2017-10-19

The NTR system is the major regulator of nitrogen metabolism in Bacteria. Despite its broad and well-known role in the assimilation, biosynthesis and recycling of nitrogenous molecules, little is known about its role in carbon metabolism. In this work, we present a new facet of the NTR system in the control of NADPH concentration and the biosynthesis of molecules dependent on reduced coenzyme in Herbaspirillum seropedicae SmR1. We demonstrated that a ntrC mutant strain accumulated high levels of polyhydroxybutyrate (PHB), reaching levels up to 2-fold higher than the parental strain. In the absence of NtrC, the activity of glucose-6-phosphate dehydrogenase (encoded by zwf) increased by 2.8-fold, consequently leading to a 2.1-fold increase in the NADPH/NADP + ratio. A GFP fusion showed that expression of zwf is likewise controlled by NtrC. The increase in NADPH availability stimulated the production of polyhydroxybutyrate regardless the C/N ratio in the medium. The mutant ntrC was more resistant to H 2 O 2 exposure and controlled the propagation of ROS when facing the oxidative condition, a phenotype associated with the increase in PHB content.

Analysis of the Light-harvesting Pigment-Protein Complex of Wild Type and a Chlorophyll-b-less Mutant of Barley 1

PubMed Central

Burke, John J.; Steinback, Katherine E.; Arntzen, Charles J.

1979-01-01

we have compared chloroplast lamellae isolated from a chlorophyll-b-less mutant and wild type barley (Hordeum vulgare). The results demonstrate that: (a) one of the two major polypeptides comprising the lightharvesting complex (LHC) is present in the chlorophyll-b-less mutant; (b) higher cation concentrations are required to maintain grana stacks in the mutant; and (c) cation effects on excitation energy distribution are present in the chlorophyll-b-less mutant but are reduced in amount and are dependent on higher concentrations of cations. We interpret these data to support the concept that the LHC mediates cation-induced grana stacking and cation regulation of excitation energy distribution between photosystems I and Ii in chloroplast lamellae. A partial LHC complement in the mutant alters the quantitative cation requirement for both phenomena, but not the over-all qualitative response. Images PMID:16660704

High Resolution Analysis of Meiotic Chromosome Structure and Behaviour in Barley (Hordeum vulgare L.)

PubMed Central

Phillips, Dylan; Nibau, Candida; Wnetrzak, Joanna; Jenkins, Glyn

2012-01-01

Reciprocal crossing over and independent assortment of chromosomes during meiosis generate most of the genetic variation in sexually reproducing organisms. In barley, crossovers are confined primarily to distal regions of the chromosomes, which means that a substantial proportion of the genes of this crop rarely, if ever, engage in recombination events. There is potentially much to be gained by redistributing crossovers to more proximal regions, but our ability to achieve this is dependent upon a far better understanding of meiosis in this species. This study explores the meiotic process by describing with unprecedented resolution the early behaviour of chromosomal domains, the progression of synapsis and the structure of the synaptonemal complex (SC). Using a combination of molecular cytogenetics and advanced fluorescence imaging, we show for the first time in this species that non-homologous centromeres are coupled prior to synapsis. We demonstrate that at early meiotic prophase the loading of the SC-associated structural protein ASY1, the cluster of telomeres, and distal synaptic initiation sites occupy the same polarised region of the nucleus. Through the use of advanced 3D image analysis, we show that synapsis is driven predominantly from the telomeres, and that new synaptic initiation sites arise during zygotene. In addition, we identified two different SC configurations through the use of super-resolution 3D structured illumination microscopy (3D-SIM). PMID:22761818

Molecular mechanisms of hypertension: role of Nox family NADPH oxidases.

PubMed

Sedeek, Mona; Hébert, Richard L; Kennedy, Chris R; Burns, Kevin D; Touyz, Rhian M

2009-03-01

Molecular mechanisms contributing to the pathoetiology of hypertension are complex, involving many interacting systems such as signaling through G protein-coupled receptors, the renin-angiotensin system, vascular inflammation and remodeling, vascular senescence and aging and developmental programming, as highlighted in the current issue of the journal. Common to these systems is NADPH oxidase-derived reactive oxygen species (ROS). This editorial highlights current concepts relating to the production of ROS in hypertension and focuses on the Nox family NADPH oxidases, major sources of free radicals in the cardiovascular and renal systems. ROS play a major role as intracellular signaling molecules to regulate normal biological cellular responses. In pathological conditions, loss of redox homeostasis contributes to vascular oxidative damage. Recent evidence indicates that specific enzymes, the Nox family of NADPH oxidases, have the sole function of generating ROS in a highly regulated fashion in physiological conditions, and that in disease states, hyperactivation of Noxes contributes to oxidative stress and consequent cardiovascular and renal injury. The Nox family comprises seven members, Nox1-Nox7. Nox1, Nox2 (gp91phox-containing NADPH oxidase), Nox4 and Nox5 have been identified in the cardiovascular-renal systems and have been implicated in the pathophysiology of cardiovascular and renal disease. Noxes, which are differentially regulated in hypertension, are major sources of cardiovascular and renal oxidative stress. This has evoked considerable interest because of the possibilities that therapies targeted against specific Nox isoforms to decrease ROS generation or to increase nitric oxide availability or both may be useful in minimizing vascular injury and renal dysfunction, and thereby prevent or regress target organ damage associated with hypertension.

Foeniculum vulgare essential oils: chemical composition, antioxidant and antimicrobial activities.

PubMed

Miguel, Maria Graça; Cruz, Cláudia; Faleiro, Leonor; Simões, Mariana T F; Figueiredo, Ana Cristina; Barroso, José G; Pedro, Luis G

2010-02-01

The essential oils from Foeniculum vulgare commercial aerial parts and fruits were isolated by hydrodistillation, with different distillation times (30 min, 1 h, 2 h and 3 h), and analyzed by GC and GC-MS. The antioxidant ability was estimated using four distinct methods. Antibacterial activity was determined by the agar diffusion method. Remarkable differences, and worrying from the quality and safety point of view, were detected in the essential oils. trans-Anethole (31-36%), alpha-pinene (14-20%) and limonene (11-13%) were the main components of the essentials oil isolated from F. vulgare dried aerial parts, whereas methyl chavicol (= estragole) (79-88%) was dominant in the fruit oils. With the DPPH method the plant oils showed better antioxidant activity than the fruits oils. With the TBARS method and at higher concentrations, fennel essential oils showed a pro-oxidant activity. None of the oils showed a hydroxyl radical scavenging capacity > 50%, but they showed an ability to inhibit 5-lipoxygenase. The essential oils showed a very low antimicrobial activity. In general, the essential oils isolated during 2 h were as effective, from the biological activity point of view, as those isolated during 3 h.

Glucose regulates enzymatic sources of mitochondrial NADPH in skeletal muscle cells; a novel role for glucose-6-phosphate dehydrogenase.

PubMed

Mailloux, Ryan J; Harper, Mary-Ellen

2010-07-01

Reduced nicotinamide adenine dinucleotide (NADPH) is a functionally important metabolite required to support numerous cellular processes. However, despite the identification of numerous NADPH-producing enzymes, the mechanisms underlying how the organellar pools of NADPH are maintained remain elusive. Here, we have identified glucose-6-phosphate dehydrogenase (G6PDH) as an important source of NADPH in mitochondria. Activity analysis, submitochondrial fractionation, fluorescence microscopy, and protease sensitivity assays revealed that G6PDH is localized to the mitochondrial matrix. 6-ANAM, a specific G6PDH inhibitor, depleted mitochondrial NADPH pools and increased oxidative stress revealing the importance of G6PDH in NADPH maintenance. We also show that glucose availability and differences in metabolic state modulate the enzymatic sources of NADPH in mitochondria. Indeed, cells cultured in high glucose (HG) not only adopted a glycolytic phenotype but also relied heavily on matrix-associated G6PDH as a source of NADPH. In contrast, cells exposed to low-glucose (LG) concentrations, which displayed increased oxygen consumption, mitochondrial metabolic efficiency, and decreased glycolysis, relied predominantly on isocitrate dehydrogenase (ICDH) as the principal NADPH-producing enzyme in the mitochondria. Culturing glycolytic cells in LG for 48 h decreased G6PDH and increased ICDH protein levels in the mitochondria, further pointing to the regulatory role of glucose. 2-Deoxyglucose treatment also prevented the increase of mitochondrial G6PDH in response to HG. The role of glucose in regulating enzymatic sources of mitochondrial NADPH pool maintenance was confirmed using human myotubes from obese adults with a history of type 2 diabetes mellitus (post-T2DM). Myotubes from post-T2DM participants failed to increase mitochondrial G6PDH in response to HG in contrast to mitochondria in myotubes from control participants (non-T2DM). Hence, we not only identified a matrix

Direct Activation of NADPH Oxidase 2 by 2-Deoxyribose-1-Phosphate Triggers Nuclear Factor Kappa B-Dependent Angiogenesis

PubMed Central

Vara, Dina; Watt, Joanna M.; Fortunato, Tiago M.; Mellor, Harry; Burgess, Matthew; Wicks, Kate; Mace, Kimberly; Reeksting, Shaun; Lubben, Anneke; Wheeler-Jones, Caroline P.D.

2018-01-01

Abstract Aims: Deoxyribose-1-phosphate (dRP) is a proangiogenic paracrine stimulus released by cancer cells, platelets, and macrophages and acting on endothelial cells. The objective of this study was to clarify how dRP stimulates angiogenic responses in human endothelial cells. Results: Live cell imaging, electron paramagnetic resonance, pull-down of dRP-interacting proteins, followed by immunoblotting, gene silencing of different NADPH oxidases (NOXs), and their regulatory cosubunits by small interfering RNA (siRNA) transfection, and experiments with inhibitors of the sugar transporter glucose transporter 1 (GLUT1) were utilized to demonstrate that dRP acts intracellularly by directly activating the endothelial NOX2 complex, but not NOX4. Increased reactive oxygen species generation in response to NOX2 activity leads to redox-dependent activation of the transcription factor nuclear factor kappa B (NF-κB), which, in turn, induces vascular endothelial growth factor receptor 2 (VEGFR2) upregulation. Using endothelial tube formation assays, gene silencing by siRNA, and antibody-based receptor inhibition, we demonstrate that the activation of NF-κB and VEGFR2 is necessary for the angiogenic responses elicited by dRP. The upregulation of VEGFR2 and NOX2-dependent stimulation of angiogenesis by dRP were confirmed in excisional wound and Matrigel plug vascularization assays in vivo using NOX2−/− mice. Innovation: For the first time, we demonstrate that dRP acts intracellularly and stimulates superoxide anion generation by direct binding and activation of the NOX2 enzymatic complex. Conclusions: This study describes a novel molecular mechanism underlying the proangiogenic activity of dRP, which involves the sequential activation of NOX2 and NF-κB and upregulation of VEGFR2. Antioxid. Redox Signal. 28, 110–130. PMID:28793782

In vitro activity of origanum vulgare essential oil against candida species

PubMed Central

Cleff, Marlete Brum; Meinerz, Ana Raquel; Xavier, Melissa; Schuch, Luiz Filipe; Schuch, Luiz Filipe; Araújo Meireles, Mário Carlos; Alves Rodrigues, Maria Regina; de Mello, João Roberto Braga

2010-01-01

The aim of this study was to evaluate the in vitro activity of the essential oil extracted from Origanum vulgare against sixteen Candida species isolates. Standard strains tested comprised C. albicans (ATCC strains 44858, 4053, 18804 and 3691), C. parapsilosis (ATCC 22019), C. krusei (ATCC 34135), C. lusitaniae (ATCC 34449) and C. dubliniensis (ATCC MY646). Six Candida albicans isolates from the vaginal mucous membrane of female dogs, one isolate from the cutaneous tegument of a dog and one isolate of a capuchin monkey were tested in parallel. A broth microdilution technique (CLSI) was used, and the inoculum concentration was adjusted to 5 x 106 CFU mL-1. The essential oil was obtained by hydrodistillation in a Clevenger apparatus and analyzed by gas chromatography. Susceptibility was expressed as Minimal Inhibitory Concentration (MIC) and Minimal Fungicidal Concentration (MFC). All isolates tested in vitro were sensitive to O. vulgare essential oil. The chromatographic analysis revealed that the main compounds present in the essential oil were 4-terpineol (47.95%), carvacrol (9.42%), thymol (8.42%) and □-terpineol (7.57%). C. albicans isolates obtained from animal mucous membranes exhibited MIC and MFC values of 2.72 μL mL-1 and 5 μL mL-1, respectively. MIC and MFC values for C. albicans standard strains were 2.97 μL mL-1 and 3.54 μL mL-1, respectively. The MIC and MFC for non-albicans species were 2.10 μL mL-1 and 2.97 μL mL-1, respectively. The antifungal activity of O. vulgare essential oil against Candida spp. observed in vitro suggests its administration may represent an alternative treatment for candidiasis. PMID:24031471

Producing offspring in Armadillidium vulgare: Effects of genetic diversity and inbreeding.

PubMed

Durand, Sylvine; Loiseau, Vincent; Prigot, Cybèle; Braquart-Varnier, Christine; Beltran-Bech, Sophie

2018-03-01

Genetic diversity is known to be correlated to fitness traits, and inbred individuals often display lower values for life history traits. In this study, we attempt to quantify how inbreeding affects such traits in the terrestrial isopod Armadillidium vulgare by performing inbred and non-inbred crosses under laboratory conditions. We estimated genetic characteristics of parents and offspring, and related them to fecundity and fertility measures, as well as offspring growth and survival. Our study shows that a decrease in offspring number might result from mortality around birth, but not to changes in fecundity, fertilization rate, or developmental failure between inbred and non-inbred crosses. More heterozygous females tended to be bigger and had a higher fecundity, which could have implications in mate choice. No effect of inbreeding was detected on offspring growth and survival. These results can be related to previously observed effects of genetic characteristics on mating strategies in A. vulgare, and could shed light on mechanisms of inbreeding avoidance in this species. © 2018 Wiley Periodicals, Inc.

Crystallographic analysis and structure-guided engineering of NADPH-dependent Ralstonia sp. alcohol dehydrogenase toward NADH cosubstrate specificity.

PubMed

Lerchner, Alexandra; Jarasch, Alexander; Meining, Winfried; Schiefner, André; Skerra, Arne

2013-11-01

The NADP⁺-dependent alcohol dehydrogenase from Ralstonia sp. (RasADH) belongs to the protein superfamily of short-chain dehydrogenases/reductases (SDRs). As an enzyme that accepts different types of substrates--including bulky-bulky as well as small-bulky secondary alcohols or ketones--with high stereoselectivity, it offers potential as a biocatalyst for industrial biotechnology. To understand substrate and cosubstrate specificities of RasADH we determined the crystal structure of the apo-enzyme as well as its NADP⁺-bound state with resolutions down to 2.8 Å. RasADH displays a homotetrameric quaternary structure that can be described as a dimer of homodimers while in each subunit a seven-stranded parallel β-sheet, flanked by three α-helices on each side, forms a Rossmann fold-type dinucleotide binding domain. Docking of the well-known substrate (S)-1-phenylethanol clearly revealed the structural determinants of stereospecificity. To favor practical RasADH application in the context of established cofactor recycling systems, for example, those involving an NADH-dependent amino acid dehydrogenase, we attempted to rationally change its cosubstrate specificity from NADP⁺ to NAD⁺ utilizing the structural information that NADP⁺ specificity is largely governed by the residues Asn15, Gly37, Arg38, and Arg39. Furthermore, an extensive sequence alignment with homologous dehydrogenases that have different cosubstrate specificities revealed a modified general SDR motif ASNG (instead of NNAG) at positions 86-89 of RasADH. Consequently, we constructed mutant enzymes with one (G37D), four (N15G/G37D/R38V/R39S), and six (N15G/G37D/R38V/R39S/A86N/S88A) amino acid exchanges. RasADH (N15G/G37D/R38V/R39S) was better able to accept NAD⁺ while showing much reduced catalytic efficiency with NADP⁺, leading to a change in NADH/NADPH specificity by a factor of ∼3.6 million. © 2013 Wiley Periodicals, Inc.

Purification and Characterization of Two Distinct NAD(P)H Dehydrogenases from Onion (Allium cepa L.) Root Plasma Membrane.

PubMed Central

Serrano, A.; Cordoba, F.; Gonzalez-Reyes, J. A.; Navas, P.; Villalba, J. M.

1994-01-01

Highly purified plasma membrane fractions were obtained from onion (Allium cepa L.) roots and used as a source for purification of redox proteins. Plasma membranes solubilized with Triton X-100 contained two distinct polypeptides showing NAD(P)H-dependent dehydrogenase activities. Dehydrogenase I was purified by gel filtration in Sephacryl S-300 HR, ion-exchange chromatography in DEAE-Sepharose CL-6B, and dye-ligand affinity chromatography in Blue-Sepharose CL-6B after biospecific elution with NADH. Dehydrogenase I consisted of a single polypeptide of about 27 kD and an isoelectric point of about 6. Dehydrogenase II was purified from the DEAE-unbound fraction by chromatography in Blue-Sepharose CL-6B and affinity elution with NADH. Dehydrogenase II consisted of a single polypeptide of about 31 kD and an isoelectric point of about 8. Purified dehydrogenase I oxidized both NADPH and NADH, although higher rates of electron transfer were obtained with NADPH. Maximal activity was achieved with NADPH as donor and juglone or coenzyme Q as acceptor. Dehydrogenase II was specific for NADH and exhibited maximal activity with ferricyanide. Optimal pH for both dehydrogenases was about 6. Dehydrogenase I was moderately inhibited by dicumarol, thenoyltrifluoroacetone, and the thiol reagent N-ethyl-maleimide. A strong inhibition of dehydrogenase II was obtained with dicumarol, thenoyltrifluoroacetone, and the thiol reagent p-hydroxymercuribenzoate. PMID:12232306

Tanacetum vulgare as a bioindicator of trace-metal contamination: a study of a naturally colonized open-pit lignite mine.

PubMed

Jasion, Mateusz; Samecka-Cymerman, Aleksandra; Kolon, Krzysztof; Kempers, Alexander J

2013-10-01

We investigated the possibility of use of Tanacetum vulgare (tansy) as an ecological indicator of metal concentration in a naturally colonized open-pit lignite mine in Bełchatów (Poland). Tanacetum vulgare is the only species growing abundantly and spontaneously in the lignite mine waste dumps. Metal concentrations in roots, stems, leaves, flowers, and soil were measured in dump sites differing in type and time of reclamation and therefore differing in pollution levels. Tanacetum vulgare appeared to be an accumulator of chromium and iron in roots, whereas highest concentrations of manganese and zinc were found in leaves. A high bioaccumulation factor for cadmium (Cd) was observed in dumps and control sites, indicating that even small amounts of Cd in the environment may result in significant uptake by the plant. The lowest concentrations of metals were found in plants from sites situated on dumps reclaimed with argillaceous limestone.

Evolution of NADPH Oxidase Inhibitors: Selectivity and Mechanisms for Target Engagement.

PubMed

Altenhöfer, Sebastian; Radermacher, Kim A; Kleikers, Pamela W M; Wingler, Kirstin; Schmidt, Harald H H W

2015-08-10

Oxidative stress, an excess of reactive oxygen species (ROS) production versus consumption, may be involved in the pathogenesis of different diseases. The only known enzymes solely dedicated to ROS generation are nicotinamide adenine dinucleotide phosphate (NADPH) oxidases with their catalytic subunits (NOX). After the clinical failure of most antioxidant trials, NOX inhibitors are the most promising therapeutic option for diseases associated with oxidative stress. Historical NADPH oxidase inhibitors, apocynin and diphenylene iodonium, are un-specific and not isoform selective. Novel NOX inhibitors stemming from rational drug discovery approaches, for example, GKT137831, ML171, and VAS2870, show improved specificity for NADPH oxidases and moderate NOX isoform selectivity. Along with NOX2 docking sequence (NOX2ds)-tat, a peptide-based inhibitor, the use of these novel small molecules in animal models has provided preliminary in vivo evidence for a pathophysiological role of specific NOX isoforms. Here, we discuss whether novel NOX inhibitors enable reliable validation of NOX isoforms' pathological roles and whether this knowledge supports translation into pharmacological applications. Modern NOX inhibitors have increased the evidence for pathophysiological roles of NADPH oxidases. However, in comparison to knockout mouse models, NOX inhibitors have limited isoform selectivity. Thus, their use does not enable clear statements on the involvement of individual NOX isoforms in a given disease. The development of isoform-selective NOX inhibitors and biologicals will enable reliable validation of specific NOX isoforms in disease models other than the mouse. Finally, GKT137831, the first NOX inhibitor in clinical development, is poised to provide proof of principle for the clinical potential of NOX inhibition.

13C-flux analysis reveals NADPH-balancing transhydrogenation cycles in stationary phase of nitrogen-starving Bacillus subtilis.

PubMed

Rühl, Martin; Le Coq, Dominique; Aymerich, Stéphane; Sauer, Uwe

2012-08-10

In their natural habitat, microorganisms are typically confronted with nutritional limitations that restrict growth and force them to persevere in a stationary phase. Despite the importance of this phase, little is known about the metabolic state(s) that sustains it. Here, we investigate metabolically active but non-growing Bacillus subtilis during nitrogen starvation. In the absence of biomass formation as the major NADPH sink, the intracellular flux distribution in these resting B. subtilis reveals a large apparent catabolic NADPH overproduction of 5.0 ± 0.6 mmol g(-1)h(-1) that was partly caused by high pentose phosphate pathway fluxes. Combining transcriptome analysis, stationary (13)C-flux analysis in metabolic deletion mutants, (2)H-labeling experiments, and kinetic flux profiling, we demonstrate that about half of the catabolic excess NADPH is oxidized by two transhydrogenation cycles, i.e. isoenzyme pairs of dehydrogenases with different cofactor specificities that operate in reverse directions. These transhydrogenation cycles were constituted by the combined activities of the glyceraldehyde 3-phosphate dehydrogenases GapA/GapB and the malic enzymes MalS/YtsJ. At least an additional 6% of the overproduced NADPH is reoxidized by continuous cycling between ana- and catabolism of glutamate. Furthermore, in vitro enzyme data show that a not yet identified transhydrogenase could potentially reoxidize ∼20% of the overproduced NADPH. Overall, we demonstrate the interplay between several metabolic mechanisms that concertedly enable network-wide NADPH homeostasis under conditions of high catabolic NADPH production in the absence of cell growth in B. subtilis.

13C-flux Analysis Reveals NADPH-balancing Transhydrogenation Cycles in Stationary Phase of Nitrogen-starving Bacillus subtilis *

PubMed Central

Rühl, Martin; Le Coq, Dominique; Aymerich, Stéphane; Sauer, Uwe

2012-01-01

In their natural habitat, microorganisms are typically confronted with nutritional limitations that restrict growth and force them to persevere in a stationary phase. Despite the importance of this phase, little is known about the metabolic state(s) that sustains it. Here, we investigate metabolically active but non-growing Bacillus subtilis during nitrogen starvation. In the absence of biomass formation as the major NADPH sink, the intracellular flux distribution in these resting B. subtilis reveals a large apparent catabolic NADPH overproduction of 5.0 ± 0.6 mmol·g−1·h−1 that was partly caused by high pentose phosphate pathway fluxes. Combining transcriptome analysis, stationary 13C-flux analysis in metabolic deletion mutants, 2H-labeling experiments, and kinetic flux profiling, we demonstrate that about half of the catabolic excess NADPH is oxidized by two transhydrogenation cycles, i.e. isoenzyme pairs of dehydrogenases with different cofactor specificities that operate in reverse directions. These transhydrogenation cycles were constituted by the combined activities of the glyceraldehyde 3-phosphate dehydrogenases GapA/GapB and the malic enzymes MalS/YtsJ. At least an additional 6% of the overproduced NADPH is reoxidized by continuous cycling between ana- and catabolism of glutamate. Furthermore, in vitro enzyme data show that a not yet identified transhydrogenase could potentially reoxidize ∼20% of the overproduced NADPH. Overall, we demonstrate the interplay between several metabolic mechanisms that concertedly enable network-wide NADPH homeostasis under conditions of high catabolic NADPH production in the absence of cell growth in B. subtilis. PMID:22740702

NADPH: Protochlorophyllide Oxidoreductase-Structure, Catalytic Function, and Role in Prolamellar Body Formation and Morphogenesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Timko, Michael P

2013-02-01

The biosynthesis of chlorophyll is a critical biochemical step in the development of photosynthetic vascular plants and green algae. From photosynthetic bacteria (cyanobacteria) to algae, non-vascular plants, gymnosperms and vascular plants, mechanisms have evolved for protochlorophyllide reduction a key step in chlorophyll synthesis. Protochlorophyllide reduction is carried out by both a light-dependent (POR) and light-independent (LIPOR) mechanisms. NADPH: protochlorophyllide oxidoreductase (EC 1.3.1.33, abbreviated POR) catalyzes the light-dependent reduction of protochlorophyllide (PChlide) to chlorophyllide (Chlide). In contrast, a light-independent protochlorophyllide reductase (LIPOR) involves three plastid gene products (chlL, chlN, and chlB) and several nuclear factors. Our work focused on characterization ofmore » both the POR and LIPOR catalyzed processes.« less

Seizure activity results in calcium- and mitochondria-independent ROS production via NADPH and xanthine oxidase activation

PubMed Central

Kovac, S; Domijan, A-M; Walker, M C; Abramov, A Y

2014-01-01

Seizure activity has been proposed to result in the generation of reactive oxygen species (ROS), which then contribute to seizure-induced neuronal damage and eventually cell death. Although the mechanisms of seizure-induced ROS generation are unclear, mitochondria and cellular calcium overload have been proposed to have a crucial role. We aim to determine the sources of seizure-induced ROS and their contribution to seizure-induced cell death. Using live cell imaging techniques in glioneuronal cultures, we show that prolonged seizure-like activity increases ROS production in an NMDA receptor-dependent manner. Unexpectedly, however, mitochondria did not contribute to ROS production during seizure-like activity. ROS were generated primarily by NADPH oxidase and later by xanthine oxidase (XO) activity in a calcium-independent manner. This calcium-independent neuronal ROS production was accompanied by an increase in intracellular [Na+] through NMDA receptor activation. Inhibition of NADPH or XO markedly reduced seizure-like activity-induced neuronal apoptosis. These findings demonstrate a critical role for ROS in seizure-induced neuronal cell death and identify novel therapeutic targets. PMID:25275601

Differential Expression of NADPH Oxidases Depends on Skeletal Muscle Fiber Type in Rats.

PubMed

Loureiro, Adriano César Carneiro; do Rêgo-Monteiro, Igor Coutinho; Louzada, Ruy A; Ortenzi, Victor Hugo; de Aguiar, Angélica Ponte; de Abreu, Ewerton Sousa; Cavalcanti-de-Albuquerque, João Paulo Albuquerque; Hecht, Fabio; de Oliveira, Ariclécio Cunha; Ceccatto, Vânia Marilande; Fortunato, Rodrigo S; Carvalho, Denise P

2016-01-01

NADPH oxidases (NOX) are important sources of reactive oxygen species (ROS) in skeletal muscle, being involved in excitation-contraction coupling. Thus, we aimed to investigate if NOX activity and expression in skeletal muscle are fiber type specific and the possible contribution of this difference to cellular oxidative stress. Oxygen consumption rate, NOX activity and mRNA levels, and the activity of catalase (CAT), glutathione peroxidase (GPX), and superoxide dismutase (SOD), as well as the reactive protein thiol levels, were measured in the soleus (SOL), red gastrocnemius (RG), and white gastrocnemius (WG) muscles of rats. RG showed higher oxygen consumption flow than SOL and WG, while SOL had higher oxygen consumption than WG. SOL showed higher NOX activity, as well as NOX2 and NOX4 mRNA levels, antioxidant enzymatic activities, and reactive protein thiol contents when compared to WG and RG. NOX activity and NOX4 mRNA levels as well as antioxidant enzymatic activities were higher in RG than in WG. Physical exercise increased NOX activity in SOL and RG, specifically NOX2 mRNA levels in RG and NOX4 mRNA levels in SOL. In conclusion, we demonstrated that NOX activity and expression differ according to the skeletal muscle fiber type, as well as antioxidant defense.

Fluorescence labelling of NADPH-cytochrome P-450 reductase with the monobromomethyl derivative of syn-9,10-dioxabimane.

PubMed Central

Vogel, F; Lumper, L

1983-01-01

The kinetics of thiol-group alkylation in NADPH-cytochrome P-450 reductase during its inactivation by monobromobimane has been studied using the fluorimetric determination of S-bimane-L-cysteine by high-performance liquid chromatography. Loss of activity during the reaction of NADPH-cytochrome P-450 reductase with monobromobimane is caused by the alkylation of one single critical cysteine residue, which can be protected against thiol-specific reagents by NADP(H). The chemical stability of the bimane group allows the digestion of bimane-labelled NADPH-cytochrome P-450 reductase by CNBr. The critical cysteine residue could be located in a CNBr-cleaved peptide purified to homogeneity with Mr 10 500 +/- 1 000 and valine as N-terminus. Images Fig. 2. PMID:6414464

Engineering of a functional human NADH-dependent cytochrome P450 system

PubMed Central

Döhr, Olaf; Paine, Mark J. I.; Friedberg, Thomas; Roberts, Gordon C. K.; Wolf, C. Roland

2001-01-01

A functional human NADH-dependent cytochrome P450 system has been developed by altering the cofactor preference of human NADPH cytochrome P450 reductase (CPR), the redox partner for P450s. This has been achieved by a single amino acid change of the conserved aromatic amino acid Trp-676, which covers the re-side of the FAD isoalloxazine ring in the nicotinamide-binding site. Of the mutations made, the substitution of Trp-676 with alanine (W676A) resulted in a functional NADH-dependent enzyme, which catalyzed the reduction of cytochrome c and ferricyanide as well as facilitated the metabolism of 7-ethoxyresorufin by CYP1A2. Kinetic analysis measuring cytochrome c activity revealed that the NADH-dependent kcat of W676A is equivalent (90%) to the NADPH-dependent kcat of the wild-type enzyme, with W676A having an approximately 1,000-fold higher specificity for NADH. The apparent KMNADPH and KMNADH values of W676A are 80- and 150-fold decreased, respectively. In accordance with structural data, which show a bipartite binding mode of NADPH, substitution of Trp-676 does not affect 2′-AMP binding as seen by the inhibition of both wild-type CPR and the W676A mutant. Furthermore, NADPH was a potent inhibitor of the W676A NADH-dependent cytochrome c reduction and CYP1A2 activity. Overall, the results show that Trp-676 of human CPR plays a major role in cofactor discrimination, and substitution of this conserved aromatic residue with alanine results in an efficient NADH-dependent cytochrome P450 system. PMID:11136248

NADPH-diaphorase activity and NO synthase expression in the olfactory epithelium of the bovine.

PubMed

Wenisch, S; Arnhold, S

2010-06-01

NADPH-diaphorase (NADPH-d) staining of the bovine olfactory epithelium was compared with the immunohistochemical localization of nitric oxide synthase (NOS), soluble guanylyl cyclase, and cGMP (cyclic guanosine 3',5'-monophosphate). Out of the three isoforms, only the inducible NOS (NOS-II) was found at the epithelial surface correlating with the strong labelling for NADPH-d. In contrast, light diaphorase staining associated with deeper epithelial regions did not coincide with any NOS immunoreactivity. As there is overlapping expression of NOS-II, soluble guanylyl cyclase and cGMP at the luminal surface morphologically occupied by dendritic knobs of olfactory receptor neurons and microvillar endings of supporting cells, the nitric oxide (NO)/cGMP pathway is likely to be involved in modulating the odour signals during olfactory transduction.

An Unprecedented NADPH Domain Conformation in Lysine Monooxygenase NbtG Provides Insights into Uncoupling of Oxygen Consumption from Substrate Hydroxylation

DOE PAGES

Binda, Claudia; Robinson, Reeder M.; Martin del Campo, Julia S.; ...

2015-03-23

N-hydroxylating monooxygenases (NMOs) are involved in the biosynthesis of iron-chelating hydroxamate-containing siderophores that play a role in microbial virulence. These flavoenzymes catalyze the NADPH- and oxygen-dependent hydroxylation of amines, such as those found on the side chains of lysine and ornithine. In this work we report the biochemical and structural characterization of Nocardia farcinica Lys monooxygenase (NbtG), which has similar biochemical properties to mycobacterial homologs. NbtG is also active on D-Lys although it binds L-Lys with a higher affinity. Differently from the ornithine monooxygenases PvdA, SidA and KtzI, NbtG can use both NADH and NADPH and is highly uncoupled, producingmore » more superoxide and hydrogen peroxide than hydroxylated Lys. The crystal structure of NbtG solved at 2.4 Å resolution revealed an unexpected protein conformation with a 30° rotation of the NAD(P)H domain with respect to the FAD domain that precludes binding of the nicotinamide cofactor. This “occluded” structure may explain the biochemical properties of NbtG, specifically with regard to the substantial uncoupling and limited stabilization of the C4a-hydroperoxyflavin intermediate. We discuss the biological implications of these findings.« less

Coexistence of calbindin D-28k and NADPH-diaphorase in vagal and glossopharyngeal sensory neurons of the rat.

PubMed

Ichikawa, H; Helke, C J

1996-10-07

The presence and coexistence of calbindin D-28k-immunoreactivity (ir) and nicotinamide adenosine dinucleotide phosphate (NADPH)-diaphorase activity (a marker of neurons that are presumed to convert L-arginine to L-citrulline and nitric oxide) were examined in the glossopharyngeal and vagal sensory ganglia (jugular, petrosal and nodose ganglia) of the rat. Calbindin D-28k-ir nerve cells were found in moderate and large numbers in the petrosal and nodose ganglia, respectively. Some calbindin D-28k-ir nerve cells were also observed in the jugular ganglion. NADPH-diaphorase positive nerve cells were localized to the jugular and nodose ganglia and were rare in the petrosal ganglion. A considerable portion (33-51%) of the NADPH-diaphorase positive neurons in these ganglia colocalized calbindin D-28k-ir. The presence and colocalization of calbindin D-28k-ir and NADPH-diaphorase activity in neurotransmitter-identified subpopulations of visceral sensory neurons were also studied. In all three ganglia, calcitonin gene-related peptide (CGRP)-ir was present in many NADPH-diaphorase positive neurons, a subset of which also contained calbindin D-28k-ir. In the nodose ganglion, many (42%) of tyrosine hydroxylase (TH)-ir neurons also contained NADPH diaphorase activity but did not contain calbindin D-28k-ir. These data are consistent with a potential co-operative role for calbindin D-28k and NADPH-diaphorase in the functions of a subpopulation of vagal and glossopharyngeal sensory neurons.

High glucose condition increases NADPH oxidase activity in endothelial microparticles that promote vascular inflammation.

PubMed

Jansen, Felix; Yang, Xiaoyan; Franklin, Bernardo S; Hoelscher, Marion; Schmitz, Theresa; Bedorf, Jörg; Nickenig, Georg; Werner, Nikos

2013-04-01

Diabetes is a major risk factor for cardiovascular diseases. Circulating endothelial microparticles (EMP) are increased in diabetic patients, but their potential contribution in atherogenesis is unclear. We sought to determine the role of EMP derived under high glucose conditions in the development of atherosclerosis. EMP were generated from human coronary endothelial cells (HCAEC) exposed to high glucose concentrations in order to mimic diabetic conditions. These EMP were defined as 'injured' EMP (iEMP) and their effects were compared with EMP generated from 'healthy' untreated HCAEC. iEMP injection significantly impaired endothelial function in ApoE(-/-) mice compared with EMP and vehicle treatment. Immunofluorescent experiments showed increased macrophage infiltration and adhesion protein expression in atherosclerotic lesions of iEMP-treated ApoE(-/-) mice compared with controls. To further investigate the underlying mechanism of iEMP-induced vascular inflammation, additional in vitro experiments were performed. iEMP, but not EMP, induced activation of HCAEC in a time- and dose-dependent manner and increased monocyte adhesion. Further experiments demonstrated that iEMP induced activation of HCAEC by phosphorylation of p38 into its biologically active form phospho-p38. Inhibition of p38 activation abrogated iEMP-dependent induction of adhesion proteins and monocyte adhesion on HCAEC. Moreover, we could demonstrate that iEMP show increased NADPH oxidase activity and contain significantly higher level of reactive oxygen species (ROS) than EMP. iEMP triggered ROS production in HCAEC and thereby activate p38 in an ROS-dependent manner. High glucose condition increases NADPH oxidase activity in endothelial microparticles that amplify endothelial inflammation and impair endothelial function by promoting activation of the endothelium. These findings provide new insights into the pathogenesis of diabetes-associated atherosclerosis.

NADPH-Diaphorase Colocalizes with GPER and Is Modulated by the GPER Agonist G1 in the Supraoptic and Paraventricular Nuclei of Ovariectomized Female Rats.

PubMed

Grassi, Daniela; Lagunas, Natalia; Pinos, Helena; Panzica, GianCarlo; Garcia-Segura, Luis Miguel; Collado, Paloma

2017-01-01

Nitric oxide is produced in the brain by the neuronal nitric oxide synthase (nNOS) and carries out a wide range of functions by acting as a neurotransmitter-like molecule. Gonadal hormones are involved in the regulation of the brain nitrergic system. We have previously demonstrated that estradiol, via classical estrogen receptors (ERs), regulates NOS activity in the supraoptic (SON) and paraventricular (PVN) nuclei of the hypothalamus, acting through both ERα and ERβ. Magnocellular and parvocellular neurons in the SON and PVN also express the G protein-coupled ER (GPER). In this study, we have assessed whether GPER is also involved in the regulation of nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase in the SON and PVN. Adult female ovariectomized rats were treated with G1, a selective GPER agonist, or with G1 in combination with G15, a selective GPER antagonist. G1 treatment decreased NADPH-diaphorase expression in the SON and in all PVN subnuclei. The treatment with G1 + G15 effectively rescued the G1-dependent decrease in NADPH-diaphorase expression in both brain regions. In addition, the activation of extracellular signal-regulated kinase (ERK) 1/2, one of the kinases involved in the GPER-dependent intracellular signaling pathway and in NOS phosphorylation, was assessed in the same brain nuclei. Treatment with G1 significantly decreased the number of p-ERK 1/2-positive cells in the SON and PVN, while the treatment with G1 + G15 significantly recovered its number to control values. These findings suggest that the activation of GPER in the SON and PVN inhibits the phosphorylation of ERK 1/2, which induces a decrease in NADPH-diaphorase expression. © 2016 S. Karger AG, Basel.

Differentially regulated NADPH:cytochrome P450 oxidoreductases in parsley

PubMed Central

Koopmann, Edda; Hahlbrock, Klaus

1997-01-01

Two NADPH:cytochrome P450 oxidoreductases (CPRs) from parsley (Petroselinum crispum) were cloned, and the complete proteins were expressed and functionally identified in yeast. The two enzymes, designated CPR1 and CPR2, are 80% identical in amino acid sequence with one another and about 75% identical with CPRs from several other plant species. The mRNA accumulation patterns for CPR1 and CPR2 in fungal elicitor-treated or UV-irradiated cultured parsley cells and in developing or infected parsley plants were compared with those for cinnamate 4-hydroxylase (C4H), one of the most abundant CPR-dependent P450 enzymes in plants. All treatments strongly induced the mRNAs for C4H and CPR1 but not for CPR2, suggesting distinct metabolic roles of CPR1 and CPR2 and a functional relationship between CPR1 and C4H. PMID:9405720

Phytochemical screening, anticancer and antioxidant activities of Origanum vulgare L. ssp. viride (Boiss.) Hayek, a plant of traditional usage.

PubMed

Koldaş, Serkan; Demirtas, Ibrahim; Ozen, Tevfik; Demirci, Mehmet Ali; Behçet, Lütfi

2015-03-15

A detailed phytochemical analysis of Origanum vulgare L. ssp. viride (Boiss.) Hayek was carried out and the antioxidant activities of five different crude extracts were determined. The antiproliferative activities of the extracts were determined using the xCELLigence system (Real Time Cell Analyzer). Differences between the essential oil and volatile organic compound profiles of the plant were shown. The main component of the essential oil was caryophyllene oxide, while the main volatile organic compounds were sabinene and eucalyptol as determined by HS-GC/MS. Phenolic contents of the extracts were determined qualitatively and quantitatively by HPLC/TOF-MS. Ten phenolic compounds were found in the extracts from O. vulgare and Origanum acutidens: rosmarinic acid (in highest abundance), chicoric acid, caffeic acid, p-coumaric acid, gallic acid, quercetin, apigenin-7-glucoside, kaempferol, naringenin and 4-hydroxybenzaldehyde. This study provides first results on the antiproliferative and antioxidant properties and detailed phytochemical screening of O. vulgare ssp. viride (Boiss.) Hayek. © 2014 Society of Chemical Industry.

Activation of NADPH oxidase mediates increased endoplasmic reticulum stress and left ventricular remodeling after myocardial infarction in rabbits.

PubMed

Li, Bao; Tian, Jing; Sun, Yi; Xu, Tao-Rui; Chi, Rui-Fang; Zhang, Xiao-Li; Hu, Xin-Ling; Zhang, Yue-An; Qin, Fu-Zhong; Zhang, Wei-Fang

2015-05-01

Nicotinamide adenine dinucleotide 3-phosphate (NADPH) oxidase activity and endoplasmic reticulum (ER) stress are increased after myocardial infarction (MI). In this study, we proposed to test whether activation of the NADPH oxidase in the remote non-infarcted myocardium mediates ER stress and left ventricular (LV) remodeling after MI. Rabbits with MI or sham operation were randomly assigned to orally receive an NADPH oxidase inhibitor apocynin or placebo for 30 days. The agents were administered beginning at 1 week after surgery. MI rabbits exhibited decreases in LV fractional shortening, LV ejection fraction and the first derivative of the LV pressure rise, which were abolished by apocynin treatment. NADPH oxidase Nox2 protein and mRNA expressions were increased in the remote non-infarcted myocardium after MI. Immunolabeling further revealed that Nox2 was increased in cardiac myocytes in the remote myocardium. The apocynin treatment prevented increases in the Nox2 expression, NADPH oxidase activity, oxidative stress, myocyte apoptosis and GRP78, CHOP and cleaved caspase 12 protein expression in the remote myocardium. The apocynin treatment also attenuated increases in myocyte diameter and cardiac fibrosis. In cultured H9C2 cardiomyocytes exposed to angiotensin II, an important stimulus for post-MI remodeling, Nox2 knockdown with siRNA significantly inhibited angiotensin II-induced NADPH oxidase activation, reactive oxygen species and GRP78 and CHOP protein expression. We conclude that NADPH oxidase inhibition attenuates increased ER stress in the remote non-infarcted myocardium and LV remodeling late after MI in rabbits. These findings suggest that the activation of NADPH oxidase in the remote non-infarcted myocardium mediates increased ER stress, contributing to myocyte apoptosis and LV remodeling after MI. Copyright © 2015 Elsevier B.V. All rights reserved.

RhoA/ROCK downregulates FPR2-mediated NADPH oxidase activation in mouse bone marrow granulocytes.

PubMed

Filina, Julia V; Gabdoulkhakova, Aida G; Safronova, Valentina G

2014-10-01

Polymorphonuclear neutrophils (PMNs) express the high and low affinity receptors to formylated peptides (mFPR1 and mFPR2 in mice, accordingly). RhoA/ROCK (Rho activated kinase) pathway is crucial for cell motility and oxidase activity regulated via FPRs. There are contradictory data on RhoA-mediated regulation of NADPH oxidase activity in phagocytes. We have shown divergent Rho GTPases signaling via mFPR1 and mFPR2 to NADPH oxidase in PMNs from inflammatory site. The present study was aimed to find out the role of RhoA/ROCK in the respiratory burst activated via mFPR1 and mFPR2 in the bone marrow PMNs. Different kinetics of RhoA activation were detected with 0.1μM fMLF and 1μM WKYMVM operating via mFPR1 and mFPR2, accordingly. RhoA was translocated in fMLF-activated cells towards the cell center and juxtamembrane space versus uniform allocation in the resting cells. Specific inhibition of RhoA by CT04, Rho inhibitor I, weakly depressed the respiratory burst induced via mFPR1, but significantly increased the one induced via mFPR2. Inhibition of ROCK, the main effector of RhoA, by Y27632 led to the same effect on the respiratory burst. Regulation of mFPR2-induced respiratory response by ROCK was impossible under the cytoskeleton disruption by cytochalasin D, whereas it persisted in the case of mFPR1 activation. Thus we suggest RhoA to be one of the regulatory and signal transduction components in the respiratory burst through FPRs in the mouse bone marrow PMNs. Both mFPR1 and mFPR2 binding with a ligand trigger the activation of RhoA. FPR1 signaling through RhoA/ROCK increases NADPH-oxidase activity. But in FPR2 action RhoA/ROCK together with cytoskeleton-linked systems down-regulates NADPH-oxidase. This mechanism could restrain the reactive oxygen species dependent damage of own tissues during the chemotaxis of PMNs and in the resting cells. Copyright © 2014 Elsevier Inc. All rights reserved.

Oligo-carrageenan kappa increases NADPH, ascorbate and glutathione syntheses and TRR/TRX activities enhancing photosynthesis, basal metabolism, and growth in Eucalyptus trees.

PubMed

González, Alberto; Moenne, Fabiola; Gómez, Melissa; Sáez, Claudio A; Contreras, Rodrigo A; Moenne, Alejandra

2014-01-01

In order to analyze the effect of OC kappa in redox status, photosynthesis, basal metabolism and growth in Eucalyptus globulus, trees were treated with water (control), with OC kappa at 1 mg mL(-1), or treated with inhibitors of NAD(P)H, ascorbate (ASC), and glutathione (GSH) syntheses and thioredoxin reductase (TRR) activity, CHS-828, lycorine, buthionine sulfoximine (BSO), and auranofin, respectively, and with OC kappa, and cultivated for 4 months. Treatment with OC kappa induced an increase in NADPH, ASC, and GSH syntheses, TRR and thioredoxin (TRX) activities, photosynthesis, growth and activities of basal metabolism enzymes such as rubisco, glutamine synthetase (GlnS), adenosine 5'-phosphosulfate reductase (APR), involved in C, N, and S assimilation, respectively, Krebs cycle and purine/pyrimidine synthesis enzymes. Treatment with inhibitors and OC kappa showed that increases in ASC, GSH, and TRR/TRX enhanced NADPH synthesis, increases in NADPH and TRR/TRX enhanced ASC and GSH syntheses, and only the increase in NADPH enhanced TRR/TRX activities. In addition, the increase in NADPH, ASC, GSH, and TRR/TRX enhanced photosynthesis and growth. Moreover, the increase in NADPH, ASC and TRR/TRX enhanced activities of rubisco, Krebs cycle, and purine/pyrimidine synthesis enzymes, the increase in GSH, NADPH, and TRR/TRX enhanced APR activity, and the increase in NADPH and TRR/TRX enhanced GlnS activity. Thus, OC kappa increases NADPH, ASC, and GSH syntheses leading to a more reducing redox status, the increase in NADPH, ASC, GSH syntheses, and TRR/TRX activities are cross-talking events leading to activation of photosynthesis, basal metabolism, and growth in Eucalyptus trees.

Vitamin C prevents zidovudine-induced NAD(P)H oxidase activation and hypertension in the rat.

PubMed

Papparella, Italia; Ceolotto, Giulio; Berto, Laura; Cavalli, Maurizio; Bova, Sergio; Cargnelli, Gabriella; Ruga, Ezia; Milanesi, Ornella; Franco, Lorenzo; Mazzoni, Martina; Petrelli, Lucia; Nussdorfer, Gastone G; Semplicini, Andrea

2007-01-15

Cardiovascular risk is increased among HIV-infected patients receiving antiretroviral therapy due to the development of hypertension and metabolic abnormalities. In this study, we investigated the effects of long-term treatment with zidovudine (AZT) and vitamin C, alone and in combination, on blood pressure and on the chain of events linking oxidative stress to cardiac damage in the rat. Six adult Wistar Kyoto rats received AZT (1 mg/ml) in the drinking water for 8 months, six vitamin C (10 g/kg of food) and AZT, six vitamin C alone, and six served as controls. AZT increased systolic blood pressure, expression of gp91(phox) and p47(phox) subunits of NAD(P)H oxidase, and protein kinase C (PKC) delta activation and reduced antioxidant power of plasma and cardiac homogenates. AZT also caused morphological alterations in cardiac myocyte mitochondria, indicative of functional damage. All of these effects were prevented by vitamin C. Chronic AZT administration increases blood pressure and promotes cardiovascular damage through a NAD(P)H oxidase-dependent mechanism that involves PKC delta. Vitamin C antagonizes these adverse effects of AZT in the cardiovascular system.

NAD(H) and NADP(H) Redox Couples and Cellular Energy Metabolism.

PubMed

Xiao, Wusheng; Wang, Rui-Sheng; Handy, Diane E; Loscalzo, Joseph

2018-01-20

The nicotinamide adenine dinucleotide (NAD + )/reduced NAD + (NADH) and NADP + /reduced NADP + (NADPH) redox couples are essential for maintaining cellular redox homeostasis and for modulating numerous biological events, including cellular metabolism. Deficiency or imbalance of these two redox couples has been associated with many pathological disorders. Recent Advances: Newly identified biosynthetic enzymes and newly developed genetically encoded biosensors enable us to understand better how cells maintain compartmentalized NAD(H) and NADP(H) pools. The concept of redox stress (oxidative and reductive stress) reflected by changes in NAD(H)/NADP(H) has increasingly gained attention. The emerging roles of NAD + -consuming proteins in regulating cellular redox and metabolic homeostasis are active research topics. The biosynthesis and distribution of cellular NAD(H) and NADP(H) are highly compartmentalized. It is critical to understand how cells maintain the steady levels of these redox couple pools to ensure their normal functions and simultaneously avoid inducing redox stress. In addition, it is essential to understand how NAD(H)- and NADP(H)-utilizing enzymes interact with other signaling pathways, such as those regulated by hypoxia-inducible factor, to maintain cellular redox homeostasis and energy metabolism. Additional studies are needed to investigate the inter-relationships among compartmentalized NAD(H)/NADP(H) pools and how these two dinucleotide redox couples collaboratively regulate cellular redox states and cellular metabolism under normal and pathological conditions. Furthermore, recent studies suggest the utility of using pharmacological interventions or nutrient-based bioactive NAD + precursors as therapeutic interventions for metabolic diseases. Thus, a better understanding of the cellular functions of NAD(H) and NADP(H) may facilitate efforts to address a host of pathological disorders effectively. Antioxid. Redox Signal. 28, 251-272.

In vitro antimicrobial activity and chemical composition of the essential oil of Foeniculum vulgare Mill.

PubMed

Aprotosoaie, Ana Clara; Hăncianu, Monica; Poiată, Antonia; Tuchiluş, Cristina; Spac, A; Cioană, Oana; Gille, Elvira; Stănescu, Ursula

2008-01-01

In our study, four samples of volatile oil from Foeniculum vulgare, cultivated in different pedoclimatic conditions, were investigated for their antimicrobial activity and chemical composition. Organisms. Staphylococcus aureus ATCC 25923, Bacillus cereus, Pseudomonas aeruginosa, Escherichia coli ATCC 25922, Candida albicans were included in the report. Antimicrobial susceptibility tests. The comparative inhibitory activity of volatile oil samples with other antimicrobial agents was quantitative determined by minimum inhibitory concentration (MIC). Oil samples are the volatile oils extracted by steam distillation, from two ecological vegetative populations of Foeniculum vulgare. Gas chromatography coupled to mass spectrometry (GC-MS) was used to determine the chemical composition of the essential oils. All oil samples have a good activity against E. coli and S. aureus at low concentrations. Against B. cereus and P. aeruginosa these oil samples are less active. The oil samples were generally bactericidal at a concentration up to twofold or fourfold higher than the MIC value. Significantly synergic activity with amoxicillin or tetracycline showed all fennel samples against E. coli, Sarcina lutea and B. subtilis strains. Fennel oil samples have shown high activity against Candida albicans. No significant antimicrobial activity variations were observed for Foeniculum vulgare volatile oil samples obtained after two or three years cultivation period. The most important identified compounds in all samples of fennel volatile oils were trans-anethole, estragole, fenchone, limonene, alpha-pinene and gamma-terpinene.

Effect of the aqueous extract of Foeniculum vulgare (fennel) on the kidney in experimental PCOS female rats.

PubMed

Sadrefozalayi, Somayyeh; Farokhi, Farah

2014-03-01

Foeniculum vulgare seed (F. vulgare) is an herbal plant which is used with phytoestrogene compounds for polycystic ovary syndrome (PCOS) treatment. In this research, renoprotective effect of the aqueous extract of Foeniculum vulgare (AEF) in experimental PCOS female rats is studied. Forty female rats were randomly divided into five groups. The first group served as control, was injected with an equivalent volume (0.2 ml) of normal saline, and received normal diet. Animals in the second group were non poly cystic ovary syndrome (PCOS) rats which were treated with intragastric administration of aqueous extract of F. vulgare (150 mg/kg b.w.). In the third group, the rats were treated with intraperitoneal injection of estradiolvalerate (EV) (4 mg in 0.2 ml of sesame oil). The fourth groups were treated with EV and AEF (150mg/kg bw) with the same route. The fifth groups were treated with EV and AEF (100mg/kg bw). After 4 weeks of study, all of the rats were sacrificed, their kidneys tissues were processed for light microscopy, and some biochemical parameters of serum were measured. The mean values of blood urea nitrogen in PCOS rats treated with low dose of AEF and EV and non-treated, was significantly (p<0.05) increased compared with non-PCOS and PCOS rats treated with high dose of AEF. Moreover, histopathological changes of kidney samples were comparable in PCOS rats with respect to treated groups with AEF. Aqueous extract of fennel seed showed the beneficial effect (especially at dose of 150 mg/kg b.w.) on renal function in PCOS rats.

Method for obtaining three products with different properties from fennel (Foeniculum vulgare) seed

USDA-ARS?s Scientific Manuscript database

The objectives of this study were to determine the effects of distillation time (DT; 15-1080 min) on yield, composition, and antioxidant capacity of fennel (Foeniculum vulgare) seed essential oil (EO) as well as on the yield, composition, and properties of lipids extracted from steam-distilled fenne...

Histological study of some Echium vulgare, Pulmonaria officinalis and Symphytum officinale populations.

PubMed

Papp, Nóra; Bencsik, Tímea; Németh, Kitti; Gyergyák, Kinga; Sulc, Alexandra; Farkas, Agnes

2011-10-01

Plants living in different ecological habitats can show significant variability in their histological and phytochemical characters. The main histological features of various populations of three medicinal plants from the Boraginaceae family were studied. Stems, petioles and leaves were investigated by light microscopy in vertical and transverse sections. The outline of the epidermal cells, as well as the shape and cell number of trichomes was studied in leaf surface casts. Differences were measured among the populations of Echium vulgare in the width and height of epidermis cells in the stem, petiole and leaf, as well as in the size of palisade cells in the leaves. Among the populations of Pulmonaria officinalis significant differences were found in the length of trichomes and in the slightly or strongly wavy outline of epidermal radial cell walls. Populations of Symphytum officinale showed variance in the height of epidermal cells in leaves and stems, length of palisade cells and number of intercellular spaces in leaves, and the size of the central cavity in the stem. Boraginaceae bristles were found to be longer in plants in windy/shady habitats as opposed to sunny habitats, both in the leaves and stems ofP. officinalis and S. officinale, which might be connected to varying levels of exposure to wind. Longer epidermal cells were detected in the leaves and stems of both E. vulgare and S. officinale plants living in shady habitats, compared with shorter cells in sunny habitats. Leaf mesophyll cells were shorter in shady habitats as opposed to longer cells in sunny habitats, both in E. vulgare and S. officinale. This combination of histological characters may contribute to the plant's adaptation to various amounts of sunshine. The reported data prove the polymorphism of the studied taxa, as well as their ability to adapt to various ecological circumstances.

Identification of 6-octadecynoic acid from a methanol extract of Marrubium vulgare L. as a peroxisome proliferator-activated receptor γ agonist

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ohtera, Anna; Miyamae, Yusaku; Nakai, Naomi

Highlights: •6-ODA, a rare fatty acid with a triple bond, was identified from Marrubium vulgare. •6-ODA was synthesized from petroselinic acid as a starting material. •6-ODA stimulated lipid accumulation in HSC-T6 and 3T3-L1 cells. •The first report of a fatty acid with a triple bond functioning as a PPARγ agonist. •This study sheds light on novel functions of a fatty acid with a triple bond. -- Abstract: 6-Octadecynoic acid (6-ODA), a fatty acid with a triple bond, was identified in the methanol extract of Marrubium vulgare L. as an agonist of peroxisome proliferator-activated receptor γ (PPARγ). Fibrogenesis caused by hepaticmore » stellate cells is inhibited by PPARγ whose ligands are clinically used for the treatment of diabetes. Plant extracts of Marrubium vulgare L., were screened for activity to inhibit fibrosis in the hepatic stellate cell line HSC-T6 using Oil Red-O staining, which detects lipids that typically accumulate in quiescent hepatic stellate cells. A methanol extract with activity to stimulate accumulation of lipids was obtained. This extract was found to have PPARγ agonist activity using a luciferase reporter assay. After purification using several chromatographic methods, 6-ODA, a fatty acid with a triple bond, was identified as a candidate of PPARγ agonist. Synthesized 6-ODA and its derivative 9-octadecynoic acid (9-ODA), which both have a triple bond but in different positions, activated PPARγ in a luciferase reporter assay and increased lipid accumulation in 3T3-L1 adipocytes in a PPARγ-dependent manner. There is little information about the biological activity of fatty acids with a triple bond, and to our knowledge, this is the first report that 6-ODA and 9-ODA function as PPARγ agonists.« less

IFNβ/TNFα synergism induces a non-canonical STAT2/IRF9-dependent pathway triggering a novel DUOX2 NADPH Oxidase-mediated airway antiviral response

PubMed Central

Fink, Karin; Martin, Lydie; Mukawera, Esperance; Chartier, Stéfany; De Deken, Xavier; Brochiero, Emmanuelle; Miot, Françoise; Grandvaux, Nathalie

2013-01-01

Airway epithelial cells are key initial innate immune responders in the fight against respiratory viruses, primarily via the secretion of antiviral and proinflammatory cytokines that act in an autocrine/paracrine fashion to trigger the establishment of an antiviral state. It is currently thought that the early antiviral state in airway epithelial cells primarily relies on IFNβ secretion and the subsequent activation of the interferon-stimulated gene factor 3 (ISGF3) transcription factor complex, composed of STAT1, STAT2 and IRF9, which regulates the expression of a panoply of interferon-stimulated genes encoding proteins with antiviral activities. However, the specific pathways engaged by the synergistic action of different cytokines during viral infections, and the resulting physiological outcomes are still ill-defined. Here, we unveil a novel delayed antiviral response in the airways, which is initiated by the synergistic autocrine/paracrine action of IFNβ and TNFα, and signals through a non-canonical STAT2- and IRF9-dependent, but STAT1-independent cascade. This pathway ultimately leads to the late induction of the DUOX2 NADPH oxidase expression. Importantly, our study uncovers that the development of the antiviral state relies on DUOX2-dependent H2O2 production. Key antiviral pathways are often targeted by evasion strategies evolved by various pathogenic viruses. In this regard, the importance of the novel DUOX2-dependent antiviral pathway is further underlined by the observation that the human respiratory syncytial virus is able to subvert DUOX2 induction. PMID:23545780

NAD(P)H Oxidase Activity in the Small Intestine Is Predominantly Found in Enterocytes, Not Professional Phagocytes.

PubMed

Lindquist, Randall L; Bayat-Sarmadi, Jannike; Leben, Ruth; Niesner, Raluca; Hauser, Anja E

2018-05-04

The balance between various cellular subsets of the innate and adaptive immune system and microbiota in the gastrointestinal tract is carefully regulated to maintain tolerance to the normal flora and dietary antigens, while protecting against pathogens. The intestinal epithelial cells and the network of dendritic cells and macrophages in the lamina propria are crucial lines of defense that regulate this balance. The complex relationship between the myeloid compartment (dendritic cells and macrophages) and lymphocyte compartment (T cells and innate lymphoid cells), as well as the impact of the epithelial cell layer have been studied in depth in recent years, revealing that the regulatory and effector functions of both innate and adaptive immune compartments exhibit more plasticity than had been previously appreciated. However, little is known about the metabolic activity of these cellular compartments, which is the basic function underlying all other additional tasks the cells perform. Here we perform intravital NAD(P)H fluorescence lifetime imaging in the small intestine of fluorescent reporter mice to monitor the NAD(P)H-dependent metabolism of epithelial and myeloid cells. The majority of myeloid cells which comprise the surveilling network in the lamina propria have a low metabolic activity and remain resting even upon stimulation. Only a few myeloid cells, typically localized at the tip of the villi, are metabolically active and are able to activate NADPH oxidases upon stimulation, leading to an oxidative burst. In contrast, the epithelial cells are metabolically highly active and, although not considered professional phagocytes, are also able to activate NADPH oxidases, leading to massive production of reactive oxygen species. Whereas the oxidative burst in myeloid cells is mainly catalyzed by the NOX2 isotype, in epithelial cells other isotypes of the NADPH oxidases family are involved, especially NOX4. They are constitutively expressed by the epithelial

Phosphatidylinositol 3-Kinase Plays a Vital Role in Regulation of Rice Seed Vigor via Altering NADPH Oxidase Activity

PubMed Central

Liu, Jian; Zhou, Jun; Xing, Da

2012-01-01

Phosphatidylinositol 3-kinase (PI3K) has been reported to be important in normal plant growth and stress responses. In this study, it was verified that PI3K played a vital role in rice seed germination through regulating NADPH oxidase activity. Suppression of PI3K activity by inhibitors wortmannin or LY294002 could abate the reactive oxygen species (ROS) formation, which resulted in disturbance to the seed germination. And then, the signal cascades that PI3K promoted the ROS liberation was also evaluated. Diphenylene iodonium (DPI), an NADPH oxidase inhibitor, suppressed most of ROS generation in rice seed germination, which suggested that NADPH oxidase was the main source of ROS in this process. Pharmacological experiment and RT-PCR demonstrated that PI3K promoted the expression of Os rboh9. Moreover, functional analysis by native PAGE and the measurement of the 2, 3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazo-lium-5- carboxanilide (XTT) formazan concentration both showed that PI3K promoted the activity of NADPH oxidase. Furthermore, the western blot analysis of OsRac-1 demonstrated that the translocation of Rac-1 from cytoplasm to plasma membrane, which was known as a key factor in the assembly of NADPH oxidase, was suppressed by treatment with PI3K inhibitors, resulting in the decreased activity of NADPH oxidase. Taken together, these data favored the novel conclusion that PI3K regulated NADPH oxidase activity through modulating the recruitment of Rac-1 to plasma membrane and accelerated the process of rice seed germination. PMID:22448275

Congruence between PM H+-ATPase and NADPH oxidase during root growth: a necessary probability.

PubMed

Majumdar, Arkajo; Kar, Rup Kumar

2018-07-01

Plasma membrane (PM) H + -ATPase and NADPH oxidase (NOX) are two key enzymes responsible for cell wall relaxation during elongation growth through apoplastic acidification and production of ˙OH radical via O 2 ˙ - , respectively. Our experiments revealed a putative feed-forward loop between these enzymes in growing roots of Vigna radiata (L.) Wilczek seedlings. Thus, NOX activity was found to be dependent on proton gradient generated across PM by H + -ATPase as evident from pharmacological experiments using carbonyl cyanide m-chlorophenylhydrazone (CCCP; protonophore) and sodium ortho-vanadate (PM H + -ATPase inhibitor). Conversely, H + -ATPase activity retarded in response to different ROS scavengers [CuCl 2 , N, N' -dimethylthiourea (DMTU) and catalase] and NOX inhibitors [ZnCl 2 and diphenyleneiodonium (DPI)], while H 2 O 2 promoted PM H + -ATPase activity at lower concentrations. Repressing effects of Ca +2 antagonists (La +3 and EGTA) on the activity of both the enzymes indicate its possible mediation. Since, unlike animal NOX, the plant versions do not possess proton channel activity, harmonized functioning of PM H + -ATPase and NOX appears to be justified. Plasma membrane NADPH oxidase and H + -ATPase are functionally synchronized and they work cooperatively to maintain the membrane electrical balance while mediating plant cell growth through wall relaxation.

Exercise training decreases NADPH oxidase activity and restores skeletal muscle mass in heart failure rats.

PubMed

Cunha, Telma F; Bechara, Luiz R G; Bacurau, Aline V N; Jannig, Paulo R; Voltarelli, Vanessa A; Dourado, Paulo M; Vasconcelos, Andrea R; Scavone, Cristóforo; Ferreira, Júlio C B; Brum, Patricia C

2017-04-01

We have recently demonstrated that NADPH oxidase hyperactivity, NF-κB activation, and increased p38 phosphorylation lead to atrophy of glycolytic muscle in heart failure (HF). Aerobic exercise training (AET) is an efficient strategy to counteract skeletal muscle atrophy in this syndrome. Therefore, we tested whether AET would regulate muscle redox balance and protein degradation by decreasing NADPH oxidase hyperactivity and reestablishing NF-κB signaling, p38 phosphorylation, and proteasome activity in plantaris muscle of myocardial infarcted-induced HF (MI) rats. Thirty-two male Wistar rats underwent MI or fictitious surgery (SHAM) and were randomly assigned into untrained (UNT) and trained (T; 8 wk of AET on treadmill) groups. AET prevented HF signals and skeletal muscle atrophy in MI-T, which showed an improved exercise tolerance, attenuated cardiac dysfunction and increased plantaris fiber cross-sectional area. To verify the role of inflammation and redox imbalance in triggering protein degradation, circulating TNF-α levels, NADPH oxidase profile, NF-κB signaling, p38 protein levels, and proteasome activity were assessed. MI-T showed a reduced TNF-α levels, NADPH oxidase activity, and Nox2 mRNA expression toward SHAM-UNT levels. The rescue of NADPH oxidase activity induced by AET in MI rats was paralleled by reducing nuclear binding activity of the NF-κB, p38 phosphorylation, atrogin-1, mRNA levels, and 26S chymotrypsin-like proteasome activity. Taken together our data provide evidence for AET improving plantaris redox homeostasis in HF associated with a decreased NADPH oxidase, redox-sensitive proteins activation, and proteasome hyperactivity further preventing atrophy. These data reinforce the role of AET as an efficient therapy for muscle wasting in HF. NEW & NOTEWORTHY This study demonstrates, for the first time, the contribution of aerobic exercise training (AET) in decreasing muscle NADPH oxidase activity associated with reduced reactive oxygen

Increased symplasmic permeability in barley root epidermal cells correlates with defects in root hair development

PubMed Central

Marzec, M; Muszynska, A; Melzer, M; Sas-Nowosielska, H; Kurczynska, E U; Wick, S

2014-01-01

It is well known that the process of plant cell differentiation depends on the symplasmic isolation of cells. Before starting the differentiation programme, the individual cell or group of cells should restrict symplasmic communication with neighbouring cells. We tested the symplasmic communication between epidermal cells in the different root zones of parental barley plants Hordeum vulgare L., cv. ‘Karat’ with normal root hair development, and two root hairless mutants (rhl1.a and rhl1.b). The results clearly show that symplasmic communication was limited during root hair differentiation in the parental variety, whereas in both root hairless mutants epidermal cells were still symplasmically connected in the corresponding root zone. This paper is the first report on the role of symplasmic isolation in barley root cell differentiation, and additionally shows that a disturbance in the restriction of symplasmic communication is present in root hairless mutants. PMID:23927737

Characteristics of injury and recovery of net NO3- transport of barley seedlings from treatments of NaCl

NASA Technical Reports Server (NTRS)

Klobus, G.; Ward, M. R.; Huffaker, R. C.

1988-01-01

The nature of the injury and recovery of nitrate uptake (net uptake) from NaCl stress in young barley (Hordeum vulgare L, var CM 72) seedlings was investigated. Nitrate uptake was inhibited rapidly by NaCl, within 1 minute after exposure to 200 millimolar NaCl. The duration of exposure to saline conditions determined the time of recovery of NO3- uptake from NaCl stress. Recovery was dependent on the presence of NO3- and was inhibited by cycloheximide, 6-methylpurine, and cerulenin, respective inhibitors of protein, RNA, and sterol/fatty acid synthesis. These inhibitors also prevented the induction of the NO3- uptake system in uninduced seedlings. Uninduced seedlings exhibited endogenous NO3- transport activity that appeared to be constitutive. This constitutive activity was also inhibited by NaCl. Recovery of constitutive NO3- uptake did not require the presence of NO3-.

Mapping-by-Sequencing Identifies HvPHYTOCHROME C as a Candidate Gene for the early maturity 5 Locus Modulating the Circadian Clock and Photoperiodic Flowering in Barley

PubMed Central

Pankin, Artem; Campoli, Chiara; Dong, Xue; Kilian, Benjamin; Sharma, Rajiv; Himmelbach, Axel; Saini, Reena; Davis, Seth J; Stein, Nils; Schneeberger, Korbinian; von Korff, Maria

2014-01-01

Phytochromes play an important role in light signaling and photoperiodic control of flowering time in plants. Here we propose that the red/far-red light photoreceptor HvPHYTOCHROME C (HvPHYC), carrying a mutation in a conserved region of the GAF domain, is a candidate underlying the early maturity 5 locus in barley (Hordeum vulgare L.). We fine mapped the gene using a mapping-by-sequencing approach applied on the whole-exome capture data from bulked early flowering segregants derived from a backcross of the Bowman(eam5) introgression line. We demonstrate that eam5 disrupts circadian expression of clock genes. Moreover, it interacts with the major photoperiod response gene Ppd-H1 to accelerate flowering under noninductive short days. Our results suggest that HvPHYC participates in transmission of light signals to the circadian clock and thus modulates light-dependent processes such as photoperiodic regulation of flowering. PMID:24996910

NADPH oxidase-mediated redox signaling promotes oxidative stress resistance and longevity through memo-1 in C. elegans

PubMed Central

Ewald, Collin Yvès; Hourihan, John M; Bland, Monet S; Obieglo, Carolin; Katic, Iskra; Moronetti Mazzeo, Lorenza E; Alcedo, Joy; Blackwell, T Keith; Hynes, Nancy E

2017-01-01

Transient increases in mitochondrially-derived reactive oxygen species (ROS) activate an adaptive stress response to promote longevity. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases produce ROS locally in response to various stimuli, and thereby regulate many cellular processes, but their role in aging remains unexplored. Here, we identified the C. elegans orthologue of mammalian mediator of ErbB2-driven cell motility, MEMO-1, as a protein that inhibits BLI-3/NADPH oxidase. MEMO-1 is complexed with RHO-1/RhoA/GTPase and loss of memo-1 results in an enhanced interaction of RHO-1 with BLI-3/NADPH oxidase, thereby stimulating ROS production that signal via p38 MAP kinase to the transcription factor SKN-1/NRF1,2,3 to promote stress resistance and longevity. Either loss of memo-1 or increasing BLI-3/NADPH oxidase activity by overexpression is sufficient to increase lifespan. Together, these findings demonstrate that NADPH oxidase-induced redox signaling initiates a transcriptional response that protects the cell and organism, and can promote both stress resistance and longevity. DOI: http://dx.doi.org/10.7554/eLife.19493.001 PMID:28085666

SIRPα controls the activity of the phagocyte NADPH oxidase by restricting the expression of gp91(phox).

PubMed

van Beek, Ellen M; Zarate, Julian Alvarez; van Bruggen, Robin; Schornagel, Karin; Tool, Anton T J; Matozaki, Takashi; Kraal, Georg; Roos, Dirk; van den Berg, Timo K

2012-10-25

The phagocyte NADPH oxidase mediates oxidative microbial killing in granulocytes and macrophages. However, because the reactive oxygen species produced by the NADPH oxidase can also be toxic to the host, it is essential to control its activity. Little is known about the endogenous mechanism(s) that limits NADPH oxidase activity. Here, we demonstrate that the myeloid-inhibitory receptor SIRPα acts as a negative regulator of the phagocyte NADPH oxidase. Phagocytes isolated from SIRPα mutant mice were shown to have an enhanced respiratory burst. Furthermore, overexpression of SIRPα in human myeloid cells prevented respiratory burst activation. The inhibitory effect required interactions between SIRPα and its natural ligand, CD47, as well as signaling through the SIRPα cytoplasmic immunoreceptor tyrosine-based inhibitory motifs. Suppression of the respiratory burst by SIRPα was caused by a selective repression of gp91(phox) expression, the catalytic component of the phagocyte NADPH oxidase complex. Thus, SIRPα can limit gp91(phox) expression during myeloid development, thereby controlling the magnitude of the respiratory burst in phagocytes. Copyright © 2012 The Authors. Published by Elsevier Inc. All rights reserved.

A Dedicated Type II NADPH Dehydrogenase Performs the Penultimate Step in the Biosynthesis of Vitamin K1 in Synechocystis and Arabidopsis

PubMed Central

Fatihi, Abdelhak; Latimer, Scott; Schmollinger, Stefan; Block, Anna; Dussault, Patrick H.; Vermaas, Wim F.J.; Merchant, Sabeeha S.; Basset, Gilles J.

2015-01-01

Mutation of Arabidopsis thaliana NAD(P)H DEHYDROGENASE C1 (NDC1; At5g08740) results in the accumulation of demethylphylloquinone, a late biosynthetic intermediate of vitamin K1. Gene coexpression and phylogenomics analyses showed that conserved functional associations occur between vitamin K biosynthesis and NDC1 homologs throughout the prokaryotic and eukaryotic lineages. Deletion of Synechocystis ndbB, which encodes for one such homolog, resulted in the same defects as those observed in the cyanobacterial demethylnaphthoquinone methyltransferase knockout. Chemical modeling and assay of purified demethylnaphthoquinone methyltransferase demonstrated that, by virtue of the strong electrophilic nature of S-adenosyl-l-methionine, the transmethylation of the demethylated precursor of vitamin K is strictly dependent on the reduced form of its naphthoquinone ring. NDC1 was shown to catalyze such a prerequisite reduction by using NADPH and demethylphylloquinone as substrates and flavine adenine dinucleotide as a cofactor. NDC1 displayed Michaelis-Menten kinetics and was markedly inhibited by dicumarol, a competitive inhibitor of naphthoquinone oxidoreductases. These data demonstrate that the reduction of the demethylnaphthoquinone ring represents an authentic step in the biosynthetic pathway of vitamin K, that this reaction is enzymatically driven, and that a selection pressure is operating to retain type II NAD(P)H dehydrogenases in this process. PMID:26023160

S locus-linked F-box genes expressed in anthers of Hordeum bulbosum.

PubMed

Kakeda, Katsuyuki

2009-09-01

Diploid Hordeum bulbosum (a wild relative of cultivated barley) exhibits a two-locus self-incompatibility (SI) system gametophytically controlled by the unlinked multiallelic loci S and Z. This unique SI system is observed in the grasses (Poaceae) including the tribe Triticeae. This paper describes the identification and characterization of two F-box genes cosegregating with the S locus in H. bulbosum, named Hordeum S locus-linked F-box 1 (HSLF1) and HSLF2, which were derived from an S (3) haplotype-specific clone (HAS175) obtained by previous AMF (AFLP-based mRNA fingerprinting) analysis. Sequence analysis showed that both genes encode similar F-box proteins with a C-terminal leucine-rich repeat (LRR) domain, which are distinct from S locus (or S haplotype-specific) F-box protein (SLF/SFB), a class of F-box proteins identified as the pollen S determinant in S-RNase-based gametophytic SI systems. A number of homologous F-box genes with an LRR domain were found in the rice genome, although the functions of the gene family are unknown. One allele of the HSLF1 gene (HSLF1-S (3)) was expressed specifically in mature anthers, whereas no expression was detected from the other two alleles examined. Although the degree of sequence polymorphism among the three HSLF1 alleles was low, a frameshift mutation was found in one of the unexpressed alleles. The HSLF2 gene showed a low level of expression with no tissue specificity as well as little sequence polymorphism among the three alleles. The multiplicity of S locus-linked F-box genes is discussed in comparison with those found in the S-RNase-based SI system.

[Increasing reductant NADPH content via metabolic engineering of PHB synthesis pathway in Synechocystis sp. PCC 6803].

PubMed

Xie, Juan; Zhou, Jie; Zhang, Haifeng; Li, Yin

2011-07-01

Cyanobacteria have become attractive hosts for renewable chemicals production. The low productivity, however, prevents it from industrial application. Reductant NAD(P)H availability is a chief hurdle for the production of reductive metabolites in microbes. To increase NADPH content in Synechocystis sp. PCC 6803, PHB synthase encoding gene phaC and phaE in Synechocystis was inactivated by replacing phaC&E genes with chloromycetin resistance cassette via homologous recombination. PCR analysis showed that mutant S.delta phaC&E with complete genome segregation was generated. The comparison between growth curves of S.wt and S.delta phaC&E indicated the knockout of phaC & phaE genes did not affect obviously the cell growth. Gas chromatography analysis showed that the accumulation of PHB in wild type was about 2.3% of the dry cell weight, whereas no PHB was detected in the mutant S.delta phaC&E. The data indicated that inactivation of PHB synthase gene phaC and phaE interrupted the synthesis of PHB. Further comparative study of wild type and mutant demonstrated that NADPH content in S.delta phaC&E was obviously increased. On the third day, the NADPH content in S.delta phaC&E was up to 1.85 fold higher than that in wild type. These results indicated that deleting PHB synthase gene phaC and phaE not only can block the synthesis of PHB, but also can save NADPH to contribute reductant sink in cyanobacteria. Hence, the engineered cyanobacterial strain S.delta phaC&E, in which carbon flux was redirected and NADPH was increased, will be a potential host strain for chemicals production in cyanobacteria.

Improved NADPH Regeneration for Fungal Cytochrome P450 Monooxygenase by Co-Expressing Bacterial Glucose Dehydrogenase in Resting-Cell Biotransformation of Recombinant Yeast.

PubMed

Jeon, Hyunwoo; Durairaj, Pradeepraj; Lee, Dowoo; Ahsan, Md Murshidul; Yun, Hyungdon

2016-12-28

Fungal cytochrome P450 (CYP) enzymes catalyze versatile monooxygenase reactions and play a major role in fungal adaptations owing to their essential roles in the production avoid metabolites critical for pathogenesis, detoxification of xenobiotics, and exploitation avoid substrates. Although fungal CYP-dependent biotransformation for the selective oxidation avoid organic compounds in yeast system is advantageous, it often suffers from a shortage avoid intracellular NADPH. In this study, we aimed to investigate the use of bacterial glucose dehydrogenase (GDH) for the intracellular electron regeneration of fungal CYP monooxygenase in a yeast reconstituted system. The benzoate hydroxylase FoCYP53A19 and its homologous redox partner FoCPR from Fusarium oxysporum were co-expressed with the BsGDH from Bacillus subtilis in Saccharomyces cerevisiae for heterologous expression and biotransformations. We attempted to optimize several bottlenecks concerning the efficiency of fungal CYP-mediated whole-cell-biotransformation to enhance the conversion. The catalytic performance of the intracellular NADPH regeneration system facilitated the hydroxylation of benzoic acid to 4-hydroxybenzoic acid with high conversion in the resting-cell reaction. The FoCYP53A19 +FoCPR+BsGDH reconstituted system produced 0.47 mM 4-hydroxybenzoic acid (94% conversion) in the resting-cell biotransformations performed in 50 mM phosphate buffer (pH 6.0) containing 0.5 mM benzoic acid and 0.25% glucose for 24 h at 30°C. The "coupled-enzyme" system can certainly improve the overall performance of NADPH-dependent whole-cell biotransformations in a yeast system.

The in-vitro evaluation of antibacterial, antifungal and cytotoxic properties of Marrubium vulgare L. essential oil grown in Tunisia

PubMed Central

2011-01-01

Background In order to validate its antiseptic and anticancer properties with respect to traditional uses, we have screened for the first time the antimicrobial activity of aerial parts of M. vulgare L. essential oil against different pathogenic microorganisms and the cytotoxic activity against HeLa cell lines. Methods The agar disk diffusion method was used to study the antibacterial activity of M. vulgare essential oil against 12 bacterial and 4 fungi strains. The disc diameters of zone of inhibition (DD), the minimum inhibitory concentrations (MIC) and the concentration inhibiting 50% (IC50) were investigated to characterize the antimicrobial activities of this essential oil. The in vitro cytotoxicity of M. vulgare essential oil was examined using a modified MTT assay; the viability and the IC50 were used to evaluate this test. Results The antimicrobial activity of the essential oil was investigated in order to evaluate its efficacy against the different tested microorganisms. The present results results showed a significant activity against microorganisms especially Gram (+) bacteria with inhibition zones and minimal inhibitory concentration values in the range of 6.6-25.2 mm and 1120-2600 μg/ml, respectively, whereas Gram (-) bacteria exhibited a higher resistance. As far as the antifungal activity, among four strains tested, Botrytis cinerea exhibited the strongest activity with inhibition zones of 12.6 mm. However, Fusarium solani, Penicillium digitatum and Aspergillus niger were less sensitive to M. vulgare essential oil. About the citotoxicity assay, this finding indicate the capability of this essential oil to inhibited the proliferation of HeLa cell lines under some conditions with IC50 value of 0.258 μg/ml. Conclusion This investigation showed that the M. vulgare essential oil has a potent antimicrobial activity against some Gram (+) pathogenic bacteria and Botrytis cinerea fungi. The present studies confirm the use of this essential oil as anticancer

The in-vitro evaluation of antibacterial, antifungal and cytotoxic properties of Marrubium vulgare L. essential oil grown in Tunisia.

PubMed

Zarai, Zied; Kadri, Adel; Ben Chobba, Ines; Ben Mansour, Riadh; Bekir, Ahmed; Mejdoub, Hafedh; Gharsallah, Néji

2011-09-21

In order to validate its antiseptic and anticancer properties with respect to traditional uses, we have screened for the first time the antimicrobial activity of aerial parts of M. vulgare L. essential oil against different pathogenic microorganisms and the cytotoxic activity against HeLa cell lines. The agar disk diffusion method was used to study the antibacterial activity of M. vulgare essential oil against 12 bacterial and 4 fungi strains. The disc diameters of zone of inhibition (DD), the minimum inhibitory concentrations (MIC) and the concentration inhibiting 50% (IC50) were investigated to characterize the antimicrobial activities of this essential oil. The in vitro cytotoxicity of M. vulgare essential oil was examined using a modified MTT assay; the viability and the IC50 were used to evaluate this test. The antimicrobial activity of the essential oil was investigated in order to evaluate its efficacy against the different tested microorganisms. The present results results showed a significant activity against microorganisms especially Gram (+) bacteria with inhibition zones and minimal inhibitory concentration values in the range of 6.6-25.2 mm and 1120-2600 μg/ml, respectively, whereas Gram (-) bacteria exhibited a higher resistance. As far as the antifungal activity, among four strains tested, Botrytis cinerea exhibited the strongest activity with inhibition zones of 12.6 mm. However, Fusarium solani, Penicillium digitatum and Aspergillus niger were less sensitive to M. vulgare essential oil. About the citotoxicity assay, this finding indicate the capability of this essential oil to inhibited the proliferation of HeLa cell lines under some conditions with IC50 value of 0.258 μg/ml. This investigation showed that the M. vulgare essential oil has a potent antimicrobial activity against some Gram (+) pathogenic bacteria and Botrytis cinerea fungi. The present studies confirm the use of this essential oil as anticancer agent. Further research is required

Gentamicin differentially alters cellular metabolism of cochlear hair cells as revealed by NAD(P)H fluorescence lifetime imaging

NASA Astrophysics Data System (ADS)

Zholudeva, Lyandysha V.; Ward, Kristina G.; Nichols, Michael G.; Smith, Heather Jensen

2015-05-01

Aminoglycoside antibiotics are implicated as culprits of hearing loss in more than 120,000 individuals annually. Research has shown that the sensory cells, but not supporting cells, of the cochlea are readily damaged and/or lost after use of such antibiotics. High-frequency outer hair cells (OHCs) show a greater sensitivity to antibiotics than high- and low-frequency inner hair cells (IHCs). We hypothesize that variations in mitochondrial metabolism account for differences in susceptibility. Fluorescence lifetime microscopy was used to quantify changes in NAD(P)H in sensory and supporting cells from explanted murine cochleae exposed to mitochondrial uncouplers, inhibitors, and an ototoxic antibiotic, gentamicin (GM). Changes in metabolic state resulted in a redistribution of NAD(P)H between subcellular fluorescence lifetime pools. Supporting cells had a significantly longer lifetime than sensory cells. Pretreatment with GM increased NAD(P)H intensity in high-frequency sensory cells, as well as the NAD(P)H lifetime within IHCs. GM specifically increased NAD(P)H concentration in high-frequency OHCs, but not in IHCs or pillar cells. Variations in NAD(P)H intensity in response to mitochondrial toxins and GM were greatest in high-frequency OHCs. These results demonstrate that GM rapidly alters mitochondrial metabolism, differentially modulates cell metabolism, and provides evidence that GM-induced changes in metabolism are significant and greatest in high-frequency OHCs.

Gentamicin differentially alters cellular metabolism of cochlear hair cells as revealed by NAD(P)H fluorescence lifetime imaging

PubMed Central

Zholudeva, Lyandysha V.; Ward, Kristina G.; Nichols, Michael G.; Smith, Heather Jensen

2015-01-01

Abstract. Aminoglycoside antibiotics are implicated as culprits of hearing loss in more than 120,000 individuals annually. Research has shown that the sensory cells, but not supporting cells, of the cochlea are readily damaged and/or lost after use of such antibiotics. High-frequency outer hair cells (OHCs) show a greater sensitivity to antibiotics than high- and low-frequency inner hair cells (IHCs). We hypothesize that variations in mitochondrial metabolism account for differences in susceptibility. Fluorescence lifetime microscopy was used to quantify changes in NAD(P)H in sensory and supporting cells from explanted murine cochleae exposed to mitochondrial uncouplers, inhibitors, and an ototoxic antibiotic, gentamicin (GM). Changes in metabolic state resulted in a redistribution of NAD(P)H between subcellular fluorescence lifetime pools. Supporting cells had a significantly longer lifetime than sensory cells. Pretreatment with GM increased NAD(P)H intensity in high-frequency sensory cells, as well as the NAD(P)H lifetime within IHCs. GM specifically increased NAD(P)H concentration in high-frequency OHCs, but not in IHCs or pillar cells. Variations in NAD(P)H intensity in response to mitochondrial toxins and GM were greatest in high-frequency OHCs. These results demonstrate that GM rapidly alters mitochondrial metabolism, differentially modulates cell metabolism, and provides evidence that GM-induced changes in metabolism are significant and greatest in high-frequency OHCs. PMID:25688541

Light-quality and temperature-dependent CBF14 gene expression modulates freezing tolerance in cereals.

PubMed

Novák, Aliz; Boldizsár, Ákos; Ádám, Éva; Kozma-Bognár, László; Majláth, Imre; Båga, Monica; Tóth, Balázs; Chibbar, Ravindra; Galiba, Gábor

2016-03-01

C-repeat binding factor 14 (CBF14) is a plant transcription factor that regulates a set of cold-induced genes, contributing to enhanced frost tolerance during cold acclimation. Many CBF genes are induced by cool temperatures and regulated by day length and light quality, which affect the amount of accumulated freezing tolerance. Here we show that a low red to far-red ratio in white light enhances CBF14 expression and increases frost tolerance at 15°C in winter Triticum aesitivum and Hordeum vulgare genotypes, but not in T. monococcum (einkorn), which has a relatively low freezing tolerance. Low red to far-red ratio enhances the expression of PHYA in all three species, but induces PHYB expression only in einkorn. Based on our results, a model is proposed to illustrate the supposed positive effect of phytochrome A and the negative influence of phytochrome B on the enhancement of freezing tolerance in cereals in response to spectral changes of incident light. CBF-regulon, barley, cereals, cold acclimation, freezing tolerance, light regulation, low red/far-red ratio, phytochrome, wheat. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Purification and characterization of NADPH--cytochrome c reductase from the midgut of the southern armyworm (Spodoptera eridania).

PubMed Central

Crankshaw, D L; Hetnarski, K; Wilkinson, C F

1979-01-01

1. NADPH-cytochrome c reductase was solubilized with bromelain and purified about 400-fold from sucrose/pyrophosphate-washed microsomal fractions from southern armyworm (Spodoptera eridania) larval midguts. 2. The enzyme has a mol.wt. of 70 035 +/- 1300 and contained 2 mol of flavin/mol of enzyme consisting of almost equimolar amounts of FMN and FAD. 3. Aerobic titration of the enzyme with NADPH caused the formation of a stable half-reduced state at 0.5 mol of NADPH/mol of flavin. 4. Kinetic analysis showed that the reduction of cytochrome c proceeded by a Bi Bi Ping Pong mechanism. 5. Apparent Km values for NADPH and cytochrome c and Ki values for NADP+ and 2'-AMP were considerably higher for the insect reductase than for the mammalian liver enzyme. 6. These are discussed in relation to possible differences in the active sites of the enzymes. Images Fig. 3. PMID:117798

Purification and characterization of NADPH--cytochrome c reductase from the midgut of the southern armyworm (Spodoptera eridania).

PubMed

Crankshaw, D L; Hetnarski, K; Wilkinson, C F

1979-09-01

1. NADPH-cytochrome c reductase was solubilized with bromelain and purified about 400-fold from sucrose/pyrophosphate-washed microsomal fractions from southern armyworm (Spodoptera eridania) larval midguts. 2. The enzyme has a mol.wt. of 70 035 +/- 1300 and contained 2 mol of flavin/mol of enzyme consisting of almost equimolar amounts of FMN and FAD. 3. Aerobic titration of the enzyme with NADPH caused the formation of a stable half-reduced state at 0.5 mol of NADPH/mol of flavin. 4. Kinetic analysis showed that the reduction of cytochrome c proceeded by a Bi Bi Ping Pong mechanism. 5. Apparent Km values for NADPH and cytochrome c and Ki values for NADP+ and 2'-AMP were considerably higher for the insect reductase than for the mammalian liver enzyme. 6. These are discussed in relation to possible differences in the active sites of the enzymes.

The crystal structure of NAD(P)H oxidase from Lactobacillus sanfranciscensis: insights into the conversion of O2 into two water molecules by the flavoenzyme.

PubMed

Lountos, George T; Jiang, Rongrong; Wellborn, William B; Thaler, Tracey L; Bommarius, Andreas S; Orville, Allen M

2006-08-15

The FAD-dependent NAD(P)H oxidase from Lactobacillus sanfrancisensis (L.san-Nox2) catalyzes the oxidation of 2 equivalents of either NADH or NADPH and reduces 1 equivalent of O(2) to yield 2 equivalents of water. During steady-state turnover only 0.5% of the reducing equivalents are detected in solution as hydrogen peroxide, suggesting that it is not released from the enzyme after the oxidation of the first equivalent of NAD(P)H and reaction with O(2). Here we report the crystal structure of L.san-Nox2 to 1.8 A resolution. The enzyme crystallizes as a dimer with each monomer consisting of a FAD binding domain (residues 1-120), a NAD(P)H binding domain (residues 150-250), and a dimerization domain (residues 325-451). The electron density for the redox-active Cys42 residue located adjacent to the si-face FAD is consistent with oxidation to the sulfenic acid (Cys-SOH) state. The side chain of Cys42 is also observed in two conformations; in one the sulfenic acid is hydrogen bonded to His10 and in the other it hydrogen bonds with the FAD O2' atom. Surprisingly, the NAD(P)H binding domains each contain an ADP ligand as established by electron density maps and MALDI-TOF analysis of the ligands released from heat-denatured enzyme. The ADP ligand copurifies with the enzyme, and its presence does not inhibit enzyme activity. Consequently, we hypothesize that either NADPH or NADH substrates bind via a long channel that extends from the enzyme exterior and terminates at the FAD re-face. A homology model of the NADH oxidase from Lactococcus lactis (L.lac-Nox2) was also generated using the crystal structure of L.san-Nox2, which reveals several important similarities and differences between the two enzymes. HPLC analysis of ligands released from denatured L.lac-Nox2 indicates that it does not bind ADP, which correlates with the specificity of the enzyme for oxidation of NADH.

Rumen fermentation and production effects of Origanum vulgare L. leaves in lactating dairy cows

USDA-ARS?s Scientific Manuscript database

A lactating cow trial was conducted to study the effects of dietary addition of oregano leaf material (Origanum vulgare L.; 0, control vs. 500 g/d, OV) on ruminal fermentation, methane production, total tract digestibility, manure gas emissions, N metabolism, organoleptic characteristics of milk, an...

BIOCHEMICAL COMPOSITION AND NUTRITIONAL EVALUATION OF BARLEY RIHANE (HORDEUM VULGARE L.).

PubMed

Lahouar, Lamia; Ghrairi, Fatma; El Arem, Amira; Medimagh, Sana; El Felah, Mouledi; Salem, Hichem Ben; Achour, Lotfi

2017-01-01

Many experimental studies have suggested an important role for barley Rihane(BR)in the prevention of colon cancer and cardiovascular diseases. The objective of this study was to evaluate the physico-chemical properties and nutritional characterizations of BR compared to other varieties grown in Tunisia (Manel, Roho and Tej). Total, insoluble and soluble dietary fiber(β-glucan), total protein, ash and some minerals of BR and Tunisian barley varieties were determined. The results revealed that BR is good source of dietary fiber mainly β-glucan compared to the other varieties. This variety is a relatively rich source of phosphorous and potassium and it contains many important unsaturated fatty acids. BR has higher nutritional value than other varieties. Barley Rihane has significant nutritional characterizations compared to others Tunisian barleys varieties. Abbreviations: BR, Barley Rihane; LDL, low density lipoprotein; HDL, high density lipoprotein; AOM, azoxymethane; TBV, Tunisian barley varieties; TGW, thousand grain weight; SW, weight specific; TDF, total dietary fiber; IDF, insoluble dietary fiber; SDF, soluble dietary fiber; DM, Dry Matter.

BIOCHEMICAL COMPOSITION AND NUTRITIONAL EVALUATION OF BARLEY RIHANE (HORDEUM VULGARE L.)

PubMed Central

Lahouar, Lamia; Ghrairi, Fatma; El Arem, Amira; Medimagh, Sana; El Felah, Mouledi; Salem, Hichem Ben; Achour, Lotfi

2017-01-01

Background: Many experimental studies have suggested an important role for barley Rihane(BR)in the prevention of colon cancer and cardiovascular diseases. The objective of this study was to evaluate the physico-chemical properties and nutritional characterizations of BR compared to other varieties grown in Tunisia (Manel, Roho and Tej). Material and Methods: Total, insoluble and soluble dietary fiber(β-glucan), total protein, ash and some minerals of BR and Tunisian barley varieties were determined. Results: The results revealed that BR is good source of dietary fiber mainly β-glucan compared to the other varieties. This variety is a relatively rich source of phosphorous and potassium and it contains many important unsaturated fatty acids. BR has higher nutritional value than other varieties. Conclusion: Barley Rihane has significant nutritional characterizations compared to others Tunisian barleys varieties. Abbreviations: BR, Barley Rihane; LDL, low density lipoprotein; HDL, high density lipoprotein; AOM, azoxymethane; TBV, Tunisian barley varieties; TGW, thousand grain weight; SW, weight specific; TDF, total dietary fiber; IDF, insoluble dietary fiber; SDF, soluble dietary fiber; DM, Dry Matter. PMID:28480409

Virulent Diuraphis noxia Aphids Over-Express Calcium Signaling Proteins to Overcome Defenses of Aphid-Resistant Wheat Plants

PubMed Central

Sinha, Deepak K.; Chandran, Predeesh; Timm, Alicia E.; Aguirre-Rojas, Lina; Smith, C. Michael

2016-01-01

The Russian wheat aphid, Diuraphis noxia, an invasive phytotoxic pest of wheat, Triticum aestivum, and barley, Hordeum vulgare, causes huge economic losses in Africa, South America, and North America. Most acceptable and ecologically beneficial aphid management strategies include selection and breeding of D. noxia-resistant varieties, and numerous D. noxia resistance genes have been identified in T. aestivum and H. vulgare. North American D. noxia biotype 1 is avirulent to T. aestivum varieties possessing Dn4 or Dn7 genes, while biotype 2 is virulent to Dn4 and avirulent to Dn7. The current investigation utilized next-generation RNAseq technology to reveal that biotype 2 over expresses proteins involved in calcium signaling, which activates phosphoinositide (PI) metabolism. Calcium signaling proteins comprised 36% of all transcripts identified in the two D. noxia biotypes. Depending on plant resistance gene-aphid biotype interaction, additional transcript groups included those involved in tissue growth; defense and stress response; zinc ion and related cofactor binding; and apoptosis. Activation of enzymes involved in PI metabolism by D. noxia biotype 2 aphids allows depletion of plant calcium that normally blocks aphid feeding sites in phloem sieve elements and enables successful, continuous feeding on plants resistant to avirulent biotype 1. Inhibition of the key enzyme phospholipase C significantly reduced biotype 2 salivation into phloem and phloem sap ingestion. PMID:26815857

Absence of Proton Channels in COS-7 Cells Expressing Functional NADPH Oxidase Components

PubMed Central

Morgan, Deri; Cherny, Vladimir V.; Price, Marianne O.; Dinauer, Mary C.; DeCoursey, Thomas E.

2002-01-01

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is an enzyme of phagocytes that produces bactericidal superoxide anion (O2 −) via an electrogenic process. Proton efflux compensates for the charge movement across the cell membrane. The proton channel responsible for the H+ efflux was thought to be contained within the gp91phox subunit of NADPH oxidase, but recent data do not support this idea (DeCoursey, T.E., V.V. Cherny, D. Morgan, B.Z. Katz, and M.C. Dinauer. 2001. J. Biol. Chem. 276:36063–36066). In this study, we investigated electrophysiological properties and superoxide production of COS-7 cells transfected with all NADPH oxidase components required for enzyme function (COSphox). The 7D5 antibody, which detects an extracellular epitope of the gp91phox protein, labeled 96–98% of COSphox cells. NADPH oxidase was functional because COSphox (but not COSWT) cells stimulated by phorbol myristate acetate (PMA) or arachidonic acid (AA) produced superoxide anion. No proton currents were detected in either wild-type COS-7 cells (COSWT) or COSphox cells studied at pHo 7.0 and pHi 5.5 or 7.0. Anion currents that decayed at voltages positive to 40 mV were the only currents observed. PMA or AA did not elicit detectable H+ current in COSWT or COSphox cells. Therefore, gp91phox does not function as a proton channel in unstimulated cells or in activated cells with a demonstrably functional oxidase. PMID:12034764

Increasing anaerobic acetate consumption and ethanol yields in Saccharomyces cerevisiae with NADPH-specific alcohol dehydrogenase.

PubMed

Henningsen, Brooks M; Hon, Shuen; Covalla, Sean F; Sonu, Carolina; Argyros, D Aaron; Barrett, Trisha F; Wiswall, Erin; Froehlich, Allan C; Zelle, Rintze M

2015-12-01

Saccharomyces cerevisiae has recently been engineered to use acetate, a primary inhibitor in lignocellulosic hydrolysates, as a cosubstrate during anaerobic ethanolic fermentation. However, the original metabolic pathway devised to convert acetate to ethanol uses NADH-specific acetylating acetaldehyde dehydrogenase and alcohol dehydrogenase and quickly becomes constrained by limited NADH availability, even when glycerol formation is abolished. We present alcohol dehydrogenase as a novel target for anaerobic redox engineering of S. cerevisiae. Introduction of an NADPH-specific alcohol dehydrogenase (NADPH-ADH) not only reduces the NADH demand of the acetate-to-ethanol pathway but also allows the cell to effectively exchange NADPH for NADH during sugar fermentation. Unlike NADH, NADPH can be freely generated under anoxic conditions, via the oxidative pentose phosphate pathway. We show that an industrial bioethanol strain engineered with the original pathway (expressing acetylating acetaldehyde dehydrogenase from Bifidobacterium adolescentis and with deletions of glycerol-3-phosphate dehydrogenase genes GPD1 and GPD2) consumed 1.9 g liter(-1) acetate during fermentation of 114 g liter(-1) glucose. Combined with a decrease in glycerol production from 4.0 to 0.1 g liter(-1), this increased the ethanol yield by 4% over that for the wild type. We provide evidence that acetate consumption in this strain is indeed limited by NADH availability. By introducing an NADPH-ADH from Entamoeba histolytica and with overexpression of ACS2 and ZWF1, we increased acetate consumption to 5.3 g liter(-1) and raised the ethanol yield to 7% above the wild-type level. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Feminizing Wolbachia influence microbiota composition in the terrestrial isopod Armadillidium vulgare.

PubMed

Dittmer, Jessica; Bouchon, Didier

2018-05-03

Wolbachia are widespread heritable endosymbionts of arthropods notorious for their profound effects on host fitness as well as for providing protection against viruses and eukaryotic parasites, indicating that they can interact with other microorganisms sharing the same host environment. Using the terrestrial isopod crustacean Armadillidium vulgare, its highly diverse microbiota (>200 bacterial genera) and its three feminizing Wolbachia strains (wVulC, wVulM, wVulP) as a model system, the present study demonstrates that Wolbachia can even influence the composition of a diverse bacterial community under both laboratory and natural conditions. While host origin is the major determinant of the taxonomic composition of the microbiota in A. vulgare, Wolbachia infection affected both the presence and, more importantly, the abundance of many bacterial taxa within each host population, possibly due to competitive interactions. Moreover, different Wolbachia strains had different impacts on microbiota composition. As such, infection with wVulC affected a higher number of taxa than infection with wVulM, possibly due to intrinsic differences in virulence and titer between these two strains. In conclusion, this study shows that heritable endosymbionts such as Wolbachia can act as biotic factors shaping the microbiota of arthropods, with as yet unknown consequences on host fitness.

Cytotoxicity of the Essential Oil of Fennel (Foeniculum vulgare) from Tajikistan

PubMed Central

Valiev, Abdujabbor; Satyal, Prabodh; Gulmurodov, Isomiddin; Yusufi, Salomudin; Setzer, William N.

2017-01-01

The essential oil of fennel (Foeniculum vulgare) is rich in lipophilic secondary metabolites, which can easily cross cell membranes by free diffusion. Several constituents of the oil carry reactive carbonyl groups in their ring structures. Carbonyl groups can react with amino groups of amino acid residues in proteins or in nucleotides of DNA to form Schiff’s bases. Fennel essential oil is rich in anise aldehyde, which should interfere with molecular targets in cells. The aim of the present study was to investigate the chemical composition of the essential oil of fennel growing in Tajikistan. Gas chromatographic-mass spectrometric analysis revealed that the main components of F. vulgare oil were trans-anethole (36.8%); α-ethyl-p-methoxy-benzyl alcohol (9.1%); p-anisaldehyde (7.7%); carvone (4.9%); 1-phenyl-penta-2,4-diyne (4.8%) and fenchyl butanoate (4.2%). The oil exhibited moderate antioxidant activities. The potential cytotoxic activity was studied against HeLa (human cervical cancer), Caco-2 (human colorectal adenocarcinoma), MCF-7 (human breast adenocarcinoma), CCRF-CEM (human T lymphoblast leukaemia) and CEM/ADR5000 (adriamycin resistant leukaemia) cancer cell lines; IC50 values were between 30–210 mg L−1 and thus exhibited low cytotoxicity as compared to cytotoxic reference compounds. PMID:28846628

NADPH oxidase: an enzyme for multicellularity?

PubMed

Lalucque, Hervé; Silar, Philippe

2003-01-01

Multicellularity has evolved several times during the evolution of eukaryotes. One evolutionary pressure that permits multicellularity relates to the division of work, where one group of cells functions as nutrient providers and the other in specialized roles such as defence or reproduction. This requires signalling systems to ensure harmonious development of multicellular structures. Here, we show that NADPH oxidases are specifically present in organisms that differentiate multicellular structures during their life cycle and are absent from unicellular life forms. The biochemical properties of these enzymes make them ideal candidates for a role in intercellular signalling.

Chemical composition and bioactivity of different oregano (Origanum vulgare) extracts and essential oil.

PubMed

Teixeira, Bárbara; Marques, António; Ramos, Cristina; Serrano, Carmo; Matos, Olívia; Neng, Nuno R; Nogueira, José M F; Saraiva, Jorge Alexandre; Nunes, Maria Leonor

2013-08-30

There is a growing interest in industry to replace synthetic chemicals by natural products with bioactive properties. Aromatic plants are excellent sources of bioactive compounds that can be extracted using several processes. As far as oregano is concerned, studies are lacking addressing the effect of extraction processes in bioactivity of extracts. This study aimed to characterise the in vitro antioxidant and antibacterial properties of oregano (Origanum vulgare) essential oil and extracts (in hot and cold water, and ethanol), and the chemical composition of its essential oil. The major components of oregano essential oil were carvacrol, β-fenchyl alcohol, thymol, and γ-terpinene. Hot water extract had the strongest antioxidant properties and the highest phenolic content. All extracts were ineffective in inhibiting the growth of the seven tested bacteria. In contrast, the essential oil inhibited the growth of all bacteria, causing greater reductions on both Listeria strains (L. monocytogenes and L. innocua). O. vulgare extracts and essential oil from Portuguese origin are strong candidates to replace synthetic chemicals used by the industry. © 2013 Society of Chemical Industry.

Astragaloside IV prevents damage to human mesangial cells through the inhibition of the NADPH oxidase/ROS/Akt/NF‑κB pathway under high glucose conditions.

PubMed

Sun, Li; Li, Weiping; Li, Weizu; Xiong, Li; Li, Guiping; Ma, Rong

2014-07-01

Glomerular hypertrophy and hyperfiltration are the two major pathological characteristics of the early stages of diabetic nephropathy (DN), which are respectively related to mesangial cell (MC) proliferation and a decrease in calcium influx conducted by canonical transient receptor potential cation channel 6 (TRPC6). The marked increase in the production of reactive oxygen species (ROS) induced by hyperglycemia is the main sponsor of multiple pathological pathways in DN. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is an important source of ROS production in MCs. Astragaloside IV (AS‑IV) is an active ingredient of Radix Astragali which has a potent antioxidative effect. In this study, we aimed to investigate whether high glucose (HG)‑induced NADPH oxidase activation and ROS production contribute to MC proliferation and the downregulation of TRPC6 expression; we also wished to determine the effects of AS‑IV on MCs under HG conditions. Using a human glomerular mesangial cell line, we found that treatment with AS‑IV for 48 h markedly attenuated HG‑induced proliferation and the hypertrophy of MCs in a dose‑dependent manner. The intracellular ROS level was also markedly reduced following treatment with AS‑IV. In addition, the enhanced activity of NADPH oxidase and the expression level of NADPH oxidase 4 (Nox4) protein were decreased. Treatment with AS‑IV also inhibited the phosphorylation level of Akt and IκBα in the MCs. In addition, TRPC6 protein expression and the intracellular free calcium concentration were also markedly reduced following treatment with AS‑IV under HG conditions. These results suggest that AS‑IV inhibits HG‑induced mesangial cell proliferation and glomerular contractile dysfunction through the NADPH oxidase/ROS/Akt/nuclear factor‑κB (NF‑κB) pathway, providing a new perspective for the clinical treatment of DN.

Chemical characterization and antifungal activity of Origanum vulgare, Thymus vulgaris essential oils and carvacrol against Malassezia furfur.

PubMed

Vinciguerra, Vittorio; Rojas, Florencia; Tedesco, Viviana; Giusiano, Gustavo; Angiolella, Letizia

2018-05-04

The composition of the essential oils (EOs) of O. vulgare L. EO and T. vulgaris EO, were analyzed by GC and GC-MS. Antifungal activities of the EOs and its main component, carvacrol, were evaluated against 27 clinical isolates of Malassezia furfur. Minimum inhibitory concentrations (MICs) were measured according to the broth microdilution protocols by Clinical and Laboratory Standards Institute (CLSI) modified for Malassezia spp. EOs and carvacrol showed low MIC values ranged 450-900 μg/ml against M. furfur. No differences in EOs antifungal activity were observed in sensitive to resistant fluconazole isolates. The antifungal activity obtained showed O. vulgare EO, T. vulgaris EO and carvacrol, their compound, as potential antimicrobial agents against M. furfur, yeast associated with human mycoses.

Absence of proton channels in COS-7 cells expressing functional NADPH oxidase components.

PubMed

Morgan, Deri; Cherny, Vladimir V; Price, Marianne O; Dinauer, Mary C; DeCoursey, Thomas E

2002-06-01

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is an enzyme of phagocytes that produces bactericidal superoxide anion (O(2)(-)) via an electrogenic process. Proton efflux compensates for the charge movement across the cell membrane. The proton channel responsible for the H(+) efflux was thought to be contained within the gp91(phox) subunit of NADPH oxidase, but recent data do not support this idea (DeCoursey, T.E., V.V. Cherny, D. Morgan, B.Z. Katz, and M.C. Dinauer. 2001. J. Biol. Chem. 276:36063-36066). In this study, we investigated electrophysiological properties and superoxide production of COS-7 cells transfected with all NADPH oxidase components required for enzyme function (COS(phox)). The 7D5 antibody, which detects an extracellular epitope of the gp91(phox) protein, labeled 96-98% of COS(phox) cells. NADPH oxidase was functional because COS(phox) (but not COS(WT)) cells stimulated by phorbol myristate acetate (PMA) or arachidonic acid (AA) produced superoxide anion. No proton currents were detected in either wild-type COS-7 cells (COS(WT)) or COS(phox) cells studied at pH(o) 7.0 and pH(i) 5.5 or 7.0. Anion currents that decayed at voltages positive to 40 mV were the only currents observed. PMA or AA did not elicit detectable H(+) current in COS(WT) or COS(phox) cells. Therefore, gp91(phox) does not function as a proton channel in unstimulated cells or in activated cells with a demonstrably functional oxidase.

Differential growth response of various crop species to arbuscular mycorrhizal inoculation.

PubMed

Eo, Ju-Kyeong; Eom, Ahn-Heum

2009-03-01

To investigate the growth response of various crop species to mycorrhizal inoculation, arbuscular mycorrhizal fungi were applied to Glycine max, Vigna angularis, Senna tora, Hordeum vulgare var. hexastichon. Zea mays, Sorghum bicolor, Allium tuberosum, Solanum melongena, and Capsicum annuum. The biomass of the inoculated crops was measured every two weeks for the 12-week growth period. By measuring biomass, we calculated the mycorrhizal responsiveness of the nine crop species. Among the nine crop species, four species showed a significant response to mycorrhizal inoculation. The shoot biomasses of V. angularis, C. annuum, A. tuberosum, and S. tora significantly increased with mycorrhizal inoculation.

Mercury in mercury(II)-spiked soils is highly susceptible to plant bioaccumulation.

PubMed

Hlodák, Michal; Urík, Martin; Matúš, Peter; Kořenková, Lucia

2016-01-01

Heavy metal phytotoxicity assessments usually use soluble metal compounds in spiked soils to evaluate metal bioaccumulation, growth inhibition and adverse effects on physiological parameters. However, exampling mercury phytotoxicity for barley (Hordeum vulgare) this paper highlights unsuitability of this experimental approach. Mercury(II) in spiked soils is extremely bioavailable, and there experimentally determined bioaccumulation is significantly higher compared to reported mercury bioaccumulation efficiency from soils collected from mercury-polluted areas. Our results indicate this is not affected by soil sorption capacity, thus soil ageing and formation of more stable mercuric complexes with soil fractions is necessary for reasonable metal phytotoxicity assessments.

The Type II NADPH Dehydrogenase Facilitates Cyclic Electron Flow, Energy-Dependent Quenching, and Chlororespiratory Metabolism during Acclimation of Chlamydomonas reinhardtii to Nitrogen Deprivation1[OPEN

PubMed Central

Grossman, Arthur R.

2016-01-01

When photosynthetic organisms are deprived of nitrogen (N), the capacity to grow and assimilate carbon becomes limited, causing a decrease in the productive use of absorbed light energy and likely a rise in the cellular reduction state. Although there is a scarcity of N in many terrestrial and aquatic environments, a mechanistic understanding of how photosynthesis adjusts to low-N conditions and the enzymes/activities integral to these adjustments have not been described. In this work, we use biochemical and biophysical analyses of photoautotrophically grown wild-type and mutant strains of Chlamydomonas reinhardtii to determine the integration of electron transport pathways critical for maintaining active photosynthetic complexes even after exposure of cells to N deprivation for 3 d. Key to acclimation is the type II NADPH dehydrogenase, NDA2, which drives cyclic electron flow (CEF), chlororespiration, and the generation of an H+ gradient across the thylakoid membranes. N deprivation elicited a doubling of the rate of NDA2-dependent CEF, with little contribution from PGR5/PGRL1-dependent CEF. The H+ gradient generated by CEF is essential to sustain nonphotochemical quenching, while an increase in the level of reduced plastoquinone would promote a state transition; both are necessary to down-regulate photosystem II activity. Moreover, stimulation of NDA2-dependent chlororespiration affords additional relief from the elevated reduction state associated with N deprivation through plastid terminal oxidase-dependent water synthesis. Overall, rerouting electrons through the NDA2 catalytic hub in response to photoautotrophic N deprivation sustains cell viability while promoting the dissipation of excess excitation energy through quenching and chlororespiratory processes. PMID:26858365

The potential of Origanum vulgare L. (Lamiaceae) essential oil in inhibiting the growth of some food-related Aspergillus species

PubMed Central

Carmo, Egberto Santos; de Oliveira Lima, Edeltrudes; de Souza, Evandro Leite

2008-01-01

Origanum vulgare L. (Lamiaceae) has been currently known for their interesting antimicrobial activity being regarded as alternative antimicrobial for use is food conservation systems. This study aimed to evaluate the effectiveness of O. vulgare essential oil in inhibiting the growth of some food-related Aspergillus species (A. flavus, A. parasiticus, A. terreus, A. ochraceus, A. fumigatus and A. niger). The essential oil revealed a strong anti-Aspergillus property providing an inhibition of all assayed mould strains. MIC values were between 80 and 20 μL/mL being found a MIC50 of 40 μL/mL. The essential oil at concentration of 80 and 40 μL/mL provided a fungicidal effect on A. flavus, A. fumigatus and A. niger noted by a total inhibition of the radial mycelial growth along 14 days of interaction. In addition, the essential oil was able to inhibit the mould spores germination when assayed at concentrations of 80 and 40 μL/mL. Our results showed the interesting anti-Aspergillus activity of O. vulgare essential oil supporting their possible use as anti-mould compound in food conservation. PMID:24031231

Heterogeneity of Powdery Mildew Resistance Revealed in Accessions of the ICARDA Wild Barley Collection

PubMed Central

Dreiseitl, Antonin

2017-01-01

The primary genepool of barley comprises two subspecies – wild barley (Hordeum vulgare subsp. spontaneum) and cultivated barley H. vulgare. subsp. vulgare. The former originated 5.5 million years ago in southwest Asia and is the immediate ancestor of cultivated barley, which arose around 10,000 years ago. In this study, the specific resistance of a set of 146 wild barley accessions, maintained by the International Center for Agriculture Research in the Dry Areas (ICARDA), to 32 isolates of barley powdery mildew caused by Blumeria graminis f. sp. hordei was evaluated. The set comprised 146 heterogeneous accessions of a previously tested collection. Seed was obtained by single seed descent and each accession was usually represented by five single plant progenies. In total, 687 plant progenies were tested. There were 211 phenotypes of resistance among the accessions, 87 of which were found in single plants, while 202 plants contained the eight most common phenotypes. The most frequent phenotype was found in 56 plants that were susceptible to all pathogen isolates, whereas the second most frequent phenotype, which occurred in 46 plants, was resistant to all isolates. The broad resistance diversity that was revealed is of practical importance and is an aid to determining the extent and role of resistance in natural ecosystems. PMID:28261253

Effects of Anti-Tobacco Advertisements Based on Risk-Taking Tendencies: Realistic Fear vs. Vulgar Humor.

ERIC Educational Resources Information Center

Lee, Moon J.; Ferguson, Mary Ann

2002-01-01

Explores how college students with different risk-taking tendencies responded to different emotional appeals in anti-tobacco advertisements. Finds that rebellious participants who watched realistic fear advertisements reported higher levels of interest than those who watched the vulgar humor advertisements. Explains that impulsive participants…

Effect of lixiviated sediments affected with treated water on Selenastrum capricornutum, Printz and Origanum vulgare L.

PubMed

Ramos, Guadalupe E; Lopez, Martin H; Flores, Antonio M; Figueroa, Guadalupe T; De Leon, Fernando G

2010-01-01

Xochimilco is an area of Mexico City fulfilling important ecological functions. However, the water of the canal network in the lacustrine zone of Xochimilco is supplied by the water treatment plants of the city, implying a risk of accumulated contaminants in the sediments. This study reports the effect of lixiviates obtained from sediments collected in the canals of Xochimilco on the growth of the alga Selenastrum capricornutum and the angiosperm Origanum vulgare. Three factors were tested: (a) water source in terms of the effluent from the two water treatment plants (urban waste-water, located at Cerro de la Estrella (CE) and urban-rural waters at San Luis Tlaxialtemalco (SLT); (b) sampling season (January, dry season; May and September, rainy season; and (c) distances from the water discharge point in the Xochimilco's main canal (5200 and 1000 m for CE, and 0, 200 m for SLT). The chemical water properties analyzed were: pH, electrical conductivity, N-NO(3), N-NH(3), N(Total), P-PO(4) and P(Total). The alga was more sensitive to the contaminants than O. vulgare, showing growth inhibition of 93-100%. The effect of sampling season on the inhibition of algal growth was ordered as follows: September > May > January. Lixiviates obtained from sediment samples 200 and 1000 m from the main point of water discharge caused a higher algal growth inhibition than the samples obtained at the source point. Lixiviate promoted the growth of seedlings of O. vulgare.

Species-Level Phylogeny and Polyploid Relationships in Hordeum (Poaceae) Inferred by Next-Generation Sequencing and In Silico Cloning of Multiple Nuclear Loci.

PubMed

Brassac, Jonathan; Blattner, Frank R

2015-09-01

Polyploidization is an important speciation mechanism in the barley genus Hordeum. To analyze evolutionary changes after allopolyploidization, knowledge of parental relationships is essential. One chloroplast and 12 nuclear single-copy loci were amplified by polymerase chain reaction (PCR) in all Hordeum plus six out-group species. Amplicons from each of 96 individuals were pooled, sheared, labeled with individual-specific barcodes and sequenced in a single run on a 454 platform. Reference sequences were obtained by cloning and Sanger sequencing of all loci for nine supplementary individuals. The 454 reads were assembled into contigs representing the 13 loci and, for polyploids, also homoeologues. Phylogenetic analyses were conducted for all loci separately and for a concatenated data matrix of all loci. For diploid taxa, a Bayesian concordance analysis and a coalescent-based dated species tree was inferred from all gene trees. Chloroplast matK was used to determine the maternal parent in allopolyploid taxa. The relative performance of different multilocus analyses in the presence of incomplete lineage sorting and hybridization was also assessed. The resulting multilocus phylogeny reveals for the first time species phylogeny and progenitor-derivative relationships of all di- and polyploid Hordeum taxa within a single analysis. Our study proves that it is possible to obtain a multilocus species-level phylogeny for di- and polyploid taxa by combining PCR with next-generation sequencing, without cloning and without creating a heavy load of sequence data. © The Author(s) 2015. Published by Oxford University Press, on behalf of the Society of Systematic Biologists.

Enhancement of nitrate uptake and growth of barley seedlings by calcium under saline conditions

NASA Technical Reports Server (NTRS)

Ward, M. R.; Aslam, M.; Huffaker, R. C.

1986-01-01

The effect of Ca2+ on NO3- assimilation in young barley (Hordeum vulgare L. var CM 72) seedlings in the presence and absence of NaCl was studied. Calcium increased the activity of the NO3- transporter under saline conditions, but had little effect under nonsaline conditions. Calcium decreased the induction period for the NO3- transporter under both saline and nonsaline conditions but had little effect on its apparent Km for NO3- both in the presence and absence of NaCl. The enhancement of NO3- transport by Ca2+ under saline conditions was dependent on the presence of Ca2+ in the uptake solution along with the salt, since Ca2+ had no effect when supplied before or after salinity stress. Although Mn2+ and Mg2+ enhanced NO3- uptake under saline conditions, neither was as effective as Ca2+. In longer studies, increasing the Ca2+ concentration in saline nutrient solutions resulted in increases in NO3- assimilation and seedling growth.

Arg279 is the key regulator of coenzyme selectivity in the flavin-dependent ornithine monooxygenase SidA.

PubMed

Robinson, Reeder; Franceschini, Stefano; Fedkenheuer, Michael; Rodriguez, Pedro J; Ellerbrock, Jacob; Romero, Elvira; Echandi, Maria Paulina; Martin Del Campo, Julia S; Sobrado, Pablo

2014-04-01

Siderophore A (SidA) is a flavin-dependent monooxygenase that catalyzes the NAD(P)H- and oxygen-dependent hydroxylation of ornithine in the biosynthesis of siderophores in Aspergillus fumigatus and is essential for virulence. SidA can utilize both NADPH or NADH for activity; however, the enzyme is selective for NADPH. Structural analysis shows that R279 interacts with the 2'-phosphate of NADPH. To probe the role of electrostatic interactions in coenzyme selectivity, R279 was mutated to both an alanine and a glutamate. The mutant proteins were active but highly uncoupled, oxidizing NADPH and producing hydrogen peroxide instead of hydroxylated ornithine. For wtSidA, the catalytic efficiency was 6-fold higher with NADPH as compared to NADH. For the R279A mutant the catalytic efficiency was the same with both coenyzmes, while for the R279E mutant the catalytic efficiency was 5-fold higher with NADH. The effects are mainly due to an increase in the KD values, as no major changes on the kcat or flavin reduction values were observed. Thus, the absence of a positive charge leads to no coenzyme selectivity while introduction of a negative charge leads to preference for NADH. Flavin fluorescence studies suggest altered interaction between the flavin and NADP⁺ in the mutant enzymes. The effects are caused by different binding modes of the coenzyme upon removal of the positive charge at position 279, as no major conformational changes were observed in the structure for R279A. The results indicate that the positive charge at position 279 is critical for tight binding of NADPH and efficient hydroxylation. Copyright © 2014 Elsevier B.V. All rights reserved.

Purification and Partial Characterization of Two Soluble NAD(P)H Dehydrogenases from Arum maculatum Mitochondria 1

PubMed Central

Chauveau, Michèle; Lance, Claude

1991-01-01

Two enzyme systems carrying out the oxidation of NAD(P)H in the presence of various electron acceptors have been isolated and partially characterized from the supernatant of frozen-thawed mitochondria from Arum maculatum spadices. The two systems contain flavoproteins and differ by their ability to oxidize NADH or NADPH, optimum pH and pI values, sensitivity to Ca2+ and EGTA, denaturation by 4 molar urea, molecular mass, and number of subunits. These properties, together with methodological considerations, are compatible with the location of these enzyme activities on the outer surface of the inner mitochondrial membrane, and support the hypothesis of the existence of two separate dehydrogenases responsible for the mitochondrial oxidation of cytosolic NADH and NADPH. Images Figure 1 Figure 3 Figure 7 PMID:16668075

Conformational Changes of NADPH-Cytochrome P450 Oxidoreductase Are Essential for Catalysis and Cofactor Binding*

PubMed Central

Xia, Chuanwu; Hamdane, Djemel; Shen, Anna L.; Choi, Vivian; Kasper, Charles B.; Pearl, Naw May; Zhang, Haoming; Im, Sang-Choul; Waskell, Lucy; Kim, Jung-Ja P.

2011-01-01

The crystal structure of NADPH-cytochrome P450 reductase (CYPOR) implies that a large domain movement is essential for electron transfer from NADPH via FAD and FMN to its redox partners. To test this hypothesis, a disulfide bond was engineered between residues Asp147 and Arg514 in the FMN and FAD domains, respectively. The cross-linked form of this mutant protein, designated 147CC514, exhibited a significant decrease in the rate of interflavin electron transfer and large (≥90%) decreases in rates of electron transfer to its redox partners, cytochrome c and cytochrome P450 2B4. Reduction of the disulfide bond restored the ability of the mutant to reduce its redox partners, demonstrating that a conformational change is essential for CYPOR function. The crystal structures of the mutant without and with NADP+ revealed that the two flavin domains are joined by a disulfide linkage and that the relative orientations of the two flavin rings are twisted ∼20° compared with the wild type, decreasing the surface contact area between the two flavin rings. Comparison of the structures without and with NADP+ shows movement of the Gly631–Asn635 loop. In the NADP+-free structure, the loop adopts a conformation that sterically hinders NADP(H) binding. The structure with NADP+ shows movement of the Gly631–Asn635 loop to a position that permits NADP(H) binding. Furthermore, comparison of these mutant and wild type structures strongly suggests that the Gly631–Asn635 loop movement controls NADPH binding and NADP+ release; this loop movement in turn facilitates the flavin domain movement, allowing electron transfer from FMN to the CYPOR redox partners. PMID:21345800

Adaptive variation in offspring size in the terrestrial isopod, Armadillidium vulgare

USGS Publications Warehouse

Brody, M.S.; Lawlor, L.R.

1984-01-01

Variation in the birth size of offspring of the terrestrial isopod, Armadillidium vulgare, was observed in laboratory experiments and in field populations. In the laboratory, larger offspring were produced when the mother's food supply was reduced. In field populations, larger offspring were produced during the summer, a period of reduced food availability. Smaller offspring are produced in the spring, when food is readily available. Females may be making larger young to increase survival during the more severe conditions of the summer breeding period.

Pyrrolizidine Alkaloids from Echium vulgare in Honey Originate Primarily from Floral Nectar.

PubMed

Lucchetti, Matteo A; Glauser, Gaetan; Kilchenmann, Verena; Dübecke, Arne; Beckh, Gudrun; Praz, Christophe; Kast, Christina

2016-06-29

Pyrrolizidine alkaloids (PAs) in honey can be a potential human health risk. So far, it has remained unclear whether PAs in honey originate from pollen or floral nectar. We obtained honey, nectar, and plant pollen from two observation sites where Echium vulgare L. was naturally abundant. The PA concentration of honey was determined by targeted analysis using a high pressure liquid chromatography-mass spectrometry system (HPLC-MS/MS), allowing the quantification of six different PAs and PA-N-oxides present in E. vulgare. Echium-type PAs were detected up to 0.153 μg/g in honey. Nectar and plant pollen were analyzed by nontargeted analysis using ultrahigh pressure liquid chromatography-high resolution-mass spectrometry (UHPLC-HR-MS), allowing the detection of 10 alkaloids in small size samples. Echium-type PAs were detected between 0.3-95.1 μg/g in nectar and 500-35000 μg/g in plant pollen. The PA composition in nectar and plant pollen was compared to the composition in honey. Echimidine (+N-oxide) was the main alkaloid detected in honey and nectar samples, while echivulgarine (+N-oxide) was the main PA found in plant pollen. These results suggest that nectar contributes more significantly to PA contamination in honey than plant pollen.

The composite water and solute transport of barley (Hordeum vulgare) roots: effect of suberized barriers.

PubMed

Ranathunge, Kosala; Kim, Yangmin X; Wassmann, Friedrich; Kreszies, Tino; Zeisler, Viktoria; Schreiber, Lukas

2017-03-01

Roots have complex anatomical structures, and certain localized cell layers develop suberized apoplastic barriers. The size and tightness of these barriers depend on the growth conditions and on the age of the root. Such complex anatomical structures result in a composite water and solute transport in roots. Development of apoplastic barriers along barley seminal roots was detected using various staining methods, and the suberin amounts in the apical and basal zones were analysed using gas chromatography-mass spectometry (GC-MS). The hydraulic conductivity of roots ( Lp r ) and of cortical cells ( Lp c ) was measured using root and cell pressure probes. When grown in hydroponics, barley roots did not form an exodermis, even at their basal zones. However, they developed an endodermis. Endodermal Casparian bands first appeared as 'dots' as early as at 20 mm from the apex, whereas a patchy suberin lamellae appeared at 60 mm. The endodermal suberin accounted for the total suberin of the roots. The absolute amount in the basal zone was significantly higher than in the apical zone, which was inversely proportional to the Lp r . Comparison of Lp r and Lp c suggested that cell to cell pathways dominate for water transport in roots. However, the calculation of Lp r from Lp c showed that at least 26 % of water transport occurs through the apoplast. Roots had different solute permeabilities ( P sr ) and reflection coefficients ( σ sr ) for the solutes used. The σ sr was below unity for the solutes, which have virtually zero permeability for semi-permeable membranes. Suberized endodermis significantly reduces Lp r of seminal roots. The water and solute transport across barley roots is composite in nature and they do not behave like ideal osmometers. The composite transport model should be extended by adding components arranged in series (cortex, endodermis) in addition to the currently included components arranged in parallel (apoplastic, cell to cell pathways). © The

The composite water and solute transport of barley (Hordeum vulgare) roots: effect of suberized barriers

PubMed Central

Ranathunge, Kosala; Kim, Yangmin X.; Wassmann, Friedrich; Kreszies, Tino; Zeisler, Viktoria

2017-01-01

Abstract Background and Aims Roots have complex anatomical structures, and certain localized cell layers develop suberized apoplastic barriers. The size and tightness of these barriers depend on the growth conditions and on the age of the root. Such complex anatomical structures result in a composite water and solute transport in roots. Methods Development of apoplastic barriers along barley seminal roots was detected using various staining methods, and the suberin amounts in the apical and basal zones were analysed using gas chromatography–mass spectometry (GC-MS). The hydraulic conductivity of roots (Lpr) and of cortical cells (Lpc) was measured using root and cell pressure probes. Key Results When grown in hydroponics, barley roots did not form an exodermis, even at their basal zones. However, they developed an endodermis. Endodermal Casparian bands first appeared as ‘dots’ as early as at 20 mm from the apex, whereas a patchy suberin lamellae appeared at 60 mm. The endodermal suberin accounted for the total suberin of the roots. The absolute amount in the basal zone was significantly higher than in the apical zone, which was inversely proportional to the Lpr. Comparison of Lpr and Lpc suggested that cell to cell pathways dominate for water transport in roots. However, the calculation of Lpr from Lpc showed that at least 26 % of water transport occurs through the apoplast. Roots had different solute permeabilities (Psr) and reflection coefficients (σsr) for the solutes used. The σsr was below unity for the solutes, which have virtually zero permeability for semi-permeable membranes. Conclusions Suberized endodermis significantly reduces Lpr of seminal roots. The water and solute transport across barley roots is composite in nature and they do not behave like ideal osmometers. The composite transport model should be extended by adding components arranged in series (cortex, endodermis) in addition to the currently included components arranged in

Importance of NADPH oxidase-mediated redox signaling in the detrimental effect of CRP on pancreatic insulin secretion.

PubMed

Chan, Pei-Chi; Wang, Ya-Chin; Chen, Yi-Ling; Hsu, Wan-Ning; Tian, Yu-Feng; Hsieh, Po-Shiuan

2017-11-01

Elevations in C-reactive protein (CRP) levels are positively correlated with the progress of type 2 diabetes mellitus. However, the effect of CRP on pancreatic insulin secretion is unknown. Here, we showed that purified human CRP impaired insulin secretion in isolated mouse islets and NIT-1 insulin-secreting cells in dose- and time-dependent manners. CRP increased NADPH oxidase-mediated ROS (reactive oxygen species) production, which simultaneously promoted the production of nitrotyrosine (an indicator of RNS, reactive nitrogen species) and TNFα, to diminish cell viability, insulin secretion in islets and insulin-secreting cells. These CRP-mediated detrimental effects on cell viability and insulin secretion were significantly reversed by adding NAC (a potent antioxidant), apocynin (a selective NADPH oxidase inhibitor), L-NAME (a non-selective nitric oxide synthase (NOS) inhibitor), aminoguanidine (a selective iNOS inhibitor), PDTC (a selective NFκB inhibitor) or Enbrel (an anti-TNFα fusion protein). However, CRP-induced ROS production failed to change after adding L-NAME, aminoguanidine or PDTC. In isolated islets and NIT-1 cells, the elevated nitrotyrosine contents by CRP pretreatment were significantly suppressed by adding L-NAME but not PDTC. Conversely, CRP-induced increases in TNF-α production were significantly reversed by administration of PDTC but not L-NAME. In addition, wild-type mice treated with purified human CRP showed significant decreases in the insulin secretion index (HOMA-β cells) and the insulin stimulation index in isolated islets that were reversed by the addition of L-NAME, aminoguanidine or NAC. It is suggested that CRP-activated NADPH-oxidase redox signaling triggers iNOS-mediated RNS and NFκB-mediated proinflammatory cytokine production to cause β cell damage in state of inflammation. Copyright © 2017 Elsevier Inc. All rights reserved.

Ozone affects pollen viability and NAD(P)H oxidase release from Ambrosia artemisiifolia pollen.

PubMed

Pasqualini, Stefania; Tedeschini, Emma; Frenguelli, Giuseppe; Wopfner, Nicole; Ferreira, Fatima; D'Amato, Gennaro; Ederli, Luisa

2011-10-01

Air pollution is frequently proposed as a cause of the increased incidence of allergy in industrialised countries. We investigated the impact of ozone (O(3)) on reactive oxygen species (ROS) and allergen content of ragweed pollen (Ambrosia artemisiifolia). Pollen was exposed to acute O(3) fumigation, with analysis of pollen viability, ROS and nitric oxide (NO) content, activity of nicotinamide adenine dinucleotide phosphate (NAD[P]H) oxidase, and expression of major allergens. There was decreased pollen viability after O(3) fumigation, which indicates damage to the pollen membrane system, although the ROS and NO contents were not changed or were only slightly induced, respectively. Ozone exposure induced a significant enhancement of the ROS-generating enzyme NAD(P)H oxidase. The expression of the allergen Amb a 1 was not affected by O(3), determined from the mRNA levels of the major allergens. We conclude that O(3) can increase ragweed pollen allergenicity through stimulation of ROS-generating NAD(P)H oxidase. Copyright © 2011 Elsevier Ltd. All rights reserved.

Role of the Rho GTPase Rac in the activation of the phagocyte NADPH oxidase

PubMed Central

Pick, Edgar

2014-01-01

The superoxide-generating NADPH oxidase of phagocytes consists of the membrane-associated cytochrome b558 (a heterodimer of Nox2 and p22phox) and 4 cytosolic components: p47phox, p67phox, p40phox, and the small GTPase, Rac, in complex with RhoGDI. Superoxide is produced by the NADPH-driven reduction of molecular oxygen, via a redox gradient located in Nox2. Electron flow in Nox2 is initiated by interaction with cytosolic components, which translocate to the membrane, p67phox playing the central role. The participation of Rac is expressed in the following sequence: (1) Translocation of the RacGDP-RhoGDI complex to the membrane; (2) Dissociation of RacGDP from RhoGDI; (3) GDP to GTP exchange on Rac, mediated by a guanine nucleotide exchange factor; (4) Binding of RacGTP to p67phox; (5) Induction of a conformational change in p67phox, promoting interaction with Nox2. The particular involvement of Rac in NADPH oxidase assembly serves as a paradigm for signaling by Rho GTPases, in general. PMID:24598074

Salinity impact on yield, water use, mineral and essential oil content of fennel (Foeniculum vulgare Mill.)

USDA-ARS?s Scientific Manuscript database

The experimental study was carried out to determine the effects of salinity on water consumption, plant height, fresh and seed yields, biomass production, ion accumulation and essential oil content of fennel (Foeniculum vulgare Mill.) under greenhouse conditions. The experiment was conducted with a ...

Pharmacological inhibition of NADPH oxidase protects against cisplatin induced nephrotoxicity in mice by two step mechanism.

PubMed

Wang, Yimin; Luo, Xiao; Pan, Hao; Huang, Wei; Wang, Xueping; Wen, Huali; Shen, Kezhen; Jin, Baiye

2015-09-01

Cisplatin induced nephrotoxicity is primarily caused by ROS (Reactive Oxygen Species) induced proximal tubular cell death. NADPH oxidase is major source of ROS production by cisplatin. Here, we reported that pharmacological inhibition of NADPH oxidase by acetovanillone (obtained from medicinal herb Picrorhiza kurroa) led to reduced cisplatin nephrotoxicity in mice. In this study we used various molecular biology and biochemistry methods a clinically relevant model of nephropathy, induced by an important chemotherapeutic drug cisplatin. Cisplatin-induced nephrotoxicity was evident by histological damage from loss of the tubular structure. The damage was also marked by the increase in blood urea nitrogen, creatinine, protein nitration as well as cell death markers such as caspase 3/7 activity and DNA fragmentation. Tubular cell death by cisplatin led to pro-inflammatory response by production of TNFα and IL1β followed by leukocyte/neutrophil infiltration which resulted in new wave of ROS involving more NADPH oxidases. Cisplatin-induced markers of kidney damage such as oxidative stress, cell death, inflammatory cytokine production and nephrotoxicity were attenuated by acetovanillone. In addition to that, acetovanillone enhanced cancer cell killing efficacy of cisplatin. Thus, pharmacological inhibition of NADPH oxidase can be protective for cisplatin-induced nephrotoxicity in mice. Copyright © 2015. Published by Elsevier Ltd.

A Prenylated p47phox-p67phox-Rac1 Chimera Is a Quintessential NADPH Oxidase Activator

PubMed Central

Mizrahi, Ariel; Berdichevsky, Yevgeny; Casey, Patrick J.; Pick, Edgar

2010-01-01

The superoxide-generating NADPH oxidase complex of resting phagocytes includes cytochrome b559, a membrane-associated heterodimer composed of two subunits (Nox2 and p22phox), and four cytosolic proteins (p47phox, p67phox, Rac, and p40phox). Upon stimulation, the cytosolic components translocate to the membrane, as the result of a series of interactions among the cytosolic components and among the cytosolic components and cytochrome b559 and its phospholipid environment. We described the construction of a tripartite chimera (trimera) consisting of strategic domains of p47phox, p67phox, and Rac1, in which interactions among cytosolic components were replaced by fusion (Berdichevsky, Y., Mizrahi, A., Ugolev, Y., Molshanski-Mor, S., and Pick, E. (2007) J. Biol. Chem. 282, 22122–22139). We now fused green fluorescent protein (GFP) to the N terminus of the trimera and found the following. 1) The GFP-p47phox-p67phox-Rac1 trimera activates the oxidase in amphiphile-dependent and -independent (anionic phospholipid-enriched membrane) cell-free systems. 2) Geranylgeranylation of the GFP-trimera makes it a potent oxidase activator in unmodified (native) membranes and in the absence of amphiphile. 3) Prenylated GFP-trimera binds spontaneously to native membranes (as assessed by gel filtration and in-line fluorometry), forming a tight complex capable of NADPH-dependent, activator-independent superoxide production at rates similar to those measured in canonical cell-free systems. 4) Prenylation of the GFP-trimera supersedes completely the dependence of oxidase activation on the p47phox phox homology domain and, partially, on the Rac1 polybasic domain, but the requirement for Trp193 in p47phox persists. Prenylated GFP-p47phox-p67phox-Rac1 trimera acts as a quintessential single molecule oxidase activator of potential use in high throughput screening of inhibitors. PMID:20529851

Genetic Phagocyte NADPH Oxidase Deficiency Enhances Nonviable Candida albicans-Induced Inflammation in Mouse Lungs.

PubMed

Endo, Daiki; Fujimoto, Kenta; Hirose, Rika; Yamanaka, Hiroko; Homme, Mizuki; Ishibashi, Ken-Ichi; Miura, Noriko; Ohno, Naohito; Aratani, Yasuaki

2017-02-01

Patients with chronic granulomatous disease (CGD) have mutated phagocyte NADPH oxidase, resulting in reduced production of reactive oxygen species (ROS). While the mechanism underlying hyperinfection in CGD is well understood, the basis for inflammatory disorders that arise in the absence of evident infection has not been fully explained. This study aimed to evaluate the effect of phagocyte NADPH oxidase deficiency on lung inflammation induced by nonviable Candida albicans (nCA). Mice deficient in this enzyme (CGD mice) showed more severe neutrophilic pneumonia than nCA-treated wild-type mice, which exhibited significantly higher lung concentrations of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and keratinocyte-derived chemokine (KC). Neutralization of these proinflammatory mediators significantly reduced neutrophil infiltration. In vitro, production of IL-1β and TNF-α from neutrophils and that of KC from macrophages was enhanced in nCA-stimulated neutrophils from CGD mice. Expression of IL-1β mRNA was higher in the stimulated CGD neutrophils than in the stimulated wild-type cells, concomitant with upregulation of nuclear factor (NF)-κB and its upstream regulator extracellular-signal regulated kinase (ERK) 1/2. Pretreatment with an NADPH oxidase inhibitor significantly enhanced IL-1β production in the wild-type neutrophils stimulated with nCA. These results suggest that lack of ROS production because of NADPH oxidase deficiency results in the production of higher levels of proinflammatory mediators from neutrophils and macrophages, which may at least partly contribute to the exacerbation of nCA-induced lung inflammation in CGD mice.

The crystal structure of NADPH:ferredoxin reductase from Azotobacter vinelandii.

PubMed Central

Sridhar Prasad, G.; Kresge, N.; Muhlberg, A. B.; Shaw, A.; Jung, Y. S.; Burgess, B. K.; Stout, C. D.

1998-01-01

NADPH:ferredoxin reductase (AvFPR) is involved in the response to oxidative stress in Azotobacter vinelandii. The crystal structure of AvFPR has been determined at 2.0 A resolution. The polypeptide fold is homologous with six other oxidoreductases whose structures have been solved including Escherichia coli flavodoxin reductase (EcFldR) and spinach, and Anabaena ferredoxin:NADP+ reductases (FNR). AvFPR is overall most homologous to EcFldR. The structure is comprised of a N-terminal six-stranded antiparallel beta-barrel domain, which binds FAD, and a C-terminal five-stranded parallel beta-sheet domain, which binds NADPH/NADP+ and has a classical nucleotide binding fold. The two domains associate to form a deep cleft where the NADPH and FAD binding sites are juxtaposed. The structure displays sequence conserved motifs in the region surrounding the two dinucleotide binding sites, which are characteristic of the homologous enzymes. The folded over conformation of FAD in AvFPR is similar to that in EcFldR due to stacking of Phe255 on the adenine ring of FAD, but it differs from that in the FNR enzymes, which lack a homologous aromatic residue. The structure of AvFPR displays three unique features in the environment of the bound FAD. Two features may affect the rate of reduction of FAD: the absence of an aromatic residue stacked on the isoalloxazine ring in the NADPH binding site; and the interaction of a carbonyl group with N10 of the flavin. Both of these features are due to the substitution of a conserved C-terminal tyrosine residue with alanine (Ala254) in AvFPR. An additional unique feature may affect the interaction of AvFPR with its redox partner ferredoxin I (FdI). This is the extension of the C-terminus by three residues relative to EcFldR and by four residues relative to FNR. The C-terminal residue, Lys258, interacts with the AMP phosphate of FAD. Consequently, both phosphate groups are paired with a basic group due to the simultaneous interaction of the FMN

Method for attaining fennel (Foeniculum vulgare Mill.) seed oil fractions with different composition and antioxidant capacity

USDA-ARS?s Scientific Manuscript database

Fennel (Foeniculum vulgare Mill.) is cultivated for its seeds and foliage, which contain essential oil. We hypothesized that the collection of fennel seed oil at different time points during the distillation process may result in fennel oil with distinct composition and bioactivity. We collected ess...

The protein inhibitor of nNOS (PIN/DLC1/LC8) binding does not inhibit the NADPH-dependent heme reduction in nNOS, a key step in NO synthesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Parhad, Swapnil S.; Jaiswal, Deepa; TIFR Centre for Interdisciplinary Sciences, 21 Brundavan Colony, Narsingi, Hyderabad 500075

The neuronal nitric oxide synthase (nNOS) is an essential enzyme involved in the synthesis of nitric oxide (NO), a potent neurotransmitter. Although previous studies have indicated that the dynein light chain 1 (DLC1) binding to nNOS could inhibit the NO synthesis, the claim is challenged by contradicting reports. Thus, the mechanism of nNOS regulation remained unclear. nNOS has a heme-bearing, Cytochrome P450 core, and the functional enzyme is a dimer. The electron flow from NADPH to Flavin, and finally to the heme of the paired nNOS subunit within a dimer, is facilitated upon calmodulin (CaM) binding. Here, we show thatmore » DLC1 binding to nNOS-CaM complex does not affect the electron transport from the reductase to the oxygenase domain. Therefore, it cannot inhibit the rate of NADPH-dependent heme reduction in nNOS, which results in L-Arginine oxidation. Also, the NO release activity does not decrease with increasing DLC1 concentration in the reaction mix, which further confirmed that DLC1 does not inhibit nNOS activity. These findings suggest that the DLC1 binding may have other implications for the nNOS function in the cell. - Highlights: • The effect of interaction of nNOS with DLC1 has been debatable with contradicting reports in literature. • Purified DLC1 has no effect on electron transport between reductase and oxygenase domain of purified nNOS-CaM. • The NO release activity of nNOS was not altered by DLC1, supporting that DLC1 does not inhibit the enzyme. • These findings suggest that the DLC1 binding may have other implications for the nNOS function in the cell.« less

Serotonin Signaling Through the 5-HT1B Receptor and NADPH Oxidase 1 in Pulmonary Arterial Hypertension.

PubMed

Hood, Katie Y; Mair, Kirsty M; Harvey, Adam P; Montezano, Augusto C; Touyz, Rhian M; MacLean, Margaret R

2017-07-01

Serotonin can induce human pulmonary artery smooth muscle cell (hPASMC) proliferation through reactive oxygen species (ROS), influencing the development of pulmonary arterial hypertension (PAH). We hypothesize that in PASMCs, serotonin induces oxidative stress through NADPH-oxidase-derived ROS generation and reduced Nrf-2 (nuclear factor [erythroid-derived 2]-like 2) antioxidant systems, promoting vascular injury. HPASMCs from controls and PAH patients, and PASMCs from Nox1 -/- mice, were stimulated with serotonin in the absence/presence of inhibitors of Src kinase, the 5-HT 1B receptor, and NADPH oxidase 1 (Nox1). Markers of fibrosis were also determined. The pathophysiological significance of our findings was examined in vivo in serotonin transporter overexpressing female mice, a model of pulmonary hypertension. We confirmed thatserotonin increased superoxide and hydrogen peroxide production in these cells. For the first time, we show that serotonin increased oxidized protein tyrosine phosphatases and hyperoxidized peroxiredoxin and decreased Nrf-2 and catalase activity in hPASMCs. ROS generation was exaggerated and dependent on cellular Src-related kinase, 5-HT 1B receptor, and the serotonin transporter in human pulmonary artery smooth muscle cells from PAH subjects. Proliferation and extracellular matrix remodeling were exaggerated in human pulmonary artery smooth muscle cells from PAH subjects and dependent on 5-HT 1B receptor signaling and Nox1, confirmed in PASMCs from Nox1 -/- mice. In serotonin transporter overexpressing mice, SB216641, a 5-HT 1B receptor antagonist, prevented development of pulmonary hypertension in a ROS-dependent manner. Serotonin can induce cellular Src-related kinase-regulated Nox1-induced ROS and Nrf-2 dysregulation, contributing to increased post-translational oxidative modification of proteins and activation of redox-sensitive signaling pathways in hPASMCs, associated with mitogenic responses. 5-HT 1B receptors contribute to

Serotonin Signaling Through the 5-HT1B Receptor and NADPH Oxidase 1 in Pulmonary Arterial Hypertension

PubMed Central

Hood, Katie Y.; Mair, Kirsty M.; Harvey, Adam P.; Montezano, Augusto C.; Touyz, Rhian M.

2017-01-01

Objective— Serotonin can induce human pulmonary artery smooth muscle cell (hPASMC) proliferation through reactive oxygen species (ROS), influencing the development of pulmonary arterial hypertension (PAH). We hypothesize that in PASMCs, serotonin induces oxidative stress through NADPH-oxidase–derived ROS generation and reduced Nrf-2 (nuclear factor [erythroid-derived 2]-like 2) antioxidant systems, promoting vascular injury. Approach and Results— HPASMCs from controls and PAH patients, and PASMCs from Nox1−/− mice, were stimulated with serotonin in the absence/presence of inhibitors of Src kinase, the 5-HT1B receptor, and NADPH oxidase 1 (Nox1). Markers of fibrosis were also determined. The pathophysiological significance of our findings was examined in vivo in serotonin transporter overexpressing female mice, a model of pulmonary hypertension. We confirmed thatserotonin increased superoxide and hydrogen peroxide production in these cells. For the first time, we show that serotonin increased oxidized protein tyrosine phosphatases and hyperoxidized peroxiredoxin and decreased Nrf-2 and catalase activity in hPASMCs. ROS generation was exaggerated and dependent on cellular Src-related kinase, 5-HT1B receptor, and the serotonin transporter in human pulmonary artery smooth muscle cells from PAH subjects. Proliferation and extracellular matrix remodeling were exaggerated in human pulmonary artery smooth muscle cells from PAH subjects and dependent on 5-HT1B receptor signaling and Nox1, confirmed in PASMCs from Nox1−/− mice. In serotonin transporter overexpressing mice, SB216641, a 5-HT1B receptor antagonist, prevented development of pulmonary hypertension in a ROS-dependent manner. Conclusions— Serotonin can induce cellular Src-related kinase–regulated Nox1-induced ROS and Nrf-2 dysregulation, contributing to increased post-translational oxidative modification of proteins and activation of redox-sensitive signaling pathways in hPASMCs, associated with

Peroxisomal plant metabolism - an update on nitric oxide, Ca2+ and the NADPH recycling network.

PubMed

Corpas, Francisco J; Barroso, Juan B

2018-01-29

Plant peroxisomes are recognized organelles that - with their capacity to generate greater amounts of H 2 O 2 than other subcellular compartments - have a remarkable oxidative metabolism. However, over the last 15 years, new information has shown that plant peroxisomes contain other important molecules and enzymes, including nitric oxide (NO), peroxynitrite, a NADPH-recycling system, Ca 2+ and lipid-derived signals, such as jasmonic acid (JA) and nitro-fatty acid (NO 2 -FA). This highlights the potential for complex interactions within the peroxisomal nitro-oxidative metabolism, which also affects the status of the cell and consequently its physiological processes. In this review, we provide an update on the peroxisomal interactions between all these molecules. Particular emphasis will be placed on the generation of the free-radical NO, which requires the presence of Ca 2+ , calmodulin and NADPH redox power. Peroxisomes possess several NADPH regeneration mechanisms, such as those mediated by glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) proteins, which are involved in the oxidative phase of the pentose phosphate pathway, as well as that mediated by NADP-isocitrate dehydrogenase (ICDH). The generated NADPH is also an essential cofactor across other peroxisomal pathways, including the antioxidant ascorbate-glutathione cycle and unsaturated fatty acid β-oxidation, the latter being a source of powerful signaling molecules such as JA and NO 2 -FA. © 2018. Published by The Company of Biologists Ltd.

Identification and Characterization of Sclerotinia sclerotiorum NADPH Oxidases▿†

PubMed Central

Kim, Hyo-jin; Chen, Changbin; Kabbage, Mehdi; Dickman, Martin B.

2011-01-01

Numerous studies have shown both the detrimental and beneficial effects of reactive oxygen species (ROS) in animals, plants, and fungi. These organisms utilize controlled generation of ROS for signaling, pathogenicity, and development. Here, we show that ROS are essential for the pathogenic development of Sclerotinia sclerotiorum, an economically important fungal pathogen with a broad host range. Based on the organism's completed genome sequence, we identified two S. sclerotiorum NADPH oxidases (SsNox1 and SsNox2), which presumably are involved in ROS generation. RNA interference (RNAi) was used to examine the function of SsNox1 and SsNox2. Silencing of SsNox1 expression indicated a central role for this enzyme in both virulence and pathogenic (sclerotial) development, while inactivation of the SsNox2 gene resulted in limited sclerotial development, but the organism remained fully pathogenic. ΔSsnox1 strains had reduced ROS levels, were unable to develop sclerotia, and unexpectedly correlated with significantly reduced oxalate production. These results are in accordance with previous observations indicating that fungal NADPH oxidases are required for pathogenic development and are consistent with the importance of ROS regulation in the successful pathogenesis of S. sclerotiorum. PMID:21890677

Mitochondrial NAD(P)H In vivo: Identifying Natural Indicators of Oxidative Phosphorylation in the (31)P Magnetic Resonance Spectrum.

PubMed

Conley, Kevin E; Ali, Amir S; Flores, Brandon; Jubrias, Sharon A; Shankland, Eric G

2016-01-01

Natural indicators provide intrinsic probes of metabolism, biogenesis and oxidative protection. Nicotinamide adenine dinucleotide metabolites (NAD(P)) are one class of indicators that have roles as co-factors in oxidative phosphorylation, glycolysis, and anti-oxidant protection, as well as signaling in the mitochondrial biogenesis pathway. These many roles are made possible by the distinct redox states (NAD(P)(+) and NAD(P)H), which are compartmentalized between cytosol and mitochondria. Here we provide evidence for detection of NAD(P)(+) and NAD(P)H in separate mitochondrial and cytosol pools in vivo in human tissue by phosphorus magnetic resonance spectroscopy ((31)P MRS). These NAD(P) pools are identified by chemical standards (NAD(+), NADP(+), and NADH) and by physiological tests. A unique resonance reflecting mitochondrial NAD(P)H is revealed by the changes elicited by elevation of mitochondrial oxidation. The decline of NAD(P)H with oxidation is matched by a stoichiometric rise in the NAD(P)(+) peak. This unique resonance also provides a measure of the improvement in mitochondrial oxidation that parallels the greater phosphorylation found after exercise training in these elderly subjects. The implication is that the dynamics of the mitochondrial NAD(P)H peak provides an intrinsic probe of the reversal of mitochondrial dysfunction in elderly muscle. Thus, non-invasive detection of NAD(P)(+) and NAD(P)H in cytosol vs. mitochondria yields natural indicators of redox compartmentalization and sensitive intrinsic probes of the improvement of mitochondrial function with an intervention in human tissues in vivo. These natural indicators hold the promise of providing mechanistic insight into metabolism and mitochondrial function in vivo in a range of tissues in health, disease and with treatment.

Dual blockade of aldosterone and angiotensin II additively suppresses TGF-beta and NADPH oxidase in the hypertensive kidney.

PubMed

Onozato, Maristela Lika; Tojo, Akihiro; Kobayashi, Naohiko; Goto, Atsuo; Matsuoka, Hiroaki; Fujita, Toshiro

2007-05-01

Angiotensin II blockade and spironolactone effectively reduces proteinuria in humans. To clarify the mechanisms of the beneficial effect of blockade of both aldosterone and angiotensin II, we associated the aldosterone antagonist eplerenone to an angiotensin-converting enzyme inhibitor (ACEI) and examined the effect on renal transforming growth factor (TGF)-beta expression and oxidative stress by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in the Dahl salt-sensitive rat with heart failure (DSHF). Dahl salt-resistant control rats and DSHF rats were fed with 8% NaCl diet and at 11 weeks the DSHF rats were treated with vehicle, eplerenone (Epl), trandolapril or a combination of both drugs for 7 weeks. DSHF rats showed increased NADPH oxidase and decreased superoxide dismutase (SOD) resulting in increased oxidative stress. ACEI and Epl reduced NADPH oxidase showing an additive effect in their combination; ACEI increased manganese SOD (MnSOD) and Epl increased MnSOD, copper-zinc SOD and catalase, resulting in the lowest levels of oxidative stress with the combination therapy. Glomerulosclerosis and proteinuria were increased in the DSHF rats, and Epl suppressed them more effectively than ACEI to levels not different from the combination of both, showing a positive correlation with NADPH oxidase expression and TGF-beta. Renal TGF-beta was specifically suppressed with Epl The association of Epl to ACEI is beneficial due to further reduction of NADPH oxidase and specific inhibition of TGF-beta resulting in improvement of renal damage.

Atrial myocardial nox2 containing NADPH oxidase activity contribution to oxidative stress in mitral regurgitation: potential mechanism for atrial remodeling.

PubMed

Chang, Jen-Ping; Chen, Mien-Cheng; Liu, Wen-Hao; Yang, Cheng-Hsu; Chen, Chien-Jen; Chen, Yung-Lung; Pan, Kuo-Li; Tsai, Tzu-Hsien; Chang, Hsueh-Wen

2011-01-01

Oxidative stress is linked with several cardiovascular diseases. However, the NADPH oxidase activity in severe mitral regurgitation patients with and without atrial fibrillation has not yet been explored. This study involved 16 adult patients (eight patients with persistent atrial fibrillation and eight with sinus rhythm) with severe mitral and moderate-to-severe tricuspid regurgitation and five control patients without mitral and tricuspid disease. Atrial tissues of the right and left atrial appendages were obtained during surgery. Superoxide anion production was measured by lucigenin-enhanced chemiluminescence, and the expression of nox2 containing NADPH oxidase mRNA was measured by quantitative real-time RT-PCR. Additionally, immunohistochemical study was performed. NADPH-stimulated superoxide release was significantly higher than basal superoxide production from right [5671.9±3498.7 vs. 232.7±70.0 relative light units per second per milligram of protein (RLU s(-1) mg protein(-1)), P=.008) and left atrial homogenates (6475.1±1890.8 vs. 229.0±79.6 RLU s(-1) mg protein(-1), P=.008) in atrial fibrillation patients. The NADPH-stimulated superoxide release from right atrial homogenates was also significantly higher than basal superoxide production in sinus patients (6809.1±1327.1 vs. 244.2±65.5 RLU s(-1) mg protein(-1), P=.008). Additionally, there was a borderline significant correlation between NADPH-stimulated superoxide production from left atrial homogenates and left atrial sizes (r=0.683, P=.062) in atrial fibrillation patients. Membrane-bound nox2 containing NADPH oxidase mRNA expression was increased and was similar in both the atrial fibrillation patients and sinus patients. The NADPH-stimulated superoxide production in right atrial homogenates in control atrial samples was 1863.7±137.2 RLU s(-1) mg protein(-1). Immunohistochemical study demonstrated increased expression of nox2 in myocytes with moderate-to-severe myolysis and hypertrophy. Results of

Transhydrogenase Promotes the Robustness and Evolvability of E. coli Deficient in NADPH Production

PubMed Central

Chou, Hsin-Hung; Marx, Christopher J.; Sauer, Uwe

2015-01-01

Metabolic networks revolve around few metabolites recognized by diverse enzymes and involved in myriad reactions. Though hub metabolites are considered as stepping stones to facilitate the evolutionary expansion of biochemical pathways, changes in their production or consumption often impair cellular physiology through their system-wide connections. How does metabolism endure perturbations brought immediately by pathway modification and restore hub homeostasis in the long run? To address this question we studied laboratory evolution of pathway-engineered Escherichia coli that underproduces the redox cofactor NADPH on glucose. Literature suggests multiple possibilities to restore NADPH homeostasis. Surprisingly, genetic dissection of isolates from our twelve evolved populations revealed merely two solutions: (1) modulating the expression of membrane-bound transhydrogenase (mTH) in every population; (2) simultaneously consuming glucose with acetate, an unfavored byproduct normally excreted during glucose catabolism, in two subpopulations. Notably, mTH displays broad phylogenetic distribution and has also played a predominant role in laboratory evolution of Methylobacterium extorquens deficient in NADPH production. Convergent evolution of two phylogenetically and metabolically distinct species suggests mTH as a conserved buffering mechanism that promotes the robustness and evolvability of metabolism. Moreover, adaptive diversification via evolving dual substrate consumption highlights the flexibility of physiological systems to exploit ecological opportunities. PMID:25715029

Persistent activation of microglia and NADPH drive hippocampal dysfunction in experimental multiple sclerosis

PubMed Central

Di Filippo, Massimiliano; de Iure, Antonio; Giampà, Carmela; Chiasserini, Davide; Tozzi, Alessandro; Orvietani, Pier Luigi; Ghiglieri, Veronica; Tantucci, Michela; Durante, Valentina; Quiroga-Varela, Ana; Mancini, Andrea; Costa, Cinzia; Sarchielli, Paola; Fusco, Francesca Romana; Calabresi, Paolo

2016-01-01

Cognitive impairment is common in multiple sclerosis (MS). Unfortunately, the synaptic and molecular mechanisms underlying MS-associated cognitive dysfunction are largely unknown. We explored the presence and the underlying mechanism of cognitive and synaptic hippocampal dysfunction during the remission phase of experimental MS. Experiments were performed in a chronic-relapsing experimental autoimmune encephalomyelitis (EAE) model of MS, after the resolution of motor deficits. Immunohistochemistry and patch-clamp recordings were performed in the CA1 hippocampal area. The hole-board was utilized as cognitive/behavioural test. In the remission phase of experimental MS, hippocampal microglial cells showed signs of activation, CA1 hippocampal synapses presented an impaired long-term potentiation (LTP) and an alteration of spatial tests became evident. The activation of hippocampal microglia mediated synaptic and cognitive/behavioural alterations during EAE. Specifically, LTP blockade was found to be caused by the reactive oxygen species (ROS)-producing enzyme nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. We suggest that in the remission phase of experimental MS microglia remains activated, causing synaptic dysfunctions mediated by NADPH oxidase. Inhibition of microglial activation and NADPH oxidase may represent a promising strategy to prevent neuroplasticity impairment associated with active neuro-inflammation, with the aim to improve cognition and counteract MS disease progression. PMID:26887636

Supercritical CO₂ extraction of volatile oils from Sardinian Foeniculum vulgare ssp. vulgare (Apiaceae): chemical composition and biological activity.

PubMed

Piras, Alessandra; Falconieri, Danilo; Porcedda, Silvia; Marongiu, Bruno; Gonçalves, Maria José; Cavaleiro, Carlos; Salgueiro, Ligia

2014-01-01

This article reports the results on the composition and antifungal effect of volatile extracts obtained from the aerial parts of Sardinian wild fennel (Foeniculum vulgare Mill.), by supercritical fluid extraction (SFE) and by hydrodistillation (HD). The extracts were analysed by gas chromatography-mass spectrometry for qualitative composition and gas chromatography-flame ionisation detector to establish the percentage of constituents. The main components were fenchone (7.1% vs. 8.8%), estragole (34.9% vs. 42.6%) and (E)-anethole (24.6% vs. 43.4%) in the SFE and HD extract, respectively. Minimum inhibitory concentrations (MICs) were measured according to the reference Clinical and Laboratory Standards Institute (CLSI) broth macrodilution protocols. Minimum lethal concentrations were determined by subsequent subculturing of the same cell suspensions in solid medium. The essential oil was more active against Candida albicans, whereas the supercritical fluid extract possesses higher activity against Candida guillermondii and Cryptococcus neoformans, with MIC values of 0.32 μL/mL.

EPA:DHA 6:1 prevents angiotensin II-induced hypertension and endothelial dysfunction in rats: role of NADPH oxidase- and COX-derived oxidative stress.

PubMed

Niazi, Zahid Rasul; Silva, Grazielle C; Ribeiro, Thais Porto; León-González, Antonio J; Kassem, Mohamad; Mirajkar, Abdur; Alvi, Azhar; Abbas, Malak; Zgheel, Faraj; Schini-Kerth, Valérie B; Auger, Cyril

2017-12-01

Eicosapentaenoic acid:docosahexaenoic acid (EPA:DHA) 6:1, an omega-3 polyunsaturated fatty acid formulation, has been shown to induce a sustained formation of endothelial nitric oxide (NO) synthase-derived NO, a major vasoprotective factor. This study examined whether chronic intake of EPA:DHA 6:1 prevents hypertension and endothelial dysfunction induced by angiotensin II (Ang II) in rats. Male Wister rats received orally corn oil or EPA:DHA 6:1 (500 mg kg -1 per day) before chronic infusion of Ang II (0.4 mg kg -1 per day). Systolic blood pressure was determined by tail cuff sphingomanometry, vascular reactivity using a myograph, oxidative stress using dihydroethidium and protein expression by immunofluorescence and western blot analysis. Ang II-induced hypertension was associated with reduced acetylcholine-induced relaxations of secondary branch mesenteric artery rings affecting the endothelium-dependent hyperpolarization (EDH)- and the NO-mediated relaxations, both of which were improved by the NADPH oxidase inhibitor VAS-2870. The Ang II treatment induced also endothelium-dependent contractile responses (EDCFs), which were abolished by the cyclooxygenase (COX) inhibitor indomethacin. An increased level of vascular oxidative stress and expression of NADPH oxidase subunits (p47 phox and p22 phox ), COX-1 and COX-2, endothelial NO synthase and Ang II type 1 receptors were observed in the Ang II group, whereas SK Ca and connexin 37 were downregulated. Intake of EPA:DHA 6:1 prevented the Ang II-induced hypertension and endothelial dysfunction by improving both the NO- and EDH-mediated relaxations, and by reducing EDCFs and the expression of target proteins. The present findings indicate that chronic intake of EPA:DHA 6:1 prevented the Ang II-induced hypertension and endothelial dysfunction in rats, most likely by preventing NADPH oxidase- and COX-derived oxidative stress.

A biomimetic synthesis of stable gold nanoparticles derived from aqueous extract of Foeniculum vulgare seeds and evaluation of their catalytic activity

NASA Astrophysics Data System (ADS)

Choudhary, Manoj Kumar; Kataria, Jyoti; Sharma, Shweta

2017-10-01

A facile biomimetic approach for the synthesis of gold nanoparticles (AuNPs) using aqueous extract of fennel ( Foeniculum vulgare) seeds have been reported in this article. The seeds of F. vulgare are rich in various plant secondary metabolites (phytochemicals) such as polyphenolic acids, flavonoids, and saponins. The phytochemicals of F. vulgare seeds play dual role of reducing and stabilizing agents. The formation of gold nanoparticles was evidenced from the appearance of intense purple color at room temperature with λ max around 550 nm in the UV-Vis absorption spectra. The stable AuNPs were further characterized by X-ray diffraction (XRD), Fourier transform infrared (FT-IR) spectroscopy and transmission electron microscopy (TEM) analysis. The synthesized nanoparticles were observed to be polydispersed, spherical and ranged from 10 to 30 nm with an average size of 20 ± 2 nm, as obtained from TEM images. The catalytic activity of gold nanoparticles was investigated by studying the reduction of anthropogenic dyes such as methylene blue (MB) and rhodamine B (Rh-B) with sodium borohydride. Results showed the possible applications of biogenic AuNPs in environment related problems.

Differential roles of NADPH oxidases in vascular physiology and pathophysiology

PubMed Central

Amanso, Angelica M.; Griendling, Kathy K.

2012-01-01

Reactive oxygen species (ROS) are produced by all vascular cells and regulate the major physiological functions of the vasculature. Production and removal of ROS are tightly controlled and occur in discrete subcellular locations, allowing for specific, compartmentalized signaling. Among the many sources of ROS in the vessel wall, NADPH oxidases are implicated in physiological functions such as control of vasomotor tone, regulation of extracellular matrix and phenotypic modulation of vascular smooth muscle cells. They are involved in the response to injury, whether as an oxygen sensor during hypoxia, as a regulator of protein processing, as an angiogenic stimulus, or as a mechanism of wound healing. These enzymes have also been linked to processes leading to disease development, including migration, proliferation, hypertrophy, apoptosis and autophagy. As a result, NADPH oxidases participate in atherogenesis, systemic and pulmonary hypertension and diabetic vascular disease. The role of ROS in each of these processes and diseases is complex, and a more full understanding of the sources, targets, cell-specific responses and counterbalancing mechanisms is critical for the rational development of future therapeutics. PMID:22202108

Diversity in boron toxicity tolerance of Australian barley (Hordeum vulgare L.) genotypes.

PubMed

Hayes, Julie E; Pallotta, Margaret; Garcia, Melissa; Öz, Mehmet Tufan; Rongala, Jay; Sutton, Tim

2015-09-26

Boron (B) is an important micronutrient for plant growth, but is toxic when levels are too high. This commonly occurs in environments with alkaline soils and relatively low rainfall, including many of the cereal growing regions of southern Australia. Four major genetic loci controlling tolerance to high soil B have been identified in the landrace barley, Sahara 3771. Genes underlying two of the loci encode the B transporters HvBot1 and HvNIP2;1. We investigated sequence and expression level diversity in HvBot1 and HvNIP2;1 across barley germplasm, and identified five novel coding sequence alleles for HvBot1. Lines were identified containing either single or multiple copies of the Sahara HvBot1 allele. We established that only the tandemly duplicated Sahara allele conferred B tolerance, and this duplicated allele was found only in a set of nine lines accessioned in Australian collections as Sahara 3763-3771. HvNIP2;1 coding sequences were highly conserved across barley germplasm. We identified the likely causative SNP in the 5'UTR of Sahara HvNIP2;1, and propose that the creation of a small upstream open reading frame interferes with HvNIP2;1 translation in Sahara 3771. Similar to HvBot1, the tolerant HvNIP2;1 allele was unique to the Sahara barley accessions. We identified a new source of the 2H B tolerance allele controlling leaf symptom development, in the landrace Ethiopia 756. Ethiopia 756, as well as the cultivar Sloop Vic which carries both the 2H and HvBot1 B tolerance alleles derived from Sahara 3771, may be valuable as alternative parents in breeding programs targeted to high soil B environments. There is significant diversity in B toxicity tolerance among contemporary Australian barley varieties but this is not related to variation at any of the four known B tolerance loci, indicating that novel, as yet undiscovered, sources of tolerance exist.

Diversity and Evolution of Disease Resistance Genes in Barley (Hordeum vulgare L.)

PubMed Central

Andersen, Ethan J.; Ali, Shaukat; Reese, R. Neil; Yen, Yang; Neupane, Surendra; Nepal, Madhav P.

2016-01-01

Plant disease resistance genes (R-genes) play a critical role in the defense response to pathogens. Barley is one of the most important cereal crops, having a genome recently made available, for which the diversity and evolution of R-genes are not well understood. The main objectives of this research were to conduct a genome-wide identification of barley Coiled-coil, Nucleotide-binding site, Leucine-rich repeat (CNL) genes and elucidate their evolutionary history. We employed a Hidden Markov Model using 52 Arabidopsis thaliana CNL reference sequences and analyzed for phylogenetic relationships, structural variation, and gene clustering. We identified 175 barley CNL genes nested into three clades, showing (a) evidence of an expansion of the CNL-C clade, primarily due to tandem duplications; (b) very few members of clade CNL-A and CNL-B; and (c) a complete absence of clade CNL-D. Our results also showed that several of the previously identified mildew locus A (MLA) genes may be allelic variants of two barley CNL genes, MLOC_66581 and MLOC_10425, which respond to powdery mildew. Approximately 23% of the barley CNL genes formed 15 gene clusters located in the extra-pericentromeric regions on six of the seven chromosomes; more than half of the clustered genes were located on chromosomes 1H and 7H. Higher average numbers of exons and multiple splice variants in barley relative to those in Arabidopsis and rice may have contributed to a diversification of the CNL-C members. These results will help us understand the evolution of R-genes with potential implications for developing durable resistance in barley cultivars. PMID:27168720

Genome-wide association mapping of frost tolerance in barley (Hordeum vulgare L.)

PubMed Central

2013-01-01

Background Frost tolerance is a key trait with economic and agronomic importance in barley because it is a major component of winter hardiness, and therefore limits the geographical distribution of the crop and the effective transfer of quality traits between spring and winter crop types. Three main frost tolerance QTL (Fr-H1, Fr-H2 and Fr-H3) have been identified from bi-parental genetic mapping but it can be argued that those mapping populations only capture a portion of the genetic diversity of the species. A genetically broad dataset consisting of 184 genotypes, representative of the barley gene pool cultivated in the Mediterranean basin over an extended time period, was genotyped with 1536 SNP markers. Frost tolerance phenotype scores were collected from two trial sites, Foradada (Spain) and Fiorenzuola (Italy) and combined with the genotypic data in genome wide association analyses (GWAS) using Eigenstrat and kinship approaches to account for population structure. Results GWAS analyses identified twelve and seven positive SNP associations at Foradada and Fiorenzuola, respectively, using Eigenstrat and six and four, respectively, using kinship. Linkage disequilibrium analyses of the significant SNP associations showed they are genetically independent. In the kinship analysis, two of the significant SNP associations were tightly linked to the Fr-H2 and HvBmy loci on chromosomes 5H and 4HL, respectively. The other significant kinship associations were located in genomic regions that have not previously been associated with cold stress. Conclusions Haplotype analysis revealed that most of the significant SNP loci are fixed in the winter or facultative types, while they are freely segregating within the un-adapted spring barley genepool. Although there is a major interest in detecting new variation to improve frost tolerance of available winter and facultative types, from a GWAS perspective, working within the un-adapted spring germplasm pool is an attractive alternative strategy which would minimize statistical issues, simplify the interpretation of the data and identify phenology independent genetic determinants of frost tolerance. PMID:23802597

Landscape genomics reveal signatures of local adaptation in barley (Hordeum vulgare L.)

PubMed Central

Abebe, Tiegist D.; Naz, Ali A.; Léon, Jens

2015-01-01

Land plants are sessile organisms that cannot escape the adverse climatic conditions of a given environment. Hence, adaptation is one of the solutions to surviving in a challenging environment. This study was aimed at detecting adaptive loci in barley landraces that are affected by selection. To that end, a diverse population of barley landraces was analyzed using the genotyping by sequencing approach. Climatic data for altitude, rainfall and temperature were collected from 61 weather sites near the origin of selected landraces across Ethiopia. Population structure analysis revealed three groups whereas spatial analysis accounted significant similarities at shorter geographic distances (< 40 Km) among barley landraces. Partitioning the variance between climate variables and geographic distances indicated that climate variables accounted for most of the explainable genetic variation. Markers by climatic variables association analysis resulted in altogether 18 and 62 putative adaptive loci using Bayenv and latent factor mixed model (LFMM), respectively. Subsequent analysis of the associated SNPs revealed putative candidate genes for plant adaptation. This study highlights the presence of putative adaptive loci among barley landraces representing original gene pool of the farming communities. PMID:26483825

Quantification of the tissue-culture induced variation in barley (Hordeum vulgare L.)

PubMed Central

Bednarek, Piotr T; Orłowska, Renata; Koebner, Robert MD; Zimny, Janusz

2007-01-01

Background When plant tissue is passaged through in vitro culture, many regenerated plants appear to be no longer clonal copies of their donor genotype. Among the factors that affect this so-called tissue culture induced variation are explant genotype, explant tissue origin, medium composition, and the length of time in culture. Variation is understood to be generated via a combination of genetic and/or epigenetic changes. A lack of any phenotypic variation between regenerants does not necessarily imply a concomitant lack of genetic (or epigenetic) change, and it is therefore of interest to assay the outcomes of tissue culture at the genotypic level. Results A variant of methylation sensitive AFLP, based on the isoschizomeric combinations Acc65I/MseI and KpnI/MseI was applied to analyze, at both the sequence and methylation levels, the outcomes of regeneration from tissue culture in barley. Both sequence mutation and alteration in methylation pattern were detected. Two sets of regenerants from each of five DH donor lines were compared. One set was derived via androgenesis, and the other via somatic embryogenesis, developed from immature embryos. These comparisons delivered a quantitative assessment of the various types of somaclonal variation induced. The average level of variation was 6%, of which almost 1.7% could be accounted for by nucleotide mutation, and the remainder by changes in methylation state. The nucleotide mutation rates and the rate of epimutations were substantially similar between the andro- and embryo-derived sets of regenerants across all the donors. Conclusion We have developed an AFLP based approach that is capable of describing the qualitative and quantitative characteristics of the tissue culture-induced variation. We believe that this approach will find particular value in the study of patterns of inheritance of somaclonal variation, since non-heritable variation is of little interest for the improvement of plant species which are sexually propagated. Of significant biological interest is the conclusion that the mode of regeneration has no significant effect on the balance between sequence and methylation state change induced by the tissue culture process. PMID:17335560

Variability in mesophyll conductance between barley genotypes, and effects on transpiration efficiency and carbon isotope discrimination.

PubMed

Barbour, Margaret M; Warren, Charles R; Farquhar, Graham D; Forrester, Guy; Brown, Hamish

2010-07-01

Leaf internal, or mesophyll, conductance to CO(2) (g(m)) is a significant and variable limitation of photosynthesis that also affects leaf transpiration efficiency (TE). Genotypic variation in g(m) and the effect of g(m) on TE were assessed in six barley genotypes (four Hordeum vulgare and two H. bulbosum). Significant variation in g(m) was found between genotypes, and was correlated with photosynthetic rate. The genotype with the highest g(m) also had the highest TE and the lowest carbon isotope discrimination as recorded in leaf tissue (Delta(p)). These results suggest g(m) has unexplored potential to provide TE improvement within crop breeding programmes.

Antioxidant activity and total phenolic and flavonoids content variations of leaves extracts of white Horehound (Marrubium vulgare Linné) from three geographical origins.

PubMed

Bouterfas, K; Mehdadi, Z; Elaoufi, M M; Latreche, A; Benchiha, W

2016-11-01

To elucidate the effect of the sampling location of Marrubium vulgare L. leaves on phenolic contents and antioxidant proprieties of flavonoids extracts. M. vulgare L. leaves were collected from three different geographical locations belonging to northwest Algeria: Tessala (mountain region), M'sila forest (coastal region), and Ain Skhouna (steppe region). The flavonoid extraction was achieved using organic solvents with different polarities (methanol, chloroform, ethyl acetate, and hexane). Folin-Ciocalteu colorimetric method was used for quantification of total phenolic contents, and aluminum chloride assay for quantification of total flavonoid contents. The antioxidant properties of flavonoids extract were studied by free l,l-diphenyl-2-picrylhydrazyl radical-scavenging technique. Total phenolic and flavonoids concentrations varied respectively between 40.7 and 160mg gallic acid equivalents/g and 27.4 and 66.3mg catechin equivalents/g. The DPPH free radical-scavenging activity shows that the antioxidant activity of the flavonoid extracts varied significantly (P<0.001) depending on the type of the organic solvent used, and the sampling location. The methanol, chloroform and ethyl acetate extracts exhibited the highest percentages of inhibition unlike to the aqueous and hexane extracts. These percentages are ranged from 54.8 to 98.8% at 1000μg/mL. In general, M'sila forest flavonoids extracts showed the highest free radical inhibition capacity; followed by those of Ain Skhouna and Tessala Mountain. The inhibitory concentration 50 (IC 50 ) ranged from 33.7 to 774μg/mL and often exceeded those recorded by phenolic standards (ascorbic acid, gallic acid, caffeic acid, tannic acid and catechin). The phytochemical screening revealed the presence of some flavonoid classes, such as flavans and flavanols. The results suggested a potent antioxidant activity of M. vulgare flavonoids extracts, which may find its application in feature research for the food and the

Time-dependent inhibition of CYP3A4 by gallic acid in human liver microsomes and recombinant systems.

PubMed

Pu, Qiang-Hong; Shi, Liang; Yu, Chao

2015-03-01

1.Gallic acid is a main polyphenol in various fruits and plants. Inhibitory characteristics of gallic acid on CYP3A4 were still unclear. The objective of this work is hence to investigate inhibitory characteristics of gallic acid on CYP3A4 using testosterone as the probe substrate in human liver microsomes (HLMs) and recombinant CYP3A4 (rCYP3A4) systems. 2.Gallic acid caused concentration-dependent loss of CYP3A4 activity with IC50 values of 615.2 μM and 669.5 μM in HLM and rCYP3A4 systems, respectively. IC50-shift experiments showed that pre-incubation with gallic acid in the absence of NADPH contributed to 12- or 14-fold reduction of IC50 in HLM and rCYP3A4 systems, respectively, supporting a time-dependent inhibition. In HLM, time-dependent inactivation variables KI and Kinact were 485.8 μM and 0.05 min(-1), respectively. 3.Compared with the presence of NADPH, pre-incubation of gallic acid in the absence of NADPH markedly increased its inhibitory effects in HLM and rCYP3A4 systems. Those results indicate that CYP3A4 inactivation by gallic acid was independent on NADPH and was mainly mediated its oxidative products. 4.In conclusion, we showed that gallic acid weakly and time-dependently inactivated CYP3A4 via its oxidative products.

Nitro-oleic acid ameliorates oxygen and glucose deprivation/re-oxygenation triggered oxidative stress in renal tubular cells via activation of Nrf2 and suppression of NADPH oxidase.

PubMed

Nie, Huibin; Xue, Xia; Liu, Gang; Guan, Guangju; Liu, Haiying; Sun, Lina; Zhao, Long; Wang, Xueling; Chen, Zhixin

2016-01-01

Nitroalkene derivative of oleic acid (OA-NO 2 ), due to its ability to mediate revisable Michael addition, has been demonstrated to have various biological properties and become a therapeutic agent in various diseases. Though its antioxidant properties have been reported in different models of acute kidney injury (AKI), the mechanism by which OA-NO 2 attenuates intracellular oxidative stress is not well investigated. Here, we elucidated the anti-oxidative mechanism of OA-NO 2 in an in vitro model of renal ischemia/reperfusion (I/R) injury. Human tubular epithelial cells were subjected to oxygen and glucose deprivation/re-oxygenation (OGD/R) injury. Pretreatment with OA-NO 2 (1.25 μM, 45 min) attenuated OGD/R triggered reactive oxygen species (ROS) generation and subsequent mitochondrial membrane potential disruption. This action was mediated via up-regulating endogenous antioxidant defense components including superoxide dismutase (SOD1), heme oxygenase 1 (HO-1), and γ-glutamyl cysteine ligase modulatory subunits (GCLM). Moreover, subcellular fractionation analyses demonstrated that OA-NO 2 promoted nuclear translocation of nuclear factor-E2- related factor-2 (Nrf2) and Nrf2 siRNA partially abrogated these protective effects. In addition, OA-NO 2 inhibited NADPH oxidase activation and NADPH oxidase 4 (NOX4), NADPH oxidase 2 (NOX2) and p22 phox up-regulation after OGD/R injury, which was not relevant to Nrf2. These results contribute to clarify that the mechanism of OA-NO 2 reno-protection involves both inhibition of NADPH oxidase activity and induction of SOD1, Nrf2-dependent HO-1, and GCLM.

Effect of γ-irradiation on structure and nutraceutical potential of β-D-glucan from barley (Hordeum vulgare).

PubMed

Shah, Asima; Ahmad, Mudasir; Ashwar, Bilal Ahmad; Gani, Adil; Masoodi, Farooq Ahmad; Wani, Idrees Ahmed; Wani, Sajad Mohd; Gani, Asir

2015-01-01

This paper reports the characterization and potential antioxidant activity of β-D-glucan isolated from barley treated with γ-rays. The β-D-glucan was irradiated with 0, 2, 4 and 8 kGy by gamma ray. The samples were characterized by Fourier transform-infrared spectroscopy, gel permeation chromatography (GPC) and quantitative estimation by Megazyme β-D-glucan assay kit. The average molecular weight of non-irradiated β-D-glucan was 177 kDa that decreased to 79 kDa at 8 kGy. Antioxidant activity was evaluated by five complementary assays including DPPH, lipid peroxidation, reducing power, metal chelating ability and oxidative DNA damage assays. Further, the antiproliferative potential of irradiated β-D-glucan was tested against three human cancer cell lines including Colo-205, T47D and MCF7 using MTT assay. Irradiated β-D-glucan exhibited dose dependent cancer cell growth inhibition. In conclusion, the present study demonstrates that irradiation leads to the formation of low molecular weight β-D-glucan with enhanced antioxidant and antiproliferative activities. Copyright © 2014 Elsevier B.V. All rights reserved.

[Induction of NAD(P)H: quinone reductase by anticarcinogenic ingredients of tea].

PubMed

Qi, L; Han, C

1998-09-30

By assaying the activity of NAD(P)H: quinone reductase (QR) in Hep G2 cells exposed to inducing agents, a variety of ingredients in tea, we compared their abilities on inducing QR and preventing cancer. The results showed that tea polyphenols, tea pigments and mixed tea were all able to induce the activity of QR significantly. The single-component ingredients of tea polyphenols and tea pigments, including thearubigens, EGCG and ECG, also enhanced the activity of QR. But EGC, EC, theaflavins, tea polysaccharide and tea caffeine, showed no apparent induction of QR. We found that among those tea ingredients studied, the multi-component ingredients were more effective than the single-component ones. So we thought that the abilities of antioxidation and cancer prevention of tea depended on the combined effects of several kinds of active ingredients, which mainly include tea polyphenols and tea pigments.

NADPH Oxidase Signaling Pathway Mediates Mesenchymal Stem Cell-Induced Inhibition of Hepatic Stellate Cell Activation.

PubMed

Qiao, Haowen; Zhou, Yu; Qin, Xingping; Cheng, Jing; He, Yun; Jiang, Yugang

2018-01-01

Bone marrow-derived mesenchymal stem cells (BMSCs) have blossomed into an effective approach with great potential for the treatment of liver fibrosis. The aim of this study was to investigate the underlying antifibrosis mechanisms by which the BMSC inhibit activated hepatic stellate cells (HSCs) in vivo and in vitro. To study the effect of human bone marrow-derived mesenchymal stem cells (hBM-MSCs) on activated HSCs, we used HSCs and the coculture systems to evaluate the inhibition of activated HSCs from the aspects of the apoptosis of activated HSCs. In addition, activation of NADPH oxidase pathway and the changes in liver histopathology were tested by using the carbon tetrachloride- (CCl 4 -) induced liver fibrosis in mice. Introduction of hBM-MSCs significantly inhibited the proliferation of activated HSCs by inducing the apoptosis process of activated HSCs. The effect of hBM-MSCs reduced the signaling pathway of NADPH oxidase in activated HSCs. Besides, the signaling pathway of NADPH oxidase mediated hBM-MSC upregulation of the expression of the peroxisome proliferator-activated receptor γ and downregulation of the expression of α 1(I) collagen and alpha-smooth muscle actin ( α -SMA) in activated HSCs. Moreover, the hBM-MSC-induced decrease in the signaling pathway of NADPH oxidase was accompanied by the decrease of the activated HSC number and liver fibrosis in a mouse model of CCl 4 -induced liver fibrosis. The hBM-MSCs act as a promising drug source against liver fibrosis development with respect to hepatopathy as a therapeutic target.

The senescence-accelerated mouse prone-8 (SAM-P8) oxidative stress is associated with upregulation of renal NADPH oxidase system.

PubMed

Baltanás, Ana; Solesio, Maria E; Zalba, Guillermo; Galindo, María F; Fortuño, Ana; Jordán, Joaquín

2013-12-01

Herein, we investigate whether the NADPH oxidase might be playing a key role in the degree of oxidative stress in the senescence-accelerated mouse prone-8 (SAM-P8). To this end, the activity and expression of the NADPH oxidase, the ratio of glutathione and glutathione disulfides (GSH/GSSG), and the levels of malonyl dialdehyde (MDA) and nitrotyrosine (NT) were determined in renal tissue from SAM-P8 mice at the age of 1 and 6 months. The senescence-accelerated-resistant mouse (SAM-R1) was used as control. At the age of 1 month, NADPH oxidase activity and Nox2 protein expression were higher in SAM-P8 than in SAM-R1 mice. However, we found no differences in the GSH/GSSG ratio, MDA, NT, and Nox4 levels between both groups of animals. At the age of 6 months, SAM-R1 mice in comparison to SAM-P8 mice showed an increase in NADPH oxidase activity, which is associated with higher levels of NT and increased Nox4 and Nox2 expression levels. Furthermore, we found oxidative stress hallmarks including depletion in GSH/GSSG ratio and increase in MDA levels in the kidney of SAM-P8 mice. Finally, NADPH oxidase activity positively correlated with Nox2 expression in all the animals (r = 0.382, P < 0.05). Taken together, our data allow us to suggest that an increase in NADPH oxidase activity might be an early hallmark to predict future oxidative stress in renal tissue during the aging process that takes place in SAM-P8 mice.

Differential levels of metabolic activity in isolated versus confluent/partially confluent HeLa cells are analyzed by autofluorescent NAD(P)H using multi-photon FLIM microscopy

NASA Astrophysics Data System (ADS)

Chandler, Andrea; Chandler, Aaron; Wallrabe, Horst; Periasamy, Ammasi

2017-02-01

NAD(P)H is a known biomarker for cellular metabolism; a higher ratio of enzyme-bound NAD(P)H to free/unbound NAD(P)H indicates an increase in metabolic activity. Free NADH has a shorter fluorescence lifetime (τ1), the bound version (τ2) a longer lifetime. FLIM's unique capability to establish inter alia the relative fractions of τ1 (a1%) and τ2 (a2%) in each pixel, determines the level of metabolic activity. The relative abundances of bound NAD(P)H were analyzed for single cells, confluent and partially confluent cells within 3 Fields-of-View (FoVs). A gradient of increasing a 2% levels of bound NAD(P)H from single, partially confluent to confluent cells was observed.

Regional yield predictions of malting barley by remote sensing and ancillary data

NASA Astrophysics Data System (ADS)

Weissteiner, Christof J.; Braun, Matthias; Kuehbauch, Walter

2004-02-01

Yield forecasts are of high interest to the malting and brewing industry in order to allow the most convenient purchasing policy of raw materials. Within this investigation, malting barley yield forecasts (Hordeum vulgare L.) were performed for typical growing regions in South-Western Germany. Multisensoral and multitemporal Remote Sensing data on one hand and ancillary meteorological, agrostatistical, topographical and pedological data on the other hand were used as input data for prediction models, which were based on an empirical-statistical modeling approach. Since spring barley production is depending on acreage and on the yield per area, classification is needed, which was performed by a supervised multitemporal classification algorithm, utilizing optical Remote Sensing data (LANDSAT TM/ETM+). Comparison between a pixel-based and an object-oriented classification algorithm was carried out. The basic version of the yield estimation model was conducted by means of linear correlation of Remote Sensing data (NOAA-AVHRR NDVI), CORINE land cover data and agrostatistical data. In an extended version meteorological data (temperature, precipitation, etc.) and soil data was incorporated. Both, basic and extended prediction systems, led to feasible results, depending on the selection of the time span for NDVI accumulation.

Plant exudates may stabilize or weaken soil depending on species, origin and time

PubMed Central

Naveed, M.; Brown, L. K.; Raffan, A. C.; George, T. S.; Bengough, A. G.; Roose, T.; Sinclair, I.; Koebernick, N.; Cooper, L.; Hackett, C. A.

2017-01-01

Summary We hypothesized that plant exudates could either gel or disperse soil depending on their chemical characteristics. Barley (Hordeum vulgare L. cv. Optic) and maize (Zea mays L. cv. Freya) root exudates were collected using an aerated hydroponic method and compared with chia (Salvia hispanica L.) seed exudate, a commonly used root exudate analogue. Sandy loam soil was passed through a 500‐μm mesh and treated with each exudate at a concentration of 4.6 mg exudate g−1 dry soil. Two sets of soil samples were prepared. One set of treated soil samples was maintained at 4°C to suppress microbial processes. To characterize the effect of decomposition, the second set of samples was incubated at 16°C for 2 weeks at −30 kPa matric potential. Gas chromatography–mass spectrometry (GC–MS) analysis of the exudates showed that barley had the largest organic acid content and chia the largest content of sugars (polysaccharide‐derived or free), and maize was in between barley and chia. Yield stress of amended soil samples was measured by an oscillatory strain sweep test with a cone plate rheometer. When microbial decomposition was suppressed at 4°C, yield stress increased 20‐fold for chia seed exudate and twofold for maize root exudate compared with the control, whereas for barley root exudate decreased to half. The yield stress after 2 weeks of incubation compared with soil with suppressed microbial decomposition increased by 85% for barley root exudate, but for chia and maize it decreased by 87 and 54%, respectively. Barley root exudation might therefore disperse soil and this could facilitate nutrient release. The maize root and chia seed exudates gelled soil, which could create a more stable soil structure around roots or seeds. Highlights Rheological measurements quantified physical behaviour of plant exudates and effect on soil stabilization.Barley root exudates dispersed soil, which could release nutrients and carbon.Maize root and chia seed exudates

NADPH oxidase inhibition reduces tubular sodium transport and improves kidney oxygenation in diabetes.

PubMed

Persson, Patrik; Hansell, Peter; Palm, Fredrik

2012-06-15

Sustained hyperglycemia is associated with increased oxidative stress resulting in decreased intrarenal oxygen tension (Po(2)) due to increased oxygen consumption (Qo(2)). Chronic blockade of the main superoxide radicals producing system, the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, normalizes Qo(2) by isolated proximal tubular cells (PTC) and reduces proteinuria in diabetes. The aim was to investigate the effects of acute NADPH oxidase inhibition on tubular Na(+) transport and kidney Po(2) in vivo. Glomerular filtration rate (GFR), renal blood flow (RBF), filtration fraction (FF), Na(+) excretion, fractional Li(+) excretion, and intrarenal Po(2) was measured in control and streptozotocin-diabetic rats during baseline and after acute NADPH oxidase inhibition using apocynin. The effects on tubular transporters were investigated using freshly isolated PTC. GFR was increased in diabetics compared with controls (2.2 ± 0.3 vs. 1.4 ± 0.1 ml·min(-1)·kidney(-1)). RBF was similar in both groups, resulting in increased FF in diabetics. Po(2) was reduced in cortex and medulla in diabetic kidneys compared with controls (34.4 ± 0.7 vs. 42.5 ± 1.2 mmHg and 15.7 ± 1.2 vs. 25.5 ± 2.3 mmHg, respectively). Na(+) excretion was increased in diabetics compared with controls (24.0 ± 4.7 vs. 9.0 ± 2.0 μm·min(-1)·kidney(-1)). In controls, all parameters were unaffected. However, apocynin increased Na(+) excretion (+112%) and decreased fractional lithium reabsorption (-10%) in diabetics, resulting in improved cortical (+14%) and medullary (+28%) Po(2). Qo(2) was higher in PTC isolated from diabetic rats compared with control. Apocynin, dimethylamiloride, and ouabain reduced Qo(2), but the effects of combining apocynin with either dimethylamiloride or ouabain were not additive. In conclusion, NADPH oxidase inhibition reduces tubular Na(+) transport and improves intrarenal Po(2) in diabetes.

Characterisation of marrubenol, a diterpene extracted from Marrubium vulgare, as an L-type calcium channel blocker

PubMed Central

El Bardai, Sanae; Wibo, Maurice; Hamaide, Marie-Christine; Lyoussi, Badiaa; Quetin-Leclercq, Joëlle; Morel, Nicole

2003-01-01

The objective of the present study was to investigate the mechanism of the relaxant activity of marrubenol, a diterpenoid extracted from Marrubium vulgare. In rat aorta, marrubenol was a more potent inhibitor of the contraction evoked by 100 mM KCl (IC50: 11.8±0.3 μM, maximum relaxation: 93±0.6%) than of the contraction evoked by noradrenaline (maximum relaxation: 30±1.5%). In fura-2-loaded aorta, marrubenol simultaneously inhibited the Ca2+ signal and the contraction evoked by 100 mM KCl, and decreased the quenching rate of fura-2 fluorescence by Mn2+. Patch-clamp data obtained in aortic smooth muscle cells (A7r5) indicated that marrubenol inhibited Ba2+ inward current in a voltage-dependent manner (KD: 8±2 and 40±6 μM at holding potentials of −50 and −100 mV, respectively). These results showed that marrubenol inhibits smooth muscle contraction by blocking L-type calcium channels. PMID:14597602

Role of xanthine oxidoreductase and NAD(P)H oxidase in endothelial superoxide production in response to oscillatory shear stress

NASA Technical Reports Server (NTRS)

McNally, J. Scott; Davis, Michael E.; Giddens, Don P.; Saha, Aniket; Hwang, Jinah; Dikalov, Sergey; Jo, Hanjoong; Harrison, David G.

2003-01-01

Oscillatory shear stress occurs at sites of the circulation that are vulnerable to atherosclerosis. Because oxidative stress contributes to atherosclerosis, we sought to determine whether oscillatory shear stress increases endothelial production of reactive oxygen species and to define the enzymes responsible for this phenomenon. Bovine aortic endothelial cells were exposed to static, laminar (15 dyn/cm2), and oscillatory shear stress (+/-15 dyn/cm2). Oscillatory shear increased superoxide (O2.-) production by more than threefold over static and laminar conditions as detected using electron spin resonance (ESR). This increase in O2*- was inhibited by oxypurinol and culture of endothelial cells with tungsten but not by inhibitors of other enzymatic sources. Oxypurinol also prevented H2O2 production in response to oscillatory shear stress as measured by dichlorofluorescin diacetate and Amplex Red fluorescence. Xanthine-dependent O2*- production was increased in homogenates of endothelial cells exposed to oscillatory shear stress. This was associated with decreased xanthine dehydrogenase (XDH) protein levels and enzymatic activity resulting in an elevated ratio of xanthine oxidase (XO) to XDH. We also studied endothelial cells lacking the p47phox subunit of the NAD(P)H oxidase. These cells exhibited dramatically depressed O2*- production and had minimal XO protein and activity. Transfection of these cells with p47phox restored XO protein levels. Finally, in bovine aortic endothelial cells, prolonged inhibition of the NAD(P)H oxidase with apocynin decreased XO protein levels and prevented endothelial cell stimulation of O2*- production in response to oscillatory shear stress. These data suggest that the NAD(P)H oxidase maintains endothelial cell XO levels and that XO is responsible for increased reactive oxygen species production in response to oscillatory shear stress.

A de novo NADPH generation pathway for improving lysine production of Corynebacterium glutamicum by rational design of the coenzyme specificity of glyceraldehyde 3-phosphate dehydrogenase.

PubMed

Bommareddy, Rajesh Reddy; Chen, Zhen; Rappert, Sugima; Zeng, An-Ping

2014-09-01

Engineering the cofactor availability is a common strategy of metabolic engineering to improve the production of many industrially important compounds. In this work, a de novo NADPH generation pathway is proposed by altering the coenzyme specificity of a native NAD-dependent glyceraldehyde 3-phosphate dehydrogenase (GAPDH) to NADP, which consequently has the potential to produce additional NADPH in the glycolytic pathway. Specifically, the coenzyme specificity of GAPDH of Corynebacterium glutamicum is systematically manipulated by rational protein design and the effect of the manipulation for cellular metabolism and lysine production is evaluated. By a combinatorial modification of four key residues within the coenzyme binding sites, different GAPDH mutants with varied coenzyme specificity were constructed. While increasing the catalytic efficiency of GAPDH towards NADP enhanced lysine production in all of the tested mutants, the most significant improvement of lysine production (~60%) was achieved with the mutant showing similar preference towards both NAD and NADP. Metabolic flux analysis with (13)C isotope studies confirmed that there was no significant change of flux towards the pentose phosphate pathway and the increased lysine yield was mainly attributed to the NADPH generated by the mutated GAPDH. The present study highlights the importance of protein engineering as a key strategy in de novo pathway design and overproduction of desired products. Copyright © 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Understanding rhizosphere processes to enhance phytoextraction of germanium and rare earth elements

NASA Astrophysics Data System (ADS)

Wiche, Oliver

2017-04-01

Germanium (Ge) and rare earth elements (REEs) are economically valuable raw materials that are not actually rare in terms of concentrations in soils but they are hardly available for plant uptake due to interactions with organic matter (SOM), secondary soil constituents such as Fe/Mn oxides and P bearing soil fractions. Processes in the rhizosphere might influence availability of Ge and REEs in the soil-plant system, since lowering of the pH and presence of carboxylates and siderophores (small molecules that strongly chelate Fe and other elements) strongly influences the chemical speciation of Ge and REEs in soil and consequently this comprehensive knowledge helps us to improve phytomining. In a series of field and greenhouse experiments 16 plant species from the functional groups of grasses, herbs and legumes were tested with regard to their accumulation efficiency of Ge and REEs in shoots. Subsequently, we conducted mixed culture experiments in which inefficient species (e.g. cereals like Avena sativa, Hordeum vulgare, Panicum miliaceum) were cultivated in mixed cultures with efficient species (Lupinus albus, Lupinus angustifolius). Based on the plant concentrations a principal component analysis (PCA) was performed to identify significant factors that explain the accumulation behavior of different plant species with regard to Ge, REEs, Si, Fe and Mn. In this analysis Mn was used to identify plant species with efficient mechanisms to access sparingly available P-resources in soils. Particularly in nonmycorrhizal species concentrations of Mn in leaves often indicate a carboxylate based P-mobilising strategy. Herbaceous plant species accumulated significantly higher amounts of REEs while grasses accumulated significantly higher amounts of Ge. Concentrations of Ge in shoots of grasses correlated significantly positive with Si, but negatively with concentrations of Mn. Indeed, the results of the PCA clearly show that plants with high Mn concentrations tend to have

Glucose-6-phosphate dehydrogenase, NADPH, and cell survival.

PubMed

Stanton, Robert C

2012-05-01

Glucose-6-phosphate dehydrogenase (G6PD) is the rate-limiting enzyme of the pentose phosphate pathway. Many scientists think that the roles and regulation of G6PD in physiology and pathophysiology have been well established as the enzyme was first identified 80 years ago. And that G6PD has been extensively studied especially with respect to G6PD deficiency and its association with hemolysis, and with respect to the role G6PD plays in lipid metabolism. But there has been a growing understanding of the central importance of G6PD to cellular physiology as it is a major source of NADPH that is required by many essential cellular systems including the antioxidant pathways, nitric oxide synthase, NADPH oxidase, cytochrome p450 system, and others. Indeed G6PD is essential for cell survival. It has also become evident that G6PD is highly regulated by many signals that affect transcription, post-translation, intracellular location, and interactions with other protein. Pathophysiologic roles for G6PD have also been identified in such disease processes as diabetes, aldosterone-induced endothelial dysfunction, cancer, and others. It is now clear that G6PD is under complex regulatory control and of central importance to many cellular processes. In this review the biochemistry, regulatory signals, physiologic roles, and pathophysiologic roles for G6PD that have been elucidated over the past 20 years are discussed. Copyright © 2012 Wiley Periodicals, Inc.

Low-Dose Dextromethorphan, a NADPH Oxidase Inhibitor, Reduces Blood Pressure and Enhances Vascular Protection in Experimental Hypertension

PubMed Central

Wu, Tao-Cheng; Chao, Chih-Yu; Lin, Shing-Jong; Chen, Jaw-Wen

2012-01-01

Background Vascular oxidative stress may be increased with age and aggravate endothelial dysfunction and vascular injury in hypertension. This study aimed to investigate the effects of dextromethorphan (DM), a NADPH oxidase inhibitor, either alone or in combination treatment, on blood pressure (BP) and vascular protection in aged spontaneous hypertensive rats (SHRs). Methodology/Principal Findings Eighteen-week-old WKY rats and SHRs were housed for 2 weeks. SHRs were randomly assigned to one of the 12 groups: untreated; DM monotherapy with 1, 5 or 25 mg/kg/day; amlodipine (AM, a calcium channel blocker) monotherapy with 1 or 5 mg/kg/day; and combination therapy of DM 1, 5 or 25 mg/kg/day with AM 1 or 5 mg/kg/day individually for 4 weeks. The in vitro effects of DM were also examined. In SHRs, AM monotherapy dose-dependently reduced arterial systolic BP. DM in various doses significantly and similarly reduced arterial systolic BP. Combination of DM with AM gave additive effects on BP reduction. DM, either alone or in combination with AM, improved aortic endothelial function indicated by ex vivo acetylcholine-induced relaxation. The combination of low-dose DM with AM gave most significant inhibition on aortic wall thickness in SHRs. Plasma total antioxidant status was significantly increased by all the therapies except for the combination of high-dose DM with high-dose AM. Serum nitrite and nitrate level was significantly reduced by AM but not by DM or the combination of DM with AM. Furthermore, in vitro treatment with DM reduced angiotensin II-induced reactive oxygen species and NADPH oxidase activation in human aortic endothelial cells. Conclusions/Significance Treatment of DM reduced BP and enhanced vascular protection probably by inhibiting vascular NADPH oxidase in aged hypertensive animals with or without AM treatment. It provides the potential rationale to a novel combination treatment with low-dose DM and AM in clinical hypertension. PMID:23049937

Micro-RNA 21 inhibition of SMAD7 enhances fibrogenesis via leptin-mediated NADPH oxidase in experimental and human nonalcoholic steatohepatitis.

PubMed

Dattaroy, Diptadip; Pourhoseini, Sahar; Das, Suvarthi; Alhasson, Firas; Seth, Ratanesh Kumar; Nagarkatti, Mitzi; Michelotti, Gregory A; Diehl, Anna Mae; Chatterjee, Saurabh

2015-02-15

Hepatic fibrosis in nonalcoholic steatohepatitis (NASH) is the common pathophysiological process resulting from chronic liver inflammation and oxidative stress. Although significant research has been carried out on the role of leptin-induced NADPH oxidase in fibrogenesis, the molecular mechanisms that connect the leptin-NADPH oxidase axis in upregulation of transforming growth factor (TGF)-β signaling have been unclear. We aimed to investigate the role of leptin-mediated upregulation of NADPH oxidase and its subsequent induction of micro-RNA 21 (miR21) in fibrogenesis. Human NASH livers and a high-fat (60% kcal) diet-fed chronic mouse model, where hepatotoxin bromodichloromethane was used to induce NASH, were used for this study. To prove the role of the leptin-NADPH oxidase-miR21 axis, mice deficient in genes for leptin, p47phox, and miR21 were used. Results showed that wild-type mice and human livers with NASH had increased oxidative stress, increased p47phox expression, augmented NF-κB activation, and increased miR21 levels. These mice and human livers showed increased TGF-β, SMAD2/3-SMAD4 colocalizations in the nucleus, increased immunoreactivity against Col1α, and α-SMA with a concomitant decrease in protein levels of SMAD7. Mice that were deficient in leptin or p47phox had decreased activated NF-κB and miR21 levels, suggesting the role of leptin and NADPH oxidase in inducing NF-κB-mediated miR21 expression. Further miR21 knockout mice had decreased colocalization events of SMAD2/3-SMAD4 in the nucleus, increased SMAD7 levels, and decreased fibrogenesis. Taken together, the studies show the novel role of leptin-NADPH oxidase induction of miR21 as a key regulator of TGF-β signaling and fibrogenesis in experimental and human NASH. Copyright © 2015 the American Physiological Society.

NADPH Oxidase-Mediated ROS Production Determines Insulin's Action on the Retinal Microvasculature.

PubMed

Kida, Teruyo; Oku, Hidehiro; Horie, Taeko; Matsuo, Junko; Kobayashi, Takatoshi; Fukumoto, Masanori; Ikeda, Tsunehiko

2015-10-01

To determine whether insulin induces nitric oxide (NO) formation in retinal microvessels and to examine the effects of high glucose on the formation of NO. Freshly isolated rat retinal microvessels were incubated in normal (5.5 mM) or high (20 mM) glucose with or without insulin (100 nM). The levels of insulin-induced NO and reactive oxygen species (ROS) in the retinal microvessels were determined semiquantitatively using fluorescent probes, 4,5-diaminofluorescein diacetate, and hydroethidine, respectively, and a laser scanning confocal microscope. The insulin-induced changes of NO in rat retinal endothelial cells and pericytes cultured at different glucose concentrations (5.5 and 25 mM) were determined using flow cytometry. Nitric oxide synthase (NOS) protein levels were determined by Western blot analysis; intracellular levels of ROS were determined using fluorescence-activated cell sorting (FACS) analysis of ethidium fluorescence; and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase RNA expression was quantified using real-time PCR. Exposure of microvessels to insulin under normal glucose conditions led to a significant increase in NO levels; however, this increase was significantly suppressed when the microvessels were incubated under high glucose conditions. Intracellular levels of ROS were significantly increased in both retinal microvessels and cultured microvascular cells under high glucose conditions. The expression of NOS and NADPH oxidase were significantly increased in endothelial cells and pericytes under high glucose conditions. The increased formation of NO by insulin and its suppression by high glucose conditions suggests that ROS production mediated by NADPH oxidase is important by insulin's effect on the retinal microvasculature.

Structure and function of NADPH-cytochrome P450 reductase and nitric oxide synthase reductase domain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Iyanagi, Takashi

2005-12-09

NADPH-cytochrome P450 reductase (CPR) and the nitric oxide synthase (NOS) reductase domains are members of the FAD-FMN family of proteins. The FAD accepts two reducing equivalents from NADPH (dehydrogenase flavin) and FMN acts as a one-electron carrier (flavodoxin-type flavin) for the transfer from NADPH to the heme protein, in which the FMNH {sup {center_dot}}/FMNH{sub 2} couple donates electrons to cytochrome P450 at constant oxidation-reduction potential. Although the interflavin electron transfer between FAD and FMN is not strictly regulated in CPR, electron transfer is activated in neuronal NOS reductase domain upon binding calmodulin (CaM), in which the CaM-bound activated form canmore » function by a similar mechanism to that of CPR. The oxygenated form and spin state of substrate-bound cytochrome P450 in perfused rat liver are also discussed in terms of stepwise one-electron transfer from CPR. This review provides a historical perspective of the microsomal mixed-function oxidases including CPR and P450. In addition, a new model for the redox-linked conformational changes during the catalytic cycle for both CPR and NOS reductase domain is also discussed.« less

Mechanism and characteristics of stimuli-dependent ROS generation in undifferentiated HL-60 cells.

PubMed

Muranaka, Shikibu; Fujita, Hirofumi; Fujiwara, Takuzo; Ogino, Tetsuya; Sato, Eisuke F; Akiyama, Jitsuo; Imada, Isuke; Inoue, Masayasu; Utsumi, Kozo

2005-01-01

It has been widely believed that undifferentiated human promyelocytic leukemia cells (HL-60) have no ability to generate reactive oxygen species (ROS) responding to stimuli. We report here that undifferentiated HL-60 cells possess NADPH oxidase and that generation of superoxide can be measured using a highly sensitive chemiluminescence dye, L-012. Five subunits of NADPH oxidase, namely, gp91(phox), p22(phox), p67(phox), p47(phox), and Rac 2, were detected in undifferentiated HL-60 cells by immunoblotting analysis. The contents of these NADPH oxidase components in the cells were increased with the differentiation induced by phorbol myristate acetate (PMA), except for p22(phox). Messenger RNAs of these subunits were also detected by the RT-PCR method, and their expressions increased except that of p22(phox) with the differentiation induced by PMA. Kinetic analysis using L-012 revealed that HL-60 cells generated substantial amounts of ROS by various stimulants, including formylmethionyl-leucyl-phenylalanine, PMA, myristic acid, and a Ca2+ ionophore, A23187. Both diphenyleneiodonium (an inhibitor of FAD-dependent oxidase) and apocynin (a specific inhibitor of NADPH oxidase) suppressed this stimuli-dependent ROS generation. Genistein, staurosporine, uric acid, and sodium azide inhibited the ROS generation in undifferentiated HL-60 cells in a similar way to that in undifferentiated neutrophils. These results suggested that the mechanism of ROS generation in undifferentiated HL-60 cells is the same as that in primed neutrophils.

Role of cytosolic NADP+-dependent isocitrate dehydrogenase in ischemia-reperfusion injury in mouse kidney.

PubMed

Kim, Jinu; Kim, Ki Young; Jang, Hee-Seong; Yoshida, Takumi; Tsuchiya, Ken; Nitta, Kosaku; Park, Jeen-Woo; Bonventre, Joseph V; Park, Kwon Moo

2009-03-01

Cytosolic NADP+-dependent isocitrate dehydrogenase (IDPc) synthesizes reduced NADP (NADPH), which is an essential cofactor for the generation of reduced glutathione (GSH), the most abundant and important antioxidant in mammalian cells. We investigated the role of IDPc in kidney ischemia-reperfusion (I/R) in mice. The activity and expression of IDPc were highest in the cortex, modest in the outer medulla, and lowest in the inner medulla. NADPH levels were greatest in the cortex. IDPc expression in the S1 and S2 segments of proximal tubules was higher than in the S3 segment, which is much more susceptible to I/R. IDPc protein was also highly expressed in the mitochondrion-rich intercalated cells of the collecting duct. IDPc activity was 10- to 30-fold higher than the activity of glucose-6-phosphate dehydrogenase, another producer of cytosolic NADPH, in various kidney regions. This study identifies that IDPc may be the primary source of NADPH in the kidney. I/R significantly reduced IDPc expression and activity and NADPH production and increased the ratio of oxidized glutathione to total glutathione [GSSG/(GSH+GSSG)], resulting in kidney dysfunction, tubular cell damage, and lipid peroxidation. In LLC-PK(1) cells, upregulation of IDPc by IDPc gene transfer protected the cells against hydrogen peroxide, enhancing NADPH production, inhibiting the increase of GSSG/(GSH+GSSG), and reducing lipid peroxidation. IDPc downregulation by small interference RNA treatment presented results contrasting with the upregulation. In conclusion, these results demonstrate that IDPc is expressed differentially along tubules in patterns that may contribute to differences in susceptibility to injury, is a major enzyme in cytosolic NADPH generation in kidney, and is downregulated with I/R.

Role of cytosolic NADP+-dependent isocitrate dehydrogenase in ischemia-reperfusion injury in mouse kidney

PubMed Central

Kim, Jinu; Kim, Ki Young; Jang, Hee-Seong; Yoshida, Takumi; Tsuchiya, Ken; Nitta, Kosaku; Park, Jeen-Woo; Bonventre, Joseph V.; Park, Kwon Moo

2009-01-01

Cytosolic NADP+-dependent isocitrate dehydrogenase (IDPc) synthesizes reduced NADP (NADPH), which is an essential cofactor for the generation of reduced glutathione (GSH), the most abundant and important antioxidant in mammalian cells. We investigated the role of IDPc in kidney ischemia-reperfusion (I/R) in mice. The activity and expression of IDPc were highest in the cortex, modest in the outer medulla, and lowest in the inner medulla. NADPH levels were greatest in the cortex. IDPc expression in the S1 and S2 segments of proximal tubules was higher than in the S3 segment, which is much more susceptible to I/R. IDPc protein was also highly expressed in the mitochondrion-rich intercalated cells of the collecting duct. IDPc activity was 10- to 30-fold higher than the activity of glucose-6-phosphate dehydrogenase, another producer of cytosolic NADPH, in various kidney regions. This study identifies that IDPc may be the primary source of NADPH in the kidney. I/R significantly reduced IDPc expression and activity and NADPH production and increased the ratio of oxidized glutathione to total glutathione [GSSG/(GSH+GSSG)], resulting in kidney dysfunction, tubular cell damage, and lipid peroxidation. In LLC-PK1 cells, upregulation of IDPc by IDPc gene transfer protected the cells against hydrogen peroxide, enhancing NADPH production, inhibiting the increase of GSSG/(GSH+GSSG), and reducing lipid peroxidation. IDPc downregulation by small interference RNA treatment presented results contrasting with the upregulation. In conclusion, these results demonstrate that IDPc is expressed differentially along tubules in patterns that may contribute to differences in susceptibility to injury, is a major enzyme in cytosolic NADPH generation in kidney, and is downregulated with I/R. PMID:19106211

NADPH oxidase inhibitor, diphenyleneiodonium prevents necroptosis in HK-2 cells.

PubMed

Dong, Wei; Li, Zhilian; Chen, Yuanhan; Zhang, Li; Ye, Zhiming; Liang, Huaban; Li, Ruizhao; Xu, Lixia; Zhang, Bin; Liu, Shuangxin; Wang, Weidong; Li, Chunling; Luo, Jialun; Shi, Wei; Liang, Xinling

2017-09-01

The aim of the present study was to investigate the protective effect of the NADPH oxidase inhibitor, diphenyleneiodonium (DPI) against necroptosis in renal tubular epithelial cells. A necroptosis model of HK-2 cells was established using tumor necrosis factor-α, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone and antimycin A (collectively termed TZA), as in our previous research. The necroptosis inhibitor, necrostatin-1 (Nec-1) or the NADPH oxidase inhibitor, DPI were administered to the necroptosis model. Production of reactive oxygen species (ROS) was detected by dichlorodihydrofluorescein diacetate in the different groups, and the manner of cell death was identified by flow cytometry. Western blot analysis was used to determine the levels of phosphorylation of receptor-interacting protein kinase 3 (RIP-3) and mixed lineage kinase domain-like (MLKL), which are essential to necroptosis. The results revealed that TZA increased the percentages of propidium iodide-positive HK-2 cells from 1.22±0.69 to 8.98±0.73% (P<0.001), and augmented the phosphorylation of RIP-3 and MLKL. ROS levels were increased in the TZA group compared with the control group (27.74±1.60×10 4 vs. 18.51±1.10×10 4 , respectively; P<0.001), and could be inhibited by Nec-1 (TZA + Nec-1 group, 22.90±2.22×10 4 vs. TZA group, 27.74±1.60×10 4 ; P=0.01). DPI decreased ROS production (TZA + DPI group, 22.13±1.86×10 4 vs. TZA group, 27.74±1.60×10 4 ; P<0.001) and also reduced the proportions of necrosis in the necroptosis model (TZA + DPI group, 4.40±1.51% vs. TZA group, 8.98±0.73%; P<0.001). Phosphorylated RIP-3 and MLKL were also decreased by DPI treatment. The results indicate that ROS production increases in HK-2 cells undergoing necroptosis, and that the NADPH oxidase inhibitor, DPI may protect HK-2 cells from necroptosis via inhibition of ROS production.

Reactive Oxygen Species and Inhibitors of Inflammatory Enzymes, NADPH Oxidase, and iNOS in Experimental Models of Parkinson's Disease

PubMed Central

Koppula, Sushruta; Kumar, Hemant; Kim, In Su; Choi, Dong-Kug

2012-01-01

Reactive oxygen species (ROSs) are emerging as important players in the etiology of neurodegenerative disorders including Parkinson's disease (PD). Out of several ROS-generating systems, the inflammatory enzymes nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and inducible nitric oxide synthase (iNOS) were believed to play major roles. Mounting evidence suggests that activation of NADPH oxidase and the expression of iNOS are directly linked to the generation of highly reactive ROS which affects various cellular components and preferentially damage midbrain dopaminergic neurons in PD. Therefore, appropriate management or inhibition of ROS generated by these enzymes may represent a therapeutic target to reduce neuronal degeneration seen in PD. Here, we have summarized recently developed agents and patents claimed as inhibitors of NADPH oxidase and iNOS enzymes in experimental models of PD. PMID:22577256

NADPH Thioredoxin Reductase C and Thioredoxins Act Concertedly in Seedling Development.

PubMed

Ojeda, Valle; Pérez-Ruiz, Juan Manuel; González, Maricruz; Nájera, Victoria A; Sahrawy, Mariam; Serrato, Antonio J; Geigenberger, Peter; Cejudo, Francisco Javier

2017-07-01

Thiol-dependent redox regulation of enzyme activity plays a central role in the rapid acclimation of chloroplast metabolism to ever-fluctuating light availability. This regulatory mechanism relies on ferredoxin reduced by the photosynthetic electron transport chain, which fuels reducing power to thioredoxins (Trxs) via a ferredoxin-dependent Trx reductase. In addition, chloroplasts harbor an NADPH-dependent Trx reductase, which has a joint Trx domain at the carboxyl terminus, termed NTRC. Thus, a relevant issue concerning chloroplast function is to establish the relationship between these two redox systems and its impact on plant development. To address this issue, we generated Arabidopsis ( Arabidopsis thaliana ) mutants combining the deficiency of NTRC with those of Trxs f , which participate in metabolic redox regulation, and that of Trx x , which has antioxidant function. The ntrc-trxf1f2 and, to a lower extent, ntrc-trxx mutants showed severe growth-retarded phenotypes, decreased photosynthesis performance, and almost abolished light-dependent reduction of fructose-1,6-bisphosphatase. Moreover, the combined deficiency of both redox systems provokes aberrant chloroplast ultrastructure. Remarkably, both the ntrc-trxf1f2 and ntrc-trxx mutants showed high mortality at the seedling stage, which was overcome by the addition of an exogenous carbon source. Based on these results, we propose that NTRC plays a pivotal role in chloroplast redox regulation, being necessary for the activity of diverse Trxs with unrelated functions. The interaction between the two thiol redox systems is indispensable to sustain photosynthesis performed by cotyledons chloroplasts, which is essential for early plant development. © 2017 American Society of Plant Biologists. All Rights Reserved.

Antioxidant capacity and total phenolic contents of oregano (Origanum vulgare), lavender (Lavandula angustifolia) and lemon balm (Melissa officinalis) from Romania.

PubMed

Spiridon, Iuliana; Colceru, Svetlana; Anghel, Narcis; Teaca, Carmen Alice; Bodirlau, Ruxanda; Armatu, Alice

2011-10-01

The study reported here presents a comparative screening of three medicinal plants including oregano (Origanum vulgare L.), lavender (Lavandula angustifolia) and lemon balm (Melissa officinalis) having the same geographical origin, the Southeast region of Romania, and growing in the same natural conditions. The contents of total phenolics and total flavonoids for the extracts of these were determined. Furthermore, the total antioxidant capacity was also evaluated. It was found that Origanum vulgare and Melissa officinalis extracts present the most effective antioxidant capacity in scavenging DPPH radicals, while Lavandula angustifolia is less active. High performance liquid chromatography-mass spectrometry analysis was used to identify the components of extracts. Major phenolic acids identified in the analysed species were ferulic, rosmarinic, p-coumaric and caffeic, while predominant flavonoids were quercetin, apigenin kaempherol, which were present as glucosides.

Molecular cloning and functional characterization of multiple NADPH-cytochrome P450 reductases from Andrographis paniculata.

PubMed

Lin, Huixin; Wang, Jian; Qi, Mengdie; Guo, Juan; Rong, Qixian; Tang, Jinfu; Wu, Yisheng; Ma, Xiaojing; Huang, Luqi

2017-09-01

Andrographis paniculata (Burm.f.) Wall. ex Nees is widely used as medicinal herb in Southern and Southeastern Asia and andrographolide is its main medicinal constituent. Based on the structure of andrographolide, it has been proposed that cytochrome P450 enzymes play vital roles on its biosynthesis. NADPH:cytochrome P450 reductase (CPR) is the most important redox partner of multiple P450s. In this study, three CPRs were identified in the genomic data of A. paniculata (namely ApCPR1, ApCPR2, and ApCPR3), and their coding regions were cloned. They varied from 62% to 70% identities to each other at the amino acid sequence level. ApCPR1 belongs to Class I of dicotyledonous CPR while both ApCPR2 and ApCPR3 are grouped to Class II. The recombinant enzymes ApCPR1 and ApCPR2 reduced cytochrome c and ferricyanide in an NADPH-dependent manner. In yeast, they supported the activity of CYP76AH1, a ferruginol-forming enzyme. However, ApCPR3 did not show any enzymatic activities either in vitro or in vivo. Quantitative real-time PCR analysis showed that both ApCPR1 and ApCPR2 expressed in all tissues examined, but ApCPR2 showed higher expression in leaves. Expression of ApCPR2 was inducible by MeJA and its pattern matched with andrographolide accumulation. Present investigation suggested ApCPR2 involves in the biosynthesis of secondary metabolites including andrographolide. Copyright © 2017. Published by Elsevier B.V.

The arachidonic acid-binding protein S100A8/A9 promotes NADPH oxidase activation by interaction with p67phox and Rac-2.

PubMed

Kerkhoff, Claus; Nacken, Wolfgang; Benedyk, Malgorzata; Dagher, Marie Claire; Sopalla, Claudia; Doussiere, Jacques

2005-03-01

The Ca2+- and arachidonic acid-binding S100A8/A9 protein complex was recently identified by in vitro studies as a novel partner of the phagocyte NADPH oxidase. The present study demonstrated its functional relevance by the impaired oxidase activity in neutrophil-like NB4 cells, after specific blockage of S100A9 expression, and bone marrow polymorphonuclear neutrophils from S100A9-/- mice. The impaired oxidase activation could also be mimicked in a cell-free system by pretreatment of neutrophil cytosol with an S100A9-specific antibody. Further analyses gave insights into the molecular mechanisms by which S100A8/A9 promoted NADPH oxidase activation. In vitro analysis of oxidase activation as well as protein-protein interaction studies revealed that S100A8 is the privileged interaction partner for the NADPH oxidase complex since it bound to p67phox and Rac, whereas S100A9 did interact with neither p67phox nor p47phox. Moreover, S100A8/A9 transferred the cofactor arachidonic acid to NADPH oxidase as shown by the impotence of a mutant S100A8/A9 complex unable to bind arachidonic acid to enhance NADPH oxidase activity. It is concluded that S100A8/A9 plays an important role in phagocyte NADPH oxidase activation.

The NADPH oxidase inhibitor apocynin (acetovanillone) induces oxidative stress

DOE Office of Scientific and Technical Information (OSTI.GOV)

Riganti, Chiara; Costamagna, Costanzo; Bosia, Amalia

Apocynin (acetovanillone) is often used as a specific inhibitor of NADPH oxidase. In N11 glial cells, apocynin induced, in a dose-dependent way, a significant increase of both malonyldialdehyde level (index of lipid peroxidation) and lactate dehydrogenase release (index of a cytotoxic effect). Apocynin evoked also, in a significant way, an increase of H{sub 2}O{sub 2} concentration and a decrease of the intracellular glutathione/glutathione disulfide ratio, accompanied by augmented efflux of glutathione and glutathione disulfide. Apocynin induced the activation of both pentose phosphate pathway and tricarboxylic acid cycle, which was blocked when the cells were incubated with glutathione together with apocynin.more » The cell incubation with glutathione prevented also the apocynin-induced increase of malonyldialdehyde generation and lactate dehydrogenase leakage. Apocynin exerted an oxidant effect also in a cell-free system: indeed, in aqueous solution, it evoked a faster oxidation of the thiols glutathione and dithiothreitol, and elicited the generation of reactive oxygen species, mainly superoxide anions. Our results suggest that apocynin per se can induce an oxidative stress and exert a cytotoxic effect in N11 cells and other cell types, and that some effects of apocynin in in vitro and in vivo experimental models should be interpreted with caution.« less

Elevated Mitochondrial Reactive Oxygen Species and Cellular Redox Imbalance in Human NADPH-Oxidase-Deficient Phagocytes

PubMed Central

Sundqvist, Martina; Christenson, Karin; Björnsdottir, Halla; Osla, Veronica; Karlsson, Anna; Dahlgren, Claes; Speert, David P.; Fasth, Anders; Brown, Kelly L.; Bylund, Johan

2017-01-01

Chronic granulomatous disease (CGD) is caused by mutations in genes that encode the NADPH-oxidase and result in a failure of phagocytic cells to produce reactive oxygen species (ROS) via this enzyme system. Patients with CGD are highly susceptible to infections and often suffer from inflammatory disorders; the latter occurs in the absence of infection and correlates with the spontaneous production of inflammatory cytokines. This clinical feature suggests that NADPH-oxidase-derived ROS are not required for, or may even suppress, inflammatory processes. Experimental evidence, however, implies that ROS are in fact required for inflammatory cytokine production. By using a myeloid cell line devoid of a functional NADPH-oxidase and primary CGD cells, we analyzed intracellular oxidants, signs of oxidative stress, and inflammatory cytokine production. Herein, we demonstrate that phagocytes lacking a functional NADPH-oxidase, namely primary CGD phagocytes and a gp91phox-deficient cell line, display elevated levels of ROS derived from mitochondria. Accordingly, these cells, despite lacking the major source of cellular ROS, display clear signs of oxidative stress, including an induced expression of antioxidants and altered oxidation of cell surface thiols. These observed changes in redox state were not due to abnormalities in mitochondrial mass or membrane integrity. Finally, we demonstrate that increased mitochondrial ROS enhanced phosphorylation of ERK1/2, and induced production of IL8, findings that correlate with previous observations of increased MAPK activation and inflammatory cytokine production in CGD cells. Our data show that elevated baseline levels of mitochondria-derived oxidants lead to the counter-intuitive observation that CGD phagocytes are under oxidative stress and have enhanced MAPK signaling, which may contribute to the elevated basal production of inflammatory cytokines and the sterile inflammatory manifestations in CGD. PMID:29375548

Water Potential in Excised Leaf Tissue

PubMed Central

Nelsen, Charles E.; Safir, Gene R.; Hanson, Andrew D.

1978-01-01

Leaf water potential (Ψleaf) determinations were made on excised leaf samples using a commercial dew point hygrometer (Wescor Inc., Logan, Utah) and a thermocouple psychrometer operated in the isopiestic mode. With soybean leaves (Glycine max L.), there was good agreement between instruments; equilibration times were 2 to 3 hours. With cereals (Triticum aestivum L. and Hordeum vulgare L.), agreement between instruments was poor for moderately wilted leaves when 7-mm-diameter punches were used in the hygrometer and 20-mm slices were used in the psychrometer, because the Ψleaf values from the dew point hygrometer were too high. Agreement was improved by replacing the 7-mm punch samples in the hygrometer by 13-mm slices, which had a lower cut edge to volume ratio. Equilibration times for cereals were normally 6 to 8 hours. Spuriously high Ψleaf values obtained with 7-mm leaf punches may be associated with the ion release and reabsorption that occur upon tissue excision; such errors evidently depend both on the species and on tissue water status. PMID:16660227

The Barley Phytomer

PubMed Central

Forster, Brian P.; Franckowiak, Jerome D.; Lundqvist, Udda; Lyon, Jackie; Pitkethly, Ian; Thomas, William T. B.

2007-01-01

Background and Aims Morphological mutants have been useful in elucidating the phytomeric structure of plants. Recently described mutants have shed new light on the ontogeny (development of plant structures) and the phytomeric system of barley (Hordeum vulgare). Since the current model for barley phytomers was not adequate to explain the nature of some mutants, a new model is proposed. Methods New phytomer mutants were detected by visual assessment of mutant families in the Optic barley mutation grid population. This was done at various growth stages using laboratory, glasshouse and field screens. Simple explanations were adopted to account for aberrant phytomer phenotypes and a thesis for a new phytomer model was developed. Key Results and Conclusions A barley phytomer model is presented, in which the origins of vegetative and generative structures can be explained by a single repeating phytomer unit. Organs on the barley plant are divided into two classes, single or paired, depending on their origin. Paired structures are often fused together to create specific organs. The model can be applied to wheat (Triticum aestivum) and related grasses. PMID:17901062

Water potential in excised leaf tissue. Comparison of a commercial dew point hygrometer and a thermocouple psychrometer on soybean, wheat, and barley

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nelsen, C.E.; Safir, G.R.; Hanson, A.D.

1978-01-01

Leaf water potential (Psi/sub leaf/) determinations were made on excised leaf samples using a commercial dew point hygrometer (Wescor Inc., Logan, Utah) and a thermocouple psychrometer operated in the isopiestic mode. With soybean leaves (Glycine max L.), there was good agreement between instruments; equilibration times were 2 to 3 hours. With cereals (Triticum aestivum L. and Hordeum vulgare L.), agreement between instruments was poor for moderately wilted leaves when 7-mm-diameter punches were used in the hygrometer and 20-mm slices were used in the psychrometer, because the Psi/sub leaf/ values from the dew point hygrometer were too high. Agreement was improvedmore » by replacing the 7-mm punch samples in the hygrometer by 13-mm slices, which had a lower cut edge to volume ratio. Equilibration times for cereals were normally 6 to 8 hours. Spuriously high Psi/sub leaf/ values obtained with 7-mm leaf punches may be associated with the ion release and reabsorption that occur upon tissue excision; such errors evidently depend both on the species and on tissue water status.« less

Spatio-Temporal Dynamics of Fructan Metabolism in Developing Barley Grains[W

PubMed Central

Peukert, Manuela; Thiel, Johannes; Peshev, Darin; Weschke, Winfriede; Van den Ende, Wim; Mock, Hans-Peter; Matros, Andrea

2014-01-01

Barley (Hordeum vulgare) grain development follows a series of defined morphological and physiological stages and depends on the supply of assimilates (mainly sucrose) from the mother plant. Here, spatio-temporal patterns of sugar distributions were investigated by mass spectrometric imaging, targeted metabolite analyses, and transcript profiling of microdissected grain tissues. Distinct spatio-temporal sugar balances were observed, which may relate to differentiation and grain filling processes. Notably, various types of oligofructans showed specific distribution patterns. Levan- and graminan-type oligofructans were synthesized in the cellularized endosperm prior to the commencement of starch biosynthesis, while during the storage phase, inulin-type oligofructans accumulated to a high concentration in and around the nascent endosperm cavity. In the shrunken endosperm mutant seg8, with a decreased sucrose flux toward the endosperm, fructan accumulation was impaired. The tight partitioning of oligofructan biosynthesis hints at distinct functions of the various fructan types in the young endosperm prior to starch accumulation and in the endosperm transfer cells that accomplish the assimilate supply toward the endosperm at the storage phase. PMID:25271242

Water potential in excised leaf tissue: comparison of a commercial dew point hygrometer and a thermocouple psychrometer on soybean, wheat, and barley.

PubMed

Nelsen, C E; Safir, G R; Hanson, A D

1978-01-01

Leaf water potential (Psi(leaf)) determinations were made on excised leaf samples using a commercial dew point hygrometer (Wescor Inc., Logan, Utah) and a thermocouple psychrometer operated in the isopiestic mode. With soybean leaves (Glycine max L.), there was good agreement between instruments; equilibration times were 2 to 3 hours. With cereals (Triticum aestivum L. and Hordeum vulgare L.), agreement between instruments was poor for moderately wilted leaves when 7-mm-diameter punches were used in the hygrometer and 20-mm slices were used in the psychrometer, because the Psi(leaf) values from the dew point hygrometer were too high. Agreement was improved by replacing the 7-mm punch samples in the hygrometer by 13-mm slices, which had a lower cut edge to volume ratio. Equilibration times for cereals were normally 6 to 8 hours. Spuriously high Psi(leaf) values obtained with 7-mm leaf punches may be associated with the ion release and reabsorption that occur upon tissue excision; such errors evidently depend both on the species and on tissue water status.

Transcriptome profiling reveals mosaic genomic origins of modern cultivated barley.

PubMed

Dai, Fei; Chen, Zhong-Hua; Wang, Xiaolei; Li, Zefeng; Jin, Gulei; Wu, Dezhi; Cai, Shengguan; Wang, Ning; Wu, Feibo; Nevo, Eviatar; Zhang, Guoping

2014-09-16

The domestication of cultivated barley has been used as a model system for studying the origins and early spread of agrarian culture. Our previous results indicated that the Tibetan Plateau and its vicinity is one of the centers of domestication of cultivated barley. Here we reveal multiple origins of domesticated barley using transcriptome profiling of cultivated and wild-barley genotypes. Approximately 48-Gb of clean transcript sequences in 12 Hordeum spontaneum and 9 Hordeum vulgare accessions were generated. We reported 12,530 de novo assembled transcripts in all of the 21 samples. Population structure analysis showed that Tibetan hulless barley (qingke) might have existed in the early stage of domestication. Based on the large number of unique genomic regions showing the similarity between cultivated and wild-barley groups, we propose that the genomic origin of modern cultivated barley is derived from wild-barley genotypes in the Fertile Crescent (mainly in chromosomes 1H, 2H, and 3H) and Tibet (mainly in chromosomes 4H, 5H, 6H, and 7H). This study indicates that the domestication of barley may have occurred over time in geographically distinct regions.

A novel domain of amino-Nogo-A protects HT22 cells exposed to oxygen glucose deprivation by inhibiting NADPH oxidase activity.

PubMed

Guo, Fan; Wang, Huiwen; Li, Liya; Zhou, Heng; Wei, Haidong; Jin, Weilin; Wang, Qiang; Xiong, Lize

2013-04-01

This study aimed to investigate the protective effect of the M9 region (residues 290-562) of amino-Nogo-A fused to the human immunodeficiency virus trans-activator TAT in an in vitro model of ischemia-reperfusion induced by oxygen-glucose deprivation (OGD) in HT22 hippocampal neurons, and to investigate the role of NADPH oxidase in this protection. Transduction of TAT-M9 was analyzed by immunofluorescence staining and western blot. The biologic activity of TAT-M9 was assessed by its effects against OGD-induced HT22 cell damage, compared with a mutant M9 fusion protein or vehicle. Cellular viability and lactate dehydrogenase (LDH) release were assessed. Neuronal apoptosis was evaluated by flow cytometry. The Bax/Bcl-2 ratio was determined by western blotting. Reactive oxygen species (ROS) levels and NADPH oxidase activity were also measured in the presence or absence of an inhibitor or activator of NADPH oxidase. Our results confirmed the delivery of the protein into HT22 cells by immunofluorescence and western blot. Addition of 0.4 μmol/L TAT-M9 to the culture medium effectively improved neuronal cell viability and reduced LDH release induced by OGD. The fusion protein also protected HT22 cells from apoptosis, suppressed overexpression of Bax, and inhibited the reduction in Bcl-2 expression. Furthermore, TAT-M9, as well as apocynin, decreased NADPH oxidase activity and ROS content. The protective effects of the TAT-M9 were reversed by TBCA, an agonist of NADPH oxidase. In conclusion, TAT-M9 could be successfully transduced into HT22 cells, and protected HT22 cells against OGD damage by inhibiting NADPH oxidase-mediated oxidative stress. These findings suggest that the TAT-M9 protein may be an efficient therapeutic agent for neuroprotection.

NADPH Oxidase Contributes to Resistance against Aggregatibacter actinomycetemcomitans-Induced Periodontitis in Mice.

PubMed

Bast, Antje; Kubis, Helen; Holtfreter, Birte; Ribback, Silvia; Martin, Heiner; Schreiner, Helen C; Dominik, Malte J; Breitbach, Katrin; Dombrowski, Frank; Kocher, Thomas; Steinmetz, Ivo

2017-02-01

Aggregatibacter actinomycetemcomitans is a Gram-negative commensal bacterium of the oral cavity which has been associated with the pathogenesis of periodontitis with severe alveolar bone destruction. The role of host factors such as reactive oxygen and nitrogen intermediates in periodontal A. actinomycetemcomitans infection and progression to periodontitis is still ill-defined. Therefore, this study aimed to analyze the role of NADPH oxidase and inducible nitric oxide synthase (iNOS) in a murine model of A. actinomycetemcomitans-induced periodontitis. NADPH oxidase-deficient (gp91 phox knockout [KO]), iNOS-deficient (iNOS KO), and C57BL/6 wild-type mice were orally infected with A. actinomycetemcomitans and analyzed for bacterial colonization at various time points. Alveolar bone mineral density and alveolar bone volume were quantified by three-dimensional micro-computed tomography, and the degree of tissue inflammation was calculated by histological analyses. At 5 weeks after infection, A. actinomycetemcomitans persisted at significantly higher levels in the murine oral cavities of infected gp91 phox KO mice than in those of iNOS KO and C57BL/6 mice. Concomitantly, alveolar bone mineral density was significantly lower in all three infected groups than in uninfected controls, but with the highest loss of bone density in infected gp91 phox KO mice. Only infected gp91 phox KO mice revealed significant loss of alveolar bone volume and enhanced inflammatory cell infiltration, as well as an increased number of osteoclasts. Our results indicate that NADPH oxidase is important to control A. actinomycetemcomitans infection in the murine oral cavity and to prevent subsequent alveolar bone destruction and osteoclastogenesis. Copyright © 2017 American Society for Microbiology.

NADPH Oxidase Contributes to Resistance against Aggregatibacter actinomycetemcomitans-Induced Periodontitis in Mice

PubMed Central

Bast, Antje; Kubis, Helen; Holtfreter, Birte; Ribback, Silvia; Martin, Heiner; Schreiner, Helen C.; Dominik, Malte J.; Breitbach, Katrin; Dombrowski, Frank; Kocher, Thomas

2016-01-01

ABSTRACT Aggregatibacter actinomycetemcomitans is a Gram-negative commensal bacterium of the oral cavity which has been associated with the pathogenesis of periodontitis with severe alveolar bone destruction. The role of host factors such as reactive oxygen and nitrogen intermediates in periodontal A. actinomycetemcomitans infection and progression to periodontitis is still ill-defined. Therefore, this study aimed to analyze the role of NADPH oxidase and inducible nitric oxide synthase (iNOS) in a murine model of A. actinomycetemcomitans-induced periodontitis. NADPH oxidase-deficient (gp91phox knockout [KO]), iNOS-deficient (iNOS KO), and C57BL/6 wild-type mice were orally infected with A. actinomycetemcomitans and analyzed for bacterial colonization at various time points. Alveolar bone mineral density and alveolar bone volume were quantified by three-dimensional micro-computed tomography, and the degree of tissue inflammation was calculated by histological analyses. At 5 weeks after infection, A. actinomycetemcomitans persisted at significantly higher levels in the murine oral cavities of infected gp91phox KO mice than in those of iNOS KO and C57BL/6 mice. Concomitantly, alveolar bone mineral density was significantly lower in all three infected groups than in uninfected controls, but with the highest loss of bone density in infected gp91phox KO mice. Only infected gp91phox KO mice revealed significant loss of alveolar bone volume and enhanced inflammatory cell infiltration, as well as an increased number of osteoclasts. Our results indicate that NADPH oxidase is important to control A. actinomycetemcomitans infection in the murine oral cavity and to prevent subsequent alveolar bone destruction and osteoclastogenesis. PMID:27849181

The ALD6 gene product is indispensable for providing NADPH in yeast cells lacking glucose-6-phosphate dehydrogenase activity.

PubMed

Grabowska, Dorota; Chelstowska, Anna

2003-04-18

Reducing equivalents in the form of NADPH are essential for many enzymatic steps involved in the biosynthesis of cellular macromolecules. An adequate level of NADPH is also required to protect cells against oxidative stress. The major enzymatic source of NADPH in the cell is the reaction catalyzed by glucose-6-phosphate dehydrogenase, the first enzyme in the pentose phosphate pathway. Disruption of the ZWF1 gene, encoding glucose-6-phosphate dehydrogenase in the yeast Saccharomyces cerevisiae, results in methionine auxotrophy and increased sensitivity to oxidizing agents. It is assumed that both phenotypes are due to an NADPH deficiency in the zwf1Delta strain. We used a Met(-) phenotype displayed by the zwf1Delta strain to look for multicopy suppressors of this deletion. We found that overexpression of the ALD6 gene coding for cytosolic acetaldehyde dehydrogenase, which utilizes NADP(+) as its cofactor, restores the Met(+) phenotype of the zwf1Delta strain. Another multicopy suppressor identified in our screen, the ZMS1 gene encoding a putative transcription factor, regulates the level of ALD6 expression. A strain bearing a double ZWF1 ALD6 gene disruption is not viable. Thus, our results indicate the reaction catalyzed by Ald6p as an important source of reducing equivalents in the yeast cells.

Labeled Azospirillum brasilense wild type and excretion-ammonium strains in association with barley roots.

PubMed

Santos, Adrian Richard Schenberger; Etto, Rafael Mazer; Furmam, Rafaela Wiegand; Freitas, Denis Leandro de; Santos, Karina Freire d'Eça Nogueira; Souza, Emanuel Maltempi de; Pedrosa, Fábio de Oliveira; Ayub, Ricardo Antônio; Steffens, Maria Berenice Reynaud; Galvão, Carolina Weigert

2017-09-01

Soil bacteria colonization in plants is a complex process, which involves interaction between many bacterial characters and plant responses. In this work, we labeled Azospirillum brasilense FP2 (wild type) and HM053 (excretion-ammonium) strains by insertion of the reporter gene gusA-kanamycin into the dinitrogenase reductase coding gene, nifH, and evaluated bacteria colonization in barley (Hordeum vulgare). In addition, we determined inoculation effect based on growth promotion parameters. We report an uncommon endophytic behavior of A. brasilense Sp7 derivative inside the root hair cells of barley and highlight the promising use of A. brasilense HM053 as plant growth-promoting bacterium. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Ramularia collo-cygni--An Emerging Pathogen of Barley Crops.

PubMed

Havis, Neil D; Brown, James K M; Clemente, Gladys; Frei, Peter; Jedryczka, Malgorzata; Kaczmarek, Joanna; Kaczmarek, Maciej; Matusinsky, Pavel; McGrann, Graham R D; Pereyra, Sylvia; Piotrowska, Marta; Sghyer, Hind; Tellier, Aurelien; Hess, Michael

2015-07-01

Ramularia collo-cygni is the biotic factor responsible for the disease Ramularia leaf spot (RLS) of barley (Hordeum vulgare). Despite having been described over 100 years ago and being considered a minor disease in some countries, the fungus is attracting interest in the scientific community as a result of the increasing number of recorded economically damaging disease epidemics. New reports of disease spread and fungal identification using molecular diagnostics have helped redefine RLS as a global disease. This review describes recent developments in our understanding of the biology and epidemiology of the fungus, outlines advances made in the field of the genetics of both the fungus and host, and summarizes the control strategies currently available.

Priming of the neutrophil NADPH oxidase activation: role of p47phox phosphorylation and NOX2 mobilization to the plasma membrane.

PubMed

El-Benna, Jamel; Dang, Pham My-Chan; Gougerot-Pocidalo, Marie-Anne

2008-07-01

Neutrophils play an essential role in host defense against microbial pathogens and in the inflammatory reaction. Upon activation, neutrophils produce superoxide anion (O*2), which generates other reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), hydroxyl radical (OH*) and hypochlorous acid (HOCl), together with microbicidal peptides and proteases. The enzyme responsible for O2* production is called the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase or respiratory burst oxidase. This multicomponent enzyme system is composed of two trans-membrane proteins (p22phox and gp91phox/NOX2, which form the cytochrome b558), three cytosolic proteins (p47phox, p67phox, p40phox) and a GTPase (Rac1 or Rac2), which assemble at membrane sites upon cell activation. NADPH oxidase activation in phagocytes can be induced by a large number of soluble and particulate factors. Three major events accompany NAPDH oxidase activation: (1) protein phosphorylation, (2) GTPase activation, and (3) translocation of cytosolic components to the plasma membrane to form the active enzyme. Actually, the neutrophil NADPH oxidase exists in different states: resting, primed, activated, or inactivated. The resting state is found in circulating blood neutrophils. The primed state can be induced by neutrophil adhesion, pro-inflammatory cytokines, lipopolysaccharide, and other agents and has been characterized as a "ready to go" state, which results in a faster and higher response upon exposure to a second stimulus. The active state is found at the inflammatory or infection site. Activation is induced by the pathogen itself or by pathogen-derived formylated peptides and other agents. Finally, inactivation of NADPH oxidase is induced by anti-inflammatory agents to limit inflammation. Priming is a "double-edged sword" process as it contributes to a rapid and efficient elimination of the pathogens but can also induce the generation of large quantities of toxic ROS by hyperactivation of

Molecular Interface of S100A8 with Cytochrome b558 and NADPH Oxidase Activation

PubMed Central

Berthier, Sylvie; Hograindleur, Marc-André; Paclet, Marie-Hélène; Polack, Benoît; Morel, Françoise

2012-01-01

S100A8 and S100A9 are two calcium binding Myeloid Related Proteins, and important mediators of inflammatory diseases. They were recently introduced as partners for phagocyte NADPH oxidase regulation. However, the precise mechanism of their interaction remains elusive. We had for aim (i) to evaluate the impact of S100 proteins on NADPH oxidase activity; (ii) to characterize molecular interaction of either S100A8, S100A9, or S100A8/S100A9 heterocomplex with cytochrome b 558; and (iii) to determine the S100A8 consensus site involved in cytochrome b 558/S100 interface. Recombinant full length or S100A9-A8 truncated chimera proteins and ExoS-S100 fusion proteins were expressed in E. coli and in P. aeruginosa respectively. Our results showed that S100A8 is the functional partner for NADPH oxidase activation contrary to S100A9, however, the loading with calcium and a combination with phosphorylated S100A9 are essential in vivo. Endogenous S100A9 and S100A8 colocalize in differentiated and PMA stimulated PLB985 cells, with Nox2/gp91phox and p22phox. Recombinant S100A8, loaded with calcium and fused with the first 129 or 54 N-terminal amino acid residues of the P. aeruginosa ExoS toxin, induced a similar oxidase activation in vitro, to the one observed with S100A8 in the presence of S100A9 in vivo. This suggests that S100A8 is the essential component of the S100A9/S100A8 heterocomplex for oxidase activation. In this context, recombinant full-length rS100A9-A8 and rS100A9-A8 truncated 90 chimera proteins as opposed to rS100A9-A8 truncated 86 and rS100A9-A8 truncated 57 chimeras, activate the NADPH oxidase function of purified cytochrome b 558 suggesting that the C-terminal region of S100A8 is directly involved in the molecular interface with the hemoprotein. The data point to four strategic 87HEES90 amino acid residues of the S100A8 C-terminal sequence that are involved directly in the molecular interaction with cytochrome b558 and then in the phagocyte NADPH oxidase

The NADPH oxidase Cpnox1 is required for full pathogenicity of the ergot fungus Claviceps purpurea.

PubMed

Giesbert, Sabine; Schürg, Timo; Scheele, Sandra; Tudzynski, Paul

2008-05-01

The role of reactive oxygen species (ROS) in interactions between phytopathogenic fungi and their hosts is well established. An oxidative burst mainly caused by superoxide formation by membrane-associated NADPH oxidases is an essential element of plant defence reactions. Apart from primary effects, ROS play a major role as a second messenger in host response. Recently, NADPH oxidase (nox)-encoding genes have been identified in filamentous fungi. Functional analyses have shown that these fungal enzymes are involved in sexual differentiation, and there is growing evidence that they also affect developmental programmes involved in fungus-plant interactions. Here we show that in the biotrophic plant pathogen Claviceps purpurea deletion of the cpnox1 gene, probably encoding an NADPH oxidase, has impact on germination of conidia and pathogenicity: Deltacpnox1 mutants can penetrate the host epidermis, but they are impaired in colonization of the plant ovarian tissue. In the few cases where macroscopic signs of infection (honeydew) appear, they are extremely delayed and fully developed sclerotia have never been observed. C. purpurea Nox1 is important for the interaction with its host, probably by directly affecting pathogenic differentiation of the fungus.

Pre-steady-state kinetic studies of redox reactions catalysed by Bacillus subtilis ferredoxin-NADP(+) oxidoreductase with NADP(+)/NADPH and ferredoxin.

PubMed

Seo, Daisuke; Soeta, Takahiro; Sakurai, Hidehiro; Sétif, Pierre; Sakurai, Takeshi

2016-06-01

Ferredoxin-NADP(+) oxidoreductase ([EC1.18.1.2], FNR) from Bacillus subtilis (BsFNR) is a homodimeric flavoprotein sharing structural homology with bacterial NADPH-thioredoxin reductase. Pre-steady-state kinetics of the reactions of BsFNR with NADP(+), NADPH, NADPD (deuterated form) and B. subtilis ferredoxin (BsFd) using stopped-flow spectrophotometry were studied. Mixing BsFNR with NADP(+) and NADPH yielded two types of charge-transfer (CT) complexes, oxidized FNR (FNR(ox))-NADPH and reduced FNR (FNR(red))-NADP(+), both having CT absorption bands centered at approximately 600n m. After mixing BsFNR(ox) with about a 10-fold molar excess of NADPH (forward reaction), BsFNR was almost completely reduced at equilibrium. When BsFNR(red) was mixed with NADP(+), the amount of BsFNR(ox) increased with increasing NADP(+) concentration, but BsFNR(red) remained as the major species at equilibrium even with about 50-fold molar excess NADP(+). In both directions, the hydride-transfer was the rate-determining step, where the forward direction rate constant (~500 s(-1)) was much higher than the reverse one (<10 s(-1)). Mixing BsFd(red) with BsFNR(ox) induced rapid formation of a neutral semiquinone form. This process was almost completed within 1 ms. Subsequently the neutral semiquinone form was reduced to the hydroquinone form with an apparent rate constant of 50 to 70 s(-1) at 10°C, which increased as BsFd(red) increased from 40 to 120 μM. The reduction rate of BsFNR(ox) by BsFd(red) was markedly decreased by premixing BsFNR(ox) with BsFd(ox), indicating that the dissociation of BsFd(ox) from BsFNR(sq) is rate-limiting in the reaction. The characteristics of the BsFNR reactions with NADP(+)/NADPH were compared with those of other types of FNRs. Copyright © 2016 Elsevier B.V. All rights reserved.

A ROS-Assisted Calcium Wave Dependent on the AtRBOHD NADPH Oxidase and TPC1 Cation Channel Propagates the Systemic Response to Salt Stress.

PubMed

Evans, Matthew J; Choi, Won-Gyu; Gilroy, Simon; Morris, Richard J

2016-07-01

Plants exhibit rapid, systemic signaling systems that allow them to coordinate physiological and developmental responses throughout the plant body, even to highly localized and quickly changing environmental stresses. The propagation of these signals is thought to include processes ranging from electrical and hydraulic networks to waves of reactive oxygen species (ROS) and cytoplasmic Ca(2+) traveling throughout the plant. For the Ca(2+) wave system, the involvement of the vacuolar ion channel TWO PORE CHANNEL1 (TPC1) has been reported. However, the precise role of this channel and the mechanism of cell-to-cell propagation of the wave have remained largely undefined. Here, we use the fire-diffuse-fire model to analyze the behavior of a Ca(2+) wave originating from Ca(2+) release involving the TPC1 channel in Arabidopsis (Arabidopsis thaliana). We conclude that a Ca(2+) diffusion-dominated calcium-induced calcium-release mechanism is insufficient to explain the observed wave transmission speeds. The addition of a ROS-triggered element, however, is able to quantitatively reproduce the observed transmission characteristics. The treatment of roots with the ROS scavenger ascorbate and the NADPH oxidase inhibitor diphenyliodonium and analysis of Ca(2+) wave propagation in the Arabidopsis respiratory burst oxidase homolog D (AtrbohD) knockout background all led to reductions in Ca(2+) wave transmission speeds consistent with this model. Furthermore, imaging of extracellular ROS production revealed a systemic spread of ROS release that is dependent on both AtRBOHD and TPC1 These results suggest that, in the root, plant systemic signaling is supported by a ROS-assisted calcium-induced calcium-release mechanism intimately involving ROS production by AtRBOHD and Ca(2+) release dependent on the vacuolar channel TPC1. © 2016 American Society of Plant Biologists. All Rights Reserved.

Structures of Saccharomyces cerevisiae D-arabinose dehydrogenase Ara1 and its complex with NADPH: implications for cofactor-assisted substrate recognition.

PubMed

Hu, Xiao-Qian; Guo, Peng-Chao; Ma, Jin-Di; Li, Wei-Fang

2013-11-01

The primary role of yeast Ara1, previously mis-annotated as a D-arabinose dehydrogenase, is to catalyze the reduction of a variety of toxic α,β-dicarbonyl compounds using NADPH as a cofactor at physiological pH levels. Here, crystal structures of Ara1 in apo and NADPH-complexed forms are presented at 2.10 and 2.00 Å resolution, respectively. Ara1 exists as a homodimer, each subunit of which adopts an (α/β)8-barrel structure and has a highly conserved cofactor-binding pocket. Structural comparison revealed that induced fit upon NADPH binding yielded an intact active-site pocket that recognizes the substrate. Moreover, the crystal structures combined with computational simulation defined an open substrate-binding site to accommodate various substrates that possess a dicarbonyl group.

Antidepressant-like effects of young green barley leaf (Hordeum vulgare L.) in the mouse forced swimming test.

PubMed

Yamaura, Katsunori; Nakayama, Noriyuki; Shimada, Maki; Bi, Yuanyuan; Fukata, Hideki; Ueno, Koichi

2012-01-01

Young green barley leaf is one of the richest sources of antioxidants and has been widely consumed for health management in Japan. In this study, we examined whether oral administration of young green barley leaf has an antidepressant effect on the forced swimming test in mice. Mice were individually forced to swim in an open cylindrical container, one hour after oral administration of young green barley leaf (400 or 1000 mg / kg) or imipramine (100 mg / kg). Expression of mRNA for nerve growth factor (NGF), brain-derived neurotrophic factor, and glucocorticoid receptor in the brain was analyzed using real-time quantitative polymerase chain reaction (PCR). There was a significant antidepressant-like effect in the forced swimming test; both 400 and 1000 mg / kg young green barley leaves, as well as the positive control imipramine (100 mg / kg), reduced the immobility duration compared to the vehicle group. The expression of mRNA for NGF detected in the hippocampus immediately after the last swimming test was higher than that in the non-swimming group (Nil). Oral administration of imipramine suppressed this increase to the level of the Nil group. Young green barley leaf (400 and 1000 mg / kg) also showed a moderate decrease in the expression of mRNA for NGF, in a dose-dependent manner. Oral administration of young green barley leaf is able to produce an antidepressant-like effect in the forced swimming test. Consequently it is possible that the antidepressant-like effects of the young green barley leaf are, at least in part, mediated by an inhibition of the increase in the hippocampus levels of NGF.

Changes in barley (Hordeum vulgare L. subsp. vulgare) genetic diversity and structure in Jordan over a period of 31 years

USDA-ARS?s Scientific Manuscript database

In many regions of the world, the cultivation of landraces is still common, in particular in centres of crop diversity. Significant effort has been put into ex situ conservation of landraces but limited data exist on the changes in genetic diversity that occur over time in farmers’ fields. We assess...

Glucose-6-phosphate dehydrogenase and NADPH redox regulates cardiac myocyte L-type calcium channel activity and myocardial contractile function.

PubMed

Rawat, Dhwajbahadur K; Hecker, Peter; Watanabe, Makino; Chettimada, Sukrutha; Levy, Richard J; Okada, Takao; Edwards, John G; Gupte, Sachin A

2012-01-01

We recently demonstrated that a 17-ketosteroid, epiandrosterone, attenuates L-type Ca(2+) currents (I(Ca-L)) in cardiac myocytes and inhibits myocardial contractility. Because 17-ketosteroids are known to inhibit glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme in the pentose phosphate pathway, and to reduce intracellular NADPH levels, we hypothesized that inhibition of G6PD could be a novel signaling mechanism which inhibit I(Ca-L) and, therefore, cardiac contractile function. We tested this idea by examining myocardial function in isolated hearts and Ca(2+) channel activity in isolated cardiac myocytes. Myocardial function was tested in Langendorff perfused hearts and I(Ca-L) were recorded in the whole-cell patch configuration by applying double pulses from a holding potential of -80 mV and then normalized to the peak amplitudes of control currents. 6-Aminonicotinamide, a competitive inhibitor of G6PD, increased pCO(2) and decreased pH. Additionally, 6-aminonicotinamide inhibited G6PD activity, reduced NADPH levels, attenuated peak I(Ca-L) amplitudes, and decreased left ventricular developed pressure and ±dp/dt. Finally, dialyzing NADPH into cells from the patch pipette solution attenuated the suppression of I(Ca-L) by 6-aminonicotinamide. Likewise, in G6PD-deficient mice, G6PD insufficiency in the heart decreased GSH-to-GSSG ratio, superoxide, cholesterol and acetyl CoA. In these mice, M-mode echocardiographic findings showed increased diastolic volume and end-diastolic diameter without changes in the fraction shortening. Taken together, these findings suggest that inhibiting G6PD activity and reducing NADPH levels alters metabolism and leads to inhibition of L-type Ca(2+) channel activity. Notably, this pathway may be involved in modulating myocardial contractility under physiological and pathophysiological conditions during which the pentose phosphate pathway-derived NADPH redox is modulated (e.g., ischemia-reperfusion and heart failure).

Pyridine Nucleotide Complexes with Bacillus anthracis Coenzyme A-Disulfide Reductase: A Structural Analysis of Dual NAD(P)H Specificity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wallen,J.; Paige, C.; Mallett, T.

2008-01-01

We have recently reported that CoASH is the major low-molecular weight thiol in Bacillus anthracis, and we have now characterized the kinetic and redox properties of the B. anthracis coenzyme A-disulfide reductase (CoADR, BACoADR) and determined the crystal structure at 2.30 Angstroms resolution. While the Staphylococcus aureus and Borrelia burgdorferi CoADRs exhibit strong preferences for NADPH and NADH, respectively, B. anthracis CoADR can use either pyridine nucleotide equally well. Sequence elements within the respective NAD(P)H-binding motifs correctly reflect the preferences for S. aureus and Bo. burgdorferi CoADRs, but leave questions as to how BACoADR can interact with both pyridine nucleotides.more » The structures of the NADH and NADPH complexes at ca. 2.3 Angstroms resolution reveal that a loop consisting of residues Glu180-Thr187 becomes ordered and changes conformation on NAD(P)H binding. NADH and NADPH interact with nearly identical conformations of this loop; the latter interaction, however, involves a novel binding mode in which the 2'-phosphate of NADPH points out toward solvent. In addition, the NAD(P)H-reduced BACoADR structures provide the first view of the reduced form (Cys42-SH/CoASH) of the Cys42-SSCoA redox center. The Cys42-SH side chain adopts a new conformation in which the conserved Tyr367'-OH and Tyr425'-OH interact with the nascent thiol(ate) on the flavin si-face. Kinetic data with Y367F, Y425F, and Y367, 425F BACoADR mutants indicate that Tyr425' is the primary proton donor in catalysis, with Tyr367' functioning as a cryptic alternate donor in the absence of Tyr425'.« less

Cloning, functional characterization, and expression profiles of NADPH-cytochrome P450 reductase gene from the Asiatic rice striped stem borer, Chilo suppressalis (Lepidoptera: Pyralidae).

PubMed

Liu, Su; Liang, Qing-Mei; Huang, Yuan-Jie; Yuan, Xin; Zhou, Wen-Wu; Qiao, Fei; Cheng, Jiaan; Gurr, Geoff M; Zhu, Zeng-Rong

2013-01-01

NADPH-cytochrome P450 reductase (CPR) is one of the most important components of the cytochrome P450 enzyme system. It catalyzes electron transfer from NADPH to all known P450s, thus plays central roles not only in the metabolism of exogenous xenobiotics but also in the regulation of endogenous hormones in insects. In this study, a full-length cDNA encoding of a CPR (named CsCPR) was isolated from the Asiatic rice striped stem borer, Chilo suppressalis, by using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods. The cDNA contains a 2061 bp open reading frame, which encodes an enzyme of 686 amino acid residues, with a calculated molecular mass of 77.6 kDa. The deduced peptide has hallmarks of typical CPR, including an N-terminal membrane anchor and the FMN, FAD and NADPH binding domains. The N-terminal-truncated protein fused with a 6 × His·tag was heterologously expressed in Escherichia coli Rosetta (DE3) cells and purified, specific activity and the Km values of the recombinant enzyme were determined. Tissue- and developmental stage-dependent expression of CsCPR mRNA was investigated by real-time quantitative PCR. The CsCPR mRNA was noticeably expressed in the digestive, metabolic, and olfactory organs of the larvae and adults of C. suppressalis. Our initial results would provide valuable information for further study on the interactions between CPR and cytochrome P450 enzyme systems. © 2013.

Two NADPH oxidase isoforms are required for sexual reproduction and ascospore germination in the filamentous fungus Podospora anserina.

PubMed

Malagnac, Fabienne; Lalucque, Hervé; Lepère, Gersende; Silar, Philippe

2004-11-01

NADPH oxidases are enzymes that produce reactive oxygen species (ROS) using electrons derived from intracellular NADPH. In plants and mammals, ROS have been proposed to be second messengers that signal defence responses or cell proliferation. By inactivating PaNox1 and PaNox2, two genes encoding NADPH oxidases, we demonstrate the crucial role of these enzymes in the control of two key steps of the filamentous fungus Podospora anserina life cycle. PaNox1 mutants are impaired in the differentiation of fruiting bodies from their progenitor cells, and the deletion of the PaNox2 gene specifically blocks ascospore germination. Furthermore, we show that PaNox1 likely acts upstream of PaASK1, a MAPKKK previously implicated in stationary phase differentiation and cell degeneration. Using nitro blue tetrazolium (NBT) and diaminobenzidine (DAB) assays, we detect a regulated secretion of both superoxide and peroxide during P. anserina vegetative growth. In addition, two oxidative bursts are shown to occur during fruiting body development and ascospore germination. Analysis of mutants establishes that PaNox1, PaNox2, and PaASK1, as well as a still unknown additional source of ROS, modulate these secretions. Altogether, our data point toward a role for NADPH oxidases in signalling fungal developmental transitions with respect to nutrient availability. These enzymes are conserved in other multicellular eukaryotes, suggesting that early eukaryotes were endowed with a redox network used for signalling purposes.

Positive correlation between decreased cellular uptake, NADPH-glutathione reductase activity and adriamycin resistance in Ehrlich ascites tumor lines.

PubMed

Scheulen, M E; Hoensch, H; Kappus, H; Seeber, S; Schmidt, C G

1987-01-01

From a wild type strain of Ehrlich ascites tumor (EATWT) sublines resistant to daunorubicin (EATDNM), etoposide (EATETO), and cisplatinum (EATCIS) have been developed in vivo. Increase in survival and cure rate caused by adriamycin (doxorubicin) have been determined in female NMRI mice which were inoculated i.p. with EAT cells. Adriamycin concentrations causing 50% inhibition of 3H-thymidine (ICT) and 3H-uridine incorporation (ICU) and intracellular adriamycin steady-state concentrations (SSC) were measured in vitro. Adriamycin resistance increased and SSC decreased in the following sequence: EATWT - EATCIS - EATDNM - EATETO. When ICT and ICU were corrected for intracellular adriamycin concentrations in consideration of the different SSC (ICTc, ICUc), ICTc and ICUc still varied up to the 3.2 fold in EATCIS, EATDNM and EATETO in comparison to EATWT. Thus, in addition to different SSC other factors must be responsible for adriamycin resistance. Therefore, enzymes which may play a role in the cytotoxicity related to adriamycin metabolism (NADPH-cytochrome P-450 reductase, NADPH-glutathione reductase, NADP-glucose-6-phosphate dehydrogenase, NADP-isocitrate dehydrogenase) were measured. In contrast to the other parameters determined, NADPH-glutathione reductase was significantly (p less than 0.01) increased up to the 3.2 fold parallel to adriamycin resistance as determined by increase in life span, cure rate, ICTc, and ICUc, respectively. It is concluded that high activities of NADPH-glutathione reductase may contribute to an increase in adriamycin resistance of malignant tumors.

Six-rowed barley originated from a mutation in a homeodomain-leucine zipper I-class homeobox gene

PubMed Central

Komatsuda, Takao; Pourkheirandish, Mohammad; He, Congfen; Azhaguvel, Perumal; Kanamori, Hiroyuki; Perovic, Dragan; Stein, Nils; Graner, Andreas; Wicker, Thomas; Tagiri, Akemi; Lundqvist, Udda; Fujimura, Tatsuhito; Matsuoka, Makoto; Matsumoto, Takashi; Yano, Masahiro

2007-01-01

Increased seed production has been a common goal during the domestication of cereal crops, and early cultivators of barley (Hordeum vulgare ssp. vulgare) selected a phenotype with a six-rowed spike that stably produced three times the usual grain number. This improved yield established barley as a founder crop for the Near Eastern Neolithic civilization. The barley spike has one central and two lateral spikelets at each rachis node. The wild-type progenitor (H. vulgare ssp. spontaneum) has a two-rowed phenotype, with additional, strictly rudimentary, lateral rows; this natural adaptation is advantageous for seed dispersal after shattering. Until recently, the origin of the six-rowed phenotype remained unknown. In the present study, we isolated vrs1 (six-rowed spike 1), the gene responsible for the six-rowed spike in barley, by means of positional cloning. The wild-type Vrs1 allele (for two-rowed barley) encodes a transcription factor that includes a homeodomain with a closely linked leucine zipper motif. Expression of Vrs1 was strictly localized in the lateral-spikelet primordia of immature spikes, suggesting that the VRS1 protein suppresses development of the lateral rows. Loss of function of Vrs1 resulted in complete conversion of the rudimentary lateral spikelets in two-rowed barley into fully developed fertile spikelets in the six-rowed phenotype. Phylogenetic analysis demonstrated that the six-rowed phenotype originated repeatedly, at different times and in different regions, through independent mutations of Vrs1. PMID:17220272

Unexpected function of the phagocyte NADPH oxidase in supporting hyperglycolysis in stimulated neutrophils: key role of 6-phosphofructo-2-kinase.

PubMed

Baillet, Athan; Hograindleur, Marc-André; El Benna, Jamel; Grichine, Alexei; Berthier, Sylvie; Morel, Françoise; Paclet, Marie-Hélène

2017-02-01

The phagocyte NADPH oxidase 2 (Nox2) is an enzymatic complex that is involved in innate immunity, notably via its capacity to produce toxic reactive oxygen species. Recently, a proteomic analysis of the constitutively active Nox2 complex, isolated from neutrophil fractions, highlighted the presence of 6-phosphofructo-2-kinase (PFK-2). The purpose of this work was to study the relationship between PFK-2 and NADPH oxidase in neutrophils. Data have underlined a specific association of the active phosphorylated form of PFK-2 with Nox2 complex in stimulated neutrophils. In its active form, PFK-2 catalyzes the production of fructose-2,6-bisphosphate, which is the main allosteric activator of phosphofructo-1-kinase, the limiting enzyme in glycolysis. Pharmacologic inhibition of PFK-2 phosphorylation and cell depletion in PFK-2 by a small interfering RNA strategy led to a decrease in the glycolysis rate and a reduction in NADPH oxidase activity in stimulated cells. Surprisingly, alteration of Nox2 activity impacted the glycolysis rate, which indicated that Nox2 in neutrophils was not only required for reactive oxygen species production but was also involved in supporting the energetic metabolism increase that was induced by inflammatory conditions. PFK-2 seems to be a strategic element that links NADPH oxidase activation and glycolysis modulation, and, as such, is proposed as a potential therapeutic target in inflammatory diseases.-Baillet, A., Hograindleur, M.-A., El Benna, J., Grichine, A., Berthier, S., Morel, F., Paclet, M.-H. Unexpected function of the phagocyte NADPH oxidase in supporting hyperglycolysis in stimulated neutrophils: key role of 6-phosphofructo-2-kinase. © FASEB.

Sildenafil Promotes eNOS Activation and Inhibits NADPH Oxidase in the Transgenic Sickle Cell Mouse Penis

PubMed Central

Musicki, Biljana; Bivalacqua, Trinity J.; Champion, Hunter C.; Burnett, Arthur L.

2014-01-01

Introduction Sickle cell disease (SCD)-associated vasculopathy in the penis is characterized by aberrant nitric oxide and phosphodiesterase (PDE) 5 signaling, and by increased oxidative stress. Preliminary clinical trials show that continuous treatment with PDE5 inhibitor sildenafil unassociated with sexual activity decreases priapic activity in patients with SCD. However, the mechanism of its vasculoprotective effect in the penis remains unclear. Aims We evaluated whether continuous administration of PDE5 inhibitor sildenafil promotes eNOS function at posttranslational levels and decreases superoxide-producing enzyme NADPH oxidase activity in the sickle cell mouse penis. Methods SCD transgenic mice were used as an animal model of SCD. WT mice served as controls. Mice received treatment with the PDE5 inhibitor sildenafil (100 mg/kg/day) or vehicle for 3 weeks. eNOS phosphorylation on Ser-1177 (positive regulatory site), eNOS interactions with heat-shock protein 90 (HSP90) (positive regulator), phosphorylated AKT (upstream mediator of eNOS phosphorylation on Ser-1177), an NADPH oxidase catalytic subunit gp91(phox), and a marker of oxidative stress (4-hydroxy-2-nonenal [HNE]) were measured by Western blot. Main Outcome Measures Effect of continuous sildenafil treatment on eNOS posttranslational activation, NADPH oxidase catalytic subunit, and oxidative stress in the penis of the sickle cell mouse. Results Continuous treatment with sildenafil reversed (P < 0.05) the abnormalities in protein expressions of P-eNOS (Ser-1177), eNOS/HSP90 interaction, P-AKT, protein expression of gp91(phox), and 4-HNE, in the sickle cell mouse penis. Sildenafil treatment of WT mice did not affect any of these parameters. Conclusion Our findings that sildenafil enhances eNOS activation and inhibits NADPH oxidase function in the sickle cell mouse penis offers a vasculoprotective molecular basis for the therapeutic effect of sildenafil in the penis in association with SCD. PMID:24251665

Sildenafil promotes eNOS activation and inhibits NADPH oxidase in the transgenic sickle cell mouse penis.

PubMed

Musicki, Biljana; Bivalacqua, Trinity J; Champion, Hunter C; Burnett, Arthur L

2014-02-01

Sickle cell disease (SCD)-associated vasculopathy in the penis is characterized by aberrant nitric oxide and phosphodiesterase (PDE) 5 signaling, and by increased oxidative stress. Preliminary clinical trials show that continuous treatment with PDE5 inhibitor sildenafil unassociated with sexual activity decreases priapic activity in patients with SCD. However, the mechanism of its vasculoprotective effect in the penis remains unclear. We evaluated whether continuous administration of PDE5 inhibitor sildenafil promotes eNOS function at posttranslational levels and decreases superoxide-producing enzyme NADPH oxidase activity in the sickle cell mouse penis. SCD transgenic mice were used as an animal model of SCD. WT mice served as controls. Mice received treatment with the PDE5 inhibitor sildenafil (100 mg/kg/day) or vehicle for 3 weeks. eNOS phosphorylation on Ser-1177 (positive regulatory site), eNOS interactions with heat-shock protein 90 (HSP90) (positive regulator), phosphorylated AKT (upstream mediator of eNOS phosphorylation on Ser-1177), an NADPH oxidase catalytic subunit gp91(phox), and a marker of oxidative stress (4-hydroxy-2-nonenal [HNE]) were measured by Western blot. Effect of continuous sildenafil treatment on eNOS posttranslational activation, NADPH oxidase catalytic subunit, and oxidative stress in the penis of the sickle cell mouse. Continuous treatment with sildenafil reversed (P < 0.05) the abnormalities in protein expressions of P-eNOS (Ser-1177), eNOS/HSP90 interaction, P-AKT, protein expression of gp91(phox), and 4-HNE, in the sickle cell mouse penis. Sildenafil treatment of WT mice did not affect any of these parameters. Our findings that sildenafil enhances eNOS activation and inhibits NADPH oxidase function in the sickle cell mouse penis offers a vasculoprotective molecular basis for the therapeutic effect of sildenafil in the penis in association with SCD. © 2013 International Society for Sexual Medicine.

Nanometer size diesel exhaust particles are selectively toxic to dopaminergic neurons: the role of microglia, phagocytosis, and NADPH oxidase.

PubMed

Block, M L; Wu, X; Pei, Z; Li, G; Wang, T; Qin, L; Wilson, B; Yang, J; Hong, J S; Veronesi, B

2004-10-01

The contributing role of environmental factors to the development of Parkinson's disease has become increasingly evident. We report that mesencephalic neuron-glia cultures treated with diesel exhaust particles (DEP; 0.22 microM) (5-50 microg/ml) resulted in a dose-dependent decrease in dopaminergic (DA) neurons, as determined by DA-uptake assay and tyrosine-hydroxylase immunocytochemistry (ICC). The selective toxicity of DEP for DA neurons was demonstrated by the lack of DEP effect on both GABA uptake and Neu-N immunoreactive cell number. The critical role of microglia was demonstrated by the failure of neuron-enriched cultures to exhibit DEP-induced DA neurotoxicity, where DEP-induced DA neuron death was reinstated with the addition of microglia to neuron-enriched cultures. OX-42 ICC staining of DEP treated neuron-glia cultures revealed changes in microglia morphology indicative of activation. Intracellular reactive oxygen species and superoxide were produced from enriched-microglia cultures in response to DEP. Neuron-glia cultures from NADPH oxidase deficient (PHOX-/-) mice were insensitive to DEP neurotoxicity when compared with control mice (PHOX+/+). Cytochalasin D inhibited DEP-induced superoxide production in enriched-microglia cultures, implying that DEP must be phagocytized by microglia to produce superoxide. Together, these in vitro data indicate that DEP selectively damages DA neurons through the phagocytic activation of microglial NADPH oxidase and consequent oxidative insult.

Hyperglycaemia promotes human brain microvascular endothelial cell apoptosis via induction of protein kinase C-ßI and prooxidant enzyme NADPH oxidase.

PubMed

Shao, Beili; Bayraktutan, Ulvi

2014-01-01

Blood-brain barrier disruption represents a key feature in hyperglycaemia-aggravated cerebral damage after an ischaemic stroke. Although the underlying mechanisms remain largely unknown, activation of protein kinase C (PKC) is thought to play a critical role. This study examined whether apoptosis of human brain microvascular endothelial cells (HBMEC) might contribute to hyperglycaemia-evoked barrier damage and assessed the specific role of PKC in this phenomenon. Treatments with hyperglycaemia (25 mM) or phorbol myristate acetate (PMA, a protein kinase C activator, 100 nM) significantly increased NADPH oxidase activity, O2 (•-) generation, proapoptotic protein Bax expression, TUNEL-positive staining and caspase-3/7 activities. Pharmacological inhibition of NADPH oxidase, PKC-a, PKC-ß or PKC-ßI via their specific inhibitors and neutralisation of O2 (•-) by a cell-permeable superoxide dismutase mimetic, MnTBAP normalised all the aforementioned increases induced by hyperglycaemia. Suppression of these PKC isoforms also negated the stimulatory effects of hyperglycaemia on the protein expression of NADPH oxidase membrane-bound components, Nox2 and p22-phox which determine the overall enzymatic activity. Silencing of PKC-ßI gene through use of specific siRNAs abolished the effects of both hyperglycaemia and PMA on endothelial cell NADPH oxidase activity, O2 (•-) production and apoptosis and consequently improved the integrity and function of an in vitro model of human cerebral barrier comprising HBMEC, astrocytes and pericytes. Hyperglycaemia-mediated apoptosis of HBMEC contributes to cerebral barrier dysfunction and is modulated by sequential activations of PKC-ßI and NADPH oxidase.

Hyperglycaemia promotes human brain microvascular endothelial cell apoptosis via induction of protein kinase C-ßI and prooxidant enzyme NADPH oxidase

PubMed Central

Shao, Beili; Bayraktutan, Ulvi

2014-01-01

Blood–brain barrier disruption represents a key feature in hyperglycaemia-aggravated cerebral damage after an ischaemic stroke. Although the underlying mechanisms remain largely unknown, activation of protein kinase C (PKC) is thought to play a critical role. This study examined whether apoptosis of human brain microvascular endothelial cells (HBMEC) might contribute to hyperglycaemia-evoked barrier damage and assessed the specific role of PKC in this phenomenon. Treatments with hyperglycaemia (25 mM) or phorbol myristate acetate (PMA, a protein kinase C activator, 100 nM) significantly increased NADPH oxidase activity, O2•- generation, proapoptotic protein Bax expression, TUNEL-positive staining and caspase-3/7 activities. Pharmacological inhibition of NADPH oxidase, PKC-a, PKC-ß or PKC-ßI via their specific inhibitors and neutralisation of O2•- by a cell-permeable superoxide dismutase mimetic, MnTBAP normalised all the aforementioned increases induced by hyperglycaemia. Suppression of these PKC isoforms also negated the stimulatory effects of hyperglycaemia on the protein expression of NADPH oxidase membrane-bound components, Nox2 and p22-phox which determine the overall enzymatic activity. Silencing of PKC-ßI gene through use of specific siRNAs abolished the effects of both hyperglycaemia and PMA on endothelial cell NADPH oxidase activity, O2•- production and apoptosis and consequently improved the integrity and function of an in vitro model of human cerebral barrier comprising HBMEC, astrocytes and pericytes. Hyperglycaemia-mediated apoptosis of HBMEC contributes to cerebral barrier dysfunction and is modulated by sequential activations of PKC-ßI and NADPH oxidase. PMID:24936444

The Red Queen and the seed bank: pathogen resistance of ex situ and in situ conserved barley.

PubMed

Jensen, Helen R; Dreiseitl, Antonín; Sadiki, Mohammed; Schoen, Daniel J

2012-06-01

Plant geneticists have proposed that the dynamic conservation of crop plants in farm environments (in situ conservation) is complementary to static conservation in seed banks (ex situ conservation) because it may help to ensure adaptation to changing conditions. Here, we test whether collections of a traditional variety of Moroccan barley (Hordeum vulgare ssp. vulgare) conserved ex situ showed differences in qualitative and quantitative resistance to the endemic fungal pathogen, Blumeria graminis f.sp. hordei, compared to collections that were continuously cultivated in situ. In detached-leaf assays for qualitative resistance, there were some significant differences between in situ and ex situ conserved collections from the same localities. Some ex situ conserved collections showed lower resistance levels, while others showed higher resistance levels than their in situ conserved counterparts. In field trials for quantitative resistance, similar results were observed, with the highest resistance observed in situ. Overall, this study identifies some cases where the Red Queen appears to drive the evolution of increased resistance in situ. However, in situ conservation does not always result in improved adaptation to pathogen virulence, suggesting a more complex evolutionary scenario, consistent with several published examples of plant-pathogen co-evolution in wild systems.

When GIS zooms in: spatio-genetic maps of multipaternity in Armadillidium vulgare.

PubMed

Bech, Nicolas; Depeux, Charlotte; Durand, Sylvine; Debenest, Catherine; Lafitte, Alexandra; Beltran-Bech, Sophie

2017-12-01

Geographic information system (GIS) tools are designed to illustrate, analyse and integrate geographic or spatial data, usually on a macroscopic scale. By contrast, genetic tools focus on a microscopic scale. Because in reality, landscapes have no predefined scale, our original study aims to develop a new approach, combining both cartographic and genetic approaches to explore microscopic landscapes. For this, we focused on Armadillidium vulgare, a terrestrial isopod model in which evolutionary pressures imposed by terrestrial life have led to the development of internal fertilisation and, consequently, to associated physiological changes. Among these, the emergence of internal receptacles, found in many taxa ranging from mammals to arthropods, allowed females to store sperm from several partners, enabling multipaternity. Among arthropods, terrestrial isopods like the polygynandrous A. vulgare present a female structure, the marsupium, in which fertilised eggs migrate and develop into mancae (larval stage). To test our innovative combined approach, we proposed different males to four independent females, and at the end of incubation in the marsupium, we mapped (using GIS methods) and genotyped (using 12 microsatellite markers) all the incubated mancae. This methodology permitted to obtain spatio-genetic maps describing heterozygosity and spatial distribution of mancae and of multipaternity within the marsupial landscape. We discussed the interest of this kind of multidisciplinary approach which could improve in this case our understanding of sexual selection mechanisms in this terrestrial crustacean. Beyond the interesting model-focused insights, the main challenge of this study was the transfer of GIS techniques to a microscopic scale and our results appear so as pioneers rendering GIS tools available for studies involving imagery whatever their study scale.

Cytosolic NADP+-dependent isocitrate dehydrogenase plays a key role in lipid metabolism.

PubMed

Koh, Ho-Jin; Lee, Su-Min; Son, Byung-Gap; Lee, Soh-Hyun; Ryoo, Zae Young; Chang, Kyu-Tae; Park, Jeen-Woo; Park, Dong-Chan; Song, Byoung J; Veech, Richard L; Song, Hebok; Huh, Tae-Lin

2004-09-17

NADPH is an essential cofactor for many enzymatic reactions including glutathione metabolism and fat and cholesterol biosynthesis. We have reported recently an important role for mitochondrial NADP(+)-dependent isocitrate dehydrogenase in cellular defense against oxidative damage by providing NADPH needed for the regeneration of reduced glutathione. However, the role of cytosolic NADP(+)-dependent isocitrate dehydrogenase (IDPc) is still unclear. We report here for the first time that IDPc plays a critical role in fat and cholesterol biosynthesis. During differentiation of 3T3-L1 adipocytes, both IDPc enzyme activity and its protein content were increased in parallel in a time-dependent manner. Increased expression of IDPc by stable transfection of IDPc cDNA positively correlated with adipogenesis of 3T3-L1 cells, whereas decreased IDPc expression by an antisense IDPc vector retarded adipogenesis. Furthermore, transgenic mice with overexpressed IDPc exhibited fatty liver, hyperlipidemia, and obesity. In the epididymal fat pads of the transgenic mice, the expressions of adipocyte-specific genes including peroxisome proliferator-activated receptor gamma were markedly elevated. The hepatic and epididymal fat pad contents of acetyl-CoA and malonyl-CoA in the transgenic mice were significantly lower, whereas the total triglyceride and cholesterol contents were markedly higher in the liver and serum of transgenic mice compared with those measured in wild type mice, suggesting that the consumption rate of those lipogenic precursors needed for fat biosynthesis must be increased by elevated IDPc activity. Taken together, our findings strongly indicate that IDPc would be a major NADPH producer required for fat and cholesterol synthesis.

Two functionally distinct NADP+-dependent ferredoxin oxidoreductases maintain the primary redox balance of Pyrococcus furiosus.

PubMed

Nguyen, Diep M N; Schut, Gerrit J; Zadvornyy, Oleg A; Tokmina-Lukaszewska, Monika; Poudel, Saroj; Lipscomb, Gina L; Adams, Leslie A; Dinsmore, Jessica T; Nixon, William J; Boyd, Eric S; Bothner, Brian; Peters, John W; Adams, Michael W W

2017-09-01

Electron bifurcation has recently gained acceptance as the third mechanism of energy conservation in which energy is conserved through the coupling of exergonic and endergonic reactions. A structure-based mechanism of bifurcation has been elucidated recently for the flavin-based enzyme NADH-dependent ferredoxin NADP + oxidoreductase I (NfnI) from the hyperthermophillic archaeon Pyrococcus furiosus. NfnI is thought to be involved in maintaining the cellular redox balance, producing NADPH for biosynthesis by recycling the two other primary redox carriers, NADH and ferredoxin. The P. furiosus genome encodes an NfnI paralog termed NfnII, and the two are differentially expressed, depending on the growth conditions. In this study, we show that deletion of the genes encoding either NfnI or NfnII affects the cellular concentrations of NAD(P)H and particularly NADPH. This results in a moderate to severe growth phenotype in deletion mutants, demonstrating a key role for each enzyme in maintaining redox homeostasis. Despite their similarity in primary sequence and cofactor content, crystallographic, kinetic, and mass spectrometry analyses reveal that there are fundamental structural differences between the two enzymes, and NfnII does not catalyze the NfnI bifurcating reaction. Instead, it exhibits non-bifurcating ferredoxin NADP oxidoreductase-type activity. NfnII is therefore proposed to be a bifunctional enzyme and also to catalyze a bifurcating reaction, although its third substrate, in addition to ferredoxin and NADP(H), is as yet unknown. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Biophysical studies of photosystem II-related recovery processes after a heat pulse in barley seedlings (Hordeum vulgare L.).

PubMed

Tóth, Szilvia Z; Schansker, Gert; Kissimon, Judit; Kovács, László; Garab, Gyozo; Strasser, Reto J

2005-02-01

Leaves of 7-day-old barley seedlings were subjected to heat pulses at 50 degrees C for 20 or 40s to inhibit partially or fully the oxygen evolution without inducing visible symptoms. By means of biophysical techniques, we investigated the time course and mechanism of photosystem II (PSII) recovery. After the heat treatment, the samples were characterized by typical heat stress symptoms: loss of oxygen evolution activity, strong decrease of Fv/Fm, induction of the K-step in the fluorescence induction transient, emergence of the AT-thermoluminescence-band and a dramatic increase in membrane permeability. In the first 4h in the light following the heat pulse, the AT-band and the K-step disappeared in parallel, indicating the loss of this restricted activity of PSII. This phase was followed by a recovery period, during which PSII-activity was gradually restored in the light. In darkness, no recovery, except for the membrane permeability, was observed. A model is presented that accounts for (i) the damage induced by the heat pulse on the membrane architecture and on the PSII donor side, (ii) the light-dependent removal of the impaired reaction centers from the disorganized membrane, and (iii) the subsequent light-independent restoration of the membrane permeability and the de novo synthesis of the PSII reaction centers in the light.

Deficiency of Rac1 Blocks NADPH Oxidase Activation, Inhibits Endoplasmic Reticulum Stress, and Reduces Myocardial Remodeling in a Mouse Model of Type 1 Diabetes

PubMed Central

Li, Jianmin; Zhu, Huaqing; Shen, E; Wan, Li; Arnold, J. Malcolm O.; Peng, Tianqing

2010-01-01

OBJECTIVE Our recent study demonstrated that Rac1 and NADPH oxidase activation contributes to cardiomyocyte apoptosis in short-term diabetes. This study was undertaken to investigate if disruption of Rac1 and inhibition of NADPH oxidase would prevent myocardial remodeling in chronic diabetes. RESEARCH DESIGN AND METHODS Diabetes was induced by injection of streptozotocin in mice with cardiomyocyte-specific Rac1 knockout and their wild-type littermates. In a separate experiment, wild-type diabetic mice were treated with vehicle or apocynin in drinking water. Myocardial hypertrophy, fibrosis, endoplasmic reticulum (ER) stress, inflammatory response, and myocardial function were investigated after 2 months of diabetes. Isolated adult rat cardiomyocytes were cultured and stimulated with high glucose. RESULTS In diabetic hearts, NADPH oxidase activation, its subunits' expression, and reactive oxygen species production were inhibited by Rac1 knockout or apocynin treatment. Myocardial collagen deposition and cardiomyocyte cross-sectional areas were significantly increased in diabetic mice, which were accompanied by elevated expression of pro-fibrotic genes and hypertrophic genes. Deficiency of Rac1 or apocynin administration reduced myocardial fibrosis and hypertrophy, resulting in improved myocardial function. These effects were associated with a normalization of ER stress markers' expression and inflammatory response in diabetic hearts. In cultured cardiomyocytes, high glucose–induced ER stress was inhibited by blocking Rac1 or NADPH oxidase. CONCLUSIONS Rac1 via NADPH oxidase activation induces myocardial remodeling and dysfunction in diabetic mice. The role of Rac1 signaling may be associated with ER stress and inflammation. Thus, targeting inhibition of Rac1 and NADPH oxidase may be a therapeutic approach for diabetic cardiomyopathy. PMID:20522592

Cation regulation by the terrestrial isopod Armadillidium vulgare (Crustacea: Isopoda: Oniscidea) during dehydration in air.

PubMed

Koh, Huishan; Wright, Jonathan

2011-06-01

Many terrestrial arthropods display tight osmotic and ionic regulation of the hemolymph during dehydration. In this study, we sought to quantify the level of regulation of the major hemolymph cations in the terrestrial isopod Armadillidium vulgare (Isopoda, Oniscidea). Inulin space measurements showed that the hemolymph comprises 52 ± 2.2% of the hydrated water content but contributes 71 ± 9.8% of water losses during desiccation. Hemolymph concentrations of Na+, K+ and Ca²+ were measured in variably dehydrated animals using ion-selective microelectrodes and compared with predicted concentrations assuming no regulation. Na+ and Ca²+ are quite tightly regulated, showing respective concentration increases of 20.8% and 7.1% following a 50% reduction in hemolymph volume, but K+ showed no measurable regulation. The excreted cation fraction during desiccation is negligible. Sites of ion sequestration were examined by injecting ²²Na and ⁴⁵Ca into the hemolymph of hydrated animals and assaying tissue-specific activities following dehydration. Na+ is apparently sequestered non-specifically by an unknown mechanism. Ca²+ accumulates in the dorsal somatic tissues, possibly in the calcium pool of the cuticle. How A. vulgare avoids significant disruptions of E(m) and neuromuscular function in the absence of K+ regulation, and how it sequesters Na+, both pose intriguing challenges for future work. Copyright © 2011 Elsevier Inc. All rights reserved.

Essential Oil from Origanum vulgare Completely Inhibits the Growth of Multidrug-Resistant Cystic Fibrosis Pathogens.

PubMed

Pesavento, Giovanna; Maggini, Valentina; Maida, Isabel; Lo Nostro, Antonella; Calonico, Carmela; Sassoli, Chiara; Perrin, Elena; Fondi, Marco; Mengoni, Alessio; Chiellini, Carolina; Vannacci, Alfredo; Gallo, Eugenia; Gori, Luigi; Bogani, Patrizia; Bilia, Anna Rita; Campana, Silvia; Ravenni, Novella; Dolce, Daniela; Firenzuoli, Fabio; Fani, Renato

2016-06-01

Essential oils (EOs) are known to inhibit the growth of a wide range of microorganisms. Particularly interesting is the possible use of EOs to treat multidrug-resistant cystic fibrosis (CF) pathogens. We tested the essential oil (EO) from Origanum vulgare for in vitro antimicrobial activity, against three of the major human opportunistic pathogens responsible for respiratory infections in CF patients; these are methicillin-resistant Staphylococcus aureus, Stenotrophomonas maltophilia and Achromobacter xylosoxidans. Antibiotic susceptibility of each strain was previously tested by the standard disk diffusion method. Most strains were resistant to multiple antibiotics and could be defined as multi-drug-resistant (MDR). The antibacterial activity of O. vulgare EO (OEO) against a panel of 59 bacterial strains was evaluated, with MIC and MBC determined at 24, 48 and 72 hours by a microdilution method. The OEO was effective against all tested strains, although to a different extent. The MBC and MIC of OEO for S. aureus strains were either lower or equal to 0.50%, v/v, for A. xylosoxidans strains were lower or equal to 1% and 0.50%, v/v, respectively; and for S. maltophilia strains were lower or equal to 0.25%, v/v. The results from this study suggest that OEO might exert a role as an antimicrobial in the treatment of CF infections.

Treatment of experimental pythiosis with essential oils of Origanum vulgare and Mentha piperita singly, in association and in combination with immunotherapy.

PubMed

Fonseca, Anelise O S; Pereira, Daniela I B; Botton, Sônia A; Pötter, Luciana; Sallis, Elisa S V; Júnior, Sérgio F V; Filho, Fernando S M; Zambrano, Cristina Gomes; Maroneze, Beatriz P; Valente, Julia S S; Baptista, Cristiane T; Braga, Caroline Q; Ben, Vanessa Dal; Meireles, Mario C A

2015-08-05

This study investigated the in vivo antimicrobial activity of the essential oils of Origanum vulgare and Mentha piperita both singly, associated and in combination with immunotherapy to treat experimental pythiosis. The disease was reproduced in 18 rabbits divided into six groups (n=3): group 1, control; group 2, treated with essential oil of Mentha piperita; group 3, treated with essential oil of Origanum vulgare; group 4, treated with commercial immunotherapic; group 5, treated with a association of oils of M. piperita and O. vulgare and group 6, treated with a combination of both oils plus immunotherapy. Essential oils were added in a topical cream base formula, and lesions were treated daily for 45 days. The animals in groups 4 and 6 received a dose of immunotherapeutic agent every 14 days. The results revealed that the evolution of lesions in groups 5 and 6 did not differ from one another but differed from the other groups. The lesions of group 5 increased 3.16 times every measurement, while those of group 6 increased 1.83 times, indicating that the smallest growth of the lesions occurred when the combination of therapies were used. A rabbit from group 5 showed clinical cure at day 20 of treatment. This research is the pioneer in the treatment of experimental pythiosis using essential oils from medicinal plants and a combination of therapies. This study demonstrated that the use of essential oils can be a viable alternative treatment to cutaneous pythiosis, particularly when used in association or combination with immunotherapy. Copyright © 2015 Elsevier B.V. All rights reserved.

Reconstitution of the Light Harvesting Chlorophyll a/b Pigment-Protein Complex into Developing Chloroplast Membranes Using a Dialyzable Detergent 1

PubMed Central

Darr, Sylvia C.; Arntzen, Charles J.

1986-01-01

Conditions were developed to isolate the light-harvesting chlorophyll-protein complex serving photosystem II (LHC-II) using a dialyzable detergent, octylpolyoxyethylene. This LHC-II was successfully reconstituted into partially developed chloroplast thylakoids of Hordeum vulgare var Morex (barley) seedlings which were deficient in LHC-II. Functional association of LHC-II with the photosystem II (PSII) core complex was measured by two independent functional assays of PSII sensitization by LHC-II. A 3-fold excess of reconstituted LHC-II was required to equal the activity of LHC developing in vivo. We suggest that a linker component may be absent in the partially developed membranes which is required for specific association of the PSII core complex and LHC-II. Images Fig. 1 PMID:16664744

Differential gene expression patterns in the autogamous plant Hordeum euclaston (Poaceae).

PubMed

Georg-Kraemer, J E; Ferreira, C A S; Cavalli, S S

2011-02-22

Sib-seedlings of 95 strains of the strictly autogamous grass Hordeum euclaston were analyzed by horizontal polyacrylamide gel electrophoresis for four isoenzyme systems at a specific ontogenetic stage. We found differences in the activity of some genes among individuals of this species. Hence, an ontogenetic analysis was carried out to investigate 12 strains at five ontogenetic stages, to determine the patterns of expression of these genes during development. The differences in the presence versus absence of certain isoenzyme bands may be due to differential regulatory activation in response to environmental differences, as all plants showed the same structural genes, although these genes were active in different tissues and/or times of development. These results indicate the importance of differential gene activation in the metabolic phenotype variability of this strictly autogamous, highly homozygous species. The same structural alleles for isoenzymes showed the active form of the enzymes (phenotypic expression) to be present in different tissues and/or stages of development. Differential isoenzyme gene activation was shown to be directly responsible for the enzymatic variability (metabolic phenotype) presented by the plants, which seem to possess almost no heterozygosis.

Stimulation of NADH-dependent microsomal DNA strand cleavage by rifamycin SV.