The Sulfur Carrier Protein TusA Has a Pleiotropic Role in Escherichia coli That Also Affects Molybdenum Cofactor Biosynthesis*

PubMed Central

Dahl, Jan-Ulrik; Radon, Christin; Bühning, Martin; Nimtz, Manfred; Leichert, Lars I.; Denis, Yann; Jourlin-Castelli, Cécile; Iobbi-Nivol, Chantal; Méjean, Vincent; Leimkühler, Silke

2013-01-01

The Escherichia coli l-cysteine desulfurase IscS mobilizes sulfur from l-cysteine for the synthesis of several biomolecules such as iron-sulfur (FeS) clusters, molybdopterin, thiamin, lipoic acid, biotin, and the thiolation of tRNAs. The sulfur transfer from IscS to various biomolecules is mediated by different interaction partners (e.g. TusA for thiomodification of tRNAs, IscU for FeS cluster biogenesis, and ThiI for thiamine biosynthesis/tRNA thiolation), which bind at different sites of IscS. Transcriptomic and proteomic studies of a ΔtusA strain showed that the expression of genes of the moaABCDE operon coding for proteins involved in molybdenum cofactor biosynthesis is increased under aerobic and anaerobic conditions. Additionally, under anaerobic conditions the expression of genes encoding hydrogenase 3 and several molybdoenzymes such as nitrate reductase were also increased. On the contrary, the activity of all molydoenzymes analyzed was significantly reduced in the ΔtusA mutant. Characterization of the ΔtusA strain under aerobic conditions showed an overall low molybdopterin content and an accumulation of cyclic pyranopterin monophosphate. Under anaerobic conditions the activity of nitrate reductase was reduced by only 50%, showing that TusA is not essential for molybdenum cofactor biosynthesis. We present a model in which we propose that the direction of sulfur transfer for each sulfur-containing biomolecule is regulated by the availability of the interaction partner of IscS. We propose that in the absence of TusA, more IscS is available for FeS cluster biosynthesis and that the overproduction of FeS clusters leads to a modified expression of several genes. PMID:23281480

Peptidomics of Cpefat/fat mouse brain regions: Implications for neuropeptide processing

PubMed Central

Zhang, Xin; Che, Fa-Yun; Berezniuk, Iryna; Sonmez, Kemal; Toll, Lawrence; Fricker, Lloyd D.

2009-01-01

SUMMARY Quantitative peptidomics was used to compare levels of peptides in wild type and Cpefat/fat mice, which lack carboxypeptidase E (CPE) activity due to a point mutation. Six different brain regions were analyzed: amygdala, hippocampus, hypothalamus, prefrontal cortex, striatum, and thalamus. Altogether, 111 neuropeptides or other peptides derived from secretory pathway proteins were identified in wild type mouse brain extracts by tandem mass spectrometry, and another 47 peptides were tentatively identified based on mass and other criteria. Most secretory pathway peptides were much lower in Cpefat/fat mouse brain, relative to wild type mouse brain, indicating that CPE plays a major role in their biosynthesis. Other peptides were only partially reduced in the Cpefat/fat mice, indicating that another enzyme (presumably carboxypeptidase D) contributes to their biosynthesis. Approximately 10% of the secretory pathway peptides were present in the Cpefat/fat mouse brain at levels similar to those in wild type mouse brain. Many peptides were greatly elevated in the Cpefat/fat mice; these peptide processing intermediates with C-terminal Lys and/or Arg were generally not detectable in wild type mice. Taken together, these results indicate that CPE contributes, either directly or indirectly, to the production of the majority of neuropeptides. PMID:19014391

Determining Roles of Accessory Genes in Denitrification by Mutant Fitness Analyses

PubMed Central

Vaccaro, Brian J.; Thorgersen, Michael P.; Lancaster, W. Andrew; Price, Morgan N.; Wetmore, Kelly M.; Poole, Farris L.; Deutschbauer, Adam; Arkin, Adam P.

2015-01-01

Enzymes of the denitrification pathway play an important role in the global nitrogen cycle, including release of nitrous oxide, an ozone-depleting greenhouse gas. In addition, nitric oxide reductase, maturation factors, and proteins associated with nitric oxide detoxification are used by pathogens to combat nitric oxide release by host immune systems. While the core reductases that catalyze the conversion of nitrate to dinitrogen are well understood at a mechanistic level, there are many peripheral proteins required for denitrification whose basic function is unclear. A bar-coded transposon DNA library from Pseudomonas stutzeri strain RCH2 was grown under denitrifying conditions, using nitrate or nitrite as an electron acceptor, and also under molybdenum limitation conditions, with nitrate as the electron acceptor. Analysis of sequencing results from these growths yielded gene fitness data for 3,307 of the 4,265 protein-encoding genes present in strain RCH2. The insights presented here contribute to our understanding of how peripheral proteins contribute to a fully functioning denitrification pathway. We propose a new low-affinity molybdate transporter, OatABC, and show that differential regulation is observed for two MoaA homologs involved in molybdenum cofactor biosynthesis. We also propose that NnrS may function as a membrane-bound NO sensor. The dominant HemN paralog involved in heme biosynthesis is identified, and a CheR homolog is proposed to function in nitrate chemotaxis. In addition, new insights are provided into nitrite reductase redundancy, nitric oxide reductase maturation, nitrous oxide reductase maturation, and regulation. PMID:26452555

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

Vaccaro, Brian J.; Thorgersen, Michael P.; Lancaster, W. Andrew

Enzymes of the denitrification pathway play an important role in the global nitrogen cycle, including release of nitrous oxide, an ozone-depleting greenhouse gas. In addition, nitric oxide reductase, maturation factors, and proteins associated with nitric oxide detoxification are used by pathogens to combat nitric oxide release by host immune systems. While the core reductases that catalyze the conversion of nitrate to dinitrogen are well understood at a mechanistic level, there are many peripheral proteins required for denitrification whose basic function is unclear. A bar-coded transposon DNA library fromPseudomonas stutzeristrain RCH2 was grown under denitrifying conditions, using nitrate or nitrite asmore » an electron acceptor, and also under molybdenum limitation conditions, with nitrate as the electron acceptor. Analysis of sequencing results from these growths yielded gene fitness data for 3,307 of the 4,265 protein-encoding genes present in strain RCH2. The insights presented here contribute to our understanding of how peripheral proteins contribute to a fully functioning denitrification pathway. We propose a new low-affinity molybdate transporter, OatABC, and show that differential regulation is observed for two MoaA homologs involved in molybdenum cofactor biosynthesis. We also propose that NnrS may function as a membrane-bound NO sensor. Finally, the dominant HemN paralog involved in heme biosynthesis is identified, and a CheR homolog is proposed to function in nitrate chemotaxis. In addition, new insights are provided into nitrite reductase redundancy, nitric oxide reductase maturation, nitrous oxide reductase maturation, and regulation.« less

Effect of atmospheric carbon dioxide levels and nitrate fertilization on glucosinolate biosynthesis in mechanically damaged Arabidopsis plants.

PubMed

Paudel, Jamuna Risal; Amirizian, Alexandre; Krosse, Sebastian; Giddings, Jessica; Ismail, Shoieb Akaram Arief; Xia, Jianguo; Gloer, James B; van Dam, Nicole M; Bede, Jacqueline C

2016-03-22

Increased atmospheric carbon dioxide (CO2) levels predicted to occur before the end of the century will impact plant metabolism. In addition, nitrate availability will affect metabolism and levels of nitrogen-containing defense compounds, such as glucosinolates (GSLs). We compared Arabidopsis foliar metabolic profile in plants grown under two CO2 regimes (440 vs 880 ppm), nitrate fertilization (1 mM vs 10 mM) and in response to mechanical damage of rosette leaves. Constitutive foliar metabolites in nitrate-limited plants show distinct global patterns depending on atmospheric CO2 levels; in contrast, plants grown under higher nitrate fertilization under elevated atmospheric CO2 conditions have a unique metabolite signature. Nitrate fertilization dampens the jasmonate burst in response to wounding in plants grown at elevated CO2 levels. Leaf GSL profile mirrors the jasmonate burst; in particular, indole GSLs increase in response to damage in plants grown at ambient CO2 but only in nitrate-limited plants grown under elevated CO2 conditions. This may reflect a reduced capacity of C3 plants grown under enriched CO2 and nitrate levels to signal changes in oxidative stress and has implications for future agricultural management practices.

Biosynthesis and intracellular movement of the melanosomal membrane glycoprotein gp75, the human b (brown) locus product

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

Vijayasaradhi, S.; Doskoch, P.M.; Houghton, A.N.

1991-10-01

A 75-kDa melanosomal glycoprotein (gp75) is the product of a gene that maps to the b (brown) locus, a genetic locus that determines coat color in the mouse. The b locus is conserved (88% identity) between mouse and human. The mouse monoclonal antibody TA99 was used to study the biosynthesis and processing of gp75. gp75 was synthesized as a 55-kDa polypeptide, glycosylated by addition and processing of five or more Asn-linked carbohydrate chains through the cis and trans Golgi, and transported to melanosomes as a mature 75-kDa form. Synthesis and processing of gp75 was rapid (T{sub 1/2} < 30 min),more » and early steps in processing were required for efficient export of gp75 was quite stable in the melanosome. Studies with inhibitors of steps in oligosaccharide processing showed that alternative forms of gp75 were generated during trimming reactions by mannosidase IA/IB and that further maturation resulted in the two mature forms of gp75. The authors purpose that the kinetics of biosynthesis and processing reflect events in the biogenesis and maturation of melanosomes.« less

The effects of genetic manipulation of putrescine biosynthesis on transcription and activities of the other polyamine biosynthetic enzymes

Treesearch

Andrew F. Page; Sridev Mohapatra; Rakesh Minocha; Subhash C. Minocha

2007-01-01

We have studied the effects of overproduction of putrescine (Put) via transgenic expression of a mouse ornithine decarboxylase (ODC) gene on the expression of native genes for four enzymes involved in polyamine biosynthesis in hybrid poplar (Populus nigra x maximowiczii) cells. An examination of the transcript levels of arginine...

Propiconazole enhanced hepatic cell proliferation is associated with dysregulation of the cholesterol biosynthesis pathway leading to activation of Erk1/2 through Ras famesylation

EPA Science Inventory

Propiconazole is a mouse hepatotumorigenic fungicide designed to inhibit CYP51, a key enzyme in the biosynthesis of ergosterol in fungi and is widely used in agriculture to prevent fungal growth. Metabolomic studies in mice revealed that propiconazole increased levels of hepatic ...

Regiospecific cytochrome P450-catalyzed nitration of L-tryptophan in thaxtomin phytotoxin biosynthesis

USDA-ARS?s Scientific Manuscript database

Thaxtomins, phytotoxins produced by plant pathogenic Streptomyces species, contain a rare nitro group that is essential for phytotoxicity. The N,N'-dimethyldiketopiperazine core of thaxtomins is assembled from L-phenylalanine and L-4-nitrotryptophan by a nonribosomal peptide synthetase. Nitric oxide...

Determining Roles of Accessory Genes in Denitrification by Mutant Fitness Analyses

DOE PAGES

Vaccaro, Brian J.; Thorgersen, Michael P.; Lancaster, W. Andrew; ...

2015-10-09

Enzymes of the denitrification pathway play an important role in the global nitrogen cycle, including release of nitrous oxide, an ozone-depleting greenhouse gas. In addition, nitric oxide reductase, maturation factors, and proteins associated with nitric oxide detoxification are used by pathogens to combat nitric oxide release by host immune systems. While the core reductases that catalyze the conversion of nitrate to dinitrogen are well understood at a mechanistic level, there are many peripheral proteins required for denitrification whose basic function is unclear. A bar-coded transposon DNA library fromPseudomonas stutzeristrain RCH2 was grown under denitrifying conditions, using nitrate or nitrite asmore » an electron acceptor, and also under molybdenum limitation conditions, with nitrate as the electron acceptor. Analysis of sequencing results from these growths yielded gene fitness data for 3,307 of the 4,265 protein-encoding genes present in strain RCH2. The insights presented here contribute to our understanding of how peripheral proteins contribute to a fully functioning denitrification pathway. We propose a new low-affinity molybdate transporter, OatABC, and show that differential regulation is observed for two MoaA homologs involved in molybdenum cofactor biosynthesis. We also propose that NnrS may function as a membrane-bound NO sensor. Finally, the dominant HemN paralog involved in heme biosynthesis is identified, and a CheR homolog is proposed to function in nitrate chemotaxis. In addition, new insights are provided into nitrite reductase redundancy, nitric oxide reductase maturation, nitrous oxide reductase maturation, and regulation.« less

Heparan sulfate C5-epimerase is essential for heparin biosynthesis in mast cells.

PubMed

Feyerabend, Thorsten B; Li, Jin-Ping; Lindahl, Ulf; Rodewald, Hans-Reimer

2006-04-01

Biosynthesis of heparin, a mast cell-derived glycosaminoglycan with widespread importance in medicine, has not been fully elucidated. In biosynthesis of heparan sulfate (HS), a structurally related polysaccharide, HS glucuronyl C5-epimerase (Hsepi) converts D-glucuronic acid (GlcA) to L-iduronic acid (IdoA) residues. We have generated Hsepi-null mouse mutant mast cells, and we show that the same enzyme catalyzes the generation of IdoA in heparin and that 'heparin' lacking IdoA shows a distorted O-sulfation pattern.

Enhancement of antibiotic productions by engineered nitrate utilization in actinomycetes.

PubMed

Meng, Sitong; Wu, Hang; Wang, Lei; Zhang, Buchang; Bai, Linquan

2017-07-01

Nitrate is necessary for primary and secondary metabolism of actinomycetes and stimulates the production of a few antibiotics, such as lincomycin and rifamycin. However, the mechanism of this nitrate-stimulating effect was not fully understood. Two putative ABC-type nitrate transporters were identified in Streptomyces lincolnensis NRRL2936 and verified to be involved in lincomycin biosynthesis. With nitrate supplementation, the transcription of nitrogen assimilation genes, nitrate-specific ABC1 transporter genes, and lincomycin exporter gene lmrA was found to be enhanced and positively regulated by the global regulator GlnR, whose expression was also improved. Moreover, heterologous expression of ABC2 transporter genes in Streptomyces coelicolor M145 resulted in an increased actinorhodin production. Further incorporation of a nitrite-specific transporter gene nirC, as in nirC-ABC2 cassette, led to an even higher actinorhodin production. Similarly, the titers of salinomycin, ansamitocin, lincomycin, and geldanamycin were increased with the integration of this cassette to Streptomyces albus BK3-25, Actinosynnema pretiosum ATCC31280, S. lincolnensis LC-G, and Streptomyces hygroscopicus XM201, respectively. Our work expanded the nitrate-stimulating effect to many antibiotic producers by utilizing the nirC-ABC2 cassette for enhanced nitrate utilization, which could become a general tool for titer increase of antibiotics in actinomycetes.

Ripening of pepper (Capsicum annuum) fruit is characterized by an enhancement of protein tyrosine nitration.

PubMed

Chaki, Mounira; Álvarez de Morales, Paz; Ruiz, Carmelo; Begara-Morales, Juan C; Barroso, Juan B; Corpas, Francisco J; Palma, José M

2015-09-01

Pepper (Capsicum annuum, Solanaceae) fruits are consumed worldwide and are of great economic importance. In most species ripening is characterized by important visual and metabolic changes, the latter including emission of volatile organic compounds associated with respiration, destruction of chlorophylls, synthesis of new pigments (red/yellow carotenoids plus xanthophylls and anthocyanins), formation of pectins and protein synthesis. The involvement of nitric oxide (NO) in fruit ripening has been established, but more work is needed to detail the metabolic networks involving NO and other reactive nitrogen species (RNS) in the process. It has been reported that RNS can mediate post-translational modifications of proteins, which can modulate physiological processes through mechanisms of cellular signalling. This study therefore examined the potential role of NO in nitration of tyrosine during the ripening of California sweet pepper. The NO content of green and red pepper fruit was determined spectrofluorometrically. Fruits at the breaking point between green and red coloration were incubated in the presence of NO for 1 h and then left to ripen for 3 d. Profiles of nitrated proteins were determined using an antibody against nitro-tyrosine (NO2-Tyr), and profiles of nitrosothiols were determined by confocal laser scanning microscopy. Nitrated proteins were identified by 2-D electrophoresis and MALDI-TOF/TOF analysis. Treatment with NO delayed the ripening of fruit. An enhancement of nitrosothiols and nitroproteins was observed in fruit during ripening, and this was reversed by the addition of exogenous NO gas. Six nitrated proteins were identified and were characterized as being involved in redox, protein, carbohydrate and oxidative metabolism, and in glutamate biosynthesis. Catalase was the most abundant nitrated protein found in both green and red fruit. The RNS profile reported here indicates that ripening of pepper fruit is characterized by an enhancement of S-nitrosothiols and protein tyrosine nitration. The nitrated proteins identified have important functions in photosynthesis, generation of NADPH, proteolysis, amino acid biosynthesis and oxidative metabolism. The decrease of catalase in red fruit implies a lower capacity to scavenge H2O2, which would promote lipid peroxidation, as has already been reported in ripe pepper fruit. © The Author 2015. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Ripening of pepper (Capsicum annuum) fruit is characterized by an enhancement of protein tyrosine nitration

PubMed Central

Chaki, Mounira; Álvarez de Morales, Paz; Ruiz, Carmelo; Begara-Morales, Juan C.; Barroso, Juan B.; Corpas, Francisco J.; Palma, José M.

2015-01-01

Background and Aims Pepper (Capsicum annuum, Solanaceae) fruits are consumed worldwide and are of great economic importance. In most species ripening is characterized by important visual and metabolic changes, the latter including emission of volatile organic compounds associated with respiration, destruction of chlorophylls, synthesis of new pigments (red/yellow carotenoids plus xanthophylls and anthocyanins), formation of pectins and protein synthesis. The involvement of nitric oxide (NO) in fruit ripening has been established, but more work is needed to detail the metabolic networks involving NO and other reactive nitrogen species (RNS) in the process. It has been reported that RNS can mediate post-translational modifications of proteins, which can modulate physiological processes through mechanisms of cellular signalling. This study therefore examined the potential role of NO in nitration of tyrosine during the ripening of California sweet pepper. Methods The NO content of green and red pepper fruit was determined spectrofluorometrically. Fruits at the breaking point between green and red coloration were incubated in the presence of NO for 1 h and then left to ripen for 3 d. Profiles of nitrated proteins were determined using an antibody against nitro-tyrosine (NO2-Tyr), and profiles of nitrosothiols were determined by confocal laser scanning microscopy. Nitrated proteins were identified by 2-D electrophoresis and MALDI-TOF/TOF analysis. Key Results Treatment with NO delayed the ripening of fruit. An enhancement of nitrosothiols and nitroproteins was observed in fruit during ripening, and this was reversed by the addition of exogenous NO gas. Six nitrated proteins were identified and were characterized as being involved in redox, protein, carbohydrate and oxidative metabolism, and in glutamate biosynthesis. Catalase was the most abundant nitrated protein found in both green and red fruit. Conclusions The RNS profile reported here indicates that ripening of pepper fruit is characterized by an enhancement of S-nitrosothiols and protein tyrosine nitration. The nitrated proteins identified have important functions in photosynthesis, generation of NADPH, proteolysis, amino acid biosynthesis and oxidative metabolism. The decrease of catalase in red fruit implies a lower capacity to scavenge H2O2, which would promote lipid peroxidation, as has already been reported in ripe pepper fruit. PMID:25814060

Studies on the muscle-paralyzing components of the juice of the banana plant.

PubMed

Singh, Y N; Inman, W D; Johnson, A; Linnell, E J

1993-01-01

The stem juice of the banana plant (Musa species) has been used as an arrow poison by African tribesmen. Lyophilized, partially purified extracts of the juice augment and then block both directly and indirectly evoked contractions of the mouse diaphragm. We have isolated, purified and determined the chemical composition of the active ingredients, and characterized their pharmacological activity. The lyophilized sample was extracted with a methanol-water (MeOH-H2O) (50/50) mixture and vacuum filtered. The filtrate was rotary evaporated and crystallized in a MeOH-H2O mixture to yield potassium nitrate crystals (melting point 332-334 degrees C). The filtrate was concentrated and chromatographed over Sephadex LH-20 gel using MeOH-H2O (40/60) as the eluent. The active component was found to be magnesium nitrate crystals (melting point 87-89 degrees C). In the mouse isolated phrenic nerve-hemidiaphragm preparation, the pharmacological profile of the first component was similar to that for authentic potassium nitrate which augments in low concentrations, and in higher concentrations augments, and then blocks both directly evoked muscle contraction the neuromuscular transmission. The second component had a profile of activity similar to that for authentic magnesium nitrate which only blocks neuromuscular transmission. It can be concluded that the two major active principles in the banana stem juice are potassium nitrate and magnesium nitrate.

Impact of Vitamin B12 and Nitrate Availability on the Concentration of Particulate Dimethylsulfoniopropionate in Phytoplankton

NASA Astrophysics Data System (ADS)

Zavala, J.; Lee, P. A.; Schanke, N. L.; Pound, H.; Penta, W. B.; Shore, S. K.

2016-02-01

The production of particulate dimethylsulfoniopropionate (DMSPp) was examined in natural phytoplankton communities from the South Atlantic Bight near Savannah, Georgia, during an expedition in June 2015. Vitamin B12 and nitrate were added to seawater samples from a coastal and an oceanic site, both of which contained low-biomass, cyanobacteria-dominated communities. Under nitrate-limited conditions, irrespective of changes in B12 levels, DMSPp concentrations increased. DMSPp concentrations of these mixed phytoplankton communities did not appear to be limited by the availability of B12. In a laboratory experiment, DMSPp concentrations in the diatom Phaeodactylum tricornutum were measured after the removal of vitamin B12 and nitrate from a synthetic seawater culture media. DMSPp concentrations increased under nitrate-limited conditions, irrespective of changes in B12 levels, and are argued to be the result of increased biosynthesis. DMSPp concentrations in P. tricornutum were unaffected by B12 limitation. It is hypothesized that P. tricornutum is using the B12-independent methionine synthase MetE to synthesize DMSPp rather than the B12-dependent methionine synthase MetH.

Combinatorial function of velvet and AreA in transcriptional regulation of nitrate utilization and secondary metabolism.

PubMed

López-Berges, Manuel S; Schäfer, Katja; Hera, Concepción; Di Pietro, Antonio

2014-01-01

Velvet is a conserved protein complex that functions as a regulator of fungal development and secondary metabolism. In the soil-inhabiting pathogen Fusarium oxysporum, velvet governs mycotoxin production and virulence on plant and mammalian hosts. Here we report a previously unrecognized role of the velvet complex in regulation of nitrate metabolism. F. oxysporum mutants lacking VeA or LaeA, two key components of the complex, were impaired in growth on the non-preferred nitrogen sources nitrate and nitrite. Both velvet and the general nitrogen response GATA factor AreA were required for transcriptional activation of nitrate (nit1) and nitrite (nii1) reductase genes under de-repressing conditions, as well as for the nitrate-triggered increase in chromatin accessibility at the nit1 locus. AreA also contributed to chromatin accessibility and expression of two velvet-regulated gene clusters, encoding biosynthesis of the mycotoxin beauvericin and of the siderophore ferricrocin. Thus, velvet and AreA coordinately orchestrate primary and secondary metabolism as well as virulence functions in F. oxysporum. Copyright © 2013 Elsevier Inc. All rights reserved.

Extracellular biosynthesis of silver nanoparticles using a novel and non-pathogenic fungus, Neurospora intermedia: controlled synthesis and antibacterial activity.

PubMed

Hamedi, Sepideh; Shojaosadati, Seyed Abbas; Shokrollahzadeh, Soheila; Hashemi-Najafabadi, Sameereh

2014-02-01

In the present study, the biosynthesis of silver nanoparticles (AgNPs) using Neurospora intermedia, as a new non-pathogenic fungus was investigated. For determination of biomass harvesting time, the effect of fungal incubation period on nanoparticle formation was investigated using UV-visible spectroscopy. Then, AgNPs were synthesized using both culture supernatant and cell-free filtrate of the fungus. Two different volume ratios (1:100 and 1:1) of the culture supernatant to the silver nitrate were employed for AgNP synthesis. In addition, cell-free filtrate and silver nitrate were mixed in presence and absence of light. Smallest average size and highest productivity were obtained when using equal volumes of the culture supernatant and silver nitrate solution as confirmed by UV-visible spectra of colloidal AgNPs. Comparing the UV-visible spectra revealed that using cell-free filtrate for AgNP synthesis resulted in the formation of particles with higher stability and monodispersity than using culture supernatant. The absence of light in cell-free filtrate mediated synthesis led to the formation of nanoparticles with the lowest rate and the highest monodispersity. The presence of elemental silver in all prepared samples was confirmed using EDX, while the crystalline nature of synthesized particles was verified by XRD. FTIR results showed the presence of functional groups which reduce Ag(+) and stabilize AgNPs. The presence of nitrate reductase was confirmed in the cell-free filtrate of the fungus suggesting the potential role of this enzyme in AgNP synthesis. Synthesized particles showed significant antibacterial activity against E. coli as confirmed by examining the growth curve of bacterial cells exposed to AgNPs.

Lack of modulation of gastric emptying by dietary nitrate in healthy volunteers.

PubMed

Terai, Shiho; Iijima, Katsunori; Asanuma, Kiyotaka; Ara, Nobuyuki; Uno, Kaname; Abe, Yasuhiko; Koike, Tomoyuki; Imatani, Akira; Ohara, Shuichi; Shimosegawa, Tooru

2009-05-01

Nitric oxide produced endogenously in vagal neurons modulates gastrointestinal motor activity as an important non-adrenergic and non-cholinergic neurotransmitter. Other than through endogenous biosynthesis, a high concentration of nitric oxide also occurs by chemical reactions within the stomach in the presence of gastric acid through the entero-salivary re-circulation of dietary nitrate. Although dietary nitrate can be a potential source of nitric oxide in the human stomach, there has been no report on the effect of dietary nitrate on gastric motor function. The aim of this study is to investigate the effect of dietary nitrate on gastric emptying, one of the major parameters for the gastric motor function. Fifteen healthy volunteers underwent a placebo-controlled (310 mg sodium nitrate or placebo), double-blind, crossover trial. Since a sufficient amount of gastric acid is essential for dietary nitrate-derived nitric oxide generation in the stomach, the same protocol was repeated after 1-week treatment with a proton pump inhibitor, rabeprazole. Gastric emptying was evaluated by (13)C-octanoate breath test. The sodium nitrate ingestion did not affect gastric emptying either prior to or during rabeprazole treatment, although rabeprazole treatment itself significantly delayed gastric emptying, being independent of the dietary nitrate load. Confirmation of the delayed gastric emptying with rabeprazole indicates the sensitivity of the breath test employed in the present study. In conclusion, despite the potential nitrogen source of exogenous nitric oxide, the ingestion of 310 mg sodium nitrate, which is equivalent to the average daily intake of Japanese adults, does not affect gastric emptying in healthy volunteers.

Ionic liquids as novel solvents for biosynthesis of octenyl succinic anhydride-modified waxy maize starch.

PubMed

Li, Dandan; Zhang, Xiwen; Tian, Yaoqi

2016-05-01

Biosynthesis of octenyl succinic anhydride (OSA) starch was investigated using ionic liquids (ILs) as reaction media. Waxy maize starch was pretreated in 1-butyl-3-methylimidazolium chlorine and then esterified with OSA in 1-octyl-3-methylimidazolium nitrate by using Novozyme 435 as catalyst. The degree of substitution of OSA starch reached 0.0130 with 5 wt% starch concentration and 1 wt% lipase dosage based on ILs weight at 50 °C for 3h. The formation of OSA starch was confirmed by fourier transform infrared spectroscopy. Scanning electron microscopy and X-ray diffraction revealed that the morphology and crystal structure of starch were significantly destroyed. Thermogravimetric analysis showed that esterification decreased the thermal stability of starch. The successful lipase-catalyzed synthesis of OSA starch in ILs suggests that ILs are potential replacement of traditional organic solvents for starch ester biosynthesis. Copyright © 2016 Elsevier B.V. All rights reserved.

Localization and regulation of mouse pantothenate kinase 2 [The PanK2 Genes of Mouse and Human Specify Proteins with Distinct Subcellular Locations

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

Leonardi, Roberta; Zhang, Yong-Mei; Lykidis, Athanasios

2007-09-07

Coenzyme A (CoA) biosynthesis is initiated by pantothenatekinase (PanK) and CoA levels are controlled through differentialexpression and feedback regulation of PanK isoforms. PanK2 is amitochondrial protein in humans, but comparative genomics revealed thatacquisition of a mitochondrial targeting signal was limited to primates.Human and mouse PanK2 possessed similar biochemical properties, withinhibition by acetylCoA and activation by palmitoylcarnitine. Mouse PanK2localized in the cytosol, and the expression of PanK2 was higher in humanbrain compared to mouse brain. Differences in expression and subcellularlocalization should be considered in developing a mouse model for humanPanK2 deficiency.

The bZIP transcription factor MdHY5 regulates anthocyanin accumulation and nitrate assimilation in apple.

PubMed

An, Jian-Ping; Qu, Feng-Jia; Yao, Ji-Fang; Wang, Xiao-Na; You, Chun-Xiang; Wang, Xiao-Fei; Hao, Yu-Jin

2017-01-01

The basic leucine zipper (bZIP) transcription factor HY5 plays a multifaceted role in plant growth and development. Here the apple MdHY5 gene was cloned based on its homology with Arabidopsis HY5 . Expression analysis demonstrated that MdHY5 transcription was induced by light and abscisic acid treatments. Electrophoretic mobility shift assays and transient expression assays subsequently showed that MdHY5 positively regulated both its own transcription and that of MdMYB10 by binding to E-box and G-box motifs, respectively. Furthermore, we obtained transgenic apple calli that overexpressed the MdHY5 gene, and apple calli coloration assays showed that MdHY5 promoted anthocyanin accumulation by regulating expression of the MdMYB10 gene and downstream anthocyanin biosynthesis genes. In addition, the transcript levels of a series of nitrate reductase genes and nitrate uptake genes in both wild-type and transgenic apple calli were detected. In association with increased nitrate reductase activities and nitrate contents, the results indicated that MdHY5 might be an important regulator in nutrient assimilation. Taken together, these results indicate that MdHY5 plays a vital role in anthocyanin accumulation and nitrate assimilation in apple.

GYY4137, an H2S Slow-Releasing Donor, Prevents Nitrative Stress and α-Synuclein Nitration in an MPTP Mouse Model of Parkinson's Disease.

PubMed

Hou, Xiaoou; Yuan, Yuqing; Sheng, Yulan; Yuan, Baoshi; Wang, Yali; Zheng, Jiyue; Liu, Chun-Feng; Zhang, Xiaohu; Hu, Li-Fang

2017-01-01

The neuromodulator hydrogen sulfide (H 2 S) was shown to exert neuroprotection in different models of Parkinson's disease (PD) via its anti-inflammatory and anti-apoptotic properties. In this study, we evaluated the effect of an H 2 S slow-releasing compound GYY4137 (GYY) on a mouse PD model induced by acute injection with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). GYY was intraperitoneally (i.p.) injected once daily into male C57BL/6J mice 3 days before and 2 weeks after MPTP (14 mg/kg, four times at 2-h intervals, i.p.) administration. Saline was given as a control. Behavioral tests (rotarod, balance beam, and grid walking) showed that 50 mg/kg GYY significantly ameliorated MPTP-caused motor impairments. At lower doses (12.5 and 25 mg/kg) GYY exhibited a less obvious effect. Consistent with this, immunohistochemistry and western blot analysis demonstrated that 50 mg/kg GYY attenuated the loss of tyrosine hydroxylase (TH) positive neurons in the substantia nigra and the decrease of TH expression in the striatum of MPTP-treated mice. Moreover, at this regimen GYY relieved the nitrative stress, as indicated by the decreases in nitric oxide (NO) generation and neuronal NO synthase (nNOS) upregulation elicited by MPTP in the striatum. The suppression of GYY on nNOS expression was verified in vitro , and the results further revealed that Akt activation may participate in the inhibition by GYY on nNOS upregulation. More important, GYY reduced the nitrated modification of α-synuclein, a PD-related protein, in MPTP-induced mice. Overall, our findings suggest that GYY attenuated dopaminergic neuron degeneration and reduced α-synuclein nitration in the midbrain, thus exerting neuroprotection in MPTP-induced mouse model of PD.

Dancing with Hormones: A Current Perspective of Nitrate Signaling and Regulation in Arabidopsis

PubMed Central

Guan, Peizhu

2017-01-01

In nature and agriculture, nitrate availability is a main environmental cue for plant growth, development and stress responses. Nitrate signaling and regulation are hence at the center of communications between plant intrinsic programs and the environment. It is also well known that endogenous phytohormones play numerous critical roles in integrating extrinsic cues and intrinsic responses, regulating and refining almost all aspects of plant growth, development and stress responses. Therefore, interaction between nitrate and phytohormones, such as auxins, cytokinins, abscisic acid, gibberellins, and ethylene, is prevalent. The growing evidence indicates that biosynthesis, de-conjugation, transport, and signaling of hormones are partly controlled by nitrate signaling. Recent advances with nitrate signaling and transcriptional regulation in Arabidopsis give rise to new paradigms. Given the comprehensive nitrate transport, sensing, signaling and regulations at the level of the cell and organism, nitrate itself is a local and long-distance signal molecule, conveying N status at the whole-plant level. A direct molecular link between nitrate signaling and cell cycle progression was revealed with TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR1-20 (TCP20) – NIN-LIKE PROTEIN 6/7 (NLP6/7) regulatory nexus. NLPs are key regulators of nitrogen responses in plants. TCPs function as the main regulators of plant morphology and architecture, with the emerging role as integrators of plant developmental responses to the environment. By analogy with auxin being proposed as a plant morphogen, nitrate may be an environmental morphogen. The morphogen-gradient-dependent and cell-autonomous mechanisms of nitrate signaling and regulation are an integral part of cell growth and cell identification. This is especially true in root meristem growth that is regulated by intertwined nitrate, phytohormones, and glucose-TOR signaling pathways. Furthermore, the nitrate transcriptional hierarchy is emerging. Nitrate regulators in primary nitrate signaling can individually and combinatorially control downstream transcriptional networks and hormonal pathways for signal propagation and amplification. Under the new paradigms, nitrate-induced hormone metabolism and signaling deserve fresh examination. The close interplay and convergent regulation of nitrate and hormonal signaling at morphological, physiological, and molecular levels have significant effects on important agronomic traits, especially nutrient-dependent adaptive root system growth and architecture. PMID:29033968

Impact of Chemical Analogs of 4-Hydroxybenzoic Acid on Coenzyme Q Biosynthesis: From Inhibition to Bypass of Coenzyme Q Deficiency

PubMed Central

Pierrel, Fabien

2017-01-01

Coenzyme Q is a lipid that participates to important physiological functions. Coenzyme Q is synthesized in multiple steps from the precursor 4-hydroxybenzoic acid. Mutations in enzymes that participate to coenzyme Q biosynthesis result in primary coenzyme Q deficiency, a type of mitochondrial disease. Coenzyme Q10 supplementation of patients is the classical treatment but it shows limited efficacy in some cases. The molecular understanding of the coenzyme Q biosynthetic pathway allowed the design of experiments to bypass deficient biosynthetic steps with analogs of 4-hydroxybenzoic acid. These molecules provide the defective chemical group and can reactivate endogenous coenzyme Q biosynthesis as demonstrated recently in yeast, mammalian cell cultures, and mouse models of primary coenzyme Q deficiency. This mini review presents how the chemical properties of various analogs of 4-hydroxybenzoic acid dictate the effect of the molecules on CoQ biosynthesis and how the reactivation of endogenous coenzyme Q biosynthesis may achieve better results than exogenous CoQ10 supplementation. PMID:28690551

Impact of Chemical Analogs of 4-Hydroxybenzoic Acid on Coenzyme Q Biosynthesis: From Inhibition to Bypass of Coenzyme Q Deficiency.

PubMed

Pierrel, Fabien

2017-01-01

Coenzyme Q is a lipid that participates to important physiological functions. Coenzyme Q is synthesized in multiple steps from the precursor 4-hydroxybenzoic acid. Mutations in enzymes that participate to coenzyme Q biosynthesis result in primary coenzyme Q deficiency, a type of mitochondrial disease. Coenzyme Q 10 supplementation of patients is the classical treatment but it shows limited efficacy in some cases. The molecular understanding of the coenzyme Q biosynthetic pathway allowed the design of experiments to bypass deficient biosynthetic steps with analogs of 4-hydroxybenzoic acid. These molecules provide the defective chemical group and can reactivate endogenous coenzyme Q biosynthesis as demonstrated recently in yeast, mammalian cell cultures, and mouse models of primary coenzyme Q deficiency. This mini review presents how the chemical properties of various analogs of 4-hydroxybenzoic acid dictate the effect of the molecules on CoQ biosynthesis and how the reactivation of endogenous coenzyme Q biosynthesis may achieve better results than exogenous CoQ 10 supplementation.

Demonstration of neuron-glia transfer of precursors for GABA biosynthesis in a co-culture system of dissociated mouse cerebral cortex.

PubMed

Leke, Renata; Bak, Lasse K; Schousboe, Arne; Waagepetersen, Helle S

2008-12-01

Co-cultures of neurons and astrocytes were prepared from dissociated embryonic mouse cerebral cortex and cultured for 7 days. To investigate if these cultures may serve as a functional model system to study neuron-glia interaction with regard to GABA biosynthesis, the cells were incubated either in media containing [U-(13)C]glutamine (0.1, 0.3 and 0.5 mM) or 1 mM acetate plus 2.5 mM glucose plus 1 mM lactate. In the latter case one of the 3 substrates was uniformly (13)C labeled. Cellular contents and (13)C labeling of glutamate, GABA, aspartate and glutamine were determined in the cells after an incubation period of 2.5 h. The GABA biosynthetic machinery exhibited the expected complexity with regard to metabolic compartmentation and involvement of TCA cycle activity as seen in other culture systems containing GABAergic neurons. Metabolism of acetate clearly demonstrated glial synthesis of glutamine and its transfer to the neuronal compartment. It is concluded that this co-culture system serves as a reliable model in which functional and pharmacological aspects of GABA biosynthesis can be investigated.

Simulation of triacylglycerol ion profiles: bioinformatics for interpretation of triacylglycerol biosynthesis[S

PubMed Central

Han, Rowland H.; Wang, Miao; Fang, Xiaoling; Han, Xianlin

2013-01-01

Although the synthesis pathways of intracellular triacylglycerol (TAG) species have been well elucidated, assessment of the contribution of an individual pathway to TAG pools in different mammalian organs, particularly under pathophysiological conditions, is difficult, although not impossible. Herein, we developed and validated a novel bioinformatic approach to assess the differential contributions of the known pathways to TAG pools through simulation of TAG ion profiles determined by shotgun lipidomics. This powerful approach was applied to determine such contributions in mouse heart, liver, and skeletal muscle and to examine the changes of these pathways in mouse liver induced after treatment with a high-fat diet. It was clearly demonstrated that assessment of the altered TAG biosynthesis pathways under pathophysiological conditions can be readily achieved through simulation of lipidomics data. Collectively, this new development should greatly facilitate our understanding of the biochemical mechanisms underpinning TAG accumulation at the states of obesity and lipotoxicity. PMID:23365150

Placental lactogens induce serotonin biosynthesis in a subset of mouse beta cells during pregnancy

PubMed Central

Schraenen, A.; Lemaire, K.; de Faudeur, G.; Hendrickx, N.; Granvik, M.; Van Lommel, L.; Mallet, J.; Vodjdani, G.; Gilon, P.; Binart, N.; in’t Veld, P.

2010-01-01

Aims/hypothesis Upregulation of the functional beta cell mass is required to match the physiological demands of mother and fetus during pregnancy. This increase is dependent on placental lactogens (PLs) and prolactin receptors, but the mechanisms underlying these events are only partially understood. We studied the mRNA expression profile of mouse islets during pregnancy to gain a better insight into these changes. Methods RNA expression was measured ex vivo via microarrays and quantitative RT-PCR. In vivo observations were extended by in vitro models in which ovine PL was added to cultured mouse islets and MIN6 cells. Results mRNA encoding both isoforms of the rate-limiting enzyme of serotonin biosynthesis, tryptophan hydroxylase (TPH), i.e. Tph1 and Tph2, were strongly induced (fold change 25- to 200-fold) during pregnancy. This induction was mimicked by exposing islets or MIN6 cells to ovine PLs for 24 h and was dependent on janus kinase 2 and signal transducer and activator of transcription 5. Parallel to Tph1 mRNA and protein induction, islet serotonin content increased to a peak level that was 200-fold higher than basal. Interestingly, only a subpopulation of the beta cells was serotonin-positive in vitro and in vivo. The stored serotonin pool in pregnant islets and PL-treated MIN6 cells was rapidly released (turnover once every 2 h). Conclusions/interpretation A very strong lactogen-dependent upregulation of serotonin biosynthesis occurs in a subpopulation of mouse islet beta cells during pregnancy. Since the newly formed serotonin is rapidly released, this lactogen-induced beta cell function may serve local or endocrine tasks, the nature of which remains to be identified. Electronic supplementary material The online version of this article (doi:10.1007/s00125-010-1913-7) contains supplementary material, which is available to authorised users. PMID:20938637

Differential regulation of fatty acid biosynthesis in two Chlorella species in response to nitrate treatments and the potential of binary blending microalgae oils for biodiesel application.

PubMed

Cha, Thye San; Chen, Jian Woon; Goh, Eng Giap; Aziz, Ahmad; Loh, Saw Hong

2011-11-01

This study was undertaken to investigate the effects of different nitrate concentrations in culture medium on oil content and fatty acid composition of Chlorella vulgaris (UMT-M1) and Chlorella sorokiniana (KS-MB2). Results showed that both species produced significant higher (p<0.05) oil content at nitrate ranging from 0.18 to 0.66 mM with C. vulgaris produced 10.20-11.34% dw, while C. sorokiniana produced 15.44-17.32% dw. The major fatty acids detected include C16:0, C18:0, C18:1, C18:2 and C18:3. It is interesting to note that both species displayed differentially regulated fatty acid accumulation patterns in response to nitrate treatments at early stationary growth phase. Their potential use for biodiesel application could be enhanced by exploring the concept of binary blending of the two microalgae oils using developed mathematical equations to calculate the oil mass blending ratio and simultaneously estimated the weight percentage (wt.%) of desirable fatty acid compositions. Copyright © 2011 Elsevier Ltd. All rights reserved.

Dietary nitrate increases tetanic [Ca2+]i and contractile force in mouse fast-twitch muscle

PubMed Central

Hernández, Andrés; Schiffer, Tomas A; Ivarsson, Niklas; Cheng, Arthur J; Bruton, Joseph D; Lundberg, Jon O; Weitzberg, Eddie; Westerblad, Håkan

2012-01-01

Dietary inorganic nitrate has profound effects on health and physiological responses to exercise. Here, we examined if nitrate, in doses readily achievable via a normal diet, could improve Ca2+ handling and contractile function using fast- and slow-twitch skeletal muscles from C57bl/6 male mice given 1 mm sodium nitrate in water for 7 days. Age matched controls were provided water without added nitrate. In fast-twitch muscle fibres dissected from nitrate treated mice, myoplasmic free [Ca2+] was significantly greater than in Control fibres at stimulation frequencies from 20 to 150 Hz, which resulted in a major increase in contractile force at ≤50 Hz. At 100 Hz stimulation, the rate of force development was ∼35% faster in the nitrate group. These changes in nitrate treated mice were accompanied by increased expression of the Ca2+ handling proteins calsequestrin 1 and the dihydropyridine receptor. No changes in force or calsequestrin 1 and dihydropyridine receptor expression were measured in slow-twitch muscles. In conclusion, these results show a striking effect of nitrate supplementation on intracellular Ca2+ handling in fast-twitch muscle resulting in increased force production. A new mechanism is revealed by which nitrate can exert effects on muscle function with applications to performance and a potential therapeutic role in conditions with muscle weakness. PMID:22687611

Dietary nitrate increases tetanic [Ca2+]i and contractile force in mouse fast-twitch muscle.

PubMed

Hernández, Andrés; Schiffer, Tomas A; Ivarsson, Niklas; Cheng, Arthur J; Bruton, Joseph D; Lundberg, Jon O; Weitzberg, Eddie; Westerblad, Håkan

2012-08-01

Dietary inorganic nitrate has profound effects on health and physiological responses to exercise. Here, we examined if nitrate, in doses readily achievable via a normal diet, could improve Ca(2+) handling and contractile function using fast- and slow-twitch skeletal muscles from C57bl/6 male mice given 1 mm sodium nitrate in water for 7 days. Age matched controls were provided water without added nitrate. In fast-twitch muscle fibres dissected from nitrate treated mice, myoplasmic free [Ca(2+)] was significantly greater than in Control fibres at stimulation frequencies from 20 to 150 Hz, which resulted in a major increase in contractile force at ≤ 50 Hz. At 100 Hz stimulation, the rate of force development was ∼35% faster in the nitrate group. These changes in nitrate treated mice were accompanied by increased expression of the Ca(2+) handling proteins calsequestrin 1 and the dihydropyridine receptor. No changes in force or calsequestrin 1 and dihydropyridine receptor expression were measured in slow-twitch muscles. In conclusion, these results show a striking effect of nitrate supplementation on intracellular Ca(2+) handling in fast-twitch muscle resulting in increased force production. A new mechanism is revealed by which nitrate can exert effects on muscle function with applications to performance and a potential therapeutic role in conditions with muscle weakness.

Resveratrol and para-coumarate serve as ring precursors for coenzyme Q biosynthesis[S

PubMed Central

Xie, Letian X.; Williams, Kevin J.; He, Cuiwen H.; Weng, Emily; Khong, San; Rose, Tristan E.; Kwon, Ohyun; Bensinger, Steven J.; Marbois, Beth N.; Clarke, Catherine F.

2015-01-01

Coenzyme Q (Q or ubiquinone) is a redox-active polyisoprenylated benzoquinone lipid essential for electron and proton transport in the mitochondrial respiratory chain. The aromatic ring 4-hydroxybenzoic acid (4HB) is commonly depicted as the sole aromatic ring precursor in Q biosynthesis despite the recent finding that para-aminobenzoic acid (pABA) also serves as a ring precursor in Saccharomyces cerevisiae Q biosynthesis. In this study, we employed aromatic 13C6-ring-labeled compounds including 13C6-4HB, 13C6-pABA, 13C6-resveratrol, and 13C6-coumarate to investigate the role of these small molecules as aromatic ring precursors in Q biosynthesis in Escherichia coli, S. cerevisiae, and human and mouse cells. In contrast to S. cerevisiae, neither E. coli nor the mammalian cells tested were able to form 13C6-Q when cultured in the presence of 13C6-pABA. However, E. coli cells treated with 13C6-pABA generated 13C6-ring-labeled forms of 3-octaprenyl-4-aminobenzoic acid, 2-octaprenyl-aniline, and 3-octaprenyl-2-aminophenol, suggesting UbiA, UbiD, UbiX, and UbiI are capable of using pABA or pABA-derived intermediates as substrates. E. coli, S. cerevisiae, and human and mouse cells cultured in the presence of 13C6-resveratrol or 13C6-coumarate were able to synthesize 13C6-Q. Future evaluation of the physiological and pharmacological responses to dietary polyphenols should consider their metabolism to Q. PMID:25681964

The effects of pilocarpine nitrate upon the mitotic index of mouse bone marrow cells.

PubMed

Prabhu, M P; Hegde, M J

1991-11-01

Aneuploidies are the most common chromosomal causes for spontaneous abortions and constitute a major part of genetic disorders among the neonates. Aneuploidy producing agents (aneugens) pose serious genetic hazards to the human population. Therefore, testing for aneuploidy induction should be part of the requirement in drug safety guidelines. The aneugenic potential of pilocarpine nitrate, an alkaloid drug used as an ophthalmic solution was screened by chromosome analysis studies in the bone marrow cells of mice. Using the technique developed by Miller and Adler (1989) we evaluated changes in the mitotic index (MI), induction of chromatid contraction and spreading (C-mitoses) and decrease of anaphase frequencies as indicators of the aneuploidy inducing potency of the drug. Concentrations of pilocarpine nitrate of 4, 8 and 12 mg/kg body weight were administered intraperitoneally to mice. Colchicine-treated and water-treated animals formed the positive and negative controls. The data obtained in the cytogenetic analysis of both dose-response and time-response studies showed a significant induction of C-mitotic effects in the mouse bone marrow. The positive results indicated that the drug is a potential aneugen and should be further evaluated.

Dietary nitrate and nitrite modulate blood and organ nitrite and the cellular ischemic stress response

PubMed Central

Raat, Nicolaas J.H.; Noguchi, Audrey C.; Liu, Virginia B.; Raghavachari, Nalini; Liu, Delong; Xu, Xiuli; Shiva, Sruti; Munson, Peter J.; Gladwin, Mark T.

2009-01-01

Dietary nitrate, found in abundance in green vegetables, can be converted to the cytoprotective molecule nitrite by oral bacteria, suggesting that nitrate and nitrite may represent active cardioprotective constituents of the Mediterranean diet. We therefore tested the hypothesis that dietary nitrate and nitrite levels modulate tissue damage and ischemic gene expression in a mouse liver ischemia-reperfusion model. We found that stomach content, plasma, heart and liver nitrite levels were significantly reduced after dietary nitrate and nitrite depletion, and could be restored to normal levels with nitrite supplementation in water. Remarkably, we confirmed that basal nitrite levels significantly reduced liver injury after ischemia-reperfusion. Consistent with an effect of nitrite on the post-translational modification of complex I of the mitochondrial electron transport chain, the severity of liver infarction was inversely proportional to complex I activity after nitrite repletion in the diet. The transcriptional response of dietary nitrite after ischemia was more robust than after normoxia, suggesting a hypoxic potentiation of nitrite-dependent transcriptional signaling. Our studies indicate that normal dietary nitrate and nitrite levels modulate ischemic stress responses and hypoxic gene expression programs, supporting the hypothesis that dietary nitrate and nitrite are cytoprotective components of the diet. PMID:19464364

Gallium nitrate ameliorates type II collagen-induced arthritis in mice.

PubMed

Choi, Jae-Hyeog; Lee, Jong-Hwan; Roh, Kug-Hwan; Seo, Su-Kil; Choi, Il-Whan; Park, Sae-Gwang; Lim, Jun-Goo; Lee, Won-Jin; Kim, Myoung-Hun; Cho, Kwang-rae; Kim, Young-Jae

2014-05-01

Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease. Gallium nitrate has been reported to reserve immunosuppressive activities. Therefore, we assessed the therapeutic effects of gallium nitrate in the mouse model of developed type II collagen-induced arthritis (CIA). CIA was induced by bovine type II collagen with Complete Freund's adjuvant. CIA mice were intraperitoneally treated from day 36 to day 49 after immunization with 3.5mg/kg/day, 7mg/kg/day gallium nitrate or vehicle. Gallium nitrate ameliorated the progression of mice with CIA. The clinical symptoms of collagen-induced arthritis did not progress after treatment with gallium nitrate. Gallium nitrate inhibited the increase of CD4(+) T cell populations (p<0.05) and also inhibited the type II collagen-specific IgG2a-isotype autoantibodies (p<0.05). Gallium nitrate reduced the serum levels of TNF-α, IL-6 and IFN-γ (p<0.05) and the mRNA expression levels of these cytokine and MMPs (MMP2 and MMP9) in joint tissues. Western blotting of members of the NF-κB signaling pathway revealed that gallium nitrate inhibits the activation of NF-κB by blocking IκB degradation. These data suggest that gallium nitrate is a potential therapeutic agent for autoimmune inflammatory arthritis through its inhibition of the NF-κB pathway, and these results may help to elucidate gallium nitrate-mediated mechanisms of immunosuppression in patients with RA. Copyright © 2014 Elsevier B.V. All rights reserved.

Differential Regulation of Gene Expression by Cholesterol Biosynthesis Inhibitors That Reduce (Pravastatin) or Enhance (Squalestatin 1) Nonsterol Isoprenoid Levels in Primary Cultured Mouse and Rat Hepatocytes

PubMed Central

Rondini, Elizabeth A.; Duniec-Dmuchowski, Zofia; Cukovic, Daniela; Dombkowski, Alan A.

2016-01-01

Squalene synthase inhibitors (SSIs), such as squalestatin 1 (SQ1), reduce cholesterol biosynthesis but cause the accumulation of isoprenoids derived from farnesyl pyrophosphate (FPP), which can modulate the activity of nuclear receptors, including the constitutive androstane receptor (CAR), farnesoid X receptor, and peroxisome proliferator-activated receptors (PPARs). In comparison, 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (e.g., pravastatin) inhibit production of both cholesterol and nonsterol isoprenoids. To characterize the effects of isoprenoids on hepatocellular physiology, microarrays were used to compare orthologous gene expression from primary cultured mouse and rat hepatocytes that were treated with either SQ1 or pravastatin. Compared with controls, 47 orthologs were affected by both inhibitors, 90 were affected only by SQ1, and 51 were unique to pravastatin treatment (P < 0.05, ≥1.5-fold change). When the effects of SQ1 and pravastatin were compared directly, 162 orthologs were found to be differentially coregulated between the two treatments. Genes involved in cholesterol and unsaturated fatty acid biosynthesis were up-regulated by both inhibitors, consistent with cholesterol depletion; however, the extent of induction was greater in rat than in mouse hepatocytes. SQ1 induced several orthologs associated with microsomal, peroxisomal, and mitochondrial fatty acid oxidation and repressed orthologs involved in cell cycle regulation. By comparison, pravastatin repressed the expression of orthologs involved in retinol and xenobiotic metabolism. Several of the metabolic genes altered by isoprenoids were inducible by a PPARα agonist, whereas cytochrome P450 isoform 2B was inducible by activators of CAR. Our findings indicate that SSIs uniquely influence cellular lipid metabolism and cell cycle regulation, probably due to FPP catabolism through the farnesol pathway. PMID:27225895

Identification of mRNA transcript and screening of amino acids in response to interaction of salinity and nitrate in aquatic fern Azolla caroliniana.

PubMed

Tammam, A A; Mostafa, E M

2012-06-01

The mechanisms by which Azolla caroliniana respond to salt stress in absence and presence of nitrate is investigated. Screening of amino acid and differential display is used to compare overall differences in gene expression between salinity-stressed and unstressed Azolla caroliniana by quantitative reverse transcriptase polymerase chain reaction (RT-PC R). Results showed that under saline conditions, aspartic acid, glutamic acid, alanine and leucine were the amino acids found to be abundant in Azolla caroliniana, accounting for 11.26%, 8.66%, 9.43%, and 12.36%, respectively. Following salinity stress, a decrease in free glutamate concomitant with a parallel decrease in free proline was indeed evident. Interaction between nitrate and salinity stress increased proline content significantly. By screening a cDNA library, we have identified protein products by homology with known proteins. The RNA transcripts encoding protein influencing secondary metabolites and vacuolar Na+/H+ antiporter that facilitate the transport system. The databasematched under interaction of nitrate and 50 mM NaCl were associated with wall biosynthesis, disease resistance, metabolite transport and protein regulator, other gene for metabolism of steroids and secondary transport. Results obtained from this research could represent a key step in understanding the molecular mechanism of salt tolerance of Azolla caroliniana in the presence and absence of nitrate.

Biosynthesis and evaluation of the characteristics of silver nanoparticles using Cassia fistula fruit aqueous extract and its antibacterial activity

NASA Astrophysics Data System (ADS)

Ghafoori, Seyed Mohammad; Entezari, Maliheh; Taghva, Arefeh; Tayebi, Zahra

2017-12-01

There are several ways to produce nanoparticles, but the biological method of nanoparticle production is considered most efficient by researchers due to its eco-friendly and energy saving properties. In this study, the biosynthesis of silver nanoparticles (AgNPs) via Cassia fistula fruit pulp extract was examined. Furthermore, its antibacterial effects were investigated both in vitro and in vivo. To achieve biosynthesis, 10 ml of C. fistula extract was added to 90 ml of aqueous solution of 1 mM silver nitrate. The solution was incubated in darkness overnight, at room temperature. After changing the color of solution, the production of AgNPs was examined by UV-Vis spectrophotometry, XRD and DLS methods. Finally, the antibacterial activity of AgNPs was investigated by using three methods: (1) agar well diffusion, (2) MIC determining and (3) effect on prevention of infection in wound on rat models. The results revealed that synthesized silver nanoparticles have strong antibacterial activity in vitro and in vivo conditions. Undeniably, further research is required to investigate the side effects of such particles.

TRIADIMEFON INDUCES RAT THYROID TUMORS THROUGH A NON-TSH MEDIATED MODE OF ACTION

EPA Science Inventory

Conazoles are a class of fungicides used as agricultural and pharmaceutical products which inhibit ergosterol biosynthesis. Members of this class are hepatotoxic and cause mouse hepatocellular tumors and/or rat thyroid follicular cell tumors. Triadimefon-induced rat thyroid tumor...

Leghemoglobin green derivatives with nitrated hemes evidence production of highly reactive nitrogen species during aging of legume nodules.

PubMed

Navascués, Joaquín; Pérez-Rontomé, Carmen; Gay, Marina; Marcos, Manuel; Yang, Fei; Walker, F Ann; Desbois, Alain; Abián, Joaquín; Becana, Manuel

2012-02-14

Globins constitute a superfamily of proteins widespread in all kingdoms of life, where they fulfill multiple functions, such as efficient O(2) transport and modulation of nitric oxide bioactivity. In plants, the most abundant Hbs are the symbiotic leghemoglobins (Lbs) that scavenge O(2) and facilitate its diffusion to the N(2)-fixing bacteroids in nodules. The biosynthesis of Lbs during nodule formation has been studied in detail, whereas little is known about the green derivatives of Lbs generated during nodule senescence. Here we characterize modified forms of Lbs, termed Lba(m), Lbc(m), and Lbd(m), of soybean nodules. These green Lbs have identical globins to the parent red Lbs but their hemes are nitrated. By combining UV-visible, MS, NMR, and resonance Raman spectroscopies with reconstitution experiments of the apoprotein with protoheme or mesoheme, we show that the nitro group is on the 4-vinyl. In vitro nitration of Lba with excess nitrite produced several isomers of nitrated heme, one of which is identical to those found in vivo. The use of antioxidants, metal chelators, and heme ligands reveals that nitration is contingent upon the binding of nitrite to heme Fe, and that the reactive nitrogen species involved derives from nitrous acid and is most probably the nitronium cation. The identification of these green Lbs provides conclusive evidence that highly oxidizing and nitrating species are produced in nodules leading to nitrosative stress. These findings are consistent with a previous report showing that the modified Lbs are more abundant in senescing nodules and have aberrant O(2) binding.

Oleic acid derived metabolites in mouse neuroblastoma N18TG2 cells.

PubMed

Merkler, David J; Chew, Geoffrey H; Gee, Andrew J; Merkler, Kathleen A; Sorondo, Jean-Paul O; Johnson, Mitchell E

2004-10-05

Oleamide is an endogenous sleep-inducing lipid that has been isolated from the cerebrospinal fluid of sleep-deprived mammals. Oleamide is the best-understood member of the primary fatty acid amide family. One key unanswered question regarding oleamide and all other primary acid amides is the pathway by which these molecules are produced. One proposed pathway involves oleoyl-CoA and N-oleoylglycine as intermediates: oleic acid --> oleoyl-CoA --> N-oleoylglycine --> oleamide. The first and third reactions are known reactions, catalyzed by acyl-CoA synthetase and peptidylglycine alpha-amidating monooxygenase (PAM). Oleoyl-CoA formation from oleic acid has been demonstrated in vitro and in vivo while, to date, N-oleoylglycine cleavage to oleamide has been established only in vitro. PAM catalyzes the final step in alpha-amidated peptide biosynthesis, and its proposed role in primary fatty acid amide biosynthesis has been controversial. Mouse neuroblastoma N(18)TG(2) cells are an excellent model system for the study of oleamide biosynthesis because these cells convert [(14)C]-oleic acid to [(14)C]-oleamide and express PAM in a regulated fashion. We report herein that growth of the N(18)TG(2) cells in the presence of [(14)C]-oleic acid under conditions known to stimulate PAM expression generates an increase in [(14)C]-oleamide or in the presence of a PAM inhibitor generates [(14)C]-N-oleoylglycine. This represents the first identification of N-oleoylglycine from a biological source. In addition, N(18)TG(2) cell growth in the presence of N-oleoylglycine yields oleamide. These results strongly indicate that N-oleoylglycine is an intermediate in oleamide biosynthesis and provide further evidence that PAM does have a role in primary fatty acid amide production in vivo.

COMPARATIVE LIVER P450 ENZYME ACTIVITY AND HISTOPATHOLOGY IN MICE TREATED WITH THE CONAZOLE FUNGICIDES: MYCLOBUTANIL, PROPICONAZOLE AND TRIADIMETON

EPA Science Inventory

Conazoles used in agriculture and pharmaceutical products comprise a class of chemicals which inhibit ergosterol biosynthesis to act as fungicides. Both propiconazole and triadimefon are hepatotoxic and hepatotumorigenic in mice, while myclobutanil is not a mouse liver tumorigen....

Skeletal muscle as an endogenous nitrate reservoir

PubMed Central

Piknova, Barbora; Park, Ji Won; Swanson, Kathryn M.; Dey, Soumyadeep; Noguchi, Constance Tom; Schechter, Alan N

2015-01-01

The nitric oxide synthase (NOS) family of enzymes form nitric oxide (NO) from arginine in the presence of oxygen. At reduced oxygen availability NO is also generated from nitrate in a two step process by bacterial and mammalian molybdopterin proteins, and also directly from nitrite by a variety of five-coordinated ferrous hemoproteins. The mammalian NO cycle also involves direct oxidation of NO to nitrite, and both NO and nitrite to nitrate by oxy-ferrous hemoproteins. The liver and blood are considered the sites of active mammalian NO metabolism and nitrite and nitrate concentrations in the liver and blood of several mammalian species, including human, have been determined. However, the large tissue mass of skeletal muscle had not been generally considered in the analysis of the NO cycle, in spite of its long-known presence of significant levels of active neuronal NOS (nNOS or NOS1). We hypothesized that skeletal muscle participates in the NO cycle and, due to its NO oxidizing heme protein, oxymyoglobin, has high concentrations of nitrate ions. We measured nitrite and nitrate concentrations in rat and mouse leg skeletal muscle and found unusually high concentrations of nitrate but similar levels of nitrite, when compared to the liver. The nitrate reservoir in muscle is easily accessible via the bloodstream and therefore nitrate is available for transport to internal organs where it can be reduced to nitrite and NO. Nitrate levels in skeletal muscle and blood in nNOS−/− mice were dramatically lower when compared with controls, which support further our hypothesis. Although the nitrate reductase activity of xanthine oxidoreductase in muscle is less than that of liver, the residual activity in muscle could be very important in view of its total mass and the high basal level of nitrate. We suggest that skeletal muscle participates in overall NO metabolism, serving as a nitrate reservoir, for direct formation of nitrite and NO, and for determining levels of nitrate in other organs. PMID:25727730

HUMAN ALVEOLAR AND PERITONEAL MACROPHAGES MEDIATE FUNGISTASIS INDEPENDENTLY OF L-ARGININE OXIDATION TO NITRITE OR NITRATE

EPA Science Inventory

Human alveolar macrophages (HAM) from 28 normal volunteers were found to inhibit replication of Cryptoccous neoformans. onditions under which fungistasis occurred were different than those required for mouse peritoneal macrophage-mediated fungi stasis. nhibition of fungal replica...

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

Murphy, Lynea A.; Moore, Tanya; Nesnow, Stephen, E-mail: nesnow.stephen@epa.gov

Propiconazole is a mouse hepatotumorigenic fungicide designed to inhibit CYP51, a key enzyme in the biosynthesis of ergosterol in fungi and is widely used in agriculture to prevent fungal growth. Metabolomic studies in mice revealed that propiconazole increased levels of hepatic cholesterol metabolites and bile acids, and transcriptomic studies revealed that genes within the cholesterol biosynthesis, cholesterol metabolism and bile acid biosyntheses pathways were up-regulated. Hepatic cell proliferation was also increased by propiconazole. AML12 immortalized hepatocytes were used to study propiconazole's effects on cell proliferation focusing on the dysregulation of cholesterol biosynthesis and resulting effects on Ras farnesylation and Erk1/2more » activation as a primary pathway. Mevalonate, a key intermediate in the cholesterol biosynthesis pathway, increases cell proliferation in several cancer cell lines and tumors in vivo and serves as the precursor for isoprenoids (e.g. farnesyl pyrophosphate) which are crucial in the farnesylation of the Ras protein by farnesyl transferase. Farnesylation targets Ras to the cell membrane where it is involved in signal transduction, including the mitogen-activated protein kinase (MAPK) pathway. In our studies, mevalonic acid lactone (MVAL), a source of mevalonic acid, increased cell proliferation in AML12 cells which was reduced by farnesyl transferase inhibitors (L-744,832 or manumycin) or simvastatin, an HMG-CoA reductase inhibitor, indicating that this cell system responded to alterations in the cholesterol biosynthesis pathway. Cell proliferation in AML12 cells was increased by propiconazole which was reversed by co-incubation with L-744,832 or simvastatin. Increasing concentrations of exogenous cholesterol muted the proliferative effects of propiconazole and the inhibitory effects of L-733,832, results ascribed to reduced stimulation of the endogenous cholesterol biosynthesis pathway. Western blot analysis of subcellular fractions from control, MVAL or propiconazole-treated cells revealed increased Ras protein in the cytoplasmic fraction of L-744,832-treated cells, while propiconazole or MVAL reversed these effects. Western blot analysis indicated that phosphorylation of Erk1/2, a protein downstream of Ras, was increased by propiconazole. These data indicate that propiconazole increases cell proliferation by increasing the levels of cholesterol biosynthesis intermediates presumably through a negative feedback mechanism within the pathway, a result of CYP51 inhibition. This feedback mechanism increases Erk1/2 signaling through mevalonate-mediated Ras activation. These results provide an explanation for the observed effects of propiconazole on hepatic cholesterol pathways and on the increased hepatic cell proliferation induced by propiconazole in mice. Highlights: ► Propiconazole increases cell proliferation in AML12 mouse hepatocytes. ► Propiconazole increases Ras farnesylation and alters Ras membrane localization. ► Propiconazole increases Erk1/2 phosphorylation. ► Dysregulation of the cholesterol biosynthesis pathway can explain these results. ► These results can explain similar effects induced by propiconazole in mice.« less

Ornithine: the overlooked molecule in the regulation of polyamine metabolism

Treesearch

Rajtilak Majumdar; Lin Shao; Rakesh Minocha; Stephanie Long; Subhash C. Minocha

2013-01-01

We overexpressed a mouse ornithine decarboxylase gene under the control of a constitutive and an estradiol-inducible promoter in Arabidopsis thaliana to increase our understanding of the regulation of polyamine metabolism. Of particular interest was the role of the substrate ornithine not only in the regulation of polyamine biosynthesis, but also in...

TOXICITY PROFILES IN RATS TREATED WITH TUMORIGENIC AND NONTUMORIGENIC TRIAZOLE CONAZOLE FUNGICIDES: PROPICONAZOLE, TRIADIMEFON, AND MYCLOBUTANIL

EPA Science Inventory

Conazoles are a class of azole based fungicides used in agriculture and as pharmaceutical products. They have a common mode of antifungal action through inhibition of ergosterol biosynthesis. Some members of this class have been shown to be hepatotoxic and will induce mouse hepa...

Molecular adaptations to phosphorus deprivation and comparison with nitrogen deprivation responses in the diatom Phaeodactylum tricornutum.

PubMed

Alipanah, Leila; Winge, Per; Rohloff, Jens; Najafi, Javad; Brembu, Tore; Bones, Atle M

2018-01-01

Phosphorus, an essential element for all living organisms, is a limiting nutrient in many regions of the ocean due to its fast recycling. Changes in phosphate (Pi) availability in aquatic systems affect diatom growth and productivity. We investigated the early adaptive mechanisms in the marine diatom Phaeodactylum tricornutum to P deprivation using a combination of transcriptomics, metabolomics, physiological and biochemical experiments. Our analysis revealed strong induction of gene expression for proteins involved in phosphate acquisition and scavenging, and down-regulation of processes such as photosynthesis, nitrogen assimilation and nucleic acid and ribosome biosynthesis. P deprivation resulted in alterations of carbon allocation through the induction of the pentose phosphate pathway and cytosolic gluconeogenesis, along with repression of the Calvin cycle. Reorganization of cellular lipids was indicated by coordinated induced expression of phospholipases, sulfolipid biosynthesis enzymes and a putative betaine lipid biosynthesis enzyme. A comparative analysis of nitrogen- and phosphorus-deprived P. tricornutum revealed both common and distinct regulation patterns in response to phosphate and nitrate stress. Regulation of central carbon metabolism and amino acid metabolism was similar, whereas unique responses were found in nitrogen assimilation and phosphorus scavenging in nitrogen-deprived and phosphorus-deprived cells, respectively.

Nano titanium dioxide photocatalytic protein tyrosine nitration: A potential hazard of TiO{sub 2} on skin

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

Lu, Naihao; Zhu Zhening; Zhao Xuqi

Protein tyrosine nitration is a prevalent post-translational modification which occurs as a result of oxidative and nitrative stress, it may be directly involved in the onset and/or progression of diseases. Considering the existence of nano titanium dioxide (TiO{sub 2}) in environment and sunscreen products along with the high content of nitrite in sweat, the UV-exposed skin may be a significant target for the photosensitized damage. In this paper, tyrosine nitration of bovine serum albumin (BSA) was initiated in the UV-irradiated reaction mixture containing 0.2-3.0 mg/ml of three commercially nano TiO{sub 2} products and 0.25-1.0 mM NO{sub 2}{sup -}. It wasmore » found that anatase TiO{sub 2} and Degussa P25 TiO{sub 2} showed prominent photocatalytic activity on promoting the formation of protein tyrosine nitration, and the optimum condition for the reaction was around physiological pH. Meanwhile, the photocatalytic effect of rutile on protein tyrosine nitration was subtle. The potential physiological significance of nano TiO{sub 2}-photocatalytic protein nitration was also demonstrated in mouse skin homogenate. Although the relationship between photocatalytic protein tyrosine nitration and chronic cutaneous diseases needs further study, the toxicity of nano TiO{sub 2} to the skin disease should be paid more attention in the production and utilization process.« less

Biosynthesis of mercapturic acids from allyl alcohol, allyl esters and acrolein

PubMed Central

Kaye, Clive M.

1973-01-01

1. 3-Hydroxypropylmercapturic acid, i.e. N-acetyl-S-(3-hydroxypropyl)-l-cysteine, was isolated, as its dicyclohexylammonium salt, from the urine of rats after the subcutaneous injection of each of the following compounds: allyl alcohol, allyl formate, allyl propionate, allyl nitrate, acrolein and S-(3-hydroxypropyl)-l-cysteine. 2. Allylmercapturic acid, i.e. N-acetyl-S-allyl-l-cysteine, was isolated from the urine of rats after the subcutaneous injection of each of the following compounds: triallyl phosphate, sodium allyl sulphate and allyl nitrate. The sulphoxide of allylmercapturic acid was detected in the urine excreted by these rats. 3. 3-Hydroxypropylmercapturic acid was identified by g.l.c. as a metabolite of allyl acetate, allyl stearate, allyl benzoate, diallyl phthalate, allyl nitrite, triallyl phosphate and sodium allyl sulphate. 4. S-(3-Hydroxypropyl)-l-cysteine was detected in the bile of a rat dosed with allyl acetate. PMID:4762754

Extracellular biosynthesis of silver nanoparticles using the fungus Fusarium semitectum

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

Basavaraja, S.; Balaji, S.D.; Department of Chemistry, Gulbarga University, Gulbarga 585106, Karnataka

2008-05-06

Development of environmental friendly procedures for the synthesis of metal nanoparticles through biological processes is evolving into an important branch of nanobiotechnology. In this paper, we report on the use of fungus 'Fusarium semitectum' for the extracellular synthesis of silver nanoparticles from silver nitrate solution (i.e. through the reduction of Ag{sup +} to Ag{sup 0}). Highly stable and crystalline silver nanoparticles are produced in solution by treating the filtrate of the fungus F. semitectum with the aqueous silver nitrate solution. The formations of nanoparticles are understood from the UV-vis and X-ray diffraction studies. Transmission electron microscopy of the silver particlesmore » indicated that they ranged in size from 10 to 60 nm and are mostly spherical in shape. Interestingly the colloidal suspensions of silver nanoparticles are stable for many weeks. Possible medicinal applications of these silver nanoparticles are envisaged.« less

Poplar trees for phytoremediation of high levels of nitrate and applications in bioenergy.

PubMed

Castro-Rodríguez, Vanessa; García-Gutiérrez, Angel; Canales, Javier; Cañas, Rafael A; Kirby, Edward G; Avila, Concepción; Cánovas, Francisco M

2016-01-01

The utilization of high amounts of nitrate fertilizers for crop yield leads to nitrate pollution of ground and surface waters. In this study, we report the assimilation and utilization of nitrate luxuriant levels, 20 times more than the highest N fertilizer application in Europe, by transgenic poplars overexpressing a cytosolic glutamine synthetase (GS1). In comparison with the wild-type controls, transgenic plants grown under high N levels exhibited increased biomass (171.6%) and accumulated higher levels of proteins, chlorophylls and total sugars such as glucose, fructose and sucrose. These plants also exhibited greater nitrogen-use efficiency particularly in young leaves, suggesting that they are able to translocate most of the resources to the above-ground part of the plant to produce biomass. The transgenic poplar transcriptome was greatly affected in response to N availability with 1237 genes differentially regulated in high N, while only 632 genes were differentially expressed in untransformed plants. Many of these genes are essential in the adaptation and response against N excess and include those involved in photosynthesis, cell wall formation and phenylpropanoid biosynthesis. Cellulose production in the transgenic plants was fivefold higher than in control plants, indicating that transgenic poplars represent a potential feedstock for applications in bioenergy. In conclusion, our results show that GS transgenic poplars can be used not only for improving growth and biomass production but also as an important resource for potential phytoremediation of nitrate pollution. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Effects of lanthanum(III) on nitrogen metabolism of soybean seedlings under elevated UV-B radiation.

PubMed

Cao, Rui; Huang, Xiao-hua; Zhou, Qing; Cheng, Xiao-ying

2007-01-01

The hydroponic culture experiments of soybean bean seedlings were conducted to investigate the effect of lanthanum (La) on nitrogen metabolism under two different levels of elevated UV-B radiation (UV-B, 280-320 nm). The whole process of nitrogen metabolism involves uptake and transport of nitrate, nitrate assimilation, ammonium assimilation, amino acid biosynthesis, and protein synthesis. Compared with the control, UV-B radiation with the intensity of low level 0.15 W/m2 and high level 0.45 W/m2 significantly affected the whole nitrogen metabolism in soybean seedlings (p < 0.05). It restricted uptake and transport of NO3(-), inhibited activity of some key nitrogen-metabolism-related enzymes, such as: nitrate reductase (NR) to the nitrate reduction, glutamine systhetase (GS) and glutamine synthase (GOGAT) to the ammonia assimilation, while it increased the content of free amino acids and decreased that of soluble protein as well. The damage effect of high level of UV-B radiation on nitrogen metabolism was greater than that of low level. And UV-B radiation promoted the activity of the anti-adversity enzyme glutamate dehydrogenase (GDH), which reduced the toxicity of excess ammonia in plant. After pretreatment with the optimum concentration of La (20 mg/L), La could increase the activity of NR, GS, GOGAT, and GDH, and ammonia assimilation, but decrease nitrate and ammonia accumulation. In conclusion, La could relieve the damage effect of UV-B radiation on plant by regulating nitrogen metabolism process, and its alleviating effect under low level was better than that under the high one.

Synergistic Interaction of Light Alcohol Administration in the Presence of Mild Iron Overload in a Mouse Model of Liver Injury: Involvement of Triosephosphate Isomerase Nitration and Inactivation

PubMed Central

Gao, Wanxia; Zhao, Jie; Gao, Zhonghong

2017-01-01

It is well known that iron overload promotes alcoholic liver injury, but the doses of iron or alcohol used in studies are usually able to induce liver injury independently. Little attention has been paid to the coexistence of low alcohol consumption and mild iron overload when either of them is insufficient to cause obvious liver damage, although this situation is very common among some people. We studied the interactive effects and the underlining mechanism of mild doses of iron and alcohol on liver injury in a mouse model. Forty eight male Kunming mice were randomly divided into four groups: control, iron (300 mg/kg iron dextran, i.p.), alcohol (2 g/kg/day ethanol for four weeks i.g.), and iron plus alcohol group. After 4 weeks of treatment, mice were sacrificed and blood and livers were collected for biochemical analysis. Protein nitration level in liver tissue was determined by immunoprecipitation and Western blot analysis. Although neither iron overload nor alcohol consumption at our tested doses can cause severe liver injury, it was found that co-administration of the same doses of alcohol and iron resulted in liver injury and hepatic dysfunction, accompanied with elevated ratio of NADH/NAD+, reduced antioxidant ability, increased oxidative stress, and subsequent elevated protein nitration level. Further study revealed that triosephosphate isomerase, an important glycolytic enzyme, was one of the targets to be oxidized and nitrated, which was responsible for its inactivation. These data indicate that even under low alcohol intake, a certain amount of iron overload can cause significant liver oxidative damage, and the modification of triosephosphate isomerasemight be the important underlining mechanism of hepatic dysfunction. PMID:28103293

Effects of electromagnetic pulse irradiation on the mouse blood-testicle barrier.

PubMed

Hou, Wu-Gang; Zhao, Jie; Li, Zhen; Li, Wei; Li, Teng; Xiong, Li-Ze; Zhang, Yuan-Qiang

2012-07-01

To investigate the effects of electromagnetic pulse irradiation on the mouse blood-testicle barrier (BTB) and spermatogenesis. After whole body irradiation with 400 kV/m electromagnetic pulse irradiation, the mouse testicles and BTB permeability were observed using hematoxylin-eosin, Evans blue, and lanthanum nitrate as tracers. The expression of the BTB tight junction protein occludin was examined using real-time polymerase chain reaction and Western blotting. At 1, 7, and 14 days after irradiation, the BTB structure was damaged, the BTB permeability was significantly increased, numerous apoptotic or necrotic spermatogenic cells were found in the lumen, and the mRNA and protein expression levels of occludin were markedly decreased. The BTB structure and occludin expression levels had gradually recovered by 21 and 28 days after irradiation. Electromagnetic pulse irradiation damaged the structure and function of mouse BTB, resulting in apoptosis or necrosis of the spermatogenic cells. Copyright © 2012 Elsevier Inc. All rights reserved.

Proteomic analysis of the action of the Mycobacterium ulcerans toxin mycolactone: targeting host cells cytoskeleton and collagen.

PubMed

Gama, José B; Ohlmeier, Steffen; Martins, Teresa G; Fraga, Alexandra G; Sampaio-Marques, Belém; Carvalho, Maria A; Proença, Fernanda; Silva, Manuel T; Pedrosa, Jorge; Ludovico, Paula

2014-08-01

Buruli ulcer (BU) is a neglected tropical disease caused by Mycobacterium ulcerans. The tissue damage characteristic of BU lesions is known to be driven by the secretion of the potent lipidic exotoxin mycolactone. However, the molecular action of mycolactone on host cell biology mediating cytopathogenesis is not fully understood. Here we applied two-dimensional electrophoresis (2-DE) to identify the mechanisms of mycolactone's cellular action in the L929 mouse fibroblast proteome. This revealed 20 changed spots corresponding to 18 proteins which were clustered mainly into cytoskeleton-related proteins (Dync1i2, Cfl1, Crmp2, Actg1, Stmn1) and collagen biosynthesis enzymes (Plod1, Plod3, P4ha1). In line with cytoskeleton conformational disarrangements that are observed by immunofluorescence, we found several regulators and constituents of both actin- and tubulin-cytoskeleton affected upon exposure to the toxin, providing a novel molecular basis for the effect of mycolactone. Consistent with these cytoskeleton-related alterations, accumulation of autophagosomes as well as an increased protein ubiquitination were observed in mycolactone-treated cells. In vivo analyses in a BU mouse model revealed mycolactone-dependent structural changes in collagen upon infection with M. ulcerans, associated with the reduction of dermal collagen content, which is in line with our proteomic finding of mycolactone-induced down-regulation of several collagen biosynthesis enzymes. Our results unveil the mechanisms of mycolactone-induced molecular cytopathogenesis on exposed host cells, with the toxin compromising cell structure and homeostasis by inducing cytoskeleton alterations, as well as disrupting tissue structure, by impairing the extracellular matrix biosynthesis.

Asymptomatic Carriage of Group A Streptococcus Is Associated with Elimination of Capsule Production

PubMed Central

Jewell, Brittany E.; Olsen, Randall J.; Shelburne, Samuel A.; Fittipaldi, Nahuel; Beres, Stephen B.; Musser, James M.

2014-01-01

Humans commonly carry pathogenic bacteria asymptomatically, but despite decades of study, the underlying molecular contributors remain poorly understood. Here, we show that a group A streptococcus carriage strain contains a frameshift mutation in the hasA gene resulting in loss of hyaluronic acid capsule biosynthesis. This mutation was repaired by allelic replacement, resulting in restoration of capsule production in the isogenic derivative strain. The “repaired” isogenic strain was significantly more virulent than the carriage strain in a mouse model of necrotizing fasciitis and had enhanced growth ex vivo in human blood. Importantly, the repaired isogenic strain colonized the mouse oropharynx with significantly greater bacterial burden and had significantly reduced ability to internalize into cultured epithelial cells than the acapsular carriage strain. We conducted full-genome sequencing of 81 strains cultured serially from 19 epidemiologically unrelated human subjects and discovered the common theme that mutations negatively affecting capsule biosynthesis arise in vivo in the has operon. The significantly decreased capsule production is a key factor contributing to the molecular détente between pathogen and host. Our discoveries suggest a general model for bacterial pathogens in which mutations that downregulate or ablate virulence factor production contribute to carriage. PMID:25024363

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

Kawagoe, Kazuyoshi; Takeda, Junji; Kinoshita, Taroh

Many membrane proteins are anchored to the cell membrane by glycosylphosphatidylinositol (GPI). The core structure and biosynthesis of the GPI anchor are well conserved in eukaryote cells. We previously cloned a human PIGA gene that participates in GPI anchor biosynthesis. We have now cloned complementary and genomic DNA of Pig-a, the murine homologue of PIGA, and compared its function and gene structure with those of PIGA. The deduced amino acid sequence of mouse PIG-A is 88% identical with that of human PIG-A. Transfection of Pig-a cDNA complemented the defects of both a PIG-A-deficient murine cell line and a PIG-A-deficient humanmore » cell line, demonstrating that functions of mouse and human PIG-A are conserved. Like human PIGA, the chromosomal Pig-a gene has six exons and spans approximately 16 kb. Moreover, Pig-a was mapped to X-F3/4, which is syntenic to human Xp22.1, where PIGA is located. Thus, murine Pig-a provides a good animal model to study paroxysmal nocturnal hemoglobinuria, a disease caused by a somatic mutation of PIGA. Database analysis demonstrated that a yeast gene, SPT14, is homologous to Pig-a and PIGA and that these genes are members of a glycosyltransferase gene family.« less

Understanding the response of Desulfovibrio desulfuricans ATCC 27774 to the electron acceptors nitrate and sulfate - biosynthetic costs modulate substrate selection.

PubMed

Sousa, Joana R; Silveira, Célia M; Fontes, Pedro; Roma-Rodrigues, Catarina; Fernandes, Alexandra R; Van Driessche, Gonzalez; Devreese, Bart; Moura, Isabel; Moura, José J G; Almeida, M Gabriela

2017-11-01

Sulfate-reducing bacteria (SRB) are a diverse group of anaerobic microorganisms that obtain their energy from dissimilatory sulfate reduction. Some SRB species have high respiratory versatility due to the possible use of alternative electron acceptors. A good example is Desulfovibrio desulfuricans ATCC 27774, which grows in the presence of nitrate (end product: ammonium) with higher rates and yields to those observed in sulfate containing medium (end product: sulfide). In this work, the mechanisms supporting the respiratory versatility of D. desulfuricans were unraveled through the analysis of the proteome of the bacterium under different experimental conditions. The most remarkable difference in the two-dimensional gel electrophoresis maps is the high number of spots exclusively represented in the nitrate medium. Most of the proteins with increase abundance are involved in the energy metabolism and the biosynthesis of amino acids (or proteins), especially those participating in ammonium assimilation processes. qPCR analysis performed during different stages of the bacterium's growth showed that the genes involved in nitrate and nitrite reduction (napA and nrfA, respectively) have different expressions profiles: while napA did not vary significantly, nrfA was highly expressed at a 6h time point. Nitrite levels measured along the growth curve revealed a peak at 3h. Thus, the initial consumption of nitrate and concomitant production of nitrite must induce nrfA expression. The activation of alternative mechanisms for energy production, aside several N-assimilation metabolisms and detoxification processes, solves potential survival problems in adapting to different environments and contributes to higher bacterial growth rates. Copyright © 2017 Elsevier B.V. All rights reserved.

Correlation between citric acid and nitrate metabolisms during CAM cycle in the atmospheric bromeliad Tillandsia pohliana.

PubMed

Freschi, Luciano; Rodrigues, Maria Aurineide; Tiné, Marco Aurélio Silva; Mercier, Helenice

2010-12-15

Crassulacean acid metabolism (CAM) confers crucial adaptations for plants living under frequent environmental stresses. A wide metabolic plasticity can be found among CAM species regarding the type of storage carbohydrate, organic acid accumulated at night and decarboxylating system. Consequently, many aspects of the CAM pathway control are still elusive while the impact of this photosynthetic adaptation on nitrogen metabolism has remained largely unexplored. In this study, we investigated a possible link between the CAM cycle and the nitrogen assimilation in the atmospheric bromeliad Tillandsia pohliana by simultaneously characterizing the diel changes in key enzyme activities and metabolite levels of both organic acid and nitrate metabolisms. The results revealed that T. pohliana performed a typical CAM cycle in which phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase phosphorylation seemed to play a crucial role to avoid futile cycles of carboxylation and decarboxylation. Unlike all other bromeliads previously investigated, almost equimolar concentrations of malate and citrate were accumulated at night. Moreover, a marked nocturnal depletion in the starch reservoirs and an atypical pattern of nitrate reduction restricted to the nighttime were also observed. Since reduction and assimilation of nitrate requires a massive supply of reducing power and energy and considering that T. pohliana lives overexposed to the sunlight, we hypothesize that citrate decarboxylation might be an accessory mechanism to increase internal CO₂ concentration during the day while its biosynthesis could provide NADH and ATP for nocturnal assimilation of nitrate. Therefore, besides delivering photoprotection during the day, citrate might represent a key component connecting both CAM pathway and nitrogen metabolism in T. pohliana; a scenario that certainly deserves further study not only in this species but also in other CAM plants that nocturnally accumulate citrate. Copyright © 2010 Elsevier GmbH. All rights reserved.

Nitrate-Dependent Iron Oxidation: A Potential Mars Metabolism

PubMed Central

Price, Alex; Pearson, Victoria K.; Schwenzer, Susanne P.; Miot, Jennyfer; Olsson-Francis, Karen

2018-01-01

This work considers the hypothetical viability of microbial nitrate-dependent Fe2+ oxidation (NDFO) for supporting simple life in the context of the early Mars environment. This draws on knowledge built up over several decades of remote and in situ observation, as well as recent discoveries that have shaped current understanding of early Mars. Our current understanding is that certain early martian environments fulfill several of the key requirements for microbes with NDFO metabolism. First, abundant Fe2+ has been identified on Mars and provides evidence of an accessible electron donor; evidence of anoxia suggests that abiotic Fe2+ oxidation by molecular oxygen would not have interfered and competed with microbial iron metabolism in these environments. Second, nitrate, which can be used by some iron oxidizing microorganisms as an electron acceptor, has also been confirmed in modern aeolian and ancient sediment deposits on Mars. In addition to redox substrates, reservoirs of both organic and inorganic carbon are available for biosynthesis, and geochemical evidence suggests that lacustrine systems during the hydrologically active Noachian period (4.1–3.7 Ga) match the circumneutral pH requirements of nitrate-dependent iron-oxidizing microorganisms. As well as potentially acting as a primary producer in early martian lakes and fluvial systems, the light-independent nature of NDFO suggests that such microbes could have persisted in sub-surface aquifers long after the desiccation of the surface, provided that adequate carbon and nitrates sources were prevalent. Traces of NDFO microorganisms may be preserved in the rock record by biomineralization and cellular encrustation in zones of high Fe2+ concentrations. These processes could produce morphological biosignatures, preserve distinctive Fe-isotope variation patterns, and enhance preservation of biological organic compounds. Such biosignatures could be detectable by future missions to Mars with appropriate instrumentation. PMID:29616015

Nitrate-Dependent Iron Oxidation: A Potential Mars Metabolism.

PubMed

Price, Alex; Pearson, Victoria K; Schwenzer, Susanne P; Miot, Jennyfer; Olsson-Francis, Karen

2018-01-01

This work considers the hypothetical viability of microbial nitrate-dependent Fe 2+ oxidation (NDFO) for supporting simple life in the context of the early Mars environment. This draws on knowledge built up over several decades of remote and in situ observation, as well as recent discoveries that have shaped current understanding of early Mars. Our current understanding is that certain early martian environments fulfill several of the key requirements for microbes with NDFO metabolism. First, abundant Fe 2+ has been identified on Mars and provides evidence of an accessible electron donor; evidence of anoxia suggests that abiotic Fe 2+ oxidation by molecular oxygen would not have interfered and competed with microbial iron metabolism in these environments. Second, nitrate, which can be used by some iron oxidizing microorganisms as an electron acceptor, has also been confirmed in modern aeolian and ancient sediment deposits on Mars. In addition to redox substrates, reservoirs of both organic and inorganic carbon are available for biosynthesis, and geochemical evidence suggests that lacustrine systems during the hydrologically active Noachian period (4.1-3.7 Ga) match the circumneutral pH requirements of nitrate-dependent iron-oxidizing microorganisms. As well as potentially acting as a primary producer in early martian lakes and fluvial systems, the light-independent nature of NDFO suggests that such microbes could have persisted in sub-surface aquifers long after the desiccation of the surface, provided that adequate carbon and nitrates sources were prevalent. Traces of NDFO microorganisms may be preserved in the rock record by biomineralization and cellular encrustation in zones of high Fe 2+ concentrations. These processes could produce morphological biosignatures, preserve distinctive Fe-isotope variation patterns, and enhance preservation of biological organic compounds. Such biosignatures could be detectable by future missions to Mars with appropriate instrumentation.

Gene expression analysis of murine cells producing amphotropic mouse leukaemia virus at a cultivation temperature of 32 and 37 degrees C.

PubMed

Beer, Christiane; Buhr, Petra; Hahn, Heidi; Laubner, Daniela; Wirth, Manfred

2003-07-01

Cultivation of retrovirus packaging cells at 32 degrees C represents a common procedure to achieve high titres in mouse retrovirus production. Gene expression profiling of mouse NIH 3T3 cells producing amphotropic mouse leukaemia virus 4070A revealed that 10 % of the 1176 cellular genes investigated were regulated by temperature shift (37/32 degrees C), while 5 % were affected by retrovirus infection. Strikingly, retrovirus production at 32 degrees C activated the cholesterol biosynthesis/transport pathway and caused an increase in plasma membrane cholesterol levels. Furthermore, these conditions resulted in transcriptional activation of smoothened (smo), patched (ptc) and gli-1; Smo, Ptc and Gli-1, as well as cholesterol, are components of the Sonic hedgehog (Shh) signalling pathway, which directs pattern formation, diversification and tumourigenesis in mammalian cells. These findings suggest a link between cultivation at 32 degrees C, production of MLV-A and the Shh signalling pathway.

Green synthesis and characterization of silver nanoparticles by leaf extracts of Cycas circinalis, Ficus amplissima, Commelina benghalensis and Lippia nodiflora

NASA Astrophysics Data System (ADS)

Johnson, I.; Prabu, H. Joy

2015-01-01

Biosynthesis of nanoparticles is a kind of bottom-up approach where the main reaction occurring is reduction. Since silver nanoparticles (AgNPs) have been used for infection prevention in medical field, it is more relevant to reduce their size using ancient Indian herbal plants. This method is good in anti-microbial efficiency against bacteria, viruses and other microorganisms and hence clearly enhances the medicinal usage of AgNPs. This type of green biosynthesis of nanoparticles has received increasing attention due to the growing need to develop safe, cost-effective and environmental-friendly technologies for nano-materials synthesis. In the process of synthesizing AgNPs, we observed a rapid reduction of silver ions leading to the formation of stable crystalline AgNPs in the solution. Plant extracts from Cycas circinalis, Ficus amplissima, Commelina benghalensis and Lippia nodiflora were used for the synthesis of AgNPs from silver nitrate solution. AgNPs were characterized by different techniques.

Structure-Based Discovery of Nonpeptide Allatostatin Analogues for Pest Control.

PubMed

Huang, Shan-Shan; Chen, Shan-Shan; Zhang, Hong-Ling; Yang, Han; Yang, Hui-Juan; Ren, Yu-Jie; Kai, Zhen-Peng

2018-04-11

FGLamide allatostatins (ASTs) are regarded as possible insecticide candidates, although their lack of in vivo effects, rapid degradation, poor water solubility, and high production costs preclude their practical use in pest control. In contrast to previous research, the C-terminal tripeptide (FGLa) was selected as the lead compound in this study. Five nonpeptide AST analogues (2-amino-1-[3-oxo-3-(substituted-anilino)propyl]pyridinium nitrate derivatives) were designed on the basis of the structure-activity relationship and docking results of FGLa. All of the nonpeptide analogues (S1-S5) were more potent against juvenile-hormone (JH) biosynthesis than the lead compound. They significantly inhibited the biosynthesis of JH in vivo following injection. A pest-control application demonstrated that S1 and S3 have larvicidal effects following oral administration (the IC 50 values were 0.020 and 0.0016 mg/g, respectively). The good oral toxicities and excellent water solubilities of S1 and S3 suggest that they have considerable potential as insecticides for pest management.

Protopine production by fumaria cell suspension cultures: effect of light.

PubMed

Georgieva, Lidiya; Ivanov, Ivan; Marchev, Andrey; Aneva, Ina; Denev, Panteley; Georgiev, Vasil; Pavlov, Atanas

2015-05-01

Protopine biosynthesis in Fumaria rostellata and Fumaria officinalis cell suspensions was investigated. For the first time, we reported for calli and cell suspensions obtained from F. rostellata and F. officinalis. Callus induction was initiated on a Murashige and Skoog medium, supplemented with sucrose and various concentrations of plant growth regulators: 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine (BAP). The best morphological characteristics, growth behavior, and protopine biosynthesis were observed for two callus lines (5FRL14 and 12FOL1) cultivated under submerged conditions, at low concentration of 2,4-D (0.2 and 0.5 mg/L) and higher concentration of BAP (2.0 and 3.0 mg/L). The maximal yield of protopine was accumulated from cell suspension of F. rostellata (line 5FRL14) cultivated under illumination-49.6 mg/L. Time courses of utilization of sucrose, ammonium, nitrate, and phosphate ions in cultural liquid and acetylcholinesterase inhibitory activity of alkaloid extracts of studied suspensions are also presented.

Chitosan nanocomposite films based on Ag-NP and Au-NP biosynthesis by Bacillus Subtilis as packaging materials.

PubMed

Youssef, Ahmed M; Abdel-Aziz, Mohamed S; El-Sayed, Samah M

2014-08-01

Chitosan-silver (CS-Ag) and Chitosan-gold (CS-Au) nanocomposites films were synthesized by a simple chemical method. A local bacterial isolate identified as Bacillus subtilis ss subtilis was found to be capable to synthesize both silver nanoparticles (Ag-NP) and gold nanoparticles (Au-NP) from silver nitrate (AgNO3) and chloroauric acid (AuCl(4-)) solutions, respectively. The biosynthesis of both Ag-NP and Au-NP characterize using UV/vis spectroscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM) and X-ray diffraction (XRD), and then added to chitosan by different ratios (0.5, 1 and 2%). The prepared chitosan nanocomposites films were characterize using UV, XRD, SEM and TEM. Moreover, the antibacterial activity of the prepared films was evaluated against gram positive (Staphylococcus aureus) and gram negative bacteria (Pseudomonas aerugenosa), fungi (Aspergillus niger) and yeast (Candida albicans). Therefore, these materials can be potential used as antimicrobial agents in packaging applications. Copyright © 2014 Elsevier B.V. All rights reserved.

Serum resistance, gallium nitrate tolerance and extrapulmonary dissemination are linked to heme consumption in a bacteremic strain of Acinetobacter baumannii.

PubMed

de Léséleuc, Louis; Harris, Greg; KuoLee, Rhonda; Xu, H Howard; Chen, Wangxue

2014-05-01

Bacteremia caused by Acinetobacter baumannii is a highly lethal complication of hospital-acquired pneumonia. In the present study, we investigated the serum resistance, gallium nitrate tolerance and heme consumption of A. baumannii strain LAC-4 which was recently reported to display high virulence in a mouse pneumonia model with extrapulmonary dissemination leading to fatal bacteremia. This strain showed enhanced growth in mouse and fetal bovine serum that was independent of complement and was not observed with regular growth media. The LAC-4 strain was found to possess a high tolerance to gallium nitrate (GaN), whereas serum synergized with GaN in inhibiting A. baumannii strain ATCC 17978. We found that LAC-4 contains a heme oxygenase gene and expresses a highly efficient heme consumption system. This system can be fully blocked in vitro and in vivo by gallium protoporphyrin IX (GaPPIX). Inhibition of heme consumption by GaPPIX completely abrogated the growth advantage of LAC-4 in serum as well as its tolerance to GaN. More importantly, GaPPIX treatment of mice intranasally infected with LAC-4 prevented extrapulmonary dissemination and death. Thus, we propose that heme provides an additional source of iron for LAC-4 to bypass iron restriction caused by serum transferrin, lactoferrin or free gallium salts. Heme consumption systems in A. baumannii may constitute major virulence factors for lethal bacteremic isolates. Copyright © 2014 Crown Copyright and Elsevier Inc. Published by Elsevier GmbH.. All rights reserved.

Decreased steroid hormone synthesis from inorganic nitrite and nitrate: studies in vitro and in vivo.

PubMed

Panesar, N S; Chan, K W

2000-12-15

Nitrites and nitrates are consumed nonchalantly in diet. Organic nitrates are also used as vasodilators in angina pectoris, but the therapy is associated with tolerance whose mechanism remains elusive. Previously, we found inorganic nitrate inhibited steroidogenesis in vitro. Because adrenocorticoids regulate water and electrolyte metabolism, tolerance may ensue from steroid deficiency. We have studied the effects of nitrite and nitrate on in vitro synthesis and in vivo blood levels of steroid hormones. In vitro, nitrite was more potent than nitrate in inhibiting human chorionic gonadotropin (hCG)-stimulated androgen synthesis by Mouse Leydig Tumor cells. At concentrations above 42 mM, nitrite completely inhibited androgen synthesis, and, unlike nitrate, the inhibition was irreversible by increasing hCG concentration. The cAMP production remained intact but reduced with both ions. The nitric oxide (NO) scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxy-3-oxide (c-PTIO) significantly increased hCG- or cAMP-stimulated androgen synthesis in all buffers, suggesting that NO is a chemical species directly involved in the nitrite/nitrate-induced inhibition. This is further supported by c-PTIO countering the inhibitory action of methylene blue on androgen synthesis. Rats given distilled water containing 50 mg/L NaNO(2) or NaNO(3) for 4 weeks drank significantly less daily. At the end, their blood corticosterone and testosterone levels were significantly decreased. The adrenocortical histology showed bigger lipid droplets, which are pathogonomic of impaired steroidogenesis. Nitrite and nitrate are metabolized to NO, which binds heme in cytochrome P450 enzymes, thereby inhibiting steroidogenesis. Therapeutic nitrates likewise may decrease adrenal (and gonadal) steroidogenesis. Cortisol deficiency would impair water excretion causing volume expansion, and aldosterone deficiency would cause sodium loss and raised renin. Paradoxically, volume expansion without sodium retention and raised renin has all been reported in tolerance. Copyright 2000 Academic Press.

Biosynthesis of silver nanoparticles using Alternanthera sessilis (Linn.) extract and their antimicrobial, antioxidant activities.

PubMed

Niraimathi, K L; Sudha, V; Lavanya, R; Brindha, P

2013-02-01

The present work focuses the use of the aqueous extract of Alternanthera sessilis Linn. (Amaranthaceae) in producing silver nanoparticles (AgNPs) from silver nitrate aqueous. Phytochemical analysis of the extract revealed the presence of alkaloid, tannins, ascorbic acid, carbohydrates and proteins and they serve as effective reducing and capping agents for converting silver nitrate into nanoparticles. The synthesized silver nanoparticles (AgNPs) were also tested for proteins and ascorbic acid. Its pH was also determined (5.63). The AgNPs obtained was characterized by UV-vis spectroscopy, FT-IR spectroscopy, SEM, Zeta sizer and TG-DSC. SEM images which revealed the presence of various shapes and sizes. FT-IR spectrum showed the AgNPs having a coating of proteins indicating a dual role of bio-molecules responsible for capping and efficient stabilization of the silver nanoparticles. Presence of impurities and melting point profile were screened by TG-DSC analyzer. AgNPs were synthesized from the silver nitrate through the reducing power of ascorbic acid present in A. sessilis leaves. In this study, we also investigated antimicrobial and antioxidant activity of green synthesized AgNPs. The antimicrobial activity is investigated by Bauer et al.'s method. Antioxidant activity was done by DPPH method. Copyright © 2012 Elsevier B.V. All rights reserved.

In Vivo-Induced Genes in Pseudomonas aeruginosa

PubMed Central

Handfield, Martin; Lehoux, Dario E.; Sanschagrin, François; Mahan, Michael J.; Woods, Donald E.; Levesque, Roger C.

2000-01-01

In vivo expression technology was used for testing Pseudomonas aeruginosa in the rat lung model of chronic infection and in a mouse model of systemic infection. Three of the eight ivi proteins found showed sequence identity to known virulence factors involved in iron acquisition via an open reading frame (called pvdI) implicated in pyoverdine biosynthesis, membrane biogenesis (FtsY), and adhesion (Hag2). PMID:10722644

High efficiency capillary column-gas chromatography mass spectrometry: analysis of the lipoxygenase pathway in eukaryot cells.

PubMed

Rabinovitch-Chable, H; Durand, J; Aldigier, J C; Chebroux, P; Gualde, N; Beneytout, J L; Rigaud, M

1984-01-01

Lipoxygenases are ubiquitous enzymes able to oxygenate polyunsaturated fatty acids. This metabolic pathway leads to hydroperoxides, hydroxyepoxyene compounds and leukotrienes. Using high performance gas chromatography prior to mass spectrometry, we studied the activity of the lipoxygenases from mouse peritoneal macrophages. Further studies on mechanism of biosynthesis of hydroxyepoxyene compounds were successfully carried out using 18O2 labelled precursors.

Purification of simulated waste water using green synthesized silver nanoparticles of Piliostigma thonningii aqueous leave extract

NASA Astrophysics Data System (ADS)

Shittu, K. O.; Ihebunna, O.

2017-12-01

Synthesis of nanoparticles from various biological systems has been reported, but among all such systems, biosynthesis of nanoparticles from plants is considered the most suitable method. The use of plant material not only makes the process eco-friendly, but also the abundance makes it more economical. The aim of this study was to biologically synthesize silver nanoparticle using Piliostigma thonningii aqueous leaf extract and applied in the purification of laboratory stimulated waste with optimization using the different conditions of silver nanoparticle production such as time, temperature, pH, concentration of silver nitrate and volume of the aqueous extract. The biosynthesized silver nanoparticles were characterized by UV-visible spectrophotometry, nanosizer, energy dispersive x-ray analysis (EDX), transmission electron microscopy (TEM) and Fourier transform infrared (FTIR) spectroscopy. The time intervals for the reaction with aqueous silver nitrate solution shows an increase in the absorbance with time and became constant giving a maximum absorbance at 415 nm at 60 min of incubation. The pH of 6.5, temperature 65 °C, 1.25 mM of silver nitrate and 5 ml of plant extract was the best condition with maximum absorbance. The results from nanosizer, UV-vis and TEM suggested the biosynthesis silver nanoparticle to be spherical ranging from 50 nm to 114 nm. The EDX confirmed the elemental synthesis of silver at 2.60 keV and FTIR suggested the capping agent to be hydroxyl (OH) group with -C=C stretching vibrations. The synthesized silver nanoparticle also shows heavy metal removal activity in laboratory simulated waste water. The safety toxicity studies show no significant difference between the orally administered silver nanoparticles treated water group and control group, while the histopathological studies show well preserved hepatic architecture for the orally administered silver nanoparticle treated waste water group when compared with the control group. Therefore, it can be concluded that the biosynthesized silver nanoparticles have efficient ability in heavy metal removal without sub chronic adverse effects in experimental rats.

A novel role for antizyme inhibitor 2 as a regulator of serotonin and histamine biosynthesis and content in mouse mast cells.

PubMed

Acosta-Andrade, Carlos; Lambertos, Ana; Urdiales, José L; Sánchez-Jiménez, Francisca; Peñafiel, Rafael; Fajardo, Ignacio

2016-10-01

Antizymes and antizyme inhibitors are key regulatory proteins of polyamine levels by affecting ornithine decarboxylase and polyamine uptake. Our previous studies indicated a metabolic interplay among polyamines, histamine and serotonin in mast cells, and demonstrated that polyamines are present in mast cell secretory granules, being important for histamine storage and serotonin levels. Recently, the novel antizyme inhibitor-2 (AZIN2) was proposed as a local regulator of polyamine biosynthesis in association with mast cell serotonin-containing granules. To gain insight into the role of AZIN2 in the biosynthesis and storage of serotonin and histamine, we have generated bone marrow derived mast cells (BMMCs) from both wild-type and transgenic Azin2 hypomorphic mice, and have analyzed polyamines, serotonin and histamine contents, and some elements of their metabolisms. Azin2 hypomorphic BMMCs did not show major mast cell phenotypic alterations as judged by morphology and specific mast cell proteases. However, compared to wild-type controls, these cells showed reduced spermidine and spermine levels, and diminished growth rate. Serotonin levels were also reduced, whereas histamine levels tended to increase. Accordingly, tryptophan hydroxylase-1 (TPH1; the key enzyme for serotonin biosynthesis) mRNA expression and protein levels were reduced, whereas histidine decarboxylase (the enzyme responsible for histamine biosynthesis) enzymatic activity was increased. Furthermore, microphtalmia-associated transcription factor, an element involved in the regulation of Tph1 expression, was reduced. Taken together, our results show, for the first time, an element of polyamine metabolism -AZIN2-, so far described as exclusively devoted to the control of polyamine concentrations, involved in regulating the biosynthesis and content of other amines like serotonin and histamine.

Embryonic lung morphogenesis in organ culture: experimental evidence for a proteoglycan function in the extracellular matrix

NASA Technical Reports Server (NTRS)

Spooner, B. S.; Bassett, K. E.; Spooner, B. S. Jr

1993-01-01

The lung rudiment, isolated from mid-gestation (11 day) mouse embryos, can undergo morphogenesis in organ culture. Observation of living rudiments, in culture, reveals both growth and ongoing bronchiolar branching activity. To detect proteoglycan (PG) biosynthesis, and deposition in the extracellular matrix, rudiments were metabolically labeled with radioactive sulfate, then fixed, embedded, sectioned and processed for autoradiography. The sulfated glycosaminoglycan (GAG) types, composing the carbohydrate component of the proteoglycans, were evaluated by selective GAG degradative approaches that showed chondroitin sulfate PG principally associated with the interstitial matrix, and heparan sulfate PG principally associated with the basement membrane. Experiments using the proteoglycan biosynthesis disrupter, beta-xyloside, suggest that when chondroitin sulfate PG deposition into the ECM is perturbed, branching morphogenesis is compromised.

Observations on gas exchange and element recycle within a gas-closed algal-mouse system

NASA Technical Reports Server (NTRS)

Smernoff, D. T.; Wharton, R. A., Jr.; Averner, M. M.

1986-01-01

Life support systems based on bioregeneration rely on the control and manipulation of organisms. Algae are potentially useful for a variety of Closed Ecological Life Support System (CELSS) functions including the revitalization of atmospheres, production of food and for nitrogen fixation. The results of experiments conducted with a gas-closed algal-mouse system designed to investigate gas exchange phenomena under varying algal environmental conditions, and the ability of algae to utilize oxidized mouse solid waste are reported. Inherent instabilities exist between the uptake and release of carbon dioxide (CO2) and oxygen (O2) by the mouse and algae in a gas-closed system. Variations in light intensity and cell density alter the photosynthetic rate of the algae and enable short-term steady-state concentrations of atmospheric CO2 and O2. Different nitrogen sources (urea and nitrate) result in different algal assimilatory quotients (AQ). Combinations of photosynthetic rate and AQ ratio manipulations were examined for their potential in stabilizing atmospheric gas concentrations in the gas-closed algal-mouse system.

Cytoprotective function of heme oxygenase 1 induced by a nitrated cyclic nucleotide formed during murine salmonellosis.

PubMed

Zaki, Mohammad Hasan; Fujii, Shigemoto; Okamoto, Tatsuya; Islam, Sabrina; Khan, Shahzada; Ahmed, Khandaker Ahtesham; Sawa, Tomohiro; Akaike, Takaaki

2009-03-15

Signaling mechanisms of NO-mediated host defense are yet to be elucidated. In this study, we report a unique signal pathway for cytoprotection during Salmonella infection that involves heme oxygenase 1 (HO-1) induced by a nitrated cyclic nucleotide, 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP). Wild-type C57BL/6 mice and C57BL/6 mice lacking inducible NO synthase (iNOS) were infected with Salmonella enterica serovar Typhimurium LT2. HO-1 was markedly up-regulated during the infection, the level being significantly higher in wild-type mice than in iNOS-deficient mice. HO-1 up-regulation was associated with 8-nitro-cGMP formation detected immunohistochemically in Salmonella-infected mouse liver and peritoneal macrophages. 8-Nitro-cGMP either exogenously added or formed endogenously induced HO-1 in cultured macrophages infected with Salmonella. HO-1 inhibition by polyethylene glycol-conjugated zinc-protoporphyrin IX impaired intracellular killing of bacteria in mouse liver and in both RAW 264 cells and peritoneal macrophages. Infection-associated apoptosis was also markedly increased in polyethylene glycol-conjugated zinc-protoporphyrin IX-treated mouse liver cells and cultured macrophages. This effect of HO-1 inhibition was further confirmed by using HO-1 short interfering RNA in peritoneal macrophages. Our results suggest that HO-1 induced by NO-mediated 8-nitro-cGMP formation contributes, via its potent cytoprotective function, to host defense during murine salmonellosis.

Nutritional control of antibiotic production by Streptomyces platensis MA7327: importance of l-aspartic acid.

PubMed

Falzone, Maria; Crespo, Emmanuel; Jones, Klarissa; Khan, Gulaba; Korn, Victoria L; Patel, Amreen; Patel, Mira; Patel, Krishnaben; Perkins, Carrie; Siddiqui, Sana; Stenger, Drew; Yu, Eileen; Gelber, Michael; Scheffler, Robert; Nayda, Vasyl; Ravin, Ariela; Komal, Ronica; Rudolf, Jeffrey D; Shen, Ben; Gullo, Vincent; Demain, Arnold L

2017-07-01

Streptomyces platensis MA7327 is a bacterium producing interesting antibiotics, which act by the novel mechanism of inhibiting fatty acid biosynthesis. The antibiotics produced by this actinomycete are platensimycin and platencin plus some minor related antibiotics. Platensimycin and platencin have activity against antibiotic-resistant bacteria such as methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus; they also lack toxicity in animal models. Platensimycin also has activity against diabetes in a mouse model. We have been interested in studying the effects of primary metabolites on production of these antibiotics in our chemically defined production medium. In the present work, we tested 32 primary metabolites for their effect. They included 20 amino acids, 7 vitamins and 5 nucleic acid derivatives. Of these, only l-aspartic acid showed stimulation of antibiotic production. We conclude that the stimulatory effect of aspartic acid is due to its role as a precursor involved in the biosynthesis of aspartate-4-semialdehyde, which is the starting point for the biosynthesis of the 3-amino-2,4-dihydroxy benzoic acid portion of the platensimycin molecule.

Nutritional control of antibiotic production by Streptomyces platensis MA7327: importance of L-aspartic acid

PubMed Central

Falzone, Maria; Crespo, Emmanuel; Jones, Klarissa; Khan, Gulaba; Korn, Victoria L; Patel, Amreen; Patel, Mira; Patel, Krishnaben; Perkins, Carrie; Siddiqui, Sana; Stenger, Drew; Yu, Eileen; Gelber, Michael; Scheffler, Robert; Nayda, Vasyl; Ravin, Ariela; Komal, Ronica; Rudolf, Jeffrey D; Shen, Ben; Gullo, Vincent; Demain, Arnold L

2017-01-01

Streptomyces platensis MA7327 is a bacterium producing interesting antibiotics, which act by the novel mechanism of inhibiting fatty acid biosynthesis. The antibiotics produced by this actinomycete are platensimycin and platencin plus some minor related antibiotics. Platensimycin and platencin have activity against antibiotic-resistant bacteria such as methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus; they also lack toxicity in animal models. Platensimycin also has activity against diabetes in a mouse model. We have been interested in studying the effects of primary metabolites on production of these antibiotics in our chemically defined production medium. In the present work, we tested 32 primary metabolites for their effect. They included 20 amino acids, 7 vitamins and 5 nucleic acid derivatives. Of these, only L-aspartic acid showed stimulation of antibiotic production. We conclude that the stimulatory effect of aspartic acid is due to its role as a precursor involved in the biosynthesis of aspartate-4-semialdehyde, which is the starting point for the biosynthesis of the 3-amino-2,4-dihydroxy benzoic acid portion of the platensimycin molecule. PMID:28465627

Curcumin improves alcoholic fatty liver by inhibiting fatty acid biosynthesis.

PubMed

Guo, Chang; Ma, Jingfan; Zhong, Qionghong; Zhao, Mengyuan; Hu, Tianxing; Chen, Tong; Qiu, Longxin; Wen, Longping

2017-08-01

Alcoholic fatty liver is a threat to human health. It has been long known that abstinence from alcohol is the most effective therapy, other effective therapies are not available for the treatment in humans. Curcumin has a great potential for anti-oxidation and anti-inflammation, but the effect on metabolic reconstruction remains little known. Here we performed metabolomic analysis by gas chromatography/mass spectrometry and explored ethanol pathogenic insight as well as curcumin action pattern. We identified seventy-one metabolites in mouse liver. Carbohydrates and lipids were characteristic categories. Pathway analysis results revealed that ethanol-induced pathways including biosynthesis of unsaturated fatty acids, fatty acid biosynthesis and pentose and glucuronate interconversions were suppressed by curcumin. Additionally, ethanol enhanced galactose metabolism and pentose phosphate pathway. Glyoxylate and dicarboxylate metabolism and pyruvate metabolism were inhibited in mice fed ethanol diet plus curcumin. Stearic acid, oleic acid and linoleic acid were disease biomarkers and therapical biomarkers. These results reflect the landscape of hepatic metabolism regulation. Our findings illustrate ethanol pathological pathway and metabolic mechanism of curcumin therapy. Copyright © 2017. Published by Elsevier Inc.

Chemical Sensitization of Clostridium botulinum Spores to Radiation in Meat1

PubMed Central

Krabbenhoft, K. L.; Corlett, D. A.; Anderson, A. W.; Elliker, P. R.

1964-01-01

Beef ground round inoculated with 1,000,000 spores of Clostridium botulinum 33-A per gram and containing various additives was exposed to gamma radiation. Spores were inactivated in samples (irradiated at 2.0, 2.5, and 3.0 Mrad) which contained sodium nitrate (1,000 ppm) plus sodium chloride (2.5%). Similar results were obtained when sodium nitrite (200 ppm) was substituted for sodium nitrate, except that there was evidence of spore survival in 1 of 120 cans irradiated at 2.0 Mrad. Spore destruction was based upon the absence of spores and mouse-lethal toxin in meat subcultures made from cans incubated at 35 C for 120 days. Spores were not destroyed when exposed to 2.5 or 3.0 Mrad in the absence of sodium nitrate, sodium nitrite, or sodium chloride. Furthermore, the use of these chemicals individually, together with radiation, was ineffective. The additives alone in the absence of radiation also did not cause spore destruction. Radiation levels of 2.0, 2.5, and 3.0 Mrad, when used with sodium chloride at 1.5 or 2.0% and sodium nitrate at 500 ppm or sodium nitrite at 100 ppm, were ineffective. PMID:14215973

Pharmacological and Behavioral Enhancement of Neuroplasticity in the MPTP-Lesioned Mouse and Nonhuman Primate

DTIC Science & Technology

2008-05-01

effect of amphetamine in mice neonatally lesioned with 6- hydroxydopamine. J Neurosci Res. 78, 289-96. 22 Berger-Sweeney, J., Stearns, N. A., Frick...through its effect on pre- and post- synaptic dopamine biosynthesis, uptake and receptor expression as well as glutamatergic synapses. This hypothesis...These studies were designed to be complementary in that both non-pharmacological and pharmacological effects of neuroplasticity are being

Biosynthesis of nanoparticles using microbes- a review.

PubMed

Hulkoti, Nasreen I; Taranath, T C

2014-09-01

The biosynthesis of nanoparticles by microorganism is a green and eco-friendly technology. This review focuses on the use of consortium of diverse microorganisms belonging to both prokaryotes and eukaryotes for the synthesis of metallic nanoparticles viz. silver, gold, platinum, zirconium, palladium, iron, cadmium and metal oxides such as titanium oxide, zinc oxide, etc. These microorganisms include bacteria, actinomycetes, fungi and algae. The synthesis of nanoparticles may be intracellular or extracellular. The several workers have reported that NADH dependent nitrate reductase enzyme plays a vital role in the conversion of metallic ions to nanoparticles. The FTIR study reveals that diverse biomolecules viz. carboxyl group, primary and secondary amines, amide I, II, and III bands etc serve as a tool for bioreduction and capping agents there by offering stability to particles by preventing agglomeration and growth. The size and shape of the nanoparticles vary with the organism employed and conditions employed during the synthesis which included pH, temperature and substrate concentration. The microorganisms provide diverse environment for biosynthesis of nanoparticles. These particles are safe and eco-friendly with a lot of applications in medicine, agriculture, cosmetic industry, drug delivery and biochemical sensors. The challenges for redressal include optimal production and minimal time to obtain desired size and shape, to enhance the stability of nanoparticles and optimization of specific microorganisms for specific application. Copyright © 2014 Elsevier B.V. All rights reserved.

The master regulator PhoP coordinates phosphate and nitrogen metabolism, respiration, cell differentiation and antibiotic biosynthesis: comparison in Streptomyces coelicolor and Streptomyces avermitilis.

PubMed

Martín, Juan F; Rodríguez-García, Antonio; Liras, Paloma

2017-05-01

Phosphate limitation is important for production of antibiotics and other secondary metabolites in Streptomyces. Phosphate control is mediated by the two-component system PhoR-PhoP. Following phosphate depletion, PhoP stimulates expression of genes involved in scavenging, transport and mobilization of phosphate, and represses the utilization of nitrogen sources. PhoP reduces expression of genes for aerobic respiration and activates nitrate respiration genes. PhoP activates genes for teichuronic acid formation and reduces expression of genes for phosphate-rich teichoic acid biosynthesis. In Streptomyces coelicolor, PhoP repressed several differentiation and pleiotropic regulatory genes, which affects development and indirectly antibiotic biosynthesis. A new bioinformatics analysis of the putative PhoP-binding sequences in Streptomyces avermitilis was made. Many sequences in S. avermitilis genome showed high weight values and were classified according to the available genetic information. These genes encode phosphate scavenging proteins, phosphate transporters and nitrogen metabolism genes. Among of the genes highlighted in the new studies was aveR, located in the avermectin gene cluster, encoding a LAL-type regulator, and afsS, which is regulated by PhoP and AfsR. The sequence logo for S. avermitilis PHO boxes is similar to that of S. coelicolor, with differences in the weight value for specific nucleotides in the sequence.

Attempting to Compensate for Reduced Neuronal Nitric Oxide Synthase Protein with Nitrate Supplementation Cannot Overcome Metabolic Dysfunction but Rather Has Detrimental Effects in Dystrophin-Deficient mdx Muscle.

PubMed

Timpani, Cara A; Trewin, Adam J; Stojanovska, Vanesa; Robinson, Ainsley; Goodman, Craig A; Nurgali, Kulmira; Betik, Andrew C; Stepto, Nigel; Hayes, Alan; McConell, Glenn K; Rybalka, Emma

2017-04-01

Duchenne muscular dystrophy arises from the loss of dystrophin and is characterized by calcium dysregulation, muscular atrophy, and metabolic dysfunction. The secondary reduction of neuronal nitric oxide synthase (nNOS) from the sarcolemma reduces NO production and bioavailability. As NO modulates glucose uptake, metabolism, and mitochondrial bioenergetics, we investigated whether an 8-week nitrate supplementation regimen could overcome metabolic dysfunction in the mdx mouse. Dystrophin-positive control (C57BL/10) and dystrophin-deficient mdx mice were supplemented with sodium nitrate (85 mg/l) in drinking water. Following the supplementation period, extensor digitorum longus and soleus were excised and radioactive glucose uptake was measured at rest (basal) and during contraction. Gastrocnemius was excised and mitochondrial respiration was measured using the Oroboros Oxygraph. Tibialis anterior was analyzed immunohistochemically for the presence of dystrophin, nNOS, nitrotyrosine, IgG and CD45+ cells, and histologically to assess areas of damage and regeneration. Glucose uptake in the basal and contracting states was normal in unsupplemented mdx muscles but was reduced following nitrate supplementation in mdx muscles only. The mitochondrial utilization of substrates was also impaired in mdx gastrocnemius during phosphorylating and maximal uncoupled respiration, and nitrate could not improve respiration in mdx muscle. Although nitrate supplementation reduced mitochondrial hydrogen peroxide emission, it induced mitochondrial uncoupling in red gastrocnemius, increased muscle fiber peroxynitrite (nitrotyrosine), and promoted skeletal muscle damage. Our novel data suggest that despite lower nNOS protein expression and likely lower NO production in mdx muscle, enhancing NO production with nitrate supplementation in these mice has detrimental effects on skeletal muscle. This may have important relevance for those with DMD.

Rapid Biosynthesis of AgNPs Using Soil Bacterium Azotobacter vinelandii With Promising Antioxidant and Antibacterial Activities for Biomedical Applications

NASA Astrophysics Data System (ADS)

Karunakaran, Gopalu; Jagathambal, Matheswaran; Gusev, Alexander; Torres, Juan Antonio Lopez; Kolesnikov, Evgeny; Kuznetsov, Denis

2017-07-01

Silver nanoparticles (AgNPs) are applied in various fields from electronics to biomedical applications as a result of their high surface-to-volume ratio. Even though different approaches are available for synthesis of AgNPs, a nontoxic method for the synthesis has not yet been developed. Thus, this study focused on developing an easy and ecofriendly approach to synthesize AgNPs using Azotobacter vinelandii culture extracts. The biosynthesized nanoparticles were further characterized by ultraviolet-visible (UV-Vis) spectroscopy, x-ray diffraction (XRD), Fourier transform infrared (FTIR), energy-dispersive spectrum, particle size distribution (PSD), and transmission electron microscopy (TEM). UV absorption noticed at 435 nm showed formation of AgNPs. The XRD pattern showed a face-centered cubic structure with broad peaks of 28.2°, 32.6°, 46.6°, 55.2°, 57.9°, and 67.8°. The FTIR confirmed the involvement of various functional groups in the biosynthesis of AgNPs. The PSD and TEM analyses showed spherical, well-distributed nanoparticles with an average size of 20-70 nm. The elemental studies confirmed the existence of pure AgNPs. The bacterial extract containing extracellular enzyme nitrate reductase converted silver nitrate into AgNPs. AgNPs significantly inhibited the growth of pathogenic bacteria such as Streptomyces fradiae (National Collection of Industrial Microorganisms (NCIM) 2419), Staphylococcus aureus (NCIM 2127), Escherichia coli (NCIM 2065), and Serratia marcescens (NCIM 2919). In addition, biosynthesized AgNPs were found to possess strong antioxidant activity. Thus, the results of this study revealed that biosynthesized AgNPs could serve as a lead in the development of nanomedicine.

Horizontal acquisition of a hypoxia-responsive molybdenum cofactor biosynthesis pathway contributed to Mycobacterium tuberculosis pathoadaptation.

PubMed

Levillain, Florence; Poquet, Yannick; Mallet, Ludovic; Mazères, Serge; Marceau, Michael; Brosch, Roland; Bange, Franz-Christoph; Supply, Philip; Magalon, Axel; Neyrolles, Olivier

2017-11-01

The unique ability of the tuberculosis (TB) bacillus, Mycobacterium tuberculosis, to persist for long periods of time in lung hypoxic lesions chiefly contributes to the global burden of latent TB. We and others previously reported that the M. tuberculosis ancestor underwent massive episodes of horizontal gene transfer (HGT), mostly from environmental species. Here, we sought to explore whether such ancient HGT played a part in M. tuberculosis evolution towards pathogenicity. We were interested by a HGT-acquired M. tuberculosis-specific gene set, namely moaA1-D1, which is involved in the biosynthesis of the molybdenum cofactor. Horizontal acquisition of this gene set was striking because homologues of these moa genes are present all across the Mycobacterium genus, including in M. tuberculosis. Here, we discovered that, unlike their paralogues, the moaA1-D1 genes are strongly induced under hypoxia. In vitro, a M. tuberculosis moaA1-D1-null mutant has an impaired ability to respire nitrate, to enter dormancy and to survive in oxygen-limiting conditions. Conversely, heterologous expression of moaA1-D1 in the phylogenetically closest non-TB mycobacterium, Mycobacterium kansasii, which lacks these genes, improves its capacity to respire nitrate and grants it with a marked ability to survive oxygen depletion. In vivo, the M. tuberculosis moaA1-D1-null mutant shows impaired survival in hypoxic granulomas in C3HeB/FeJ mice, but not in normoxic lesions in C57BL/6 animals. Collectively, our results identify a novel pathway required for M. tuberculosis resistance to host-imposed stress, namely hypoxia, and provide evidence that ancient HGT bolstered M. tuberculosis evolution from an environmental species towards a pervasive human-adapted pathogen.

Negative feedback regulation of wild-type p53 biosynthesis.

PubMed Central

Mosner, J; Mummenbrauer, T; Bauer, C; Sczakiel, G; Grosse, F; Deppert, W

1995-01-01

When growth-arrested mouse fibroblasts re-entered the cell-cycle, the rise in tumour suppressor p53 mRNA level markedly preceded the rise in expression of the p53 protein. Furthermore, gamma-irradiation of such cells led to a rapid increase in p53 protein biosynthesis even in the presence of the transcription inhibitor actinomycin D. Both findings strongly suggest that p53 biosynthesis in these cells is regulated at the translational level. We present evidence for an autoregulatory control of p53 expression by a negative feed-back loop: p53 mRNA has a predicted tendency to form a stable stem-loop structure that involves the 5'-untranslated region (5'-UTR) plus some 280 nucleotides of the coding sequence. p53 binds tightly to the 5'-UTR region and inhibits the translation of its own mRNA, most likely mediated by the p53-intrinsic RNA re-annealing activity. The inhibition of p53 biosynthesis requires wild-type p53, as it is not observed with MethA mutant p53, p53-catalysed translational inhibition is selective; it might be restricted to p53 mRNA and a few other mRNAs that are able to form extensive stem-loop structures. Release from negative feed-back regulation of p53 biosynthesis, e.g. after damage-induced nuclear transport of p53, might provide a means for rapidly increasing p53 protein levels when p53 is required to act as a cell-cycle checkpoint determinant after DNA damage. Images PMID:7556087

[Characteristics of polyamine biosynthesis regulation and tumor growth rate in hormone-dependant grafted breast tumors of mice and rats].

PubMed

Orlovskiĭ, A A

2007-01-01

Effect of the inhibitors of polyamines biosynthesis on completely or partially hormone-dependant breast tumors (mouse Ca755 carcinoma and Walker W-256 carcinosarcoma) is essentially special: in contrary to hormone-dependant tumors, this effect may be not only breaking but stimulating as well. Change-over from one to another mode of reaction is conditioned, most probable, by hormonal status, which is determined by one or another estral cycle phase. Biochemical mechanisms of this change-over are closely connected with polyamines metabolism, namely the degree of polyamines (especially spermine) interconvertion and physiological reactivity level of the system controlling expression of ornithin-decarboxilase. At that, the first of these pathways is predominant for completely hormone-dependant Ca755 and the second one -for partially hormone-dependant W-256.

New Treatments for Drug-Resistant Epilepsy that Target Presynaptic Transmitter Release

DTIC Science & Technology

2012-05-01

mouse strain to pilocarpine-induced status epilepticus and seizure- induced excitotoxic cell death (Schauwecker, 2012). Therefore, in close...kind gift of Dr. Venkatesh Murthy, Harvard University). For the induction of status epilepticus animals received methylscopolamine nitrate (0.1 mg...weight, 5 animals per group). The dose used was most efficient, yielding 90% of animals entering status epilepticus and 80% survival rate. After

New Treatments for Drug-Resistant Epilepsy that Target Presynaptic Transmitter Release

DTIC Science & Technology

2015-07-01

susceptibility of mouse strain to pilocarpine-induced status epilepticus and seizure-induced excitotoxic cell death[1]. Therefore, we optimized and fully...University). For the induction of status epilepticus animals received methylscopolamine nitrate (0.1 mg/kg in saline, s.c.) thirty minutes before...animals entering status epilepticus and 80% survival rate. After animals entered status epilepticus , a second dose of methylscopolamine was

New Treatments for Drug-Resistant Epilepsy that Target Presynaptic Transmitter Release

DTIC Science & Technology

2015-07-01

mouse strain to pilocarpine-induced status epilepticus and seizure- induced excitotoxic cell death [1]. Therefore, in close collaboration with the...Laboratory (kind gift of Dr. Venkatesh Murthy, Harvard University). For the induction of status epilepticus animals received methylscopolamine nitrate...The dose used was most efficient, yielding 90% of animals entering status epilepticus and 80% survival rate. After animals entered status epilepticus

Vitamin K2 biosynthetic enzyme, UBIAD1 is essential for embryonic development of mice.

PubMed

Nakagawa, Kimie; Sawada, Natsumi; Hirota, Yoshihisa; Uchino, Yuri; Suhara, Yoshitomo; Hasegawa, Tomoka; Amizuka, Norio; Okamoto, Tadashi; Tsugawa, Naoko; Kamao, Maya; Funahashi, Nobuaki; Okano, Toshio

2014-01-01

UbiA prenyltransferase domain containing 1 (UBIAD1) is a novel vitamin K2 biosynthetic enzyme screened and identified from the human genome database. UBIAD1 has recently been shown to catalyse the biosynthesis of Coenzyme Q10 (CoQ10) in zebrafish and human cells. To investigate the function of UBIAD1 in vivo, we attempted to generate mice lacking Ubiad1, a homolog of human UBIAD1, by gene targeting. Ubiad1-deficient (Ubiad1(-/-)) mouse embryos failed to survive beyond embryonic day 7.5, exhibiting small-sized body and gastrulation arrest. Ubiad1(-/-) embryonic stem (ES) cells failed to synthesize vitamin K2 but were able to synthesize CoQ9, similar to wild-type ES cells. Ubiad1(+/-) mice developed normally, exhibiting normal growth and fertility. Vitamin K2 tissue levels and synthesis activity were approximately half of those in the wild-type, whereas CoQ9 tissue levels and synthesis activity were similar to those in the wild-type. Similarly, UBIAD1 expression and vitamin K2 synthesis activity of mouse embryonic fibroblasts prepared from Ubiad1(+/-) E15.5 embryos were approximately half of those in the wild-type, whereas CoQ9 levels and synthesis activity were similar to those in the wild-type. Ubiad1(-/-) mouse embryos failed to be rescued, but their embryonic lifespans were extended to term by oral administration of MK-4 or CoQ10 to pregnant Ubiad1(+/-) mice. These results suggest that UBIAD1 is responsible for vitamin K2 synthesis but may not be responsible for CoQ9 synthesis in mice. We propose that UBIAD1 plays a pivotal role in embryonic development by synthesizing vitamin K2, but may have additional functions beyond the biosynthesis of vitamin K2.

Loss of lysophosphatidylcholine acyltransferase 1 leads to photoreceptor degeneration in rd11 mice

PubMed Central

Friedman, James S.; Chang, Bo; Krauth, Daniel S.; Lopez, Irma; Waseem, Naushin H.; Hurd, Ron E.; Feathers, Kecia L.; Branham, Kari E.; Shaw, Manessa; Thomas, George E.; Brooks, Matthew J.; Liu, Chunqiao; Bakeri, Hirva A.; Campos, Maria M.; Maubaret, Cecilia; Webster, Andrew R.; Rodriguez, Ignacio R.; Thompson, Debra A.; Bhattacharya, Shomi S.; Koenekoop, Robert K.; Heckenlively, John R.; Swaroop, Anand

2010-01-01

Retinal degenerative diseases, such as retinitis pigmentosa and Leber congenital amaurosis, are a leading cause of untreatable blindness with substantive impact on the quality of life of affected individuals and their families. Mouse mutants with retinal dystrophies have provided a valuable resource to discover human disease genes and helped uncover pathways critical for photoreceptor function. Here we show that the rd11 mouse mutant and its allelic strain, B6-JR2845, exhibit rapid photoreceptor dysfunction, followed by degeneration of both rods and cones. Using linkage analysis, we mapped the rd11 locus to mouse chromosome 13. We then identified a one-nucleotide insertion (c.420–421insG) in exon 3 of the Lpcat1 gene. Subsequent screening of this gene in the B6-JR2845 strain revealed a seven-nucleotide deletion (c.14–20delGCCGCGG) in exon 1. Both sequence changes are predicted to result in a frame-shift, leading to premature truncation of the lysophosphatidylcholine acyltransferase-1 (LPCAT1) protein. LPCAT1 (also called AYTL2) is a phospholipid biosynthesis/remodeling enzyme that facilitates the conversion of palmitoyl-lysophosphatidylcholine to dipalmitoylphosphatidylcholine (DPPC). The analysis of retinal lipids from rd11 and B6-JR2845 mice showed substantially reduced DPPC levels compared with C57BL/6J control mice, suggesting a causal link to photoreceptor dysfunction. A follow-up screening of LPCAT1 in retinitis pigmentosa and Leber congenital amaurosis patients did not reveal any obvious disease-causing mutations. Previously, LPCAT1 has been suggested to be critical for the production of lung surfactant phospholipids and biosynthesis of platelet-activating factor in noninflammatory remodeling pathway. Our studies add another dimension to an essential role for LPCAT1 in retinal photoreceptor homeostasis. PMID:20713727

Nitrate, nitrite and nitric oxide reductases: from the last universal common ancestor to modern bacterial pathogens

PubMed Central

Vázquez-Torres, Andrés; Bäumler, Andreas

2016-01-01

The electrochemical gradient that ensues from the enzymatic activity of cytochromes such as nitrate reductase, nitric oxide reductase, and quinol oxidase contributes to the bioenergetics of the bacterial cell. Reduction of nitrogen oxides by bacterial pathogens can, however, be uncoupled from proton translocation and biosynthesis of ATP or NH4+, but still linked to quinol and NADH oxidation. Ancestral nitric oxide reductases, as well as cytochrome coxidases and quinol bo oxidases evolved from the former, are capable of binding and detoxifying nitric oxide to nitrous oxide. The NO-metabolizing activity associated with these cytochromes can be a sizable source of antinitrosative defense in bacteria during their associations with host cells. Nitrosylation of terminal cytochromes arrests respiration, reprograms bacterial metabolism, stimulates antioxidant defenses and alters antibiotic cytotoxicity. Collectively, the bioenergetics and regulation of redox homeostasis that accompanies the utilization of nitrogen oxides and detoxification of nitric oxide by cytochromes of the electron transport chain increases fitness of many Gram-positive and –negative pathogens during their associations with invertebrate and vertebrate hosts. PMID:26426528

Fractionation of Hydrogen Isotopes by Sulfate- and Nitrate-Reducing Bacteria.

PubMed

Osburn, Magdalena R; Dawson, Katherine S; Fogel, Marilyn L; Sessions, Alex L

2016-01-01

Hydrogen atoms from water and food are incorporated into biomass during cellular metabolism and biosynthesis, fractionating the isotopes of hydrogen-protium and deuterium-that are recorded in biomolecules. While these fractionations are often relatively constant in plants, large variations in the magnitude of fractionation are observed for many heterotrophic microbes utilizing different central metabolic pathways. The correlation between metabolism and lipid δ(2)H provides a potential basis for reconstructing environmental and ecological parameters, but the calibration dataset has thus far been limited mainly to aerobes. Here we report on the hydrogen isotopic fractionations of lipids produced by nitrate-respiring and sulfate-reducing bacteria. We observe only small differences in fractionation between oxygen- and nitrate-respiring growth conditions, with a typical pattern of variation between substrates that is broadly consistent with previously described trends. In contrast, fractionation by sulfate-reducing bacteria does not vary significantly between different substrates, even when autotrophic and heterotrophic growth conditions are compared. This result is in marked contrast to previously published observations and has significant implications for the interpretation of environmental hydrogen isotope data. We evaluate these trends in light of metabolic gene content of each strain, growth rate, and potential flux and reservoir-size effects of cellular hydrogen, but find no single variable that can account for the differences between nitrate- and sulfate-respiring bacteria. The emerging picture of bacterial hydrogen isotope fractionation is therefore more complex than the simple correspondence between δ(2)H and metabolic pathway previously understood from aerobes. Despite the complexity, the large signals and rich variability of observed lipid δ(2)H suggest much potential as an environmental recorder of metabolism.

GATA1 and PU.1 Bind to Ribosomal Protein Genes in Erythroid Cells: Implications for Ribosomopathies

PubMed Central

Amanatiadou, Elsa P.; Papadopoulos, Giorgio L.; Strouboulis, John; Vizirianakis, Ioannis S.

2015-01-01

The clear connection between ribosome biogenesis dysfunction and specific hematopoiesis-related disorders prompted us to examine the role of critical lineage-specific transcription factors in the transcriptional regulation of ribosomal protein (RP) genes during terminal erythroid differentiation. By applying EMSA and ChIP methodologies in mouse erythroleukemia cells we show that GATA1 and PU.1 bind in vitro and in vivo the proximal promoter region of the RPS19 gene which is frequently mutated in Diamond-Blackfan Anemia. Moreover, ChIPseq data analysis also demonstrates that several RP genes are enriched as potential GATA1 and PU.1 gene targets in mouse and human erythroid cells, with GATA1 binding showing an association with higher ribosomal protein gene expression levels during terminal erythroid differentiation in human and mouse. Our results suggest that RP gene expression and hence balanced ribosome biosynthesis may be specifically and selectively regulated by lineage specific transcription factors during hematopoiesis, a finding which may be clinically relevant to ribosomopathies. PMID:26447946

Polyamines and Nonmelanoma Skin Cancer

PubMed Central

Gilmour, Susan K.

2007-01-01

Elevated levels of polyamines have long been associated with skin tumorigenesis. Tightly regulated metabolism of polyamines is critical for cell survival and normal skin homeostasis, and these controls are dysregulated in skin tumorigenesis. A key enzyme in polyamine biosynthesis, ornithine decarboxylase (ODC) is upregulated in skin tumors compared to normal skin. Use of transgenic mouse models has demonstrated that polyamines play an essential role in the early promotional phase of skin tumorigenesis. The formation of skin tumors in these transgenic mice is dependent upon polyamine biosynthesis, especially putrescine, since treatment with inhibitors of ODC activity blocks the formation of skin tumors and causes the rapid regression of existing tumors. Although the mechanism by which polyamines promote skin tumorigenesis are not well understood, elevated levels of polyamines have been shown to stimulate epidermal proliferation, alter keratinocyte differentiation status, increase neovascularization, and increase synthesis of extracellular matrix proteins in a manner similar to that seen in wound healing. It is becoming increasingly apparent that elevated polyamine levels activate not only epidermal cells but also underlying stromal cells in the skin to promote the development and progression of skin tumors. The inhibition of polyamine biosynthesis has potential to be an effective chemoprevention strategy for nonmelanoma skin cancer. PMID:17234230

Endogenous hydrogen sulfide regulates histone demethylase JMJD3-mediated inflammatory response in LPS-stimulated macrophages and in a mouse model of LPS-induced septic shock.

PubMed

Liu, Siyu; Wang, Xiling; Pan, Lilong; Wu, Weijun; Yang, Di; Qin, Ming; Jia, Wanwan; Xiao, Chenxi; Long, Fen; Ge, Junbo; Liu, Xinhua; Zhu, YiZhun

2018-03-01

Overproduction of inflammatory mediators contributes to uncontrolled inflammation during endotoxin shock. Cystathionine-γ-lyase (CSE), an enzyme involved in hydrogen sulfide (H 2 S) biosynthesis, has potential anti-inflammatory activity in a variety of inflammatory diseases. Jumonji domain-containing protein 3 (JMJD3), a histone 3 Lys27 (H3K27) demethylase, has been implicated in macrophage activation, but its function in CSE-mediated anti-inflammatory activities remains unknown. In the present study CSE was found to be upregulated in macrophages and mouse lipopolysaccharide (LPS) challenge models. LPS stimulation also enhanced the activation of JMJD3 and decreased H3K27me3 levels. JMJD3 knockdown upregulated H3K27me3 levels and attenuated the LPS-mediated inflammatory response. CSE knockout amplified the inflammatory cascade by increasing JMJD3 expression in septic mice. Similarly, enhanced production of inflammatory mediators by macrophages was mitigated by CSE overexpression via inhibition of JMJD3 expression. This is the first report indicating that inflammation enhanced CSE/H 2 S system biosynthesis, that in turn attenuated the LPS-triggered inflammatory response by regulating JMJD3 expression. Thus, the CSE/H 2 S system represents an epigenetic-based modification mechanism to prevent uncontrolled inflammation. Copyright © 2017 Elsevier Inc. All rights reserved.

Functional characterization of enzymes catalyzing ceramide phosphoethanolamine biosynthesis in mice.

PubMed

Bickert, Andreas; Ginkel, Christina; Kol, Matthijs; vom Dorp, Katharina; Jastrow, Holger; Degen, Joachim; Jacobs, René L; Vance, Dennis E; Winterhager, Elke; Jiang, Xian-Cheng; Dörmann, Peter; Somerharju, Pentti; Holthuis, Joost C M; Willecke, Klaus

2015-04-01

Besides bulk amounts of SM, mammalian cells produce small quantities of the SM analog ceramide phosphoethanolamine (CPE). Little is known about the biological role of CPE or enzymes responsible for CPE production. Heterologous expression studies revealed that SM synthase (SMS)2 is a bifunctional enzyme producing both SM and CPE, whereas SMS-related protein (SMSr) serves as monofunctional CPE synthase. Acute disruption of SMSr catalytic activity in cultured cells causes a rise in endoplasmic reticulum (ER) ceramides, fragmentation of ER exit sites, and induction of mitochondrial apoptosis. To address the relevance of CPE biosynthesis in vivo, we analyzed the tissue-specific distribution of CPE in mice and generated mouse lines lacking SMSr and SMS2 catalytic activity. We found that CPE levels were >300-fold lower than SM in all tissues examined. Unexpectedly, combined inactivation of SMSr and SMS2 significantly reduced, but did not eliminate, tissue-specific CPE pools and had no obvious impact on mouse development or fertility. While SMSr is widely expressed and serves as the principal CPE synthase in the brain, blocking its catalytic activity did not affect ceramide levels or secretory pathway integrity in the brain or any other tissue. Our data provide a first inventory of CPE species and CPE-biosynthetic enzymes in mammals. Copyright © 2015 by the American Society for Biochemistry and Molecular Biology, Inc.

Molecular structure and differential function of choline kinases CHKα and CHKβ in musculoskeletal system and cancer.

PubMed

Chen, Xi; Qiu, Heng; Wang, Chao; Yuan, Yu; Tickner, Jennifer; Xu, Jiake; Zou, Jun

2017-02-01

Choline, a hydrophilic cation, has versatile physiological roles throughout the body, including cholinergic neurotransmission, memory consolidation and membrane biosynthesis and metabolism. Choline kinases possess enzyme activity that catalyses the conversion of choline to phosphocholine, which is further converted to cytidine diphosphate-coline (CDP-choline) in the biosynthesis of phosphatidylcholine (PC). PC is a major constituent of the phospholipid bilayer which constitutes the eukaryotic cell membrane, and regulates cell signal transduction. Choline Kinase consists of three isoforms, CHKα1, CHKα2 and CHKβ, encoded by two separate genes (CHKA(Human)/Chka(Mouse) and CHKB(Human)/Chkb(Mouse)). Both isoforms have similar structures and enzyme activity, but display some distinct molecular structural domains and differential tissue expression patterns. Whilst Choline Kinase was discovered in early 1950, its pivotal role in the development of muscular dystrophy, bone deformities, and cancer has only recently been identified. CHKα has been proposed as a cancer biomarker and its inhibition as an anti-cancer therapy. In contrast, restoration of CHKβ deficiency through CDP-choline supplements like citicoline may be beneficial for the treatment of muscular dystrophy, bone metabolic diseases, and cognitive conditions. The molecular structure and expression pattern of Choline Kinase, the differential roles of Choline Kinase isoforms and their potential as novel therapeutic targets for muscular dystrophy, bone deformities, cognitive conditions and cancer are discussed. Copyright © 2016. Published by Elsevier Ltd.

Elevated prostacyclin biosynthesis in mice impacts memory and anxiety-like behavior.

PubMed

Vollert, Craig; Ohia, Odochi; Akasaka, Hironari; Berridge, Casey; Ruan, Ke-He; Eriksen, Jason L

2014-01-01

Prostacyclin is an endogenous lipid metabolite with properties of vasodilation and anti-platelet aggregation. While the effects of prostacyclin on the vascular protection have been well-documented, the role of this eicosanoid in the central nervous system has not been extensively studied. Recently, a transgenic mouse containing a hybrid enzyme, of cyclooxygenase-1 linked to prostacyclin synthase, was developed that produces elevated levels of prostacyclin in vivo. The goal of this study was to investigate whether increased prostacyclin biosynthesis could affect behavioral phenotypes in mice. Our results uncovered that elevated levels of prostacyclin broadly affect both cognitive and non-cognitive behaviors, including decreased anxiety-like behavior and improved learning in the fear-conditioning memory test. This study demonstrates that prostacyclin plays an important, but previously unrecognized, role in central nervous system function and behavior. Copyright © 2013 Elsevier B.V. All rights reserved.

Potassium deficiency affects the carbon-nitrogen balance in cotton leaves.

PubMed

Hu, Wei; Coomer, Taylor D; Loka, Dimitra A; Oosterhuis, Derrick M; Zhou, Zhiguo

2017-06-01

Potassium (K) plays important roles in the metabolism of carbon (C) and nitrogen (N), but studies of K deficiency affecting C-N balance are lacking. This study explored the influence of K deficiency on C-N interaction in cotton leaves by conducting a field experiment with cotton cultivar DP0912 under two K rates (K0: 0 kg K 2 O ha -1 and K67: 67 kg K 2 O ha -1 ) and a controlled environment experiment with K-deficient solution (K1: 0 mM K + ) and K-sufficient solution (K2: 6 mM K + ). The results showed that leaf K content, leaf number, leaf area, boll number, reproductive dry weight and total dry weight were significant lower under K deficiency (K0 or K1). Lower total chlorophyll content and Chl a/b ratio, and decreased Pn along with lower Gs and higher Ci were measured under K deficiency, suggesting that the decrease in Pn was resulted from non-stomatal limitation. Leaf glucose, fructose, sucrose and starch contents were higher under K deficiency, because lower sucrose export was detected in phloem. Although leaf nitrate and ammonium contents significantly decreased, free amino acid content was increased by 40-63% under K deficiency, since lower amino acid export was also measured in phloem. K deficiency also induced lower soluble protein content in leaves. Leaf ATP level was significantly increased under K deficiency, indicating ATP utilization was lower, so that less energy was supplied to C and N metabolism. The ratio of soluble sugar to free amino acid and the C/N ratio markedly increased under K deficiency, and one reason was that the phloem export reduced more prominent for sucrose (54.6-78.0%) than amino acid (36.7-85.4%) under K deficiency. In addition, lower phosphoenolpyruvate carboxylase activity limited malate and citrate biosynthesis under K deficiency, causing a decrease of C flux into the amino acids, which was not beneficial for maintaining C-N balance. Sucrose phosphate synthase and nitrate reductase activities were lower under K deficiency, which would limit sucrose biosynthesis and nitrate assimilation. This was another factor altering soluble sugar to free amino acid ratio and C/N ratio in the K-deficient leaves. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Effect of melatonin and tetrapeptide on gene expression in mouse brain.

PubMed

Anisimov, S V; Khavinson, V Kh; Anisimov, V N

2004-11-01

A microchip technique was used to study expression of 16,897 clones from a cDNA library in the brain of mice receiving melatonin or tetrapeptide Epithalon (Ala-Glu-Asp-Gly). Expression of 53 transcripts in mouse brain underwent significant changes after treatment with the preparations. Melatonin and Epithalon modified expression of 38 and 22 transcripts, respectively. These preparations produced similar changes in the expression of 6 transcripts. Expression of 1 transcript (Rp119) was inhibited by melatonin, but induced by Epithalon. The target genes are physiologically related to the cell cycle, apoptosis, biosynthesis, processing, and transport of nucleic acids. Comparative study of gene expression in the brain and heart of CBA mice receiving melatonin and Epithalon suggest that these preparations have a tissue-specific biological effect.

Photoprotective effect of vitamins A and E on polyamine and oxygenated free radical metabolism in hairless mouse epidermis.

PubMed

Khettab, N; Amory, M C; Briand, G; Bousquet, B; Combre, A; Forlot, P; Barey, M

1988-12-01

The purpose of this study was to confirm the photoprotective effect on skin of vitamins A and E, due to inhibition of polyamine synthesis and production of free radicals. These variables were measured in the lumbar epidermis of the female hairless mouse subjected to UVA + B irradiation. Polyamines were assayed in epidermal homogenate by HPLC, and production of oxygenated free radicals was determined by spectrofluorometric assay of malonyl dialdehyde. It was determined that butyl-hydroxy-toluene and vitamin E inhibited production of free radicals (56% and 60%, respectively) and caused a significant reduction in polyamine biosynthesis (P less than 0.01), whereas the inhibitory effect of malonyl dialdehyde induced by vitamin A (30%) had no associated effect on polyamine metabolism.

Differential gene expression in tomato fruit and Colletotrichum gloeosporioides during colonization of the RNAi-SlPH tomato line with reduced fruit acidity and higher pH.

PubMed

Barad, Shiri; Sela, Noa; Dubey, Amit K; Kumar, Dilip; Luria, Neta; Ment, Dana; Cohen, Shahar; Schaffer, Arthur A; Prusky, Dov

2017-08-04

The destructive phytopathogen Colletotrichum gloeosporioides causes anthracnose disease in fruit. During host colonization, it secretes ammonia, which modulates environmental pH and regulates gene expression, contributing to pathogenicity. However, the effect of host pH environment on pathogen colonization has never been evaluated. Development of an isogenic tomato line with reduced expression of the gene for acidity, SlPH (Solyc10g074790.1.1), enabled this analysis. Total RNA from C. gloeosporioides colonizing wild-type (WT) and RNAi-SlPH tomato lines was sequenced and gene-expression patterns were compared. C. gloeosporioides inoculation of the RNAi-SlPH line with pH 5.96 compared to the WT line with pH 4.2 showed 30% higher colonization and reduced ammonia accumulation. Large-scale comparative transcriptome analysis of the colonized RNAi-SlPH and WT lines revealed their different mechanisms of colonization-pattern activation: whereas the WT tomato upregulated 13-LOX (lipoxygenase), jasmonic acid and glutamate biosynthesis pathways, it downregulated processes related to chlorogenic acid biosynthesis II, phenylpropanoid biosynthesis and hydroxycinnamic acid tyramine amide biosynthesis; the RNAi-SlPH line upregulated UDP-D-galacturonate biosynthesis I and free phenylpropanoid acid biosynthesis, but mainly downregulated pathways related to sugar metabolism, such as the glyoxylate cycle and L-arabinose degradation II. Comparison of C. gloeosporioides gene expression during colonization of the WT and RNAi-SlPH lines showed that the fungus upregulates ammonia and nitrogen transport and the gamma-aminobutyric acid metabolic process during colonization of the WT, while on the RNAi-SlPH tomato, it mainly upregulates the nitrate metabolic process. Modulation of tomato acidity and pH had significant phenotypic effects on C. gloeosporioides development. The fungus showed increased colonization on the neutral RNAi-SlPH fruit, and limited colonization on the WT acidic fruit. The change in environmental pH resulted in different defense responses for the two tomato lines. Interestingly, the WT line showed upregulation of jasmonate pathways and glutamate accumulation, supporting the reduced symptom development and increased ammonia accumulation, as the fungus might utilize glutamate to accumulate ammonia and increase environmental pH for better expression of pathogenicity factors. This was not found in the RNAi-SlPH line which downregulated sugar metabolism and upregulated the phenylpropanoid pathway, leading to host susceptibility.

Kaempferol increases levels of coenzyme Q in kidney cells and serves as a biosynthetic ring precursor.

PubMed

Fernández-Del-Río, Lucía; Nag, Anish; Gutiérrez Casado, Elena; Ariza, Julia; Awad, Agape M; Joseph, Akil I; Kwon, Ohyun; Verdin, Eric; de Cabo, Rafael; Schneider, Claus; Torres, Jorge Z; Burón, María I; Clarke, Catherine F; Villalba, José M

2017-09-01

Coenzyme Q (Q) is a lipid-soluble antioxidant essential in cellular physiology. Patients with Q deficiencies, with few exceptions, seldom respond to treatment. Current therapies rely on dietary supplementation with Q 10 , but due to its highly lipophilic nature, Q 10 is difficult to absorb by tissues and cells. Plant polyphenols, present in the human diet, are redox active and modulate numerous cellular pathways. In the present study, we tested whether treatment with polyphenols affected the content or biosynthesis of Q. Mouse kidney proximal tubule epithelial (Tkpts) cells and human embryonic kidney cells 293 (HEK 293) were treated with several types of polyphenols, and kaempferol produced the largest increase in Q levels. Experiments with stable isotope 13 C-labeled kaempferol demonstrated a previously unrecognized role of kaempferol as an aromatic ring precursor in Q biosynthesis. Investigations of the structure-function relationship of related flavonols showed the importance of two hydroxyl groups, located at C3 of the C ring and C4' of the B ring, both present in kaempferol, as important determinants of kaempferol as a Q biosynthetic precursor. Concurrently, through a mechanism not related to the enhancement of Q biosynthesis, kaempferol also augmented mitochondrial localization of Sirt3. The role of kaempferol as a precursor that increases Q levels, combined with its ability to upregulate Sirt3, identify kaempferol as a potential candidate in the design of interventions aimed on increasing endogenous Q biosynthesis, particularly in kidney. Copyright © 2017 Elsevier Inc. All rights reserved.

Proinsulin atypical maturation and disposal induces extensive defects in mouse Ins2+/Akita β-cells.

PubMed

Yuan, Qingxin; Tang, Wei; Zhang, Xiaoping; Hinson, Jack A; Liu, Chao; Osei, Kwame; Wang, Jie

2012-01-01

Because of its low relative folding rate and plentiful manufacture in β-cells, proinsulin maintains a homeostatic balance of natively and plentiful non-natively folded states (i.e., proinsulin homeostasis, PIHO) through the integration of maturation and disposal processes. PIHO is susceptible to genetic and environmental influences, and its disorder has been critically linked to defects in β-cells in diabetes. To explore this hypothesis, we performed polymerase chain reaction (PCR), metabolic-labeling, immunoblotting, and histological studies to clarify what defects result from primary disorder of PIHO in model Ins2(+/Akita) β-cells. We used T antigen-transformed Ins2(+/Akita) and control Ins2(+/+) β-cells established from Akita and wild-type littermate mice. In Ins2(+/Akita) β-cells, we found no apparent defect at the transcriptional and translational levels to contribute to reduced cellular content of insulin and its precursor and secreted insulin. Glucose response remained normal in proinsulin biosynthesis but was impaired for insulin secretion. The size and number of mature insulin granules were reduced, but the size/number of endoplasmic reticulum, Golgi, mitochondrion, and lysosome organelles and vacuoles were expanded/increased. Moreover, cell death increased, and severe oxidative stress, which manifested as increased reactive oxygen species, thioredoxin-interacting protein, and protein tyrosine nitration, occurred in Ins2(+/Akita) β-cells and/or islets. These data show the first clear evidence that primary PIHO imbalance induces severe oxidative stress and impairs glucose-stimulated insulin release and β-cell survival as well as producing other toxic consequences. The defects disclosed/clarified in model Ins2(+/Akita) β-cells further support a role of the genetic and stress-susceptible PIHO disorder in β-cell failure and diabetes.

Proinsulin Atypical Maturation and Disposal Induces Extensive Defects in Mouse Ins2+/Akita β-Cells

PubMed Central

Zhang, Xiaoping; Hinson, Jack A.; Liu, Chao; Osei, Kwame; Wang, Jie

2012-01-01

Because of its low relative folding rate and plentiful manufacture in β-cells, proinsulin maintains a homeostatic balance of natively and plentiful non-natively folded states (i.e., proinsulin homeostasis, PIHO) through the integration of maturation and disposal processes. PIHO is susceptible to genetic and environmental influences, and its disorder has been critically linked to defects in β-cells in diabetes. To explore this hypothesis, we performed polymerase chain reaction (PCR), metabolic-labeling, immunoblotting, and histological studies to clarify what defects result from primary disorder of PIHO in model Ins2+/Akita β-cells. We used T antigen-transformed Ins2+/Akita and control Ins2+/+ β-cells established from Akita and wild-type littermate mice. In Ins2+/Akita β-cells, we found no apparent defect at the transcriptional and translational levels to contribute to reduced cellular content of insulin and its precursor and secreted insulin. Glucose response remained normal in proinsulin biosynthesis but was impaired for insulin secretion. The size and number of mature insulin granules were reduced, but the size/number of endoplasmic reticulum, Golgi, mitochondrion, and lysosome organelles and vacuoles were expanded/increased. Moreover, cell death increased, and severe oxidative stress, which manifested as increased reactive oxygen species, thioredoxin-interacting protein, and protein tyrosine nitration, occurred in Ins2+/Akita β-cells and/or islets. These data show the first clear evidence that primary PIHO imbalance induces severe oxidative stress and impairs glucose-stimulated insulin release and β-cell survival as well as producing other toxic consequences. The defects disclosed/clarified in model Ins2+/Akita β-cells further support a role of the genetic and stress-susceptible PIHO disorder in β-cell failure and diabetes. PMID:22509386

Enzymes involved in L-carnitine biosynthesis are expressed by small intestinal enterocytes in mice: implications for gut health.

PubMed

Shekhawat, Prem S; Sonne, Srinivas; Carter, A Lee; Matern, Dietrich; Ganapathy, Vadivel

2013-07-01

Carnitine is essential for mitochondrial β-oxidation of long-chain fatty acids. Deficiency of carnitine leads to severe gut atrophy, ulceration and inflammation in animal models of carnitine deficiency. Genetic studies in large populations have linked mutations in the carnitine transporters OCTN1 and OCTN2 with Crohn's disease (CD), while other studies at the same time have failed to show a similar association and report normal serum carnitine levels in CD patients. In this report, we have studied the expression of carnitine-synthesizing enzymes in intestinal epithelial cells to determine the capability of these cells to synthesize carnitine de novo. We studied expression of five enzymes involved in carnitine biosynthesis, namely 6-N-trimethyllysine dioxygenase (TMLD), 4-trimethylaminobutyraldehyde dehydrogenase (TMABADH), serine hydroxymethyltransferase 1 and 2 (SHMT1 and 2) and γ-butyrobetaine hydroxylase (BBH) by real-time PCR in mice (C3H strain). We also measured activity of γ-BBH in the intestine using an ex vivo assay and localized its expression by in situ hybridization. Our investigations show that mouse intestinal epithelium expresses all five enzymes required for de novo carnitine biosynthesis; the expression is localized mainly in villous surface epithelial cells throughout the intestine. The final rate-limiting enzyme γ-BBH is highly active in the small intestine; its activity was 9.7 ± 3.5 pmol/mg/min, compared to 22.7 ± 7.3 pmol/mg/min in the liver. We conclude that mouse gut epithelium is able to synthesize carnitine de novo. This capacity to synthesize carnitine in the intestine may play an important role in gut health and can help explain lack of clinical carnitine deficiency signs in subjects with mutations with OCTN transporters. Copyright © 2012 European Crohn's and Colitis Organisation. Published by Elsevier B.V. All rights reserved.

Alleviation of Nitrogen and Sulfur Deficiency and Enhancement of Photosynthesis in Arabidopsis thaliana by Overexpression of Uroporphyrinogen III Methyltransferase (UPM1)

PubMed Central

Garai, Sampurna; Tripathy, Baishnab C.

2018-01-01

Siroheme, an iron-containing tetrapyrrole, is the prosthetic group of nitrite reductase (NiR) and sulfite reductase (SiR); it is synthesized from uroporphyrinogen III, an intermediate of chlorophyll biosynthesis, and is required for nitrogen (N) and sulfur (S) assimilation. Further, uroporphyrinogen III methyltransferase (UPM1), responsible for two methylation reactions to form dihydrosirohydrochlorin, diverts uroporphyrinogen III from the chlorophyll biosynthesis pathway toward siroheme synthesis. AtUPM1 [At5g40850] was used to produce both sense and antisense plants of Arabidopsis thaliana in order to modulate siroheme biosynthesis. In our experiments, overexpression of AtUPM1 signaled higher NiR (NII) and SiR gene and gene product expression. Increased NII expression was found to regulate and enhance the transcript and protein abundance of nitrate reductase (NR). We suggest that elevated NiR, NR, and SiR expression must have contributed to the increased synthesis of S containing amino acids in AtUPM1overexpressors, observed in our studies. We note that due to higher N and S assimilation in these plants, total protein content had increased in these plants. Consequently, chlorophyll biosynthesis increased in these sense plants. Higher chlorophyll and protein content of plants upregulated photosynthetic electron transport and carbon assimilation in the sense plants. Further, we have observed increased plant biomass in these plants, and this must have been due to increased N, S, and C assimilation. On the other hand, in the antisense plants, the transcript abundance, and protein content of NiR, and SiR was shown to decrease, resulting in reduced total protein and chlorophyll content. This led to a decrease in photosynthetic electron transport rate, carbon assimilation and plant biomass in these antisense plants. Under nitrogen or sulfur starvation conditions, the overexpressors had higher protein content and photosynthetic electron transport rate than the wild type (WT). Conversely, the antisense plants had lower protein content and photosynthetic efficiency in N-deficient environment. Our results clearly demonstrate that upregulation of siroheme biosynthesis leads to increased nitrogen and sulfur assimilation, and this imparts tolerance to nitrogen and sulfur deficiency in Arabidopsis thaliana plants. PMID:29472934

Pharmacological and Behavioral Enhancement of Neuroplasticity in the MPTP Lesioned Mouse and Nonhuman Primate

DTIC Science & Technology

2007-05-01

and post - synaptic dopamine biosynthesis, uptake and receptor expression as well as glutamatergic synapses. This hypothesis will be tested through...0.05) compared to mice at 7 days (9.6 ± 3.2%) or 30 days post -MPTP (16.5 ± 7.3%). The tail suspension test showed a significant increase in percent of...were compared using one-way analysis of variance (ANOVA), followed by the Fisher post hoc test for comparison of multiple means for the following

Involvement of interleukin-1 in lead nitrate-induced hypercholesterolemia in mice.

PubMed

Kojima, Misaki; Ashino, Takashi; Yoshida, Takemi; Iwakura, Yoichiro; Degawa, Masakuni

2012-01-01

Hepatic 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) and cholesterol 7α-hydroxylase (Cyp7a1) are rate-limiting enzymes for cholesterol biosynthesis and catabolism, respectively. Involvement of inflammatory cytokines, particularly interleukin-1 (IL-1), in alterations of HMGR and Cyp7a1 gene expression during development of lead nitrate (LN)-induced hypercholesterolemia was examined in IL-1α/β-knockout (IL-1-KO) and wild-type (WT) mice. Lead nitrate treatment of WT mice led to not only a marked downregulation of the Cyp7a1 gene at 6-12 h, but also a significant upregulation of the HMGR gene at 12 h. However, such changes were not observed at significant levels in IL-1-KO mice, although a slight, transient downregulation of the Cyp7a1 gene and a minimal upregulation of the HMGR gene occurred at 6 h and 24 h, respectively. Consequently, LN treatment led to development of hypercholesterolemia at 24 h in WT mice, but not in IL-1-KO mice. Furthermore, in WT mice, significant LN-mediated increases were observed at 3-6 h in hepatic IL-1 levels, which can modulate gene expression of Cyp7a1 and HMGR. These findings indicate that, in mice, LN-mediated increases in hepatic IL-1 levels contribute, at least in part, to altered expressions of Cyp7a1 and HMGR genes, and eventually to hypercholesterolemia development.

Extracellular biosynthesis of silver nanoparticles using Bacillus sp. GP-23 and evaluation of their antifungal activity towards Fusarium oxysporum

NASA Astrophysics Data System (ADS)

Gopinath, V.; Velusamy, P.

2013-04-01

In last few decades nanoparticles have attracted and emerged as a field in biomedical research due to their incredible applications. The current research was focused on extracellular synthesis of silver nanoparticles (AgNPs) using cell free culture supernatant of strain GP-23. It was found that the strain GP-23 belonged to Bacillus species by 16S rRNA sequence analysis. Biosynthesis of AgNPs was achieved by addition of culture supernatant with aqueous silver nitrate solution, after 24 h it turned to brown color solution with a peak at 420 nm corresponding to the Plasmon absorbance of AgNPs by UV-Vis Spectroscopy. The nanoparticles were characterized by FTIR, XRD, HRTEM, EDX and AFM. The synthesized nanoparticles were found to be spherical in shape with size in the range of 7-21 nm. It was stable in aqueous solution for five months period of storage at room temperature under dark condition. The biosynthesized AgNPs exhibited strong antifungal activity against plant pathogenic fungus, Fusarium oxysporum at the concentration of 8 μg ml-1. The results suggest that the synthesized AgNPs act as an effective antifungal agent/fungicide.

A nuclear-receptor-dependent phosphatidylcholine pathway with antidiabetic effects.

PubMed

Lee, Jae Man; Lee, Yoon Kwang; Mamrosh, Jennifer L; Busby, Scott A; Griffin, Patrick R; Pathak, Manish C; Ortlund, Eric A; Moore, David D

2011-05-25

Nuclear hormone receptors regulate diverse metabolic pathways and the orphan nuclear receptor LRH-1 (also known as NR5A2) regulates bile acid biosynthesis. Structural studies have identified phospholipids as potential LRH-1 ligands, but their functional relevance is unclear. Here we show that an unusual phosphatidylcholine species with two saturated 12 carbon fatty acid acyl side chains (dilauroyl phosphatidylcholine (DLPC)) is an LRH-1 agonist ligand in vitro. DLPC treatment induces bile acid biosynthetic enzymes in mouse liver, increases bile acid levels, and lowers hepatic triglycerides and serum glucose. DLPC treatment also decreases hepatic steatosis and improves glucose homeostasis in two mouse models of insulin resistance. Both the antidiabetic and lipotropic effects are lost in liver-specific Lrh-1 knockouts. These findings identify an LRH-1 dependent phosphatidylcholine signalling pathway that regulates bile acid metabolism and glucose homeostasis.

Backdoor pathway for dihydrotestosterone biosynthesis: implications for normal and abnormal human sex development.

PubMed

Fukami, Maki; Homma, Keiko; Hasegawa, Tomonobu; Ogata, Tsutomu

2013-04-01

We review the current knowledge about the "backdoor" pathway for the biosynthesis of dihydrotestosterone (DHT). While DHT is produced from cholesterol through the conventional "frontdoor" pathway via testosterone, recent studies have provided compelling evidence for the presence of an alternative "backdoor" pathway to DHT without testosterone intermediacy. This backdoor pathway is known to exist in the tammar wallaby pouch young testis and the immature mouse testis, and has been suggested to be present in the human as well. Indeed, molecular analysis has identified pathologic mutations of genes involved in the backdoor pathway in genetic male patients with undermasculinized external genitalia, and urine steroid profile analysis has argued for the relevance of the activated backdoor pathway to abnormal virilization in genetic females with cytochrome P450 oxidoreductase deficiency and 21-hydroxylase deficiency. It is likely that the backdoor pathway is primarily operating in the fetal testis in a physiological condition to produce a sufficient amount of DHT for male sex development, and that the backdoor pathway is driven with a possible interaction between fetal and permanent adrenals in pathologic conditions with increased 17-hydroxyprogesterone levels. These findings provide novel insights into androgen biosynthesis in both physiological and pathological conditions. Copyright © 2012 Wiley Periodicals, Inc., a Wiley company.

Biosynthesis, Antimicrobial and Cytotoxic Effect of Silver Nanoparticles Using a Novel Nocardiopsis sp. MBRC-1

PubMed Central

Manivasagan, Panchanathan; Senthilkumar, Kalimuthu; Sivakumar, Kannan; Kim, Se-Kwon

2013-01-01

The biosynthesis of nanoparticles has been proposed as a cost effective environmental friendly alternative to chemical and physical methods. Microbial synthesis of nanoparticles is under exploration due to wide biomedical applications, research interest in nanotechnology and microbial biotechnology. In the present study, an ecofriendly process for the synthesis of nanoparticles using a novel Nocardiopsis sp. MBRC-1 has been attempted. We used culture supernatant of Nocardiopsis sp. MBRC-1 for the simple and cost effective green synthesis of silver nanoparticles. The reduction of silver ions occurred when silver nitrate solution was treated with the Nocardiopsis sp. MBRC-1 culture supernatant at room temperature. The nanoparticles were characterized by UV-visible, TEM, FE-SEM, EDX, FTIR, and XRD spectroscopy. The nanoparticles exhibited an absorption peak around 420 nm, a characteristic surface plasmon resonance band of silver nanoparticles. They were spherical in shape with an average particle size of 45 ± 0.15 nm. The EDX analysis showed the presence of elemental silver signal in the synthesized nanoparticles. The FTIR analysis revealed that the protein component in the form of enzyme nitrate reductase produced by the isolate in the culture supernatant may be responsible for reduction and as capping agents. The XRD spectrum showed the characteristic Bragg peaks of 1 2 3, 2 0 4, 0 4 3, 1 4 4, and 3 1 1 facets of the face centered cubic silver nanoparticles and confirms that these nanoparticles are crystalline in nature. The prepared silver nanoparticles exhibited strong antimicrobial activity against bacteria and fungi. Cytotoxicity of biosynthesized AgNPs against in vitro human cervical cancer cell line (HeLa) showed a dose-response activity. IC50 value was found to be 200 μg/mL of AgNPs against HeLa cancer cells. Further studies are needed to elucidate the toxicity and the mechanism involved with antimicrobial and anticancer activity of the synthesized AgNPs as nanomedicine. PMID:23936787

Biosynthesis, antimicrobial and cytotoxic effect of silver nanoparticles using a novel Nocardiopsis sp. MBRC-1.

PubMed

Manivasagan, Panchanathan; Venkatesan, Jayachandran; Senthilkumar, Kalimuthu; Sivakumar, Kannan; Kim, Se-Kwon

2013-01-01

The biosynthesis of nanoparticles has been proposed as a cost effective environmental friendly alternative to chemical and physical methods. Microbial synthesis of nanoparticles is under exploration due to wide biomedical applications, research interest in nanotechnology and microbial biotechnology. In the present study, an ecofriendly process for the synthesis of nanoparticles using a novel Nocardiopsis sp. MBRC-1 has been attempted. We used culture supernatant of Nocardiopsis sp. MBRC-1 for the simple and cost effective green synthesis of silver nanoparticles. The reduction of silver ions occurred when silver nitrate solution was treated with the Nocardiopsis sp. MBRC-1 culture supernatant at room temperature. The nanoparticles were characterized by UV-visible, TEM, FE-SEM, EDX, FTIR, and XRD spectroscopy. The nanoparticles exhibited an absorption peak around 420 nm, a characteristic surface plasmon resonance band of silver nanoparticles. They were spherical in shape with an average particle size of 45 ± 0.15 nm. The EDX analysis showed the presence of elemental silver signal in the synthesized nanoparticles. The FTIR analysis revealed that the protein component in the form of enzyme nitrate reductase produced by the isolate in the culture supernatant may be responsible for reduction and as capping agents. The XRD spectrum showed the characteristic Bragg peaks of 1 2 3, 2 0 4, 0 4 3, 1 4 4, and 3 1 1 facets of the face centered cubic silver nanoparticles and confirms that these nanoparticles are crystalline in nature. The prepared silver nanoparticles exhibited strong antimicrobial activity against bacteria and fungi. Cytotoxicity of biosynthesized AgNPs against in vitro human cervical cancer cell line (HeLa) showed a dose-response activity. IC50 value was found to be 200 μg/mL of AgNPs against HeLa cancer cells. Further studies are needed to elucidate the toxicity and the mechanism involved with antimicrobial and anticancer activity of the synthesized AgNPs as nanomedicine.

Metagenomics Analysis of Gut Microbiota in a High Fat Diet-Induced Obesity Mouse Model Fed with (-)-Epigallocatechin 3-O-(3-O-Methyl) Gallate (EGCG3''Me).

PubMed

Zhang, Xin; Chen, Yuhui; Zhu, Jieyu; Zhang, Man; Ho, Chi-Tang; Huang, Qingrong; Cao, Jinxuan

2018-05-15

(-)-Epigallocatechin 3-O-(3-O-methyl) gallate (EGCG3''Me) has been shown to have a modulatory effect on human intestinal microbiota, and the relationship between intestinal flora and obesity has attracted more and more attention recently. Here, we investigated the potential link between EGCG3''Me and gut microbiota composition, as well as the underlying mechanisms of the anti-obesity activity of EGCG3''Me. EGCG3''Me was prepared from oolong tea by column chromatography, and the influence of EGCG3''Me on intestinal microbiota was analysed using a human flora-associated high fat diet (HFD)-induced obesity mouse model by metagenomics. EGCG3''Me showed a weight reducing effect, ameliorated the HFD-induced gut dysbiosis, and significantly decreased the ratio of Firmicutes/Bacteroidetes. Moreover, the Kyoto Encyclopedia of Genes and Genomes (KEGG) database provided significant differences in differentially expressed genes in response to EGCG3''Me treatment. The results showed enrichment of genes involved in the biosynthesis of amino acids, the two-component system, ATP-binding cassette (ABC) transporters, purine metabolism and carbon metabolism. An EGCG3''Me supplemented diet produces promising effects on gut micro-ecology by enhancing beneficial microbial populations and by affecting metabolic pathways including amino acids biosynthesis, the two-component system, and ABC transporters, contributing to the improvement of host health. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Chrysopogon zizanioides aqueous extract mediated synthesis, characterization of crystalline silver and gold nanoparticles for biomedical applications

PubMed Central

Arunachalam, Kantha D; Annamalai, Sathesh Kumar

2013-01-01

The exploitation of various plant materials for the biosynthesis of nanoparticles is considered a green technology as it does not involve any harmful chemicals. The aim of this study was to develop a simple biological method for the synthesis of silver and gold nanoparticles using Chrysopogon zizanioides. To exploit various plant materials for the biosynthesis of nanoparticles was considered a green technology. An aqueous leaf extract of C. zizanioides was used to synthesize silver and gold nanoparticles by the bioreduction of silver nitrate (AgNO3) and chloroauric acid (HAuCl4) respectively. Water-soluble organics present in the plant materials were mainly responsible for reducing silver or gold ions to nanosized Ag or Au particles. The synthesized silver and gold nanoparticles were characterized by ultraviolet (UV)-visible spectroscopy, scanning electron microscopy (SEM), energy dispersive X-ray analysis (EDAX), Fourier transform infrared spectroscopy (FTIR), and X-ray diffraction (XRD) analysis. The kinetics decline reactions of aqueous silver/gold ion with the C. zizanioides crude extract were determined by UV-visible spectroscopy. SEM analysis showed that aqueous gold ions, when exposed to the extract were reduced and resulted in the biosynthesis of gold nanoparticles in the size range 20–50 nm. This eco-friendly approach for the synthesis of nanoparticles is simple, can be scaled up for large-scale production with powerful bioactivity as demonstrated by the synthesized silver nanoparticles. The synthesized nanoparticles can have clinical use as antibacterial, antioxidant, as well as cytotoxic agents and can be used for biomedical applications. PMID:23861583

Atmosphere stabilization and element recycle in an experimental mouse-algal system

NASA Technical Reports Server (NTRS)

Smernoff, David T.

1986-01-01

Life support systems based on bioregeneration rely on the control and manipulation of organisms. Experiments conducted with a gas-closed mouse-algal system designed to investigate principles of photosynthetic gas exchange focus primarily on observing gas exchange phenomena under varying algal environmental conditions and secondarily on studying element cycling through compartments of the experimental system. Inherent instabilities exit between the uptake and release of carbon dioxide CO2 and oxygen O2 by the mouse and algae. Variations in light intensity and cell density alter the photosynthetic rate of the algae and enable maintenance of physiologic concentrations of CO2 and O2. Different nitrogen sources (urea and nitrate) result in different algal assimilatory quotients (AQ). Combinations of photosynthetic rate and AQ ratio manipulations have been examined for their potential in stabilizing atmospheric gas concentrations in the gas-closed algal-mouse system. Elemental mass balances through the experimental systems compartments are being studied with the concurrent development of a mathematical simulation model. Element cycling experiments include quantification of elemental flows through system compartments and wet oxidation of system waste materials for use as an algal nutrient source. Oxidized waste products demonstrate inhibitory properties although dilution has been shown to allow normal growth.

In situ generation, metabolism and immunomodulatory signaling actions of nitro-conjugated linoleic acid in a murine model of inflammation.

PubMed

Villacorta, Luis; Minarrieta, Lucia; Salvatore, Sonia R; Khoo, Nicholas K; Rom, Oren; Gao, Zhen; Berman, Rebecca C; Jobbagy, Soma; Li, Lihua; Woodcock, Steven R; Chen, Y Eugene; Freeman, Bruce A; Ferreira, Ana M; Schopfer, Francisco J; Vitturi, Dario A

2018-05-01

Conjugated linoleic acid (CLA) is a prime substrate for intra-gastric nitration giving rise to the formation of nitro-conjugated linoleic acid (NO 2 -CLA). Herein, NO 2 -CLA generation is demonstrated within the context of acute inflammatory responses both in vitro and in vivo. Macrophage activation resulted in dose- and time-dependent CLA nitration and also in the production of secondary electrophilic and non-electrophilic derivatives. Both exogenous NO 2 -CLA as well as that generated in situ, attenuated NF-κB-dependent gene expression, decreased pro-inflammatory cytokine production and up-regulated Nrf2-regulated proteins. Importantly, both CLA nitration and the corresponding downstream anti-inflammatory actions of NO 2 -CLA were recapitulated in a mouse peritonitis model where NO 2 -CLA administration decreased pro-inflammatory cytokines and inhibited leukocyte recruitment. Taken together, our results demonstrate that the formation of NO 2 -CLA has the potential to function as an adaptive response capable of not only modulating inflammation amplitude but also protecting neighboring tissues via the expression of Nrf2-dependent genes. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Protoporphyrinogen oxidase: high affinity tetrahydrophthalimide radioligand for the inhibitor/herbicide-binding site in mouse liver mitochondria.

PubMed

Birchfield, N B; Casida, J E

1996-01-01

Protoporphyrinogen oxidase (protox), the last common enzyme in heme and chlorophyll biosynthesis, is the target of several classes of herbicides acting as inhibitors in both plants and mammals. N-(4-Chloro-2-fluoro-5-(propargyloxy)phenyl)-3,4,5,6-tetrahydro phthalimide (a potent protox inhibitor referred to as THP) was synthesized as a candidate radioligand ([3H]-THP) by selective catalytic reduction of 3,6-dihydrophthalic anhydride (DHPA) with tritium gas followed by condensation in 45% yield with 4-chloro-2-fluoro-5-(propargyloxy)aniline. Insertion of tritium at the 3 and 6 carbons of DHPA as well as the expected 4 and 5 carbons resulted in high specific activity [3H]THP (92 Ci/mmol). This radioligand undergoes rapid, specific, saturable, and reversible binding to the inhibitor/herbicide binding site of the protox component of cholate-solubilized mouse liver mitochondria with an apparent Kd of 0.41 nM and Bmax of 0.40 pmol/mg of protein. In the standard assay, mouse preparation (150 micrograms of protein) and [3H]THP (0.5 nM) are incubated in 500 microL of phosphate buffer at pH 7.2 for 15 min at 25 degrees C followed by addition of ammonium sulfate and filtration with glass fiber filters. The potencies of five nitrodiphenyl ethers and two other herbicides as inhibitors of [3H]THP binding correlate well with those for inhibition of protox activity (r2 = 0.97, n = 7), thus validating the binding assay as relevant to enzyme inhibition. It is also suitable to determine in vivo block as illustrated by an approximately 50% decrease in [3H]THP binding in liver mitochondria from mice treated ip with oxyfluorfen at 4 mg/kg. This is the first report of a binding assay for protox in mammals. The high affinity and specific activity of [3H]THP facilitate quantitation of protox and therefore research on a sensitive inhibition site for porphyrin biosynthesis.

Biosynthesis of CdS nanoparticles in banana peel extract.

PubMed

Zhou, Guang Ju; Li, Shuo Hao; Zhang, Yu Cang; Fu, Yun Zhi

2014-06-01

Cadmium sulfide (CdS) nanoparticles (NPs) were synthesized by using banana peel extract as a convenient, non-toxic, eco-friendly 'green' capping agent. Cadmium nitrate and sodium sulfide are main reagents. A variety of CdS NPs are prepared through changing reaction conditions (banana extracts, the amount of banana peel extract, solution pH, concentration and reactive temperature). The prepared CdS colloid displays strong fluorescence spectrum. X-ray diffraction analysis demonstrates the successful formation of CdS NPs. Fourier transform infra-red (FTIR) spectrogram indicates the involvement of carboxyl, amine and hydroxyl groups in the formation of CdS NPs. Transmission electron microscope (TEM) result reveals that the average size of the NPs is around 1.48 nm.

Recent insights into the Smith-Lemli-Opitz syndrome.

PubMed

Yu, H; Patel, S B

2005-11-01

Recent insights into the Smith-Lemli-Opitz syndrome. The Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive multiple congenital anomaly/mental retardation disorder caused by an inborn error of post-squalene cholesterol biosynthesis. Deficient cholesterol synthesis in SLOS is caused by inherited mutations of 3beta-hydroxysterol-Delta7 reductase gene (DHCR7). DHCR7 deficiency impairs both cholesterol and desmosterol production, resulting in elevated 7DHC/8DHC levels, typically decreased cholesterol levels and, importantly, developmental dysmorphology. The discovery of SLOS has led to new questions regarding the role of the cholesterol biosynthesis pathway in human development. To date, a total of 121 different mutations have been identified in over 250 patients with SLOS who represent a continuum of clinical severity. Two genetic mouse models have been generated which recapitulate some of the developmental abnormalities of SLOS and have been useful in elucidating the pathogenesis. This mini review summarizes the recent insights into SLOS genetics, pathophysiology and potential therapeutic approaches for the treatment of SLOS.

Heme deficiency in erythroid lineage causes differentiation arrest and cytoplasmic iron overload.

PubMed Central

Nakajima, O; Takahashi, S; Harigae, H; Furuyama, K; Hayashi, N; Sassa, S; Yamamoto, M

1999-01-01

Erythroid 5-aminolevulinate synthase (ALAS-E) catalyzes the first step of heme biosynthesis in erythroid cells. Mutation of human ALAS-E causes the disorder X-linked sideroblastic anemia. To examine the roles of heme during hematopoiesis, we disrupted the mouse ALAS-E gene. ALAS-E-null embryos showed no hemoglobinized cells and died by embryonic day 11.5, indicating that ALAS-E is the principal isozyme contributing to erythroid heme biosynthesis. In the ALAS-E-null mutant embryos, erythroid differentiation was arrested, and an abnormal hematopoietic cell fraction emerged that accumulated a large amount of iron diffusely in the cytoplasm. In contrast, we found typical ring sideroblasts that accumulated iron mostly in mitochondria in adult mice chimeric for ALAS-E-null mutant cells, indicating that the mode of iron accumulation caused by the lack of ALAS-E is different in primitive and definitive erythroid cells. These results demonstrate that ALAS-E, and hence heme supply, is necessary for differentiation and iron metabolism of erythroid cells. PMID:10562540

Inhibition of cholesterol biosynthesis through RNF145-dependent ubiquitination of SCAP.

PubMed

Zhang, Li; Rajbhandari, Prashant; Priest, Christina; Sandhu, Jaspreet; Wu, Xiaohui; Temel, Ryan; Castrillo, Antonio; de Aguiar Vallim, Thomas Q; Sallam, Tamer; Tontonoz, Peter

2017-10-25

Cholesterol homeostasis is maintained through concerted action of the SREBPs and LXRs. Here, we report that RNF145, a previously uncharacterized ER membrane ubiquitin ligase, participates in crosstalk between these critical signaling pathways. RNF145 expression is induced in response to LXR activation and high-cholesterol diet feeding. Transduction of RNF145 into mouse liver inhibits the expression of genes involved in cholesterol biosynthesis and reduces plasma cholesterol levels. Conversely, acute suppression of RNF145 via shRNA-mediated knockdown, or chronic inactivation of RNF145 by genetic deletion, potentiates the expression of cholesterol biosynthetic genes and increases cholesterol levels both in liver and plasma. Mechanistic studies show that RNF145 triggers ubiquitination of SCAP on lysine residues within a cytoplasmic loop essential for COPII binding, potentially inhibiting its transport to Golgi and subsequent processing of SREBP-2. These findings define an additional mechanism linking hepatic sterol levels to the reciprocal actions of the SREBP-2 and LXR pathways.

Sphingolipids Are Required for Efficient Triacylglycerol Loss in Conjugated Linoleic Acid Treated Adipocytes

PubMed Central

Wang, Wei; Fromm, Michael

2015-01-01

Conjugated linoleic acid (CLA) reduces adiposity in human and mouse adipocytes. This outcome is achieved through a variety of biological responses including increased energy expenditure and fatty acid oxidation, increased inflammation, repression of fatty acid biosynthesis, attenuated glucose transport, and apoptosis. In the current study, profiling of 261 metabolites was conducted to gain new insights into the biological pathways responding to CLA in 3T3-L1 adipocytes. Sphinganine and sphingosine levels were observed to be highly elevated in CLA treated adipocytes. Exogenous chemicals that increased endogenous ceramide levels decreased lipid levels in adipocytes, and activated AMP-activated protein kinase (AMPK) as well as NF-κB, both of which are typically activated in CLA treated adipocytes. Concurrent inhibition of ceramide de novo biosynthesis and recycling from existing sphingolipid pools attenuated the lipid lowering effect normally associated with responses to CLA, implicating ceramides as an important component of the lipid lowering response in CLA treated adipocytes. PMID:25906159

Biosynthesis and Regulation of Sulfomenaquinone, a Metabolite Associated with Virulence in Mycobacterium tuberculosis.

PubMed

Sogi, Kimberly M; Holsclaw, Cynthia M; Fragiadakis, Gabriela K; Nomura, Daniel K; Leary, Julie A; Bertozzi, Carolyn R

2016-11-11

Sulfomenaquinone (SMK) is a recently identified metabolite that is unique to the Mycobacterium tuberculosis (M. tuberculosis) complex and is shown to modulate its virulence. Here, we report the identification of the SMK biosynthetic operon that, in addition to a previously identified sulfotransferase stf3, includes a putative cytochrome P450 gene (cyp128) and a gene of unknown function, rv2269c. We demonstrate that cyp128 and stf3 are sufficient for the biosynthesis of SMK from menaquinone and rv2269c exhibits promoter activity in M. tuberculosis. Loss of Stf3 expression, but not that of Cyp128, is correlated with elevated levels of menaquinone-9, an essential component in the electron-transport chain in M. tuberculosis. Finally, we showed in a mouse model of infection that the loss of cyp128 exhibits a hypervirulent phenotype similar to that in previous studies of the stf3 mutant. These findings provide a platform for defining the molecular basis of SMK's role in M. tuberculosis pathogenesis.

Radiolabelling and positron emission tomography of PT70, a time-dependent inhibitor of InhA, the Mycobacterium tuberculosis enoyl-ACP reductase

DOE PAGES

Wang, Hui; Liu, Li; Lu, Yang; ...

2015-07-14

PT70 is a diaryl ether inhibitor of InhA, the enoyl-ACP reductase in the Mycobacterium tuberculosis fatty acid biosynthesis pathway. It has a residence time of 24 min on the target, and also shows antibacterial activity in a mouse model of tuberculosis infection. Due to the interest in studying target tissue pharmacokinetics of PT70, we developed a method to radiolabel PT70 with carbon-11 and have studied its pharmacokinetics in mice and baboons using positron emission tomography.

Radiolabelling and positron emission tomography of PT70, a time-dependent inhibitor of InhA, the Mycobacterium tuberculosis enoyl-ACP reductase

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

Wang, Hui; Liu, Li; Lu, Yang

PT70 is a diaryl ether inhibitor of InhA, the enoyl-ACP reductase in the Mycobacterium tuberculosis fatty acid biosynthesis pathway. It has a residence time of 24 min on the target, and also shows antibacterial activity in a mouse model of tuberculosis infection. Due to the interest in studying target tissue pharmacokinetics of PT70, we developed a method to radiolabel PT70 with carbon-11 and have studied its pharmacokinetics in mice and baboons using positron emission tomography.

Antidiabetic actions of a phosphatidylcholine ligand for nuclear receptor LRH-1

PubMed Central

Lee, Jae Man; Lee, Yoon Kwang; Mamrosh, Jennifer L.; Busby, Scott A.; Griffin, Patrick R.; Pathak, Manish C.; Ortlund, Eric A.; Moore, David D.

2011-01-01

Nuclear hormone receptors regulate diverse metabolic pathways and the orphan nuclear receptor LRH-1 (NR5A2) regulates bile acid biosynthesis1,2. Structural studies have identified phospholipids as potential LRH-1 ligands3–5, but their functional relevance is unclear. Here we show that an unusual phosphatidylcholine species with two saturated 12 carbon fatty acid acyl side chains (dilauroyl phosphatidylcholine, DLPC) is an LRH-1 agonist ligand in vitro. DLPC treatment induces bile acid biosynthetic enzymes in mouse liver, increases bile acid levels, and lowers hepatic triglycerides and serum glucose. DLPC treatment also decreases hepatic steatosis and improves glucose homeostasis in two mouse models of insulin resistance. Both the antidiabetic and lipotropic effects are lost in liver specific Lrh-1 knockouts. These findings identify an LRH-1 dependent phosphatidylcholine signaling pathway that regulates bile acid metabolism and glucose homeostasis. PMID:21614002

Metabolic drift in the aging brain.

PubMed

Ivanisevic, Julijana; Stauch, Kelly L; Petrascheck, Michael; Benton, H Paul; Epstein, Adrian A; Fang, Mingliang; Gorantla, Santhi; Tran, Minerva; Hoang, Linh; Kurczy, Michael E; Boska, Michael D; Gendelman, Howard E; Fox, Howard S; Siuzdak, Gary

2016-05-01

Brain function is highly dependent upon controlled energy metabolism whose loss heralds cognitive impairments. This is particularly notable in the aged individuals and in age-related neurodegenerative diseases. However, how metabolic homeostasis is disrupted in the aging brain is still poorly understood. Here we performed global, metabolomic and proteomic analyses across different anatomical regions of mouse brain at different stages of its adult lifespan. Interestingly, while severe proteomic imbalance was absent, global-untargeted metabolomics revealed an energymetabolic drift or significant imbalance in core metabolite levels in aged mouse brains. Metabolic imbalance was characterized by compromised cellular energy status (NAD decline, increased AMP/ATP, purine/pyrimidine accumulation) and significantly altered oxidative phosphorylation and nucleotide biosynthesis and degradation. The central energy metabolic drift suggests a failure of the cellular machinery to restore metabostasis (metabolite homeostasis) in the aged brain and therefore an inability to respond properly to external stimuli, likely driving the alterations in signaling activity and thus in neuronal function and communication.

A proteomics study of hyperhomocysteinemia injury of the hippocampal neurons using iTRAQ.

PubMed

Fang, Min; Wang, Jing; Yan, Han; Zhao, Yan-Xin; Liu, Xue-Yuan

2014-11-01

High levels of homocysteine, caused by abnormal methionine metabolism, can induce degeneration of mouse hippocampal neurons. iTRAQ™ technology has been widely used in the field of proteomics research and through employing this technology, the present study identified that hyperhomocysteinemia induced the downregulation of 52 proteins and upregulation of 44 proteins in the mouse hippocampus. Through gene ontology and pathway analysis, the upregulation of components of the cytoskeleton, actin, regulators of focal adhesion, calcium signaling pathways, tight junctions, ErbB and gonadotrophin‑releasing hormone signaling, leukocyte, transendothelial migration, propanoate and pyruvate metabolism, valine, leucine and isoleucine biosynthesis, synthesis and degradation of ketone bodies and benzoate degradation via CoA ligation pathway, was identified. It was additionally verified that tau protein was highly expressed in the hyperhomocysteinemic neurons. Further analysis revealed that tau network proteins played functional roles in homocysteine‑induced neuronal damage.

Cannabidiol protects against hepatic ischemia/reperfusion injury by attenuating inflammatory signaling and response, oxidative/nitrative stress, and cell death

PubMed Central

Mukhopadhyay, Partha; Rajesh, Mohanraj; Horváth, Béla; Bátkai, Sándor; Park, Ogyi; Tanashian, Galin; Gao, Rachel Y; Patel, Vivek; Wink, David A.; Liaudet, Lucas; Haskó, György; Mechoulam, Raphael; Pacher, Pál

2011-01-01

Ischemia-reperfusion (I/R) is a pivotal mechanism of liver damage following liver transplantation or hepatic surgery. We have investigated the effects of cannabidiol(CBD), the non-psychotropic constituent of marijuana, in a mouse model of hepatic I/R injury. I/R triggered time-dependent increases/changes in markers of liver injury (serum transaminases), hepatic oxidative/nitrative stress (4-hydroxy-2-nonenal, nitrotyrosine content/staining, gp91phox and inducible nitric oxide synthase mRNA), mitochondrial dysfunction (decreased complex I activity), inflammation (tumor necrosis factor alpha (TNF-α), cyclooxygenase 2, macrophage inflammatory protein-1α/2, inter-cellular adhesion molecule 1 mRNA levels, tissue neutrophil infiltration, nuclear factor kappa B (NF-KB) activation), stress signaling (p38MAPK and JNK) and cell death (DNA fragmentation, PARP activity, and TUNEL). CBD significantly reduced the extent of liver inflammation, oxidative/nitrative stress and cell death, and also attenuated the bacterial endotoxin-triggered NF-KB activation and TNF-α production in isolated Kupffer cells, likewise the adhesion molecules expression in primary human liver sinusoidal endothelial cells stimulated with TNF-α, and attachment of human neutrophils to the activated endothelium. These protective effects were preserved in CB2 knockout mice and were not prevented by CB1/2 antagonists in vitro. Thus, CBD may represent a novel, protective strategy against I/R injury by attenuating key inflammatory pathways and oxidative/nitrative tissue injury, independent from classical CB1/2 receptors. PMID:21362471

Odorant Sensory Input Modulates DNA Secondary Structure Formation and Heterogeneous Ribonucleoprotein Recruitment on the Tyrosine Hydroxylase and Glutamic Acid Decarboxylase 1 Promoters in the Olfactory Bulb.

PubMed

Wang, Meng; Cai, Elizabeth; Fujiwara, Nana; Fones, Lilah; Brown, Elizabeth; Yanagawa, Yuchio; Cave, John W

2017-05-03

Adaptation of neural circuits to changes in sensory input can modify several cellular processes within neurons, including neurotransmitter biosynthesis levels. For a subset of olfactory bulb interneurons, activity-dependent changes in GABA are reflected by corresponding changes in Glutamate decarboxylase 1 ( Gad1 ) expression levels. Mechanisms regulating Gad1 promoter activity are poorly understood, but here we show that a conserved G:C-rich region in the mouse Gad1 proximal promoter region both recruits heterogeneous nuclear ribonucleoproteins (hnRNPs) that facilitate transcription and forms single-stranded DNA secondary structures associated with transcriptional repression. This promoter architecture and function is shared with Tyrosine hydroxylase ( Th ), which is also modulated by odorant-dependent activity in the olfactory bulb. This study shows that the balance between DNA secondary structure formation and hnRNP binding on the mouse Th and Gad1 promoters in the olfactory bulb is responsive to changes in odorant-dependent sensory input. These findings reveal that Th and Gad1 share a novel transcription regulatory mechanism that facilitates sensory input-dependent regulation of dopamine and GABA expression. SIGNIFICANCE STATEMENT Adaptation of neural circuits to changes in sensory input can modify several cellular processes within neurons, including neurotransmitter biosynthesis levels. This study shows that transcription of genes encoding rate-limiting enzymes for GABA and dopamine biosynthesis ( Gad1 and Th , respectively) in the mammalian olfactory bulb is regulated by G:C-rich regions that both recruit heterogeneous nuclear ribonucleoproteins (hnRNPs) to facilitate transcription and form single-stranded DNA secondary structures associated with repression. hnRNP binding and formation of DNA secondary structure on the Th and Gad1 promoters are mutually exclusive, and odorant sensory input levels regulate the balance between these regulatory features. These findings reveal that Th and Gad1 share a transcription regulatory mechanism that facilitates odorant-dependent regulation of dopamine and GABA expression levels. Copyright © 2017 the authors 0270-6474/17/374778-12$15.00/0.

Chemical Genetic Analysis and Functional Characterization of Staphylococcal Wall Teichoic Acid 2-Epimerases Reveals Unconventional Antibiotic Drug Targets.

PubMed

Mann, Paul A; Müller, Anna; Wolff, Kerstin A; Fischmann, Thierry; Wang, Hao; Reed, Patricia; Hou, Yan; Li, Wenjin; Müller, Christa E; Xiao, Jianying; Murgolo, Nicholas; Sher, Xinwei; Mayhood, Todd; Sheth, Payal R; Mirza, Asra; Labroli, Marc; Xiao, Li; McCoy, Mark; Gill, Charles J; Pinho, Mariana G; Schneider, Tanja; Roemer, Terry

2016-05-01

Here we describe a chemical biology strategy performed in Staphylococcus aureus and Staphylococcus epidermidis to identify MnaA, a 2-epimerase that we demonstrate interconverts UDP-GlcNAc and UDP-ManNAc to modulate substrate levels of TarO and TarA wall teichoic acid (WTA) biosynthesis enzymes. Genetic inactivation of mnaA results in complete loss of WTA and dramatic in vitro β-lactam hypersensitivity in methicillin-resistant S. aureus (MRSA) and S. epidermidis (MRSE). Likewise, the β-lactam antibiotic imipenem exhibits restored bactericidal activity against mnaA mutants in vitro and concomitant efficacy against 2-epimerase defective strains in a mouse thigh model of MRSA and MRSE infection. Interestingly, whereas MnaA serves as the sole 2-epimerase required for WTA biosynthesis in S. epidermidis, MnaA and Cap5P provide compensatory WTA functional roles in S. aureus. We also demonstrate that MnaA and other enzymes of WTA biosynthesis are required for biofilm formation in MRSA and MRSE. We further determine the 1.9Å crystal structure of S. aureus MnaA and identify critical residues for enzymatic dimerization, stability, and substrate binding. Finally, the natural product antibiotic tunicamycin is shown to physically bind MnaA and Cap5P and inhibit 2-epimerase activity, demonstrating that it inhibits a previously unanticipated step in WTA biosynthesis. In summary, MnaA serves as a new Staphylococcal antibiotic target with cognate inhibitors predicted to possess dual therapeutic benefit: as combination agents to restore β-lactam efficacy against MRSA and MRSE and as non-bioactive prophylactic agents to prevent Staphylococcal biofilm formation.

Metabolic Network for the Biosynthesis of Intra- and Extracellular α-Glucans Required for Virulence of Mycobacterium tuberculosis

PubMed Central

van de Weerd, Robert; Chandra, Govind; Appelmelk, Ben; Alber, Marina; Ioerger, Thomas R.; Jacobs, William R.; Geurtsen, Jeroen; Bornemann, Stephen

2016-01-01

Mycobacterium tuberculosis synthesizes intra- and extracellular α-glucans that were believed to originate from separate pathways. The extracellular glucose polymer is the main constituent of the mycobacterial capsule that is thought to be involved in immune evasion and virulence. However, the role of the α-glucan capsule in pathogenesis has remained enigmatic due to an incomplete understanding of α-glucan biosynthetic pathways preventing the generation of capsule-deficient mutants. Three separate and potentially redundant pathways had been implicated in α-glucan biosynthesis in mycobacteria: the GlgC-GlgA, the Rv3032 and the TreS-Pep2-GlgE pathways. We now show that α-glucan in mycobacteria is exclusively assembled intracellularly utilizing the building block α-maltose-1-phosphate as the substrate for the maltosyltransferase GlgE, with subsequent branching of the polymer by the branching enzyme GlgB. Some α-glucan is exported to form the α-glucan capsule. There is an unexpected convergence of the TreS-Pep2 and GlgC-GlgA pathways that both generate α-maltose-1-phosphate. While the TreS-Pep2 route from trehalose was already known, we have now established that GlgA forms this phosphosugar from ADP-glucose and glucose 1-phosphate 1000-fold more efficiently than its hitherto described glycogen synthase activity. The two routes are connected by the common precursor ADP-glucose, allowing compensatory flux from one route to the other. Having elucidated this unexpected configuration of the metabolic pathways underlying α-glucan biosynthesis in mycobacteria, an M. tuberculosis double mutant devoid of α-glucan could be constructed, showing a direct link between the GlgE pathway, α-glucan biosynthesis and virulence in a mouse infection model. PMID:27513637

From masochistic enzymology to mechanistic physiology and disease.

PubMed

Vance, Dennis E

2017-10-20

The pioneering work of Eugene Kennedy in the 1950s established the choline pathway for phosphatidylcholine (PC) biosynthesis. However, the regulation of PC biosynthesis was poorly understood at that time. When I started my lab at the University of British Columbia in the 1970s, this was the focus of my research. This article provides my reflections on these studies that began with enzymology and the use of cultured mammalian cells, and progressed to utilize the techniques of molecular biology and gene-targeted mice. The research in my lab and others demonstrated that the regulated and rate-limiting step in the choline pathway for PC biosynthesis was catalyzed by CTP:phosphocholine cytidylyltransferase. This enzyme is regulated by its movement from a soluble form (largely in the nucleus) to a membrane-associated form where the enzyme becomes activated. Gene targeting in mice subsequently demonstrated that this gene is essential for development of mouse embryos. The other mammalian pathway for PC biosynthesis is catalyzed by phosphatidylethanolamine N -methyltransferase (PEMT) that converts phosphatidylethanolamine to PC. Understanding of the regulation and function of the integral membrane protein PEMT was improved when the enzyme was purified (a masochistic endeavor) in 1987, leading to the cloning of the Pemt cDNA. Generation of knock-out mice that lacked PEMT showed that they were protected from atherosclerosis, diet-induced obesity, and insulin resistance. The protection from atherosclerosis appears to be due to decreased secretion of lipoproteins from the liver. We continue to investigate the mechanism(s) by which Pemt -/- mice are protected from weight gain and insulin resistance. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Chemical Genetic Analysis and Functional Characterization of Staphylococcal Wall Teichoic Acid 2-Epimerases Reveals Unconventional Antibiotic Drug Targets

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

Mann, Paul A.; Müller, Anna; Wolff, Kerstin A.

Here we describe a chemical biology strategy performed in Staphylococcus aureus and Staphylococcus epidermidis to identify MnaA, a 2-epimerase that we demonstrate interconverts UDP-GlcNAc and UDP-ManNAc to modulate substrate levels of TarO and TarA wall teichoic acid (WTA) biosynthesis enzymes. Genetic inactivation of mnaA results in complete loss of WTA and dramatic in vitro β-lactam hypersensitivity in methicillin-resistant S. aureus (MRSA) and S. epidermidis (MRSE). Likewise, the β-lactam antibiotic imipenem exhibits restored bactericidal activity against mnaA mutants in vitro and concomitant efficacy against 2-epimerase defective strains in a mouse thigh model of MRSA and MRSE infection. Interestingly, whereas MnaA servesmore » as the sole 2-epimerase required for WTA biosynthesis in S. epidermidis, MnaA and Cap5P provide compensatory WTA functional roles in S. aureus. We also demonstrate that MnaA and other enzymes of WTA biosynthesis are required for biofilm formation in MRSA and MRSE. We further determine the 1.9Å crystal structure of S. aureus MnaA and identify critical residues for enzymatic dimerization, stability, and substrate binding. Finally, the natural product antibiotic tunicamycin is shown to physically bind MnaA and Cap5P and inhibit 2-epimerase activity, demonstrating that it inhibits a previously unanticipated step in WTA biosynthesis. In summary, MnaA serves as a new Staphylococcal antibiotic target with cognate inhibitors predicted to possess dual therapeutic benefit: as combination agents to restore β-lactam efficacy against MRSA and MRSE and as non-bioactive prophylactic agents to prevent Staphylococcal biofilm formation.« less

Insights into glycogen metabolism in Lactobacillus acidophilus: impact on carbohydrate metabolism, stress tolerance and gut retention.

PubMed

Goh, Yong Jun; Klaenhammer, Todd R

2014-11-20

In prokaryotic species equipped with glycogen metabolism machinery, the co-regulation of glycogen biosynthesis and degradation has been associated with the synthesis of energy storage compounds and various crucial physiological functions, including global cellular processes such as carbon and nitrogen metabolism, energy sensing and production, stress response and cell-cell communication. In addition, the glycogen metabolic pathway was proposed to serve as a carbon capacitor that regulates downstream carbon fluxes, and in some microorganisms the ability to synthesize intracellular glycogen has been implicated in host persistence. Among lactobacilli, complete glycogen metabolic pathway genes are present only in select species predominantly associated with mammalian hosts or natural environments. This observation highlights the potential involvement of glycogen biosynthesis in probiotic activities and persistence of intestinal lactobacilli in the human gastrointestinal tract. In this review, we summarize recent findings on (i) the presence and potential ecological distribution of glycogen metabolic pathways among lactobacilli, (ii) influence of carbon substrates and growth phases on glycogen metabolic gene expression and glycogen accumulation in L. acidophilus, and (iii) the involvement of glycogen metabolism on growth, sugar utilization and bile tolerance. Our present in vivo studies established the significance of glycogen biosynthesis on the competitive retention of L. acidophilus in the mouse intestinal tract, demonstrating for the first time that the ability to synthesize intracellular glycogen contributes to gut fitness and retention among probiotic microorganisms.

Role of glutathione biosynthesis in endothelial dysfunction and fibrosis.

PubMed

Espinosa-Díez, Cristina; Miguel, Verónica; Vallejo, Susana; Sánchez, Francisco J; Sandoval, Elena; Blanco, Eva; Cannata, Pablo; Peiró, Concepción; Sánchez-Ferrer, Carlos F; Lamas, Santiago

2018-04-01

Glutathione (GSH) biosynthesis is essential for cellular redox homeostasis and antioxidant defense. The rate-limiting step requires glutamate-cysteine ligase (GCL), which is composed of the catalytic (GCLc) and the modulatory (GCLm) subunits. To evaluate the contribution of GCLc to endothelial function we generated an endothelial-specific Gclc haplo-insufficient mouse model (Gclc e/+ mice). In murine lung endothelial cells (MLEC) derived from these mice we observed a 50% reduction in GCLc levels compared to lung fibroblasts from the same mice. MLEC obtained from haplo-insufficient mice showed significant reduction in GSH levels as well as increased basal and stimulated ROS levels, reduced phosphorylation of eNOS (Ser 1177) and increased eNOS S-glutathionylation, compared to MLEC from wild type (WT) mice. Studies in mesenteric arteries demonstrated impaired endothelium-dependent vasodilation in Gclc(e/+) male mice, which was corrected by pre-incubation with GSH-ethyl-ester and BH 4 . To study the contribution of endothelial GSH synthesis to renal fibrosis we employed the unilateral ureteral obstruction model in WT and Gclc(e/+) mice. We observed that obstructed kidneys from Gclc(e/+) mice exhibited increased deposition of fibrotic markers and reduced Nrf2 levels. We conclude that the preservation of endothelial GSH biosynthesis is not only critical for endothelial function but also in anti-fibrotic responses. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Sequential induction of prostaglandin E and D synthases in inflammation

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

Schuligoi, Rufina; Grill, Magdalena; Heinemann, Akos

Enhanced biosynthesis of prostaglandin (PG)D{sub 2} and subsequent formation of 15-deoxy-{delta}{sup 12,14}-PGJ{sub 2} has been suggested to contribute to resolution of inflammation. The primary aim of the present study in mouse heart was, therefore, to determine at the transcriptional level if there is sequential induction of PGE and PGD synthases (S) during inflammation. Expression of interleukin (IL)-1{beta} in heart was enhanced 4 h after systemic inflammation and declined thereafter within 3-5 days to basal levels. In contrast to cyclooxygenase-2 and membrane-bound (m)-PGES-1, which both peaked 4 h after endotoxin administration, hematopoietic (H)-PGDS expression was enhanced only 48 h after endotoxin.more » The expression of lipocalin-type (L)-PGDS was not significantly influenced. mRNA encoding the putative target of 15-deoxy-{delta}{sup 12,14}-PGJ{sub 2}, peroxisome proliferator-activated receptor {gamma}, was enhanced between 4 and 24 h after induction of inflammation. Treatment of mice with acetylsalicylic acid or indomethacin at doses effective to cause near-complete inhibition of PGE{sub 2} and PGD{sub 2} biosynthesis in heart ex vivo resulted in enhanced expression of IL-1{beta} 24 h after endotoxin administration. These results provide additional support for the hypothesis of a shift towards PGD{sub 2} biosynthesis during resolution of inflammation.« less

Antioxidant supplementation ameliorates molecular deficits in Smith-Lemli-Opitz Syndrome (SLOS)

PubMed Central

Korade, Zeljka; Xu, Libin; Harrison, Fiona E.; Ahsen, Refayat; Hart, Sarah E; Folkes, Oakleigh M; Mirnics, Karoly; Porter, Ned A

2013-01-01

Background Smith-Lemli-Opitz syndrome (SLOS) is an inborn error of cholesterol biosynthesis characterized by diminished cholesterol and increased 7-dehydrocholesterol (7-DHC) levels. 7-DHC is highly reactive, giving rise to biologically active oxysterols. Methods 7-DHC-derived oxysterols were measured in fibroblasts from SLOS patients and an in vivo SLOS rodent model using HPLC-MS-MS. Expression of lipid biosynthesis genes was ascertained by qPCR and Western blot. The effects of an antioxidant mixture, vitamin A, coenzyme Q10, vitamin C and vitamin E were evaluated for their potential to reduce formation of 7-DHC oxysterols in fibroblast from SLOS patients. Finally, the effect of maternal feeding of vitamin E enriched diet was ascertained in the brain and liver of newborn SLOS mice. Results In cultured human SLOS fibroblasts the antioxidant mixture led to decreased levels of the 7-DHC-derived oxysterol, DHCEO. Furthermore, gene expression changes in SLOS human fibroblasts were normalized with antioxidant treatment. The active ingredient appeared to be vitamin E, as even at low concentrations, it significantly decreased DHCEO levels. In addition, analyzing a mouse SLOS model revealed that feeding a vitamin E enriched diet to pregnant females led to a decrease in oxysterol formation in brain and liver tissues of the newborn Dhcr7-knockout pups. Conclusions Considering the adverse effects of 7-DHC-derived oxysterols in neuronal and glial cultures, and the positive effects of antioxidants in patient cell cultures and the transgenic mouse model, we believe that preventing formation of 7-DHC oxysterols is critical for countering the detrimental effects of Dhcr7 mutations. PMID:23896203

Mechanism of bisphenol AF-induced progesterone inhibition in human chorionic gonadotrophin-stimulated mouse Leydig tumor cell line (mLTC-1) cells.

PubMed

Feng, Yixing; Shi, Jiachen; Jiao, Zhihao; Duan, Hejun; Shao, Bing

2018-06-01

Bisphenol AF (BPAF) has been shown to inhibit testicular steroidogenesis in male rats. However, the precise mechanisms related to the toxic effects of BPAF on reproduction remain poorly understood. In the present study, a mouse Leydig tumor cell line (mLTC-1) was used as a model to investigate the mechanism of steroidogenic inhibition and to identify the molecular target of BPAF. Levels of progesterone and the concentration of cyclic adenosine monophosphate (cAMP) in cells exposed to BPAF were detected, and expression of key genes and proteins in steroid biosynthesis was assessed. The results showed that BPAF exposure decreased human chorionic gonadotrophin (hCG)-stimulated progesterone production in a dose-dependent manner. The 24-h IC 50 (half maximal inhibitory concentration) value for BPAF regarding progesterone production was 70.2 µM. A dramatic decrease in cellular cAMP concentration was also observed. Furthermore, BPAF exposure inhibited expression of genes and proteins involved in cholesterol transport and progesterone biosynthesis. Conversely, the protein levels of steroidogenic acute regulatory protein (StAR) were not altered, and those of progesterone were still decreased upon 22R-hydroxycholesterol treatment of cells exposed to higher doses of BPAF. Together, these data indicate that BPAF exposure inhibits progesterone secretion in hCG-stimulated mLTC-1 cells by reducing expression of scavenger receptor class B type I (SR-B1) and cytochrome P450 (P450scc) due to the adverse effects of cAMP. However, StAR might not be the molecular target in this process. © 2018 Wiley Periodicals, Inc.

Characterization of mouse and human GTP cyclohydrolase I genes. Mutations in patients with GTP cyclohydrolase I deficiency.

PubMed

Ichinose, H; Ohye, T; Matsuda, Y; Hori, T; Blau, N; Burlina, A; Rouse, B; Matalon, R; Fujita, K; Nagatsu, T

1995-04-28

GTP cyclohydrolase I is the first and rate-limiting enzyme for the biosynthesis of tetrahydrobiopterin in mammals. Previously, we reported three species of human GTP cyclohydrolase I cDNA in a human liver cDNA library (Togari, A., Ichinose, H., Matsumoto, S., Fujita, K., and Nagatsu, T. (1992) Biochem. Biophys. Res. Commun. 187, 359-365). Furthermore, very recently, we found that the GTP cyclohydrolase I gene is causative for hereditary progressive dystonia with marked diurnal fluctuation, also known as DOPA-responsive dystonia (Ichinose, H., Ohye, T., Takahashi, E., Seki, N., Hori, T., Segawa, M., Nomura, Y., Endo, K., Tanaka, H., Tsuji, S., Fujita, K., and Nagatsu, T. (1994) Nature Genetics 8, 236-242). To clarify the mechanisms that regulate transcription of the GTP cyclohydrolase I gene and to generate multiple species of mRNA, we isolated genomic DNA clones for the human and mouse GTP cyclohydrolase I genes. Structural analysis of the isolated clones revealed that the GTP cyclohydrolase I gene is encoded by a single copy gene and is composed of six exons spanning approximately 30 kilobases. We sequenced all exon/intron boundaries of the human and mouse genes. Structural analysis also demonstrated that the heterogeneity of GTP cyclohydrolase I mRNA is caused by an alternative usage of the splicing acceptor site at the sixth exon. The transcription start site of the mouse GTP cyclohydrolase I gene and the 5'-flanking sequences of the mouse and human genes were determined. We performed regional mapping of the mouse gene by fluorescence in situ hybridization, and the mouse GTP cyclohydrolase I gene was assigned to region C2-3 of mouse chromosome 14. We identified missense mutations in patients with GTP cyclohydrolase I deficiency and expressed mutated enzymes in Escherichia coli to confirm alterations in the enzyme activity.

Identification of N-Acyl Phosphatidylserine Molecules in Eukaryotic Cells

PubMed Central

Guan, Ziqiang; Li, Shengrong; Smith, Dale C.; Shaw, Walter A.; Raetz, Christian R. H.

2008-01-01

While profiling the lipidome of the mouse brain by mass spectrometry, we discovered a novel family of N-acyl phosphatidylserine (N-acyl-PS) molecules. These N-acyl-PS species were enriched by DEAE-cellulose column chromatography, and they were then characterized by accurate mass measurements, tandem mass spectrometry, liquid chromatography/mass spectrometry, and comparison to an authentic standard. Mouse brain N-acyl-PS molecules are heterogeneous and constitute about 0.1 % of the total lipid. In addition to various ester-linked fatty acyl chains on their glycerol backbones, the complexity of the N-acyl-PS series is further increased by the presence of diverse amide-linked N-acyl chains, which include saturated, mono-unsaturated and poly-unsaturated species. N-acyl-PS molecular species were also detected in the lipids of pig brain, mouse RAW264.7 macrophage tumor cells and yeast, but not E. coli. N-acyl-PSs may be biosynthetic precursors of N-acyl serine molecules, such as the recently reported signaling lipid N-arachidonoyl serine from bovine brain. We suggest that a phospholipase D might cleave N-acyl-PS to generate N-acyl serine, in analogy to the biosynthesis of the endocannabinoid N-arachidonoyl ethanolamine (anadamide) from N-arachidonoyl phosphatidylethanolamine. PMID:18031065

In vivo metabolic labeling of sialoglycans in the mouse brain by using a liposome-assisted bioorthogonal reporter strategy

PubMed Central

Xie, Ran; Dong, Lu; Du, Yifei; Zhu, Yuntao; Hua, Rui; Zhang, Chen; Chen, Xing

2016-01-01

Mammalian brains are highly enriched with sialoglycans, which have been implicated in brain development and disease progression. However, in vivo labeling and visualization of sialoglycans in the mouse brain remain a challenge because of the blood−brain barrier. Here we introduce a liposome-assisted bioorthogonal reporter (LABOR) strategy for shuttling 9-azido sialic acid (9AzSia), a sialic acid reporter, into the brain to metabolically label sialoglycoconjugates, including sialylated glycoproteins and glycolipids. Subsequent bioorthogonal conjugation of the incorporated 9AzSia with fluorescent probes via click chemistry enabled fluorescence imaging of brain sialoglycans in living animals and in brain sections. Newly synthesized sialoglycans were found to widely distribute on neuronal cell surfaces, in particular at synaptic sites. Furthermore, large-scale proteomic profiling identified 140 brain sialylated glycoproteins, including a wealth of synapse-associated proteins. Finally, by performing a pulse−chase experiment, we showed that dynamic sialylation is spatially regulated, and that turnover of sialoglycans in the hippocampus is significantly slower than that in other brain regions. The LABOR strategy provides a means to directly visualize and monitor the sialoglycan biosynthesis in the mouse brain and will facilitate elucidating the functional role of brain sialylation. PMID:27125855

Cardiac-specific overexpression of catalase prevents diabetes-induced pathological changes by inhibiting NF-κB signaling activation in the heart.

PubMed

Cong, Weitao; Ruan, Dandan; Xuan, Yuanhu; Niu, Chao; Tao, Youli; Wang, Yang; Zhan, Kungao; Cai, Lu; Jin, Litai; Tan, Yi

2015-12-01

Catalase is an antioxidant enzyme that specifically catabolizes hydrogen peroxide (H2O2). Overexpression of catalase via a heart-specific promoter (CAT-TG) was reported to reduce diabetes-induced accumulation of reactive oxygen species (ROS) and further prevent diabetes-induced pathological abnormalities, including cardiac structural derangement and left ventricular abnormity in mice. However, the mechanism by which catalase overexpression protects heart function remains unclear. This study found that activation of a ROS-dependent NF-κB signaling pathway was downregulated in hearts of diabetic mice overexpressing catalase. In addition, catalase overexpression inhibited the significant increase in nitration levels of key enzymes involved in energy metabolism, including α-oxoglutarate dehydrogenase E1 component (α-KGD) and ATP synthase α and β subunits (ATP-α and ATP-β). To assess the effects of the NF-κB pathway activation on heart function, Bay11-7082, an inhibitor of the NF-κB signaling pathway, was injected into diabetic mice, protecting mice against the development of cardiac damage and increased nitrative modifications of key enzymes involved in energy metabolism. In conclusion, these findings demonstrated that catalase protects mouse hearts against diabetic cardiomyopathy, partially by suppressing NF-κB-dependent inflammatory responses and associated protein nitration. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

Abel, E.L.; Boulware, S.; Fields, T.

Mustard gas, used in chemical warfare since 1917, is a mutagenic and carcinogenic agent that produces severe dermal lesions for which there are no effective therapeutics; it is currently seen as a potential terrorist threat to civilian populations. Sulforaphane, found in cruciferous vegetables, is known to induce enzymes that detoxify compounds such as the sulfur mustards that react through electrophilic intermediates. Here, we observe that a single topical treatment with sulforaphane induces mouse epidermal levels of the regulatory subunit of glutamate-cysteine ligase, the rate-limiting enzyme in glutathione biosynthesis, and also increases epidermal levels of reduced glutathione. Furthermore, a glutathione S-transferase,more » GSTA4, is also induced in mouse skin by sulforaphane. In an in vivo model in which mice are given a single mutagenic application of the sulfur mustard analog 2-(chloroethyl) ethyl sulfide (CEES), we now show that therapeutic treatment with sulforaphane abolishes the CEES-induced increase in mutation frequency in the skin, measured four days after exposure. Sulforaphane, a natural product currently in clinical trials, shows promise as an effective therapeutic against mustard gas. -- Highlights: ► Sulforaphane induces increased levels of glutathione in mouse skin. ► Sulforaphane induces increased levels of GSTA4 in mouse skin. ► Sulforaphane, applied after CEES-treatment, completely abolishes CEES-mutagenesis. ► The therapeutic effect may suggest a long biological half-life for CEES in vivo.« less

Biosynthesis of silver nanoparticles using Bacillus subtilis EWP-46 cell-free extract and evaluation of its antibacterial activity.

PubMed

Velmurugan, Palanivel; Iydroose, Mahudunan; Mohideen, Mohmed Hanifa Abdul Kader; Mohan, Thankiah Selva; Cho, Min; Oh, Byung-Taek

2014-08-01

This study highlights the ability of nitrate-reducing Bacillus subtilis EWP-46 cell-free extract used for preparation of silver nanoparticles (AgNPs) by reduction of silver ions into nano silver. The production of AgNPs was optimized with several parameters such as hydrogen ion concentration, temperature, silver ion (Ag(+) ion) and time. The maximum AgNPs production was achieved at pH 10.0, temperature 60 °C, 1.0 mM Ag(+) ion and 720 min. The UV-Vis spectrum showed surface plasmon resonance peak at 420 nm, energy-dispersive X-ray spectroscopy (SEM-EDX) spectra showed the presence of element silver in pure form. Atomic force microscopy (AFM) and transmission electron microscopy images illustrated the nanoparticle size, shape, and average particle size ranging from 10 to 20 nm. Fourier transform infrared spectroscopy provided the evidence for the presence of biomolecules responsible for the reduction of silver ion, and X-ray diffraction analysis confirmed that the obtained nanoparticles were in crystalline form. SDS-PAGE was performed to identify the proteins and its molecular mass in the purified nitrate reductase from the cell-free extract. In addition, the minimum inhibitory concentration and minimum bactericidal concentration of AgNPs were investigated against gram-negative (Pseudomonas fluorescens) and gram-positive (Staphylococcus aureus) bacteria.

A-Type Carrier Protein ErpA Is Essential for Formation of an Active Formate-Nitrate Respiratory Pathway in Escherichia coli K-12

PubMed Central

Pinske, Constanze

2012-01-01

A-type carrier (ATC) proteins of the Isc (iron-sulfur cluster) and Suf (sulfur mobilization) iron-sulfur ([Fe-S]) cluster biogenesis pathways are proposed to traffic preformed [Fe-S] clusters to apoprotein targets. In this study, we analyzed the roles of the ATC proteins ErpA, IscA, and SufA in the maturation of the nitrate-inducible, multisubunit anaerobic respiratory enzymes formate dehydrogenase N (Fdh-N) and nitrate reductase (Nar). Mutants lacking SufA had enhanced activities of both enzymes. While both Fdh-N and Nar activities were strongly reduced in an iscA mutant, both enzymes were inactive in an erpA mutant and in a mutant unable to synthesize the [Fe-S] cluster scaffold protein IscU. It could be shown for both Fdh-N and Nar that loss of enzyme activity correlated with absence of the [Fe-S] cluster-containing small subunit. Moreover, a slowly migrating form of the catalytic subunit FdnG of Fdh-N was observed, consistent with impeded twin arginine translocation (TAT)-dependent transport. The highly related Fdh-O enzyme was also inactive in the erpA mutant. Although the Nar enzyme has its catalytic subunit NarG localized in the cytoplasm, it also exhibited aberrant migration in an erpA iscA mutant, suggesting that these modular enzymes lack catalytic integrity due to impaired cofactor biosynthesis. Cross-complementation experiments demonstrated that multicopy IscA could partially compensate for lack of ErpA with respect to Fdh-N activity but not Nar activity. These findings suggest that ErpA and IscA have overlapping roles in assembly of these anaerobic respiratory enzymes but demonstrate that ErpA is essential for the production of active enzymes. PMID:22081393

Magnetic resonance imaging of the submandibular-sublingual complex.

PubMed

Sbarbati, A; Baldassarri, A; Leclercq, F; Merigo, F; Antonakis, K; Boicelli, A

1994-01-01

The submandibular-sublingual complex (SSC) was studied in vivo by magnetic resonance imaging (MRI) at 4.7 and 7.05 Tesla in rat and mouse. A correlation was found between histology and MRI signal. The mainly mucous sublingual gland emitted a more intense signal than the mainly serous submandibular gland. Ventral to the glands, cutis, subcutaneous adipose tissue and two planes of muscular tissue separated by connective laminae were visible in vivo. Autopsy and histology confirmed the in vivo description provided by MRI. The reactivity of the salivary system after pharmacological stimulation was studied in mice at 7.05 Tesla. Stimulation of salivary secretion by pilocarpine nitrate injected in the subcutaneous space ventrally to the SSC resulted in an augmentation of the salivary liquid visible in the oral cavity by MRI. The diffusion of pilocarpine nitrate in the connective tissue located ventrally the SSC and in the glandular parenchyma was also followed in vivo. These results show that MRI is a potentially useful tool for studying the salivary glands in vivo.

NAMPT-Mediated NAD(+) Biosynthesis Is Essential for Vision In Mice.

PubMed

Lin, Jonathan B; Kubota, Shunsuke; Ban, Norimitsu; Yoshida, Mitsukuni; Santeford, Andrea; Sene, Abdoulaye; Nakamura, Rei; Zapata, Nicole; Kubota, Miyuki; Tsubota, Kazuo; Yoshino, Jun; Imai, Shin-Ichiro; Apte, Rajendra S

2016-09-27

Photoreceptor death is the endpoint of many blinding diseases. Identifying unifying pathogenic mechanisms in these diseases may offer global approaches for facilitating photoreceptor survival. We found that rod or cone photoreceptor-specific deletion of nicotinamide phosphoribosyltransferase (Nampt), the rate-limiting enzyme in the major NAD(+) biosynthetic pathway beginning with nicotinamide, caused retinal degeneration. In both cases, we could rescue vision with nicotinamide mononucleotide (NMN). Significantly, retinal NAD(+) deficiency was an early feature of multiple mouse models of retinal dysfunction, including light-induced degeneration, streptozotocin-induced diabetic retinopathy, and age-associated dysfunction. Mechanistically, NAD(+) deficiency caused metabolic dysfunction and consequent photoreceptor death. We further demonstrate that the NAD(+)-dependent mitochondrial deacylases SIRT3 and SIRT5 play important roles in retinal homeostasis and that NAD(+) deficiency causes SIRT3 dysfunction. These findings demonstrate that NAD(+) biosynthesis is essential for vision, provide a foundation for future work to further clarify the mechanisms involved, and identify a unifying therapeutic target for diverse blinding diseases. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Trehalose Biosynthesis Promotes Pseudomonas aeruginosa Pathogenicity in Plants

PubMed Central

Djonović, Slavica; Urbach, Jonathan M.; Drenkard, Eliana; Bush, Jenifer; Feinbaum, Rhonda; Ausubel, Jonathan L.; Traficante, David; Risech, Martina; Kocks, Christine; Fischbach, Michael A.; Priebe, Gregory P.; Ausubel, Frederick M.

2013-01-01

Pseudomonas aeruginosa strain PA14 is a multi-host pathogen that infects plants, nematodes, insects, and vertebrates. Many PA14 factors are required for virulence in more than one of these hosts. Noting that plants have a fundamentally different cellular architecture from animals, we sought to identify PA14 factors that are specifically required for plant pathogenesis. We show that synthesis by PA14 of the disaccharide trehalose is required for pathogenesis in Arabidopsis, but not in nematodes, insects, or mice. In-frame deletion of two closely-linked predicted trehalose biosynthetic operons, treYZ and treS, decreased growth in Arabidopsis leaves about 50 fold. Exogenously co-inoculated trehalose, ammonium, or nitrate, but not glucose, sulfate, or phosphate suppressed the phenotype of the double ΔtreYZΔtreS mutant. Exogenous trehalose or ammonium nitrate does not suppress the growth defect of the double ΔtreYZΔtreS mutant by suppressing the plant defense response. Trehalose also does not function intracellularly in P. aeruginosa to ameliorate a variety of stresses, but most likely functions extracellularly, because wild-type PA14 rescued the in vivo growth defect of the ΔtreYZΔtreS in trans. Surprisingly, the growth defect of the double ΔtreYZΔtreS double mutant was suppressed by various Arabidopsis cell wall mutants that affect xyloglucan synthesis, including an xxt1xxt2 double mutant that completely lacks xyloglucan, even though xyloglucan mutants are not more susceptible to pathogens and respond like wild-type plants to immune elicitors. An explanation of our data is that trehalose functions to promote the acquisition of nitrogen-containing nutrients in a process that involves the xyloglucan component of the plant cell wall, thereby allowing P. aeruginosa to replicate in the intercellular spaces in a leaf. This work shows how P. aeruginosa, a multi-host opportunistic pathogen, has repurposed a highly conserved “house-keeping” anabolic pathway (trehalose biosynthesis) as a potent virulence factor that allows it to replicate in the intercellular environment of a leaf. PMID:23505373

Biosynthesis of silver nanoparticles using bark extracts of Butea monosperma (Lam.) Taub. and study of their antimicrobial activity

NASA Astrophysics Data System (ADS)

Das, Manoja; Smita, Soumya Shuvra

2018-03-01

Biosynthesis of silver nanoparticles was achieved using bark extract of Butea monosperma (Lam.) Taub., a native plant of Indian subcontinent and southeast Asia. The plant parts are familiar for ailment of different diseases. The bioactive compounds present in bark of the plant were extracted with Soxhlet extractor. Silver nitrate (AgNO3) was used as a raw material for preparation of silver nanoparticles (AgNPs). The ratio of bark extract and silver nitrate solution for synthesis of AgNPs was standardized as 3:5. The change in colour of the solution from pale yellow to deep brown can be correlated to reduction reaction catalyzed by plant bioactive compounds. The formation of AgNPs was confirmed by UV-Vis spectrophotometer. The surface plasmon resonance (SPR) maxima, λmax, were recorded at 452 nm. SPR indicates the nature and type of particles present in the solution. The suitable concentration of AgNO3 was found to be 10 mM to carry out reduction reaction with the bark extract. Alkaline environment (pH 9) suitably promotes the reaction. FTIR graph of synthesized AgNPs shows the shifting peak of 3265.0 wavelength/cm and 1635.40 wavelength/cm indicates that AgNPs were coated with plant biomolecules, which is attributed to the stabilization of AgNPs. XRD and SEM photograph of the AgNPs showed that they were spherical in shape and capped with bioactive compounds. Thus, the synthesized AgNPs are more stable, less toxic and homogenous in shape. The average diameter of the nanoparticles was 81 nm. The synthesized AgNPs had efficacy against a Gram-negative bacteria (Escherichia coli), a Gram-positive bacteria (Staphylococcus aureus), and a mold (Aspergillus niger). The maximum conversion was 66%. From the present investigation, it can be concluded that the bioactive compounds present in the bark of Butea have the capacity to reduce silver ion into silver nanoparticles in aqueous condition and the synthesized AgNPs are stabilized and loss toxic. Moreover, they also possess antimicrobial properties against human pathogens.

Lipophilic Lysine-Spermine Conjugates are Potent Polyamine Transport Inhibitors for use in Combination with a Polyamine Biosynthesis Inhibitor

PubMed Central

Burns, Mark R.; Graminski, Gerard F.; Weeks, Reitha S.; Chen, Yan; O’Brien, Thomas G.

2009-01-01

Cancer cells can overcome the ability of polyamine biosynthesis inhibitors from completely depleting their internal polyamines by the importation polyamines from external sources. We have developed a group of lipophilic polyamine analogs that potently inhibit the cellular polyamine uptake system and greatly increase the effectiveness of polyamine depletion when used in combination with DFMO, a well-studied polyamine biosynthesis inhibitor. By the attachment of an length-optimized C16 lipophilic substituent to the epsilon-nitrogen atom of our earlier lead compound, d-Lys-Spm (5), we have produced an analog, d-Lys(C16acyl)-Spm (11) with several orders of magnitude more potent cell growth inhibition on a variety of cultured cancer cell types including breast (MDA-MB-231), prostate (PC-3), melanoma (A375) and ovarian (SK-OV-3), among others. We discuss these results in the context of a possible membrane-catalyzed interaction with the extracellular polyamine transport apparatus. The resulting novel two-drug combination therapy targeting cellular polyamine metabolism has shown exceptional efficacy against cutaneous squamous cell carcinomas (SCC) in a transgenic ornithine decarboxylase (ODC) mouse model of skin cancer. A majority (88%) of large, aggressive SCCs exhibited complete or near-complete remission to this combination therapy, while responses to each agent alone were poor. The availability of a potent polyamine transport inhibitor allows, for the first time, for a real test of the hypothesis that starving cells of polyamines will lead to objective clinical response. PMID:19281226

5-thiomannosides block the biosynthesis of dolichol-linked oligosaccharides and mimic class I congenital disorders of glycosylation.

PubMed

Zandberg, Wesley F; Gao, Ningguo; Kumarasamy, Jayakanthan; Lehrman, Mark A; Seidah, Nabil G; Pinto, B Mario

2012-02-13

In a cell-based assay for novel inhibitors, we have discovered that two glycosides of 5-thiomannose, each containing an interglycosidic nitrogen atom, prevented the correct zymogen processing of the prohormone proopiomelanocortinin (POMC) and the transcription factor sterol-regulatory element-binding protein-2 (SREBP-2) in mouse pituitary cells and Chinese hamster ovary (CHO) cells, respectively. In the case of SREBP-2, these effects were correlated with the altered N-linked glycosylation of subtilisin/kexin-like isozyme-1 (SKI-1), the protease responsible for SREBP-2 processing under sterol-limiting conditions. Further examination of the effects of these compounds in CHO cells showed that they cause extensive protein hypoglycosylation in a manner similar to type I congenital disorders of glycosylation (CDGs) since the remaining N-glycans in treated cells were complete (normal) structures. The under-glycosylation of glycoproteins in 5-thiomannoside-treated cells is now shown to be caused by the compromised biosynthesis of the dolichol-linked oligosaccharide (DLO) N-glycosylation donor, although the nucleotide sugars required for the synthesis of DLOs were neither reduced under these conditions, nor were their effects reversed upon the addition of exogenous mannose. Analysis of DLO intermediates by fluorophore-assisted carbohydrate electrophoresis demonstrated that 5-thiomannose-containing glycosides block DLO biosynthesis most likely at a stage prior to the GlcNAc(2) Man(3) intermediate, on the cytosolic face of the endoplasmic reticulum. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Circadian rhythm genes mediate fenvalerate-induced inhibition of testosterone synthesis in mouse Leydig cells.

PubMed

Guo, Yichen; Shen, Ouxi; Han, Jingjing; Duan, Hongyu; Yang, Siyuan; Zhu, Zhenghong; Tong, Jian; Zhang, Jie

2017-01-01

Fenvalerate (Fen), a widely used pesticide, is known to impair male reproductive functions by mechanisms that remain to be elucidated. Recent studies indicated that circadian clock genes may play an important role in successful male reproduction. The aim of this study was to determine the effects of Fen on circadian clock genes involved in the biosynthesis of testosterone using TM3 cells derived from mouse Leydig cells. Data demonstrated that the circadian rhythm of testosterone synthesis in TM3 cells was disturbed following Fen treatment as evidenced by changes in the circadian rhythmicity of core clock genes (Bmal1, Rev-erbα, Rorα). Further, the observed altered rhythms were accompanied by increased intracellular Ca 2+ levels and modified steroidogenic acute regulatory (StAR) mRNA expression. Thus, data suggested that Fen inhibits testosterone synthesis via pathways involving intracellular Ca 2+ and clock genes (Bmal1, Rev-Erbα, Rorα) as well as StAR mRNA expression in TM3 cells.

Metabolic drift in the aging brain

PubMed Central

Ivanisevic, Julijana; Stauch, Kelly L.; Petrascheck, Michael; Benton, H. Paul; Epstein, Adrian A.; Fang, Mingliang; Gorantla, Santhi; Tran, Minerva; Hoang, Linh; Kurczy, Michael E.; Boska, Michael D.; Gendelman, Howard E.; Fox, Howard S.; Siuzdak, Gary

2016-01-01

Brain function is highly dependent upon controlled energy metabolism whose loss heralds cognitive impairments. This is particularly notable in the aged individuals and in age-related neurodegenerative diseases. However, how metabolic homeostasis is disrupted in the aging brain is still poorly understood. Here we performed global, metabolomic and proteomic analyses across different anatomical regions of mouse brain at different stages of its adult lifespan. Interestingly, while severe proteomic imbalance was absent, global-untargeted metabolomics revealed an energy metabolic drift or significant imbalance in core metabolite levels in aged mouse brains. Metabolic imbalance was characterized by compromised cellular energy status (NAD decline, increased AMP/ATP, purine/pyrimidine accumulation) and significantly altered oxidative phosphorylation and nucleotide biosynthesis and degradation. The central energy metabolic drift suggests a failure of the cellular machinery to restore metabostasis (metabolite homeostasis) in the aged brain and therefore an inability to respond properly to external stimuli, likely driving the alterations in signaling activity and thus in neuronal function and communication. PMID:27182841

Statins Inhibit Monocyte Chemotactic Protein 1 Expression in Endometriosis

PubMed Central

Cakmak, Hakan; Basar, Murat; Seval-Celik, Yasemin; Osteen, Kevin G.; Duleba, Antoni J.; Taylor, Hugh S.; Lockwood, Charles J.; Arici, Aydin

2012-01-01

Statins are potent inhibitors of the endogenous mevalonate pathway. Besides inhibiting cholesterol biosynthesis, statins may also demonstrate anti-inflammatory properties. Inflammation is implicated in the attachment and invasion of endometrial cells to the peritoneal surface and growth of ectopic endometrium by inducing proliferation and angiogenesis. In this study, the effect of statins on monocyte chemotactic protein 1 (MCP-1) expression in endometriotic implants in nude mouse model and in cultured endometriotic cells was evaluated. In mouse model, simvastatin decreased MCP-1 expression in a dose-dependent manner in endometriotic implants (P < .05). Similarly, both simvastatin and mevastatin revealed a dose-dependent inhibition of MCP-1 production in cultured endometriotic cells (P < .01). This inhibitory effect of the statins on MCP-1 production was reversed by the downstream substrates of the mevalonate pathway. Moreover, statins decreased MCP-1 messenger RNA expression in cultured endometriotic cells (P < .05). In conclusion, statins exert anti-inflammatory effect in endometriotic cells and could provide a potential treatment of endometriosis in the future. PMID:22267540

ERG2 and ERG24 Are Required for Normal Vacuolar Physiology as Well as Candida albicans Pathogenicity in a Murine Model of Disseminated but Not Vaginal Candidiasis

PubMed Central

Luna-Tapia, Arturo; Peters, Brian M.; Eberle, Karen E.; Kerns, Morgan E.; Foster, Timothy P.; Marrero, Luis; Noverr, Mairi C.; Fidel, Paul L.

2015-01-01

Several important classes of antifungal agents, including the azoles, act by blocking ergosterol biosynthesis. It was recently reported that the azoles cause massive disruption of the fungal vacuole in the prevalent human pathogen Candida albicans. This is significant because normal vacuolar function is required to support C. albicans pathogenicity. This study examined the impact of the morpholine antifungals, which inhibit later steps of ergosterol biosynthesis, on C. albicans vacuolar integrity. It was found that overexpression of either the ERG2 or ERG24 gene, encoding C-8 sterol isomerase or C-14 sterol reductase, respectively, suppressed C. albicans sensitivity to the morpholines. In addition, both erg2Δ/Δ and erg24Δ/Δ mutants were hypersensitive to the morpholines. These data are consistent with the antifungal activity of the morpholines depending upon the simultaneous inhibition of both Erg2p and Erg24p. The vacuoles within both erg2Δ/Δ and erg24Δ/Δ C. albicans strains exhibited an aberrant morphology and accumulated large quantities of the weak base quinacrine, indicating enhanced vacuolar acidification compared with that of control strains. Both erg mutants exhibited significant defects in polarized hyphal growth and were avirulent in a mouse model of disseminated candidiasis. Surprisingly, in a mouse model of vaginal candidiasis, both mutants colonized mice at high levels and induced a pathogenic response similar to that with the controls. Thus, while targeting Erg2p or Erg24p alone could provide a potentially efficacious therapy for disseminated candidiasis, it may not be an effective strategy to treat vaginal infections. The potential value of drugs targeting these enzymes as adjunctive therapies is discussed. PMID:26231054

ERG2 and ERG24 Are Required for Normal Vacuolar Physiology as Well as Candida albicans Pathogenicity in a Murine Model of Disseminated but Not Vaginal Candidiasis.

PubMed

Luna-Tapia, Arturo; Peters, Brian M; Eberle, Karen E; Kerns, Morgan E; Foster, Timothy P; Marrero, Luis; Noverr, Mairi C; Fidel, Paul L; Palmer, Glen E

2015-10-01

Several important classes of antifungal agents, including the azoles, act by blocking ergosterol biosynthesis. It was recently reported that the azoles cause massive disruption of the fungal vacuole in the prevalent human pathogen Candida albicans. This is significant because normal vacuolar function is required to support C. albicans pathogenicity. This study examined the impact of the morpholine antifungals, which inhibit later steps of ergosterol biosynthesis, on C. albicans vacuolar integrity. It was found that overexpression of either the ERG2 or ERG24 gene, encoding C-8 sterol isomerase or C-14 sterol reductase, respectively, suppressed C. albicans sensitivity to the morpholines. In addition, both erg2Δ/Δ and erg24Δ/Δ mutants were hypersensitive to the morpholines. These data are consistent with the antifungal activity of the morpholines depending upon the simultaneous inhibition of both Erg2p and Erg24p. The vacuoles within both erg2Δ/Δ and erg24Δ/Δ C. albicans strains exhibited an aberrant morphology and accumulated large quantities of the weak base quinacrine, indicating enhanced vacuolar acidification compared with that of control strains. Both erg mutants exhibited significant defects in polarized hyphal growth and were avirulent in a mouse model of disseminated candidiasis. Surprisingly, in a mouse model of vaginal candidiasis, both mutants colonized mice at high levels and induced a pathogenic response similar to that with the controls. Thus, while targeting Erg2p or Erg24p alone could provide a potentially efficacious therapy for disseminated candidiasis, it may not be an effective strategy to treat vaginal infections. The potential value of drugs targeting these enzymes as adjunctive therapies is discussed. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Dietary docosahexaenoic acid supplementation modulates hippocampal development in the Pemt-/- mouse.

PubMed

da Costa, Kerry-Ann; Rai, Kiranmai S; Craciunescu, Corneliu N; Parikh, Komal; Mehedint, Mihai G; Sanders, Lisa M; McLean-Pottinger, Audrey; Zeisel, Steven H

2010-01-08

The development of fetal brain is influenced by nutrients such as docosahexaenoic acid (DHA, 22:6) and choline. Phosphatidylethanolamine-N-methyltransferase (PEMT) catalyzes the biosynthesis of phosphatidylcholine from phosphatidylethanolamine enriched in DHA and many humans have functional genetic polymorphisms in the PEMT gene. Previously, it was reported that Pemt(-/-) mice have altered hippocampal development. The present study explores whether abnormal phosphatidylcholine biosynthesis causes altered incorporation of DHA into membranes, thereby influencing brain development, and determines whether supplemental dietary DHA can reverse some of these changes. Pregnant C57BL/6 wild type (WT) and Pemt(-/-) mice were fed a control diet, or a diet supplemented with 3 g/kg of DHA, from gestational day 11 to 17. Brains from embryonic day 17 fetuses derived from Pemt(-/-) dams fed the control diet had 25-50% less phospholipid-DHA as compared with WT (p < 0.05). Also, they had 60% more neural progenitor cell proliferation (p < 0.05), 60% more neuronal apoptosis (p < 0.01), and 30% less calretinin expression (p < 0.05; a marker of neuronal differentiation) in the hippocampus compared with WT. The DHA-supplemented diet increased fetal brain Pemt(-/-) phospholipid-DHA to WT levels, and abrogated the neural progenitor cell proliferation and apoptosis differences. Although this diet did not change proliferation in the WT group, it halved the rate of apoptosis (p < 0.05). In both genotypes, the DHA-supplemented diet increased calretinin expression 2-fold (p < 0.05). These results suggest that the changes in hippocampal development in the Pemt(-/-) mouse could be mediated by altered DHA incorporation into membrane phospholipids, and that maternal dietary DHA can influence fetal brain development.

A novel fluorophosphonate inhibitor of the biosynthesis of the endocannabinoid 2-arachidonoylglycerol with potential anti-obesity effects.

PubMed

Bisogno, Tiziana; Mahadevan, Anu; Coccurello, Roberto; Chang, Jae Won; Allarà, Marco; Chen, Yugang; Giacovazzo, Giacomo; Lichtman, Aron; Cravatt, Benjamin; Moles, Anna; Di Marzo, Vincenzo

2013-06-01

The development of potent and selective inhibitors of the biosynthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG) via DAG lipases (DAGL) α and β is just starting to be considered as a novel and promising source of pharmaceuticals for the treatment of disorders that might benefit from a reduction in endocannabinoid tone, such as hyperphagia in obese subjects. Three new fluorophosphonate compounds O-7458, O-7459 and O-7460 were synthesized and characterized in various enzymatic assays. The effects of O-7460 on high-fat diet intake were tested in mice. Of the new compounds, O-7460 exhibited the highest potency (IC₅₀ = 690 nM) against the human recombinant DAGLα, and selectivity (IC₅₀ > 10 μM) towards COS-7 cell and human monoacylglycerol lipase (MAGL), and rat brain fatty acid amide hydrolase. Competitive activity-based protein profiling confirmed that O-7460 inhibits mouse brain MAGL only at concentrations ≥ 10 μM, and showed that this compound has only one major 'off-target', that is, the serine hydrolase KIAA1363. O-7460 did not exhibit measurable affinity for human recombinant CB₁ or CB₂ cannabinoid receptors (Ki > 10 μM). In mouse neuroblastoma N18TG2 cells stimulated with ionomycin, O-7460 (10 μM) reduced 2-AG levels. When administered to mice, O-7460 dose-dependently (0-12 mg·kg⁻¹, i.p.) inhibited the intake of a high-fat diet over a 14 h observation period, and, subsequently, slightly but significantly reduced body weight. O-7460 might be considered a useful pharmacological tool to investigate further the role played by 2-AG both in vitro and in vivo under physiological as well as pathological conditions. © 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.

Competitive Fitness of Fluconazole-Resistant Clinical Candida albicans Strains.

PubMed

Popp, Christina; Hampe, Irene A I; Hertlein, Tobias; Ohlsen, Knut; Rogers, P David; Morschhäuser, Joachim

2017-07-01

The pathogenic yeast Candida albicans can develop resistance to the widely used antifungal agent fluconazole, which inhibits ergosterol biosynthesis. Resistance is often caused by gain-of-function mutations in the transcription factors Mrr1 and Tac1, which result in constitutive overexpression of multidrug efflux pumps, and Upc2, which result in constitutive overexpression of ergosterol biosynthesis genes. However, the deregulated gene expression that is caused by hyperactive forms of these transcription factors also reduces the fitness of the cells in the absence of the drug. To investigate whether fluconazole-resistant clinical C. albicans isolates have overcome the fitness costs of drug resistance, we assessed the relative fitness of C. albicans isolates containing resistance mutations in these transcription factors in competition with matched drug-susceptible isolates from the same patients. Most of the fluconazole-resistant isolates were outcompeted by the corresponding drug-susceptible isolates when grown in rich medium without fluconazole. On the other hand, some resistant isolates with gain-of-function mutations in MRR1 did not exhibit reduced fitness under these conditions. In a mouse model of disseminated candidiasis, three out of four tested fluconazole-resistant clinical isolates did not exhibit a significant fitness defect. However, all four fluconazole-resistant isolates were outcompeted by the matched susceptible isolates in a mouse model of gastrointestinal colonization, demonstrating that the effects of drug resistance on in vivo fitness depend on the host niche. Collectively, our results indicate that the fitness costs of drug resistance in C. albicans are not easily remediated, especially when proper control of gene expression is required for successful adaptation to life within a mammalian host. Copyright © 2017 American Society for Microbiology.

A relative quantitative positive/negative ion switching method for untargeted lipidomics via high resolution LC-MS/MS from any biological source

PubMed Central

Breitkopf, Susanne B.; Ricoult, Stéphane J. H.; Yuan, Min; Xu, Ying; Peake, David A.; Manning, Brendan D.

2017-01-01

Introduction Advances in high-resolution mass spectrometry have created renewed interest for studying global lipid biochemistry in disease and biological systems. Objectives Here, we present an untargeted 30 min. LC-MS/MS platform that utilizes positive/negative polarity switching to perform unbiased data dependent acquisitions (DDA) via higher energy collisional dissociation (HCD) fragmentation to profile more than 1000–1500 lipid ions mainly from methyl-tert-butyl ether (MTBE) or chloroform:methanol extractions. Methods The platform uses C18 reversed-phase chromatography coupled to a hybrid QExactive Plus/HF Orbitrap mass spectrometer and the entire procedure takes ~10 h from lipid extraction to identification/quantification for a data set containing 12 samples (~4 h for a single sample). Lipids are identified by both accurate precursor ion mass and fragmentation features and quantified using Lipid-Search and Elements software. Results Using this approach, we are able to profile intact lipid ions from up to 18 different main lipid classes and 66 subclasses. We show several studies from different biological sources, including cultured cancer cells, resected tissues from mice such as lung and breast tumors and biological fluids such as plasma and urine. Conclusions Using mouse embryonic fibroblasts, we showed that TSC2−/− KD significantly abrogates lipid biosynthesis and that rapamycin can rescue triglyceride (TG) lipids and we show that SREBP−/− shuts down lipid biosynthesis significantly via mTORC1 signaling pathways. We show that in mouse EGFR driven lung tumors, a large number of TGs and phosphatidylmethanol (PMe) lipids are elevated while some phospholipids (PLs) show some of the largest decrease in lipid levels from ~ 2000 identified lipid ions. In addition, we identified more than 1500 unique lipid species from human blood plasma. PMID:28496395

Proteome-based systems biology analysis of the diabetic mouse aorta reveals major changes in fatty acid biosynthesis as potential hallmark in diabetes mellitus-associated vascular disease.

PubMed

Husi, Holger; Van Agtmael, Tom; Mullen, William; Bahlmann, Ferdinand H; Schanstra, Joost P; Vlahou, Antonia; Delles, Christian; Perco, Paul; Mischak, Harald

2014-04-01

Macrovascular complications of diabetes mellitus are a major risk factor for cardiovascular morbidity and mortality. Currently, studies only partially described the molecular pathophysiology of diabetes mellitus-associated effects on vasculature. However, better understanding of systemic effects is essential in unraveling key molecular events in the vascular tissue responsible for disease onset and progression. Our overall aim was to get an all-encompassing view of diabetes mellitus-induced key molecular changes in the vasculature. An integrative proteomic and bioinformatics analysis of data from aortic vessels in the low-dose streptozotocin-induced diabetic mouse model (10 animals) was performed. We observed pronounced dysregulation of molecules involved in myogenesis, vascularization, hypertension, hypertrophy (associated with thickening of the aortic wall), and a substantial reduction of fatty acid storage. A novel finding is the pronounced downregulation of glycogen synthase kinase-3β (Gsk3β) and upregulation of molecules linked to the tricarboxylic acid cycle (eg, aspartate aminotransferase [Got2] and hydroxyacid-oxoacid transhydrogenase [Adhfe1]). In addition, pathways involving primary alcohols and amino acid breakdown are altered, potentially leading to ketone-body production. A number of these findings were validated immunohistochemically. Collectively, the data support the hypothesis that in this diabetic model, there is an overproduction of ketone-bodies within the vessels using an alternative tricarboxylic acid cycle-associated pathway, ultimately leading to the development of atherosclerosis. Streptozotocin-induced diabetes mellitus in animals leads to a reduction of fatty acid biosynthesis and an upregulation of an alternative ketone-body formation pathway. This working hypothesis could form the basis for the development of novel therapeutic intervention and disease management approaches.

Differentiation by hydroquinone of relaxations induced by exogenous and endogenous nitrates in non-vascular smooth muscle: role of superoxide anions.

PubMed Central

Hobbs, A. J.; Tucker, J. F.; Gibson, A.

1991-01-01

1. The influence of hydroquinone on relaxations induced by nitric oxide (NO), nitrovasodilator drugs, and non-adrenergic, non-cholinergic (NANC) field stimulation has been investigated in three tissues in which endogenous nitrates have been implicated in the NANC response; the mechanism of action of hydroquinone was also studied. 2. In mouse anococcygeus, hydroquinone (10-100 microM) produced a concentration-dependent inhibition of relaxations induced by 15 microM NO. Hydroquinone, 100 microM, which reduced responses to NO by 85%, had no effect on relaxations induced by NANC field stimulation (10 Hz; 20s trains), hydroxylamine (10 microM), sodium nitroprusside (1 microM) or sodium azide (20 microM). 3. In guinea-pig trachea, 100 microM hydroquinone reduced relaxations to 150 microM NO by 75%, but had no effect on those to NANC stimulation (10 Hz; 30 s trains) or sodium azide (5 microM). 4. In rat gastric fundus, 100 microM hydroquinone reduced relaxations to 1 microM NO by 85%, but had no effect on those to NANC stimulation (0.5 Hz; 15 s trains) or sodium azide (2 microM). 5. Superoxide dismutase (SOD; 50 u ml-1) had no effect on relaxations of the mouse anococcygeus in response to 15 microM NO or 10 Hz NANC stimulation. Further, the inhibition of responses to NO by hydroquinone was unaffected in the presence of SOD. 6. Hydroquinone (10-100 microM) failed to generate superoxide anions, as detected by a chemiluminescent assay. However, 100 microM hydroquinone, like SOD (50 u ml-1), produced almost complete inhibition of superoxide anion chemiluminescence induced by xanthine (500 microM): xanthine oxidase (0.07 u ml-1). 7. It is concluded that, in our system, hydroquinone inhibits NO by acting as a free radical scavenger rather than by generating superoxide anions. The ability of hydroquinone to block relaxations to NO, but not NANC stimulation, may suggest that the endogenous nitrate substance released by these NANC nerves may not be free NO, but may be an NO-containing, or NO-generating, molecule. PMID:1665746

Alpha-synuclein aggregation induced by brief ischemia negatively impacts neuronal survival in vivo: a study in [A30P]alpha-synuclein transgenic mouse

PubMed Central

Unal-Cevik, Isin; Gursoy-Ozdemir, Yasemin; Yemisci, Muge; Lule, Sevda; Gurer, Gunfer; Can, Alp; Müller, Veronica; Kahle, Philip J; Dalkara, Turgay

2011-01-01

Alpha-synuclein oligomerization and aggregation are considered to have a role in the pathogenesis of neurodegenerative diseases. However, despite numerous in vitro studies, the impact of aggregates in the intact brain is unclear. In vitro, oxidative/nitrative stress and acidity induce α-synuclein oligomerization. These conditions favoring α-synuclein fibrillization are present in the ischemic brain, which may serve as an in vivo model to study α-synuclein aggregation. In this study, we show that 30-minute proximal middle cerebral artery (MCA) occlusion and 72 hours reperfusion induce oligomerization of wild-type α-synuclein in the ischemic mouse brain. The nonamyloidogenic isoform β-synuclein did not form oligomers. Alpha-synuclein aggregates were confined to neurons and colocalized with ubiquitin immunoreactivity. We also found that 30 minutes proximal MCA occlusion and 24 hours reperfusion induced larger infarcts in C57BL/6(Thy1)-h[A30P]alphaSYN transgenic mice, which have an increased tendency to form synuclein fibrils. Trangenics also developed more selective neuronal necrosis when subjected to 20 minutes distal MCA occlusion and 72 hours reperfusion. Enhanced 3-nitrotyrosine immunoreactivity in transgenic mice suggests that oxidative/nitrative stress may be one of the mechanisms mediating aggregate toxicity. Thus, the increased vulnerability of transgenic mice to ischemia suggests that α-synuclein aggregates not only form during ischemia but also negatively impact neuronal survival, supporting the idea that α-synuclein misfolding may be neurotoxic. PMID:20877387

Biosynthesis of silver nanoparticles using Sida acuta extract for antimicrobial actions and corrosion inhibition potential.

PubMed

Idrees, Muhammad; Batool, Saima; Kalsoom, Tanzila; Raina, Sadaf; Sharif, Hafiz Muhammad Adeel; Yasmeen, Summera

2018-02-12

Nanotechnology exhibits a multidisciplinary area and gained interests for researchers. Nanoparticles produced via physical and chemical methods affects ecosystem drastically. Green synthesis is the charming technique that is inexpensive and safe for the environment. This study aimed to explore the antibacterial actions of as-synthesized silver nanoparticles (Ag-NPs) against Escherichia coli, Staphylococcus aureus and Streptococcus faecalis. Also, the anti-corrosion actions confirmed that the Ag-NPs proved as good inhibitors. In this way, Ag-NPs were prepared via biosynthesis technique by consuming the ground leaves and stem of 'Sida acuta' as a capping agent. The Ag-NPs were formed by irradiation of the aqueous solution of silver nitrate (AgNO 3 ) with extract of S. acuta stem and leaves. The as-synthesized reaction mixture of Ag-NPs was found to exhibit an absorbance band at 446-447 nm, by an UV/VIS spectrophotometer, which is a characteristic of Ag-NPs due to the surface plasmon resonance absorption band. The X-ray diffraction and transmission electron microscopy (TEM) were used for the confirmation of Ag-NPs' variety dimension, morphology and dispersion. The infrared spectra confirmed the bio-fabrication of the Ag-NPs displayed the existence of conceivable functional groups responsible for the bio-reduction and capping. The antimicrobial actions were measured and the zone of inhibition was compared with standard antibiotics.

Overexpression of PSP1 enhances growth of transgenic Arabidopsis plants under ambient air conditions.

PubMed

Han, Xiaofang; Peng, Keli; Wu, Haixia; Song, Shanshan; Zhu, Yerong; Bai, Yanling; Wang, Yong

2017-07-01

The importance of the phosphorylated pathway (PPSB) of L-serine (Ser) biosynthesis in plant growth and development has been demonstrated, but its specific role in leaves and interaction with photorespiration, the main leaf Ser biosynthetic pathway at daytime, are still unclear. To investigate whether changes in biosynthesis of Ser by the PPSB in leaves could have an impact on photorespiration and plant growth, we overexpressed PSP1, the last enzyme of this pathway, under control of the Cauliflower Mosaic Virus 35S promoter in Arabidopsis thaliana. Overexpressor plants grown in normal air displayed larger rosette diameter and leaf area as well as higher fresh and dry weight than the wild type. By contrast, no statistically significant differences to the wild type were observed when the overexpressor seedlings were transferred to elevated CO 2 , indicating a relationship between PSP1 overexpression and photorespiration. Additionally, the transgenic plants displayed higher photorespiration, an increase in CO 2 net-uptake and stronger expression in the light of genes encoding enzymes involved in photorespiration. We further demonstrated that expression of many genes involved in nitrogen assimilation was also promoted in leaves of transgenic plants and that leaf nitrate reductase activity increased in the light, too, although not in the dark. Our results suggest a close correlation between the function of PPSB and photorespiration, and also nitrogen metabolism in leaves.

Procurement of exogenous ammonia by the swallowtail butterfly, Papilio polytes, for protein biosynthesis and sperm production

NASA Astrophysics Data System (ADS)

Honda, Keiichi; Takase, Hiroyuki; Ômura, Hisashi; Honda, Hiroshi

2012-09-01

How to acquire sufficient quantity of nitrogen is a pivotal issue for herbivores, particularly for lepidopterans (butterflies and moths) of which diet quality greatly differs among their life stages. Male Lepidoptera often feed from mud puddles, dung, and carrion, a behavior known as puddling, which is thought to be supplementary feeding targeted chiefly at sodium. During copulation, males transfer a spermatophore to females that contains, besides sperm, nutrients (nuptial gifts) rich in sodium, proteins, and amino acids. However, it is still poorly understood how adults, mostly nectarivores, extract nitrogen from the environment. We examined the availability of two ubiquitous inorganic nitrogenous ions in nature, viz. ammonium (or ammonia) and nitrate ions, as nutrients in a butterfly, and show that exogenous ammonia ingested by adult males of the swallowtail, Papilio polytes, can serve as a resource for protein biosynthesis. Feeding experiments with 15N-labeled ammonium chloride revealed that nitrogen was incorporated into eupyrene spermatozoa, seminal protein, and thoracic muscle. Ammonia uptake by males significantly increased the number of eupyrene sperms in the reproductive tract tissues. The females also had the capacity to assimilate ammonia into egg protein. Consequently, it is evident that acquired ammonia is utilized for the replenishment of proteins allocable for reproduction and somatic maintenance. The active exploitation of exogenous ammonia as a nutrient by a butterfly would foster better understanding of the foraging and reproductive strategies in insects.

Procurement of exogenous ammonia by the swallowtail butterfly, Papilio polytes, for protein biosynthesis and sperm production.

PubMed

Honda, Keiichi; Takase, Hiroyuki; Ômura, Hisashi; Honda, Hiroshi

2012-09-01

How to acquire sufficient quantity of nitrogen is a pivotal issue for herbivores, particularly for lepidopterans (butterflies and moths) of which diet quality greatly differs among their life stages. Male Lepidoptera often feed from mud puddles, dung, and carrion, a behavior known as puddling, which is thought to be supplementary feeding targeted chiefly at sodium. During copulation, males transfer a spermatophore to females that contains, besides sperm, nutrients (nuptial gifts) rich in sodium, proteins, and amino acids. However, it is still poorly understood how adults, mostly nectarivores, extract nitrogen from the environment. We examined the availability of two ubiquitous inorganic nitrogenous ions in nature, viz. ammonium (or ammonia) and nitrate ions, as nutrients in a butterfly, and show that exogenous ammonia ingested by adult males of the swallowtail, Papilio polytes, can serve as a resource for protein biosynthesis. Feeding experiments with (15)N-labeled ammonium chloride revealed that nitrogen was incorporated into eupyrene spermatozoa, seminal protein, and thoracic muscle. Ammonia uptake by males significantly increased the number of eupyrene sperms in the reproductive tract tissues. The females also had the capacity to assimilate ammonia into egg protein. Consequently, it is evident that acquired ammonia is utilized for the replenishment of proteins allocable for reproduction and somatic maintenance. The active exploitation of exogenous ammonia as a nutrient by a butterfly would foster better understanding of the foraging and reproductive strategies in insects.

Biosynthesis of silver nanoparticles from Tribulus terrestris and its antimicrobial activity: a novel biological approach.

PubMed

Gopinath, V; MubarakAli, D; Priyadarshini, S; Priyadharsshini, N Meera; Thajuddin, N; Velusamy, P

2012-08-01

In the recent decades, increased development of green synthesis of nanoparticles is inevitable because of its incredible applications in all fields of science. There were numerous work have been produced based on the plant and its extract mediated synthesis of nanoparticles, in this present study to explore that the novel approaches for the biosynthesis of silver nanoparticles using plant fruit bodies. The plant, Tribulus terrestris L. fruit bodies are used in this study, where the dried fruit body extract was mixed with silver nitrate in order to synthesis of silver nanoparticles. The active phytochemicals present in the plant were responsible for the quick reduction of silver ion (Ag(+)) to metallic silver nanoparticles (Ag(0)). The reduced silver nanoparticles were characterized by Transmission Electron Microscope (TEM), Atomic Force Microscope (AFM), XRD, FTIR, UV-vis spectroscopy. The spherical shaped silver nanoparticles were observed and it was found to be 16-28 nm range of sizes. The diffraction pattern also confirmed that the higher percentage of silver with fine particles size. The antibacterial property of synthesized nanoparticles was observed by Kirby-Bauer method with clinically isolated multi-drug resistant bacteria such as Streptococcus pyogens, Pseudomonas aeruginosa, Escherichia coli, Bacillus subtilis and Staphylococcus aureus. The plant materials mediated synthesis of silver nanoparticles have comparatively rapid and less expensive and wide application to antibacterial therapy in modern medicine. Copyright © 2012 Elsevier B.V. All rights reserved.

The development of a green approach for the biosynthesis of silver and gold nanoparticles by using Panax ginseng root extract, and their biological applications.

PubMed

Singh, Priyanka; Kim, Yeon Ju; Wang, Chao; Mathiyalagan, Ramya; Yang, Deok Chun

2016-06-01

The biosynthesis of nanoparticles has received attention because of the development of economic and environmentally friendly technology for the synthesis of nanoparticles. The study develops a convenient method for the green synthesis of silver and gold nanoparticles by utilizing fresh root extract of the four-year old Panax ginseng plant, and evaluated the antimicrobial applications of silver nanoparticles against pathogenic microorganisms. P. ginseng is a well-known herbal medicinal plant, and its active ingredients are mainly ginsenosides. The fresh root of the 4 year old P. ginseng plant has been explored for the synthesis of silver and gold nanoparticles without the use of any additional reducing and capping agents. The reduction of silver nitrate led to the formation of silver nanoparticles within 2 h of reaction at 80°C. The gold nanoparticles were also successfully synthesized by the reduction of auric acid at 80°C, within 5 min of reaction. The biosynthesized gold and silver nanoparticles were characterized by techniques using various instruments, viz. ultraviolet-visible spectroscopy (UV-Vis spectroscopy), field emission transmission electron microscopy (FE-TEM), energy dispersive X-ray analysis (EDX), elemental mapping, and X-ray diffraction (XRD). In addition, the silver nanoparticles have shown antimicrobial potential against Bacillus anthracis, Vibrio parahaemolyticus, Staphylococcus aureus, Escherichia coli, and Bacillus cereus.

Lithium attenuates lead induced toxicity on mouse non-adherent bone marrow cells.

PubMed

Banijamali, Mahsan; Rabbani-Chadegani, Azra; Shahhoseini, Maryam

2016-07-01

Lead is a poisonous heavy metal that occurs in all parts of environment and causes serious health problems in humans. The aim of the present study was to investigate the possible protective effect of lithium against lead nitrate induced toxicity in non-adherent bone marrow stem cells. Trypan blue and MTT assays represented that exposure of the cells to different concentrations of lead nitrate decreased viability in a dose dependent manner, whereas, pretreatment of the cells with lithium protected the cells against lead toxicity. Lead reduced the number and differentiation status of bone marrow-derived precursors when cultured in the presence of colony stimulating factor (CSF), while the effect was attenuated by lithium. The cells treated with lead nitrate exhibited cell shrinkage, DNA fragmentation, anion superoxide production, but lithium prevented lead action. Moreover, apoptotic indexes such as PARP cleavage and release of HMGB1 induced by lead, were protected by lithium, suggesting anti-apoptotic effect of lithium. Immunoblot analysis of histone H3K9 acetylation indicated that lithium overcame lead effect on acetylation. In conclusion, lithium efficiently reduces lead toxicity suggesting new insight into lithium action which may contribute to increased cell survival. It also provides a potentially new therapeutic strategy for lithium and a cost-effective approach to minimize destructive effects of lead on bone marrow stem cells. Copyright © 2016 Elsevier GmbH. All rights reserved.

A mouse model for Costello syndrome reveals an Ang II–mediated hypertensive condition

PubMed Central

Schuhmacher, Alberto J.; Guerra, Carmen; Sauzeau, Vincent; Cañamero, Marta; Bustelo, Xosé R.; Barbacid, Mariano

2008-01-01

Germline activation of H-RAS oncogenes is the primary cause of Costello syndrome (CS), a neuro-cardio-facio-cutaneous developmental syndrome. Here we describe the generation of a mouse model of CS by introduction of an oncogenic Gly12Val mutation in the mouse H-Ras locus using homologous recombination in ES cells. Germline expression of the endogenous H-RasG12V oncogene, even in homozygosis, resulted in hyperplasia of the mammary gland. However, development of tumors in these mice was rare. H-RasG12V mutant mice closely phenocopied some of the abnormalities observed in patients with CS, including facial dysmorphia and cardiomyopathies. These mice also displayed alterations in the homeostasis of the cardiovascular system, including development of systemic hypertension, extensive vascular remodeling, and fibrosis in both the heart and the kidneys. This phenotype was age dependent and was a consequence of the abnormal upregulation of the renin–Ang II system. Treatment with captopril, an inhibitor of Ang II biosynthesis, prevented development of the hypertension condition, vascular remodeling, and heart and kidney fibrosis. In addition, it partially alleviated the observed cardiomyopathies. These mice should help in elucidating the etiology of CS symptoms, identifying additional defects, and evaluating potential therapeutic strategies. PMID:18483625

Inhibition of HMG CoA reductase reveals an unexpected role for cholesterol during PGC migration in the mouse

PubMed Central

Ding, Jiaxi; Jiang, DeChen; Kurczy, Michael; Nalepka, Jennifer; Dudley, Brian; Merkel, Erin I; Porter, Forbes D; Ewing, Andrew G; Winograd, Nicholas; Burgess, James; Molyneaux, Kathleen

2008-01-01

Background Primordial germ cells (PGCs) are the embryonic precursors of the sperm and eggs. Environmental or genetic defects that alter PGC development can impair fertility or cause formation of germ cell tumors. Results We demonstrate a novel role for cholesterol during germ cell migration in mice. Cholesterol was measured in living tissue dissected from mouse embryos and was found to accumulate within the developing gonads as germ cells migrate to colonize these structures. Cholesterol synthesis was blocked in culture by inhibiting the activity of HMG CoA reductase (HMGCR) resulting in germ cell survival and migration defects. These defects were rescued by co-addition of isoprenoids and cholesterol, but neither compound alone was sufficient. In contrast, loss of the last or penultimate enzyme in cholesterol biosynthesis did not alter PGC numbers or position in vivo. However embryos that lack these enzymes do not exhibit cholesterol defects at the stage at which PGCs are migrating. This demonstrates that during gestation, the cholesterol required for PGC migration can be supplied maternally. Conclusion In the mouse, cholesterol is required for PGC survival and motility. It may act cell-autonomously by regulating clustering of growth factor receptors within PGCs or non cell-autonomously by controlling release of growth factors required for PGC guidance and survival. PMID:19117526

Testis development, fertility, and survival in Ethanolamine kinase 2-deficient mice.

PubMed

Gustin, Sonja E; Western, Patrick S; McClive, Peter J; Harley, Vincent R; Koopman, Peter A; Sinclair, Andrew H

2008-12-01

Ethanolamine kinase 2 (Eki2) was previously isolated from a differential expression screen designed to identify candidate genes involved in testis development and differentiation. In mouse, Eki2 is specifically up-regulated in Sertoli cells of the developing testis at the time of sex determination. Based on this expression profile, Eki2 was considered a good candidate testis-determining gene. To investigate a possible role of Eki2 in testis development, we have generated a mouse with targeted disruption of the Eki2 gene by using an EGFP replacement strategy. No abnormalities were detected in the Eki2-deficient mice with regard to embryonic and adult testis morphology, differentiation, function, or fertility. Furthermore, no significant differences were observed in litter sizes, pup mortality rates, or distribution of the sexes among the offspring. Ethanolamine kinases are involved in the biosynthesis of phosphatidylethanolamine, a major membrane phospholipid. Expression analysis indicates that the absence of an apparent phenotype in the Eki2-deficient mice may be due to compensation by Eki2-family members or the activation of an alternative pathway to generate phosphatidylethanolamine. Expression of EGFP in this mouse model enabled the isolation of gonad cell populations, providing a useful resource from which to obtain relatively pure early steroidogenic cells for further studies.

Focused glycomic analysis of the N-linked glycan biosynthetic pathway in ovarian cancer

PubMed Central

Abbott, Karen L.; Nairn, Alison V.; Hall, Erica M.; Horton, Marc B.; McDonald, John F.; Moremen, Kelley W.; Dinulescu, Daniela M.; Pierce, Michael

2014-01-01

Epithelial ovarian cancer is the deadliest female reproductive tract malignancy in Western countries. Less than 25% of cases are diagnosed when the cancer is confined, however, pointing to the critical need for early diagnostics for ovarian cancer. Identifying the changes that occur in the glycome of ovarian cancer cells may provide an avenue to develop a new generation of potential biomarkers for early detection of this disease. We performed a glycotranscriptomic analysis of endometrioid ovarian carcinoma using human tissue, as well as a newly developed mouse model that mimics this disease. Our results show that the N-linked glycans expressed in both non-diseased mouse and human ovarian tissues are similar; moreover, malignant changes in the expression of N-linked glycans in both mouse and human endometrioid ovarian carcinoma are qualitatively similar. Lectin reactivity was used as a means for rapid validation of glycan structural changes in the carcinomas that were predicted by the glycotranscriptome analysis. Among several changes in glycan expression noted, the increase of bisected N-linked glycans and the transcripts of the enzyme responsible for its biosynthesis, GnT-III, was the most significant. This study provides evidence that glycotranscriptome analysis can be an important tool in identifying potential cancer biomarkers. PMID:18690643

A mouse diversity panel approach reveals the potential for clinical kidney injury due to DB289 not predicted by classical rodent models.

PubMed

Harrill, Alison H; Desmet, Kristina D; Wolf, Kristina K; Bridges, Arlene S; Eaddy, J Scott; Kurtz, C Lisa; Hall, J Ed; Paine, Mary F; Tidwell, Richard R; Watkins, Paul B

2012-12-01

DB289 is the first oral drug shown in clinical trials to have efficacy in treating African trypanosomiasis (African sleeping sickness). Mild liver toxicity was noted but was not treatment limiting. However, development of DB289 was terminated when several treated subjects developed severe kidney injury, a liability not predicted from preclinical testing. We tested the hypothesis that the kidney safety liability of DB289 would be detected in a mouse diversity panel (MDP) comprised of 34 genetically diverse inbred mouse strains. MDP mice received 10 days of oral treatment with DB289 or vehicle and classical renal biomarkers blood urea nitrogen (BUN) and serum creatinine (sCr), as well as urine biomarkers of kidney injury were measured. While BUN and sCr remained within reference ranges, marked elevations were observed for kidney injury molecule-1 (KIM-1) in the urine of sensitive mouse strains. KIM-1 elevations were not always coincident with elevations in alanine aminotransferase (ALT), suggesting that renal injury was not linked to hepatic injury. Genome-wide association analyses of KIM-1 elevations indicated that genes participating in cholesterol and lipid biosynthesis and transport, oxidative stress, and cytokine release may play a role in DB289 renal injury. Taken together, the data resulting from this study highlight the utility of using an MDP to predict clinically relevant toxicities, to identify relevant toxicity biomarkers that may translate into the clinic, and to identify potential mechanisms underlying toxicities. In addition, the sensitive mouse strains identified in this study may be useful in screening next-in-class compounds for renal injury.

Ganaxolone improves behavioral deficits in a mouse model of post-traumatic stress disorder

PubMed Central

Pinna, Graziano; Rasmusson, Ann M.

2014-01-01

Allopregnanolone and its equipotent stereoisomer, pregnanolone (together termed ALLO), are neuroactive steroids that positively and allosterically modulate the action of gamma-amino-butyric acid (GABA) at GABAA receptors. Levels of ALLO are reduced in the cerebrospinal fluid of female premenopausal patients with post-traumatic stress disorder (PTSD), a severe, neuropsychiatric condition that affects millions, yet is without a consistently effective therapy. This suggests that restoring downregulated brain ALLO levels in PTSD may be beneficial. ALLO biosynthesis is also decreased in association with the emergence of PTSD-like behaviors in socially isolated (SI) mice. Similar to PTSD patients, SI mice also exhibit changes in the frontocortical and hippocampal expression of GABAA receptor subunits, resulting in resistance to benzodiazepine-mediated sedation and anxiolysis. ALLO acts at a larger spectrum of GABAA receptor subunits than benzodiazepines, and increasing corticolimbic ALLO levels in SI mice by injecting ALLO or stimulating ALLO biosynthesis with a selective brain steroidogenic stimulant, such as S-norfluoxetine, at doses far below those that block serotonin reuptake, reduces PTSD-like behavior in these mice. This suggests that synthetic analogs of ALLO, such as ganaxolone, may also improve anxiety, aggression, and other PTSD-like behaviors in the SI mouse model. Consistent with this hypothesis, ganaxolone (3.75–30 mg/kg, s.c.) injected 60 min before testing of SI mice, induced a dose-dependent reduction in aggression toward a same-sex intruder and anxiety-like behavior in an elevated plus maze. The EC50 dose of ganaxolone used in these tests also normalized exaggerated contextual fear conditioning and, remarkably, enhanced fear extinction retention in SI mice. At these doses, ganaxolone failed to change locomotion in an open field test. Therefore, unlike benzodiazepines, ganaxolone at non-sedating concentrations appears to improve dysfunctional emotional behavior associated with deficits in ALLO in mice and may provide an alternative treatment for PTSD patients with deficits in the synthesis of ALLO. Selective serotonin reuptake inhibitors (SSRIs) are the only medications currently approved by the FDA for treatment of PTSD, although they are ineffective in a substantial proportion of PTSD patients. Hence, an ALLO analog such as ganaxolone may offer a therapeutic GABAergic alternative to SSRIs for the treatment of PTSD or other disorders in which ALLO biosynthesis may be impaired. PMID:25309317

Cholesterol: a novel regulatory role in myelin formation.

PubMed

Saher, Gesine; Quintes, Susanne; Nave, Klaus-Armin

2011-02-01

Myelin consists of tightly compacted membranes that form an insulating sheath around axons. The function of myelin for rapid saltatory nerve conduction is dependent on its unique composition, highly enriched in glycosphingolipids and cholesterol. Cholesterol emerged as the only integral myelin component that is essential and rate limiting for the development of CNS and PNS myelin. Experiments with conditional mouse mutants that lack cholesterol biosynthesis in oligodendrocytes revealed that only minimal changes of the CNS myelin lipid composition are tolerated. In Schwann cells of the PNS, protein trafficking and myelin compaction depend on cholesterol. In this review, the authors summarize the role of cholesterol in myelin biogenesis and myelin disease.

Floral Biosynthesis of Mn3O4 and Fe2O3 Nanoparticles Using Chaenomeles sp. Flower Extracts for Efficient Medicinal Applications

NASA Astrophysics Data System (ADS)

Karunakaran, Gopalu; Jagathambal, Matheswaran; Kolesnikov, Evgeny; Dmitry, Arkhipov; Ishteev, Artur; Gusev, Alexander; Kuznetsov, Denis

2017-08-01

Manganese oxide (Mn3O4) and iron oxide (Fe2O3) nanoparticles were successfully synthesized with the flower extracts of Chaenomeles sp. This is the first ever approach to synthesize nanoparticles from Chaenomeles sp. flower extracts. The organic molecules present in the flower extracts actively converted the nitrate precursor into its corresponding nanoparticles. The organic molecules that are involved in the synthesis of nanoparticles are identified using different phytochemical and gas chromatography-mass spectrometry analyses. The identified components are glycosides, alkaloids, terpenoids, saponins, flavonoids, quinines, and steroids. The structural and chemical compositions of the synthesized powder were also analyzed. The x-ray powder diffraction analysis revealed that the particles show tetragonal and rhombohedral crystalline phases. The Fourier transform infrared spectroscopy analysis showed the functional groups that are involved in the reduction of nitrates into the corresponding nanoparticles. Energy-dispersive x-ray spectroscopy analysis confirmed the presence of the elements in the synthesized nanoparticles. Transmission electron microscopy images showed the formation of spherical nanoparticles with an average size of 30-100 nm. Antioxidant analysis showed that the synthesized nanoparticles had excellent antioxidant potential. The antibacterial study showed that they inhibit the growth of harmful bacteria such as Pseudomonas aeruginosa and Streptococcus pyogenes. Thus, this study proposes a new eco-friendly and nontoxic method to synthesize nanoparticles for medicinal applications.

Remodeling of intermediate metabolism in the diatom Phaeodactylum tricornutum under nitrogen stress

PubMed Central

Levitan, Orly; Dinamarca, Jorge; Zelzion, Ehud; Lun, Desmond S.; Guerra, L. Tiago; Kim, Min Kyung; Kim, Joomi; Van Mooy, Benjamin A. S.; Bhattacharya, Debashish; Falkowski, Paul G.

2015-01-01

Diatoms are unicellular algae that accumulate significant amounts of triacylglycerols as storage lipids when their growth is limited by nutrients. Using biochemical, physiological, bioinformatics, and reverse genetic approaches, we analyzed how the flux of carbon into lipids is influenced by nitrogen stress in a model diatom, Phaeodactylum tricornutum. Our results reveal that the accumulation of lipids is a consequence of remodeling of intermediate metabolism, especially reactions in the tricarboxylic acid and the urea cycles. Specifically, approximately one-half of the cellular proteins are cannibalized; whereas the nitrogen is scavenged by the urea and glutamine synthetase/glutamine 2-oxoglutarate aminotransferase pathways and redirected to the de novo synthesis of nitrogen assimilation machinery, simultaneously, the photobiological flux of carbon and reductants is used to synthesize lipids. To further examine how nitrogen stress triggers the remodeling process, we knocked down the gene encoding for nitrate reductase, a key enzyme required for the assimilation of nitrate. The strain exhibits 40–50% of the mRNA copy numbers, protein content, and enzymatic activity of the wild type, concomitant with a 43% increase in cellular lipid content. We suggest a negative feedback sensor that couples photosynthetic carbon fixation to lipid biosynthesis and is regulated by the nitrogen assimilation pathway. This metabolic feedback enables diatoms to rapidly respond to fluctuations in environmental nitrogen availability. PMID:25548193

Novel drug targets in cell wall biosynthesis exploited by gene disruption in Pseudomonas aeruginosa.

PubMed

Elamin, Ayssar A; Steinicke, Susanne; Oehlmann, Wulf; Braun, Yvonne; Wanas, Hanaa; Shuralev, Eduard A; Huck, Carmen; Maringer, Marko; Rohde, Manfred; Singh, Mahavir

2017-01-01

For clinicians, Pseudomonas aeruginosa is a nightmare pathogen that is one of the top three causes of opportunistic human infections. Therapy of P. aeruginosa infections is complicated due to its natural high intrinsic resistance to antibiotics. Active efflux and decreased uptake of drugs due to cell wall/membrane permeability appear to be important issues in the acquired antibiotic tolerance mechanisms. Bacterial cell wall biosynthesis enzymes have been shown to be essential for pathogenicity of Gram-negative bacteria. However, the role of these targets in virulence has not been identified in P. aeruginosa. Here, we report knockout (k.o) mutants of six cell wall biosynthesis targets (murA, PA4450; murD, PA4414; murF, PA4416; ppiB, PA1793; rmlA, PA5163; waaA, PA4988) in P. aeruginosa PAO1, and characterized these in order to find out whether these genes and their products contribute to pathogenicity and virulence of P. aeruginosa. Except waaA k.o, deletion of cell wall biosynthesis targets significantly reduced growth rate in minimal medium compared to the parent strain. The k.o mutants showed exciting changes in cell morphology and colonial architectures. Remarkably, ΔmurF cells became grossly enlarged. Moreover, the mutants were also attenuated in vivo in a mouse infection model except ΔmurF and ΔwaaA and proved to be more sensitive to macrophage-mediated killing than the wild-type strain. Interestingly, the deletion of the murA gene resulted in loss of virulence activity in mice, and the virulence was restored in a plant model by unknown mechanism. This study demonstrates that cell wall targets contribute significantly to intracellular survival, in vivo growth, and pathogenesis of P. aeruginosa. In conclusion, these findings establish a link between cell wall targets and virulence of P. aeruginosa and thus may lead to development of novel drugs for the treatment of P. aeruginosa infection.

Impairment of heme biosynthesis induces short circadian period in body temperature rhythms in mice.

PubMed

Iwadate, Reiko; Satoh, Yoko; Watanabe, Yukino; Kawai, Hiroshi; Kudo, Naomi; Kawashima, Yoichi; Mashino, Tadahiko; Mitsumoto, Atsushi

2012-07-01

It has been demonstrated that the function of mammalian clock gene transcripts is controlled by the binding of heme in vitro. To examine the effects of heme on biological rhythms in vivo, we measured locomotor activity (LA) and core body temperature (T(b)) in a mouse model of porphyria with impaired heme biosynthesis by feeding mice a griseofulvin (GF)-containing diet. Mice fed with a 2.0% GF-containing diet (GF2.0) transiently exhibited phase advance or phase advance-like phenomenon by 1-3 h in terms of the biological rhythms of T(b) or LA, respectively (both, P < 0.05) while mice were kept under conditions of a light/dark cycle (12 h:12 h). We also observed a transient, ~0.3 h shortening of the period of circadian T(b) rhythms in mice kept under conditions of constant darkness (P < 0.01). Interestingly, the observed duration of abnormal circadian rhythms in GF2.0 mice lasted between 1 and 3 wk after the onset of GF ingestion; this finding correlated well with the extent of impairment of heme biosynthesis. When we examined the effects of therapeutic agents for acute porphyria, heme, and hypertonic glucose on the pathological status of GF2.0 mice, it was found that the intraperitoneal administration of heme (10 mg·kg(-1)·day(-1)) or glucose (9 g·kg(-1)·day(-1)) for 7 days partially reversed (50%) increases in urinary δ-aminolevulinic acids levels associated with acute porphyria. Treatment with heme, but not with glucose, suppressed the phase advance (-like phenomenon) in the diurnal rhythms (P < 0.05) and restored the decrease of heme (P < 0.01) in GF2.0 mice. These results suggest that impairments of heme biosynthesis, in particular a decrease in heme, may affect phase and period of circadian rhythms in animals.

Transcriptional profiling and biomarker identification reveal tissue specific effects of expanded ataxin-3 in a spinocerebellar ataxia type 3 mouse model.

PubMed

Toonen, Lodewijk J A; Overzier, Maurice; Evers, Melvin M; Leon, Leticia G; van der Zeeuw, Sander A J; Mei, Hailiang; Kielbasa, Szymon M; Goeman, Jelle J; Hettne, Kristina M; Magnusson, Olafur Th; Poirel, Marion; Seyer, Alexandre; 't Hoen, Peter A C; van Roon-Mom, Willeke M C

2018-06-22

Spinocerebellar ataxia type 3 (SCA3) is a progressive neurodegenerative disorder caused by expansion of the polyglutamine repeat in the ataxin-3 protein. Expression of mutant ataxin-3 is known to result in transcriptional dysregulation, which can contribute to the cellular toxicity and neurodegeneration. Since the exact causative mechanisms underlying this process have not been fully elucidated, gene expression analyses in brains of transgenic SCA3 mouse models may provide useful insights. Here we characterised the MJD84.2 SCA3 mouse model expressing the mutant human ataxin-3 gene using a multi-omics approach on brain and blood. Gene expression changes in brainstem, cerebellum, striatum and cortex were used to study pathological changes in brain, while blood gene expression and metabolites/lipids levels were examined as potential biomarkers for disease. Despite normal motor performance at 17.5 months of age, transcriptional changes in brain tissue of the SCA3 mice were observed. Most transcriptional changes occurred in brainstem and striatum, whilst cerebellum and cortex were only modestly affected. The most significantly altered genes in SCA3 mouse brain were Tmc3, Zfp488, Car2, and Chdh. Based on the transcriptional changes, α-adrenergic and CREB pathways were most consistently altered for combined analysis of the four brain regions. When examining individual brain regions, axon guidance and synaptic transmission pathways were most strongly altered in striatum, whilst brainstem presented with strongest alterations in the pi-3 k cascade and cholesterol biosynthesis pathways. Similar to other neurodegenerative diseases, reduced levels of tryptophan and increased levels of ceramides, di- and triglycerides were observed in SCA3 mouse blood. The observed transcriptional changes in SCA3 mouse brain reveal parallels with previous reported neuropathology in patients, but also shows brain region specific effects as well as involvement of adrenergic signalling and CREB pathway changes in SCA3. Importantly, the transcriptional changes occur prior to onset of motor- and coordination deficits.

A Methyl 4-Oxo-4-phenylbut-2-enoate with in Vivo Activity against MRSA that Inhibits MenB in the Bacterial Menaquinone Biosynthesis Pathway

PubMed Central

Matarlo, Joe S.; Lu, Yang; Daryaee, Fereidoon; Daryaee, Taraneh; Ruzsicska, Bela; Walker, Stephen G.; Tonge, Peter J.

2016-01-01

4-Oxo-4-phenyl-but-2-enoates inhibit MenB, the 1,4-dihydroxyl-2-naphthoyl-CoA synthase in the bacterial menaquinone (MK) biosynthesis pathway, through the formation of an adduct with coenzyme A (CoA). Here, we show that the corresponding methyl butenoates have MIC values as low as 0.35–0.75 µg/mL against drug sensitive and resistant strains of Staphylococcus aureus. Mode of action studies on the most potent compound, methyl 4-(4-chlorophenyl)-4-oxobut-2-enoate (1), reveal that 1 is converted into the corresponding CoA adduct in S. aureus cells, and that this adduct binds to the S. aureus MenB (saMenB) with a Kd value of 2 µM. The antibacterial spectrum of 1 is limited to bacteria that utilize MK for respiration, and the activity of 1 can be complemented with exogenous MK or menadione. Finally, treatment of methicillin-resistant S. aureus (MRSA) with 1 results in the small colony variant phenotype and thus 1 phenocopies knockout of the menB gene. Taken together the data indicate that the antibacterial activity of 1 results from a specific effect on MK biosynthesis. We also evaluated the in vivo efficacy of 1 using two mouse models of MRSA infection. Notably, compound 1 increased survival in a systemic infection model and resulted in a dose-dependent decrease in bacterial load in a thigh infection model, validating MenB as a target for the development of new anti-MRSA candidates. PMID:27294200

Ablation of cholesterol biosynthesis in neural stem cells increases their VEGF expression and angiogenesis but causes neuron apoptosis.

PubMed

Saito, Kanako; Dubreuil, Veronique; Arai, Yoko; Wilsch-Bräuninger, Michaela; Schwudke, Dominik; Saher, Gesine; Miyata, Takaki; Breier, Georg; Thiele, Christoph; Shevchenko, Andrej; Nave, Klaus-Armin; Huttner, Wieland B

2009-05-19

Although sufficient cholesterol supply is known to be crucial for neurons in the developing mammalian brain, the cholesterol requirement of neural stem and progenitor cells in the embryonic central nervous system has not been addressed. Here we have conditionally ablated the activity of squalene synthase (SQS), a key enzyme for endogenous cholesterol production, in the neural stem and progenitor cells of the ventricular zone (VZ) of the embryonic mouse brain. Mutant embryos exhibited a reduced brain size due to the atrophy of the neuronal layers, and died at birth. Analyses of the E11.5-E15.5 dorsal telencephalon and diencephalon revealed that this atrophy was due to massive apoptosis of newborn neurons, implying that this progeny of the SQS-ablated neural stem and progenitor cells was dependent on endogenous cholesterol biosynthesis for survival. Interestingly, the neural stem and progenitor cells of the VZ, the primary target of SQS inactivation, did not undergo significant apoptosis. Instead, vascular endothelial growth factor (VEGF) expression in these cells was strongly upregulated via a hypoxia-inducible factor-1-independent pathway, and angiogenesis in the VZ was increased. Consistent with an increased supply of lipoproteins to these cells, the level of lipid droplets containing triacylglycerides with unsaturated fatty acyl chains was found to be elevated. Our study establishes a direct link between intracellular cholesterol levels, VEGF expression, and angiogenesis. Moreover, our data reveal a hitherto unknown compensatory process by which the neural stem and progenitor cells of the developing mammalian brain evade the detrimental consequences of impaired endogenous cholesterol biosynthesis.

Ablation of cholesterol biosynthesis in neural stem cells increases their VEGF expression and angiogenesis but causes neuron apoptosis

PubMed Central

Saito, Kanako; Dubreuil, Veronique; Arai, Yoko; Wilsch-Bräuninger, Michaela; Schwudke, Dominik; Saher, Gesine; Miyata, Takaki; Breier, Georg; Thiele, Christoph; Shevchenko, Andrej; Nave, Klaus-Armin; Huttner, Wieland B.

2009-01-01

Although sufficient cholesterol supply is known to be crucial for neurons in the developing mammalian brain, the cholesterol requirement of neural stem and progenitor cells in the embryonic central nervous system has not been addressed. Here we have conditionally ablated the activity of squalene synthase (SQS), a key enzyme for endogenous cholesterol production, in the neural stem and progenitor cells of the ventricular zone (VZ) of the embryonic mouse brain. Mutant embryos exhibited a reduced brain size due to the atrophy of the neuronal layers, and died at birth. Analyses of the E11.5–E15.5 dorsal telencephalon and diencephalon revealed that this atrophy was due to massive apoptosis of newborn neurons, implying that this progeny of the SQS-ablated neural stem and progenitor cells was dependent on endogenous cholesterol biosynthesis for survival. Interestingly, the neural stem and progenitor cells of the VZ, the primary target of SQS inactivation, did not undergo significant apoptosis. Instead, vascular endothelial growth factor (VEGF) expression in these cells was strongly upregulated via a hypoxia-inducible factor-1–independent pathway, and angiogenesis in the VZ was increased. Consistent with an increased supply of lipoproteins to these cells, the level of lipid droplets containing triacylglycerides with unsaturated fatty acyl chains was found to be elevated. Our study establishes a direct link between intracellular cholesterol levels, VEGF expression, and angiogenesis. Moreover, our data reveal a hitherto unknown compensatory process by which the neural stem and progenitor cells of the developing mammalian brain evade the detrimental consequences of impaired endogenous cholesterol biosynthesis. PMID:19416849

Identification and Disruption of the proBA Locus in Listeria monocytogenes: Role of Proline Biosynthesis in Salt Tolerance and Murine Infection

PubMed Central

Sleator, Roy D.; Gahan, Cormac G. M.; Hill, Colin

2001-01-01

Intracellular accumulation of the amino acid proline has previously been linked to the salt tolerance and virulence potential of a number of bacteria. Taking advantage of the proBA mutant Escherichia coli CSH26, we identified a listerial proBA operon coding for enzymes functionally similar to the glutamyl kinase (GK) and glutamylphosphate reductase (GPR) enzyme complex which catalyzes the first and second steps of proline biosynthesis in E. coli. The first gene of the operon, proB, is predicted to encode GK, a 276-residue protein with a calculated molecular mass of 30.03 kDa and pl of 5.2. Distal to the promoter and overlapping the 3′ end of proB by 17 bp is proA, which encodes GPR, a 415-residue protein with a calculated molecular mass of 45.50 kDa (pl 5.3). Using this information, we created a chromosomal deletion mutant by allelic exchange which is auxotrophic for proline. This mutant was used to assess the contribution of proline anabolism to osmotolerance and virulence. While inactivation of proBA had no significant effect on virulence in mouse assays (either perorally or intraperitoneally), growth at low (2 to 4% NaCl) and high (>6% NaCl) salt concentrations in complex media was significantly reduced in the absence of efficient proline synthesis. We conclude that while proline biosynthesis plays little, if any, role in the intracellular life cycle and infectious nature of Listeria monocytogenes, it can play an important role in survival in osmolyte-depleted environments of elevated osmolarity. PMID:11375165

Odd Chain Fatty Acids; New Insights of the Relationship Between the Gut Microbiota, Dietary Intake, Biosynthesis and Glucose Intolerance.

PubMed

Jenkins, Benjamin J; Seyssel, Kevin; Chiu, Sally; Pan, Pin-Ho; Lin, Shih-Yi; Stanley, Elizabeth; Ament, Zsuzsanna; West, James A; Summerhill, Keith; Griffin, Julian L; Vetter, Walter; Autio, Kaija J; Hiltunen, Kalervo; Hazebrouck, Stéphane; Stepankova, Renata; Chen, Chun-Jung; Alligier, Maud; Laville, Martine; Moore, Mary; Kraft, Guillaume; Cherrington, Alan; King, Sarah; Krauss, Ronald M; de Schryver, Evelyn; Van Veldhoven, Paul P; Ronis, Martin; Koulman, Albert

2017-03-23

Recent findings have shown an inverse association between circulating C15:0/C17:0 fatty acids with disease risk, therefore, their origin needs to be determined to understanding their role in these pathologies. Through combinations of both animal and human intervention studies, we comprehensively investigated all possible contributions of these fatty acids from the gut-microbiota, the diet, and novel endogenous biosynthesis. Investigations included an intestinal germ-free study and a C15:0/C17:0 diet dose response study. Endogenous production was assessed through: a stearic acid infusion, phytol supplementation, and a Hacl1 -/- mouse model. Two human dietary intervention studies were used to translate the results. Finally, a study comparing baseline C15:0/C17:0 with the prognosis of glucose intolerance. We found that circulating C15:0/C17:0 levels were not influenced by the gut-microbiota. The dose response study showed C15:0 had a linear response, however C17:0 was not directly correlated. The phytol supplementation only decreased C17:0. Stearic acid infusion only increased C17:0. Hacl1 -/- only decreased C17:0. The glucose intolerance study showed only C17:0 correlated with prognosis. To summarise, circulating C15:0 and C17:0 are independently derived; C15:0 correlates directly with dietary intake, while C17:0 is substantially biosynthesized, therefore, they are not homologous in the aetiology of metabolic disease. Our findings emphasize the importance of the biosynthesis of C17:0 and recognizing its link with metabolic disease.

Reusable magnetic nanobiocatalyst for synthesis of silver and gold nanoparticles.

PubMed

Mazumder, Jahirul Ahmed; Ahmad, Razi; Sardar, Meryam

2016-12-01

In the present work, we describe a simple procedure for the biosynthesis of nanosilver and gold by the reduction of silver nitrate and auric chloride respectively using a nanobiocatalyst. The nanobiocatalyst was prepared by covalent coupling of alpha amylase on (3-aminopropyl)triethoxysilane (APTES) modified iron oxide magnetic nanoparticles. The nanobiocatalyst retains 77% of its activity as compared to free alpha amylase. The nanobiocatalyst can be used up to three consecutive cycles for the synthesis of nano silver and gold. The biosynthesized nanoparticles after each cycle were characterized by UV-vis spectrophotometer, Dynamic Light Spectroscopy (DLS), Transmission Electron Microscope (TEM), X-ray powder diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR). Silver and gold nanoparticles of same morphology and dimensions were formed in each cycle. The procedure for synthesis of nanoparticles using an immobilized enzyme is eco-friendly and can be used repeatedly. Copyright © 2016 Elsevier B.V. All rights reserved.

Eco-friendly biosynthesis, anticancer drug loading and cytotoxic effect of capped Ag-nanoparticles against breast cancer

NASA Astrophysics Data System (ADS)

Naz, M.; Nasiri, N.; Ikram, M.; Nafees, M.; Qureshi, M. Z.; Ali, S.; Tricoli, A.

2017-11-01

The work aimed to prepare silver nanoparticles (Ag-NPs) from silver nitrate and various concentrations of the seed extract ( Setaria verticillata) by a green synthetic route. The chemical and physical properties of the resulting Ag-NPs were investigated by X-ray diffraction (XRD), transmission electron microscopy (TEM), Fourier transform infrared (FTIR) spectrometry and ultraviolet-visible (UV-Vis) spectrophotometry. Anticancer activity of Ag-NPs (5-20 nm) had dose-dependent cytotoxic effect against breast cancer (MCF7-FLV) cells. The in vitro toxicity was studied on adult earthworms (Lumbricina) resulting in statistically significant ( P < 0.05) inhibition. The prepared NPs were loaded with hydrophilic anticancer drugs (ACD), doxorubicin (DOX) and daunorubicin (DNR), for developing a novel drug delivery carrier having significant adsorption capacity and efficiency to remove the side effects of the medicines effective for leukemia chemotherapy.

Development of a novel pink-eyed dilution mouse model showing progressive darkening of the eyes and coat hair with aging

PubMed Central

ISHIKAWA, Akira; SUGIYAMA, Makoto; HONDO, Eiichi; KINOSHITA, Keiji; YAMAGISHI, Yuki

2015-01-01

Oca2p-cas (oculocutaneous albinism II; pink-eyed dilution castaneus) is a coat color mutant gene on mouse chromosome 7 that arose spontaneously in wild Mus musculus castaneus mice. Mice homozygous for Oca2p-cas usually exhibit pink eyes and gray coat hair on the non-agouti genetic background, and this ordinary phenotype remains unchanged throughout life. During breeding of a mixed strain carrying this gene on the C57BL/6J background, we discovered a novel spontaneous mutation that causes darkening of the eyes and coat hair with aging. In this study, we developed a novel mouse model showing this unique phenotype. Gross observations revealed that the pink eyes and gray coat hair of the novel mutant young mice became progressively darker in color by approximately 3 months after birth. Light and transmission-electron microscopic observations revealed a marked increase in melanin pigmentation of coat hair shafts and choroid of the eye in the novel mice compared to that in the ordinary mice. Sequence analysis of Oca2p-cas revealed a 4.1-kb deletion involving exons 15 and 16 of its wild-type gene. However, there was no sequence difference between the two types of mutant mice. Mating experiments suggested that the novel mutant phenotype was not inherited in a simple fashion, due to incomplete penetrance. The novel spontaneous mutant mouse is the first example of progressive hair darkening animals and is an essential animal model for understanding of the regulation mechanisms of melanin biosynthesis with aging. PMID:25739360

Development of a novel pink-eyed dilution mouse model showing progressive darkening of the eyes and coat hair with aging.

PubMed

Ishikawa, Akira; Sugiyama, Makoto; Hondo, Eiichi; Kinoshita, Keiji; Yamagishi, Yuki

2015-01-01

Oca2(p-cas) (oculocutaneous albinism II; pink-eyed dilution castaneus) is a coat color mutant gene on mouse chromosome 7 that arose spontaneously in wild Mus musculus castaneus mice. Mice homozygous for Oca2(p-cas) usually exhibit pink eyes and gray coat hair on the non-agouti genetic background, and this ordinary phenotype remains unchanged throughout life. During breeding of a mixed strain carrying this gene on the C57BL/6J background, we discovered a novel spontaneous mutation that causes darkening of the eyes and coat hair with aging. In this study, we developed a novel mouse model showing this unique phenotype. Gross observations revealed that the pink eyes and gray coat hair of the novel mutant young mice became progressively darker in color by approximately 3 months after birth. Light and transmission-electron microscopic observations revealed a marked increase in melanin pigmentation of coat hair shafts and choroid of the eye in the novel mice compared to that in the ordinary mice. Sequence analysis of Oca2(p-cas) revealed a 4.1-kb deletion involving exons 15 and 16 of its wild-type gene. However, there was no sequence difference between the two types of mutant mice. Mating experiments suggested that the novel mutant phenotype was not inherited in a simple fashion, due to incomplete penetrance. The novel spontaneous mutant mouse is the first example of progressive hair darkening animals and is an essential animal model for understanding of the regulation mechanisms of melanin biosynthesis with aging.

Nitrated fatty acids reverse pulmonary fibrosis by dedifferentiating myofibroblasts and promoting collagen uptake by alveolar macrophages

PubMed Central

Reddy, Aravind T.; Lakshmi, Sowmya P.; Zhang, Yingze; Reddy, Raju C.

2014-01-01

Idiopathic pulmonary fibrosis (IPF) is a progressive, fatal disease, thought to be largely transforming growth factor β (TGFβ) driven, for which there is no effective therapy. We assessed the potential benefits in IPF of nitrated fatty acids (NFAs), which are unique endogenous agonists of peroxisome proliferator-activated receptor γ (PPARγ), a nuclear hormone receptor that exhibits wound-healing and antifibrotic properties potentially useful for IPF therapy. We found that pulmonary PPARγ is down-regulated in patients with IPF. In vitro, knockdown or knockout of PPARγ expression in isolated human and mouse lung fibroblasts induced a profibrotic phenotype, whereas treating human fibroblasts with NFAs up-regulated PPARγ and blocked TGFβ signaling and actions. NFAs also converted TGFβ to inactive monomers in cell-free solution, suggesting an additional mechanism through which they may inhibit TGFβ. In vivo, treating mice bearing experimental pulmonary fibrosis with NFAs reduced disease severity. Also, NFAs up-regulated the collagen-targeting factor milk fat globule-EGF factor 8 (MFG-E8), stimulated collagen uptake and degradation by alveolar macrophages, and promoted myofibroblast dedifferentiation. Moreover, treating mice with established pulmonary fibrosis using NFAs reversed their existing myofibroblast differentiation and collagen deposition. These findings raise the prospect of treating IPF with NFAs to halt and perhaps even reverse the progress of IPF.—Reddy, A. T., Lakshmi, S. P., Zhang, Y., Reddy, R. C. Nitrated fatty acids reverse pulmonary fibrosis by dedifferentiating myofibroblasts and promoting collagen uptake by alveolar macrophages. PMID:25252739

Mitochondrial reactive oxygen species generation triggers inflammatory response and tissue injury associated with hepatic ischemia-reperfusion: therapeutic potential of mitochondrially-targeted antioxidants

PubMed Central

Mukhopadhyay, Partha; Horváth, Bėla; Zsengellėr, Zsuzsanna; Bátkai, Sándor; Cao, Zongxian; Kechrid, Malek; Holovac, Eileen; Erdėlyi, Katalin; Tanchian, Galin; Liaudet, Lucas; Stillman, Isaac E.; Joseph, Joy; Kalyanaraman, Balaraman; Pacher, Pál

2012-01-01

Mitochondrial reactive oxygen species generation has been implicated in the pathophysiology of ischemia-reperfusion (I/R) injury, however its exact role and its spatial-temporal relationship with inflammation are elusive. Herein we explored the spatial-temporal relationship of oxidative/nitrative stress and inflammatory response during the course of hepatic I/R and the possible therapeutic potential of mitochondrial-targeted antioxidants, using a mouse model of segmental hepatic ischemia-reperfusion injury. Hepatic I/R was characterized by early (at 2 hours of reperfusion) mitochondrial injury, decreased complex I activity, increased oxidant generation in the liver or liver mitochondria, and profound hepatocellular injury/dysfunction with acute pro-inflammatory response (TNF-α, MIP-1αCCL3, MIP-2/CXCL2) without inflammatory cell infiltration, followed by marked neutrophil infiltration and more pronounced secondary wave of oxidative/nitrative stress in the liver (starting from 6 hours of reperfusion and peaking at 24 hours). Mitochondrially-targeted antioxidants, MitoQ or Mito-CP, dose-dependently attenuated I/R-induced liver dysfunction, the early and delayed oxidative and nitrative stress response (HNE/carbonyl adducts, malondialdehyde, 8-OHdG, and 3-nitrotyrosine formation), mitochondrial and histopathological injury/dysfunction, as well as delayed inflammatory cell infiltration and cell death. Mitochondrially generated oxidants play a central role in triggering the deleterious cascade of events associated with hepatic I/R, which may be targeted by novel antioxidants for therapeutic advantage. PMID:22683818

IF1, a natural inhibitor of mitochondrial ATP synthase, is not essential for the normal growth and breeding of mice.

PubMed

Nakamura, Junji; Fujikawa, Makoto; Yoshida, Masasuke

2013-09-17

IF1 is an endogenous inhibitor protein of mitochondrial ATP synthase. It is evolutionarily conserved throughout all eukaryotes and it has been proposed to play crucial roles in prevention of the wasteful reverse reaction of ATP synthase, in the metabolic shift from oxidative phosphorylation to glycolysis, in the suppression of ROS (reactive oxygen species) generation, in mitochondria morphology and in haem biosynthesis in mitochondria, which leads to anaemia. Here, we report the phenotype of a mouse strain in which IF1 gene was destroyed. Unexpectedly, individuals of this IF1-KO (knockout) mouse strain grew and bred without defect. The general behaviours, blood test results and responses to starvation of the IF1-KO mice were apparently normal. There were no abnormalities in the tissue anatomy or the autophagy. Mitochondria of the IF1-KO mice were normal in morphology, in the content of ATP synthase molecules and in ATP synthesis activity. Thus, IF1 is not an essential protein for mice despite its ubiquitous presence in eukaryotes.

Responses to Systemic Nitrogen Signaling in Arabidopsis Roots Involve trans-Zeatin in Shoots.

PubMed

Poitout, Arthur; Crabos, Amandine; Petřík, Ivan; Novák, Ondřej; Krouk, Gabriel; Lacombe, Benoît; Ruffel, Sandrine

2018-05-15

Plants face temporal and spatial variation in nitrogen (N) availability. This includes heterogeneity in soil nitrate (NO3-) content. To overcome these constraints, plants modify their gene expression and physiological processes to optimize N acquisition. This plasticity relies on a complex long-distance root-shoot-root signaling network that remains poorly understood. We previously showed that cytokinin (CK) biosynthesis is required to trigger systemic N signaling. Here, we performed split-root experiments and used a combination of CK-related mutant analyses, hormone profiling, transcriptomic analysis, NO3- uptake assays, and root growth measurements to gain insight into systemic N signaling in Arabidopsis thaliana. By comparing wild-type plants and mutants affected in CK biosynthesis and ABCG14-dependent root-to-shoot translocation of CK, we revealed an important role for active trans-Zeatin (tZ) in systemic N signaling. Both rapid sentinel gene regulation and long-term functional acclimation to heterogeneous NO3- supply, including NO3- transport and root growth regulation, are likely mediated by the integration of tZ content in shoots. Furthermore, shoot transcriptome profiling revealed that glutamate/glutamine metabolism is likely a target of tZ root-to-shoot translocation, prompting an interesting hypothesis regarding shoot-to-root communication. Finally, this study highlights tZ-independent pathways regulating gene expression in shoots as well as NO3- uptake activity in response to total N-deprivation. © 2018 American Society of Plant Biologists. All rights reserved.

Isolation and characterization of gallium resistant Pseudomonas aeruginosa mutants.

PubMed

García-Contreras, Rodolfo; Lira-Silva, Elizabeth; Jasso-Chávez, Ricardo; Hernández-González, Ismael L; Maeda, Toshinari; Hashimoto, Takahiro; Boogerd, Fred C; Sheng, Lili; Wood, Thomas K; Moreno-Sánchez, Rafael

2013-12-01

Pseudomonas aeruginosa PA14 cells resistant to the novel antimicrobial gallium nitrate (Ga) were developed using transposon mutagenesis and by selecting spontaneous mutants. The mutants showing the highest growth in the presence of Ga were selected for further characterization. These mutants showed 4- to 12-fold higher Ga minimal inhibitory growth concentrations and a greater than 8-fold increase in the minimum biofilm eliminating Ga concentration. Both types of mutants produced Ga resistant biofilms whereas the formation of wild-type biofilms was strongly inhibited by Ga. The gene interrupted in the transposon mutant was hitA, which encodes a periplasmic iron binding protein that delivers Fe³⁺ to the HitB iron permease; complementation of the mutant with the hitA gene restored the Ga sensitivity. This hitA mutant showed a 14-fold decrease in Ga internalization versus the wild-type strain, indicating that the HitAB system is also involved in the Ga uptake. Ga uptake in the spontaneous mutant was also lower, although no mutations were found in the hitAB genes. Instead, this mutant harbored 64 non-silent mutations in several genes including those of the phenazine pyocyanin biosynthesis. The spontaneous mutant produced 2-fold higher pyocyanin basal levels than the wild-type; the addition of this phenazine to wild-type cultures protected them from the Ga bacteriostatic effect. The present data indicate that mutations affecting Ga transport and probably pyocyanin biosynthesis enable cells to develop resistance to Ga. Copyright © 2013 Elsevier GmbH. All rights reserved.

Nitric oxide plays a role in stem cell niche homeostasis through its interaction with auxin.

PubMed

Sanz, Luis; Fernández-Marcos, María; Modrego, Abelardo; Lewis, Daniel R; Muday, Gloria K; Pollmann, Stephan; Dueñas, Montserrat; Santos-Buelga, Celestino; Lorenzo, Oscar

2014-12-01

Nitric oxide (NO) is a unique reactive nitrogen molecule with an array of signaling functions that modulates plant developmental processes and stress responses. To explore the mechanisms by which NO modulates root development, we used a pharmacological approach and NO-deficient mutants to unravel the role of NO in establishing auxin distribution patterns necessary for stem cell niche homeostasis. Using the NO synthase inhibitor and Arabidopsis (Arabidopsis thaliana) NO biosynthesis mutants (nitric oxide-associated1 [noa1], nitrate reductase1 [nia1] and nia2, and nia1 nia2 noa1), we show that depletion of NO in noa1 reduces primary root elongation and increases flavonol accumulation consistent with elevated reactive oxygen species levels. The elevated flavonols are required for the growth effect, because the transparent testa4 mutation reverses the noa1 mutant root elongation phenotype. In addition, noa1 and nia1 nia2 noa1 NO-deficient mutant roots display small root meristems with abnormal divisions. Concomitantly, auxin biosynthesis, transport, and signaling are perturbed. We further show that NO accumulates in cortex/endodermis stem cells and their precursor cells. In endodermal and cortical cells, the noa1 mutant acts synergistically to the effect of the wuschel-related homeobox5 mutation on the proximal meristem, suggesting that NO could play an important role in regulating stem cell decisions, which has been reported in animals. © 2014 American Society of Plant Biologists. All Rights Reserved.

Pantethine treatment is effective in recovering the disease phenotype induced by ketogenic diet in a pantothenate kinase-associated neurodegeneration mouse model

PubMed Central

Brunetti, Dario; Dusi, Sabrina; Giordano, Carla; Lamperti, Costanza; Morbin, Michela; Fugnanesi, Valeria; Marchet, Silvia; Fagiolari, Gigliola; Sibon, Ody; Moggio, Maurizio; d’Amati, Giulia

2014-01-01

Pantothenate kinase-associated neurodegeneration, caused by mutations in the PANK2 gene, is an autosomal recessive disorder characterized by dystonia, dysarthria, rigidity, pigmentary retinal degeneration and brain iron accumulation. PANK2 encodes the mitochondrial enzyme pantothenate kinase type 2, responsible for the phosphorylation of pantothenate or vitamin B5 in the biosynthesis of co-enzyme A. A Pank2 knockout (Pank2−/−) mouse model did not recapitulate the human disease but showed azoospermia and mitochondrial dysfunctions. We challenged this mouse model with a low glucose and high lipid content diet (ketogenic diet) to stimulate lipid use by mitochondrial beta-oxidation. In the presence of a shortage of co-enzyme A, this diet could evoke a general impairment of bioenergetic metabolism. Only Pank2−/− mice fed with a ketogenic diet developed a pantothenate kinase-associated neurodegeneration-like syndrome characterized by severe motor dysfunction, neurodegeneration and severely altered mitochondria in the central and peripheral nervous systems. These mice also showed structural alteration of muscle morphology, which was comparable with that observed in a patient with pantothenate kinase-associated neurodegeneration. We here demonstrate that pantethine administration can prevent the onset of the neuromuscular phenotype in mice suggesting the possibility of experimental treatment in patients with pantothenate kinase-associated neurodegeneration. PMID:24316510

Lead nitrate-induced development of hypercholesterolemia in rats: sterol-independent gene regulation of hepatic enzymes responsible for cholesterol homeostasis.

PubMed

Kojima, Misaki; Masui, Toshimitsu; Nemoto, Kiyomitsu; Degawa, Masakuni

2004-12-01

Changes in the gene expressions of hepatic enzymes responsible for cholesterol homeostasis were examined during the process of lead nitrate (LN)-induced development of hypercholesterolemia in male rats. Total cholesterol levels in the liver and serum were significantly increased at 3-72 h and 12-72 h, respectively, after LN-treatment (100 micromol/kg, i.v.). Despite the development of hypercholesterolemia, the genes for hepatic 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) and other enzymes (FPPS, farnesyl diphosphate synthase; SQS, squalene synthase; CYP51, lanosterol 14alpha-demethylase) responsible for cholesterol biosynthesis were activated at 3-24 h and 12-18 h, respectively. On the other hand, the gene expression of cholesterol 7alpha-hydroxylase (CYP7A1), a catabolic enzyme of cholesterol, was remarkably suppressed at 3-72 h. The gene expression levels of cytokines interleukin-1beta (IL-1beta) and TNF-alpha, which activate the HMGR gene and suppress the CYP7A1 gene, were significantly increased at 1-3 h and 3-24 h, respectively. Furthermore, gene activation of SREBP-2, a gene activator of several cholesterogenic enzymes, occurred before the gene activations of FPPS, SQS and CYP51. This is the first report demonstrating sterol-independent gene regulation of hepatic enzymes responsible for cholesterol homeostasis in LN-treated male rats. The mechanisms for the altered-gene expressions of hepatic enzymes in LN-treated rats are discussed.

Gender-related difference in altered gene expression of a sterol regulatory element binding protein, SREBP-2, by lead nitrate in rats: correlation with development of hypercholesterolemia.

PubMed

Kojima, Misaki; Degawa, Masakuni

2006-01-01

Changes in gene expression levels of hepatic sterol regulatory element binding protein-2 (SREBP-2) and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) after a single i.v. injection of lead nitrate (LN, 100 micromol kg(-1) body weight) were examined comparatively by real time reverse transcriptase-polymerase chain reaction (RT-PCR) in male and female rats. Significant increases in the gene expression level of SREBP-2, a transcription factor for the HMGR gene, occurred at 6-12 h in male and at 24-36 h in female rats after LN-treatment. The gene expression level of HMGR, a rate-limiting enzyme for cholesterol biosynthesis, significantly increased at 3-48 h in male rats and 12-48 h in female rats. Subsequently, significant increases in the amount of hepatic total cholesterol in male and female rats were also observed at 3-48 h and 24-48 h, respectively. The present findings demonstrate that increases in gene expressions of hepatic SREBP-2 and HMGR and the amount of hepatic total cholesterol by LN occur earlier in male rats than in the females, and that increases in the gene expression level of HMGR and the amount of hepatic total cholesterol occur prior to the increase in the gene expression level of SREBP-2 in either sex of rats. Copyright (c) 2006 John Wiley & Sons, Ltd.

Influence of the temporal and spatial variation of nitrate reductase, glutamine synthetase and soil composition in the N species content in lettuce (Lactuca sativa).

PubMed

Pinto, Edgar; Fidalgo, Fernanda; Teixeira, Jorge; Aguiar, Ana A; Ferreira, Isabel M P L V O

2014-04-01

The variation of nitrate reductase (NR), glutamine synthetase (GS) and N content in lettuce was evaluated at 5 stages of lettuce growth. Soil physicochemical properties and its N content were also assessed to elucidate the soil-to-plant transfer of inorganic N and potential leaching to groundwater. A decrease of NR activity and an increase of NO3(-) and N-Kjeldahl content in lettuces were observed during plant growth, whereas GS activity and NH4(+) increased during the first few weeks of lettuce growth and then decreased. Although the temporal variation was similar in lettuces grown in different soils, quantitative differences were observed, indicating that high NO3(-) content in soil caused a higher NO3(-) accumulation in lettuce despite the higher NR activity during the initial stage of plant growth. Higher levels of NO3(-) and NH4(+) were correlated with higher levels of N-Kjeldahl in lettuce suggesting a positive effect of these N species in the biosynthesis of organic forms of N. Soil physicochemical properties influenced the mobility of inorganic N within the groundwater-soil-plant system. Sandy soils with low OM content allowed NO3(-) leaching, which was confirmed by higher NO3(-) levels in groundwater. Therefore, lettuces grown in those soils presented lower N content and the inputs of N to the environment were higher. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Phosphoproteomics of Klebsiella pneumoniae NTUH-K2044 Reveals a Tight Link between Tyrosine Phosphorylation and Virulence*

PubMed Central

Lin, Miao-Hsia; Hsu, Tung-Li; Lin, Shu-Yu; Pan, Yi-Jiun; Jan, Jia-Tsrong; Wang, Jin-Town; Khoo, Kay-Hooi; Wu, Shih-Hsiung

2009-01-01

Encapsulated Klebsiella pneumoniae is the predominant causative agent of pyogenic liver abscess, an emerging infectious disease that often complicates metastatic meningitis or endophthalmitis. The capsular polysaccharide on K. pneumoniae surface was determined as the key to virulence. Although the regulation of capsular polysaccharide biosynthesis is largely unclear, it was found that protein-tyrosine kinases and phosphatases are involved. Therefore, the identification and characterization of such kinases, phosphatases, and their substrates would advance our knowledge of the underlying mechanism in capsule formation and could contribute to the development of new therapeutic strategies. Here, we analyzed the phosphoproteome of K. pneumoniae NTUH-K2044 with a shotgun approach and identified 117 unique phosphopeptides along with 93 in vivo phosphorylated sites corresponding to 81 proteins. Interestingly, three of the identified tyrosine phosphorylated proteins, namely protein-tyrosine kinase (Wzc), phosphomannomutase (ManB), and undecaprenyl-phosphate glycosyltransferase (WcaJ), were found to be distributed in the cps locus and thus were speculated to be involved in the converging signal transduction of capsule biosynthesis. Consequently, we decided to focus on the lesser studied ManB and WcaJ for mutation analysis. The capsular polysaccharides of WcaJ mutant (WcaJY5F) were dramatically reduced quantitatively, and the LD50 increased by 200-fold in a mouse peritonitis model compared with the wild-type strain. However, the capsular polysaccharides of ManB mutant (ManBY26F) showed no difference in quantity, and the LD50 increased by merely 6-fold in mice test. Our study provided a clear trend that WcaJ tyrosine phosphorylation can regulate the biosynthesis of capsular polysaccharides and result in the pathogenicity of K. pneumoniae NTUH-K2044. PMID:19696081

Odd Chain Fatty Acids; New Insights of the Relationship Between the Gut Microbiota, Dietary Intake, Biosynthesis and Glucose Intolerance

PubMed Central

Jenkins, Benjamin J.; Seyssel, Kevin; Chiu, Sally; Pan, Pin-Ho; Lin, Shih-Yi; Stanley, Elizabeth; Ament, Zsuzsanna; West, James A.; Summerhill, Keith; Griffin, Julian L.; Vetter, Walter; Autio, Kaija J.; Hiltunen, Kalervo; Hazebrouck, Stéphane; Stepankova, Renata; Chen, Chun-Jung; Alligier, Maud; Laville, Martine; Moore, Mary; Kraft, Guillaume; Cherrington, Alan; King, Sarah; Krauss, Ronald M.; de Schryver, Evelyn; Van Veldhoven, Paul P.; Ronis, Martin; Koulman, Albert

2017-01-01

Recent findings have shown an inverse association between circulating C15:0/C17:0 fatty acids with disease risk, therefore, their origin needs to be determined to understanding their role in these pathologies. Through combinations of both animal and human intervention studies, we comprehensively investigated all possible contributions of these fatty acids from the gut-microbiota, the diet, and novel endogenous biosynthesis. Investigations included an intestinal germ-free study and a C15:0/C17:0 diet dose response study. Endogenous production was assessed through: a stearic acid infusion, phytol supplementation, and a Hacl1−/− mouse model. Two human dietary intervention studies were used to translate the results. Finally, a study comparing baseline C15:0/C17:0 with the prognosis of glucose intolerance. We found that circulating C15:0/C17:0 levels were not influenced by the gut-microbiota. The dose response study showed C15:0 had a linear response, however C17:0 was not directly correlated. The phytol supplementation only decreased C17:0. Stearic acid infusion only increased C17:0. Hacl1−/− only decreased C17:0. The glucose intolerance study showed only C17:0 correlated with prognosis. To summarise, circulating C15:0 and C17:0 are independently derived; C15:0 correlates directly with dietary intake, while C17:0 is substantially biosynthesized, therefore, they are not homologous in the aetiology of metabolic disease. Our findings emphasize the importance of the biosynthesis of C17:0 and recognizing its link with metabolic disease. PMID:28332596

Formate supplementation enhances folate-dependent nucleotide biosynthesis and prevents spina bifida in a mouse model of folic acid-resistant neural tube defects.

PubMed

Sudiwala, Sonia; De Castro, Sandra C P; Leung, Kit-Yi; Brosnan, John T; Brosnan, Margaret E; Mills, Kevin; Copp, Andrew J; Greene, Nicholas D E

2016-07-01

The curly tail mouse provides a model for neural tube defects (spina bifida and exencephaly) that are resistant to prevention by folic acid. The major ct gene, responsible for spina bifida, corresponds to a hypomorphic allele of grainyhead-like 3 (Grhl3) but the frequency of NTDs is strongly influenced by modifiers in the genetic background. Moreover, exencephaly in the curly tail strain is not prevented by reinstatement of Grhl3 expression. In the current study we found that expression of Mthfd1L, encoding a key component of mitochondrial folate one-carbon metabolism (FOCM), is significantly reduced in ct/ct embryos compared to a partially congenic wild-type strain. This expression change is not attributable to regulation by Grhl3 or the genetic background at the Mthfd1L locus. Mitochondrial FOCM provides one-carbon units as formate for FOCM reactions in the cytosol. We found that maternal supplementation with formate prevented NTDs in curly tail embryos and also resulted in increased litter size. Analysis of the folate profile of neurulation-stage embryos showed that formate supplementation resulted in an increased proportion of formyl-THF and THF but a reduction in proportion of 5-methyl THF. In contrast, THF decreased and 5-methyl THF was relatively more abundant in the liver of supplemented dams than in controls. In embryos cultured through the period of spinal neurulation, incorporation of labelled thymidine and adenine into genomic DNA was suppressed by supplemental formate, suggesting that de novo folate-dependent biosynthesis of nucleotides (thymidylate and purines) was enhanced. We hypothesise that reduced Mthfd1L expression may contribute to susceptibility to NTDs in the curly tail strain and that formate acts as a one-carbon donor to prevent NTDs. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Obesity resistant mechanisms in the Lean polygenic mouse model as indicated by liver transcriptome and expression of selected genes in skeletal muscle

PubMed Central

2011-01-01

Background Divergently selected Lean and Fat mouse lines represent unique models for a polygenic form of resistance and susceptibility to obesity development. Previous research on these lines focused mainly on obesity-susceptible factors in the Fat line. This study aimed to examine the molecular basis of obesity-resistant mechanisms in the Lean line by analyzing various fat depots and organs, the liver transcriptome of selected metabolic pathways, plasma and lipid homeostasis and expression of selected skeletal muscle genes. Results Expression profiling using our custom Steroltalk v2 microarray demonstrated that Lean mice exhibit a higher hepatic expression of cholesterol biosynthesis genes compared to the Fat line, although this was not reflected in elevation of total plasma or liver cholesterol. However, FPLC analysis showed that protective HDL cholesterol was elevated in Lean mice. A significant difference between the strains was also found in bile acid metabolism. Lean mice had a higher expression of Cyp8b1, a regulatory enzyme of bile acid synthesis, and the Abcb11 bile acid transporter gene responsible for export of acids to the bile. Additionally, a higher content of blood circulating bile acids was observed in Lean mice. Elevated HDL and upregulation of some bile acids synthesis and transport genes suggests enhanced reverse cholesterol transport in the Lean line - the flux of cholesterol out of the body is higher which is compensated by upregulation of endogenous cholesterol biosynthesis. Increased skeletal muscle Il6 and Dio2 mRNA levels as well as increased activity of muscle succinic acid dehydrogenase (SDH) in the Lean mice demonstrates for the first time that changes in muscle energy metabolism play important role in the Lean line phenotype determination and corroborate our previous findings of increased physical activity and thermogenesis in this line. Finally, differential expression of Abcb11 and Dio2 identifies novel strong positional candidate genes as they map within the quantitative trait loci (QTL) regions detected previously in crosses between the Lean and Fat mice. Conclusion We identified novel candidate molecular targets and metabolic changes which can at least in part explain resistance to obesity development in the Lean line. The major difference between the Lean and Fat mice was in increased liver cholesterol biosynthesis gene mRNA expression, bile acid metabolism and changes in selected muscle genes' expression in the Lean line. The liver Abcb11 and muscle Dio2 were identified as novel positional candidate genes to explain part of the phenotypic difference between the Lean and Fat lines. PMID:21291556

3',5'-cyclic adenosine monophosphate response element binding protein up-regulated cytochrome P450 lanosterol 14alpha-demethylase expression involved in follicle-stimulating hormone-induced mouse oocyte maturation.

PubMed

Ning, Gang; Ouyang, Hong; Wang, Songbo; Chen, Xiufen; Xu, Baoshan; Yang, Jiange; Zhang, Hua; Zhang, Meijia; Xia, Guoliang

2008-07-01

Cytochrome P450 lanosterol 14alpha-demethylase (CYP51) is a key enzyme in sterols and steroids biosynthesis that can induce meiotic resumption in mouse oocytes. The present study investigated the expression mechanism and function of CYP51 during FSH-induced mouse cumulus oocyte complexes (COCs) meiotic resumption. FSH increased cAMP-dependent protein kinase (PKA) RIIbeta level and induced cAMP response element-binding protein (CREB) phosphorylation and CYP51 expression in cumulus cells before oocyte meiotic resumption. Moreover, CYP51 and epidermal growth factor (EGF)-like factor [amphiregulin (AR)] expression were blocked by (2)-naphthol-AS-Ephosphate (KG-501) (a drug interrupting the formation of CREB functional complex). KG-501 and RS21607 (a specific inhibitor of CYP51 activity) inhibited oocyte meiotic resumption, which can be partially rescued by progesterone. These two inhibitors also inhibited FSH-induced MAPK phosphorylation. EGF could rescue the suppression by KG-501 but not RS21607. Furthermore, type II PKA analog pairs, N(6)-monobutyryl-cAMP plus 8-bromo-cAMP, increased PKA RIIbeta level and mimicked the action of FSH, including CREB phosphorylation, AR and CYP51 expression, MAPK activation, and oocyte maturation. All these data suggest that CYP51 plays a critical role in FSH-induced meiotic resumption of mouse oocytes. CYP51 and AR gene expression in cumulus cells are triggered by FSH via a type II PKA/CREB-dependent signal pathway. Our study also implicates that CYP51 activity in cumulus cells participates in EGF receptor signaling-regulated oocyte meiotic resumption.

A Congenic Line of the C57BL/6J Mouse Strain that is Proficient in Melatonin Synthesis.

PubMed

Zhang, Zhijing; Silveyra, Eduardo; Jin, Nange; Ribelayga, Christophe P

2018-05-16

The C57BL/6J (B6) is the most common inbred mouse strain used in biomedical research in the United States. Yet, this strain is notoriously known for being deficient in the biosynthesis of melatonin, an important effector of circadian clocks in the brain and in the retina. Melatonin deficiency in this strain results from non-functional alleles of the genes coding two key enzymes of the melatonin synthesis pathway: arylalkylamine-N-acetyltransferase (Aanat) and N-acetylserotonin-O-methyltransferase (Asmt). By introducing functional alleles of the Aanat and Asmt genes from the melatonin-proficient CBA/CaJ (CBA) mouse strain to B6, we have generated a B6 congenic line that has acquired the capacity of rhythmic melatonin synthesis. In addition, the melatonin-dependent rhythm of dopamine release in the retina is restored in the B6 congenic line. Finally, we have partially characterized the Aanat and Asmt genes of the CBA strain and have identified multiple differences between CBA and B6 alleles, including single nucleotide polymorphism and deletion/insertion of DNA segments of various sizes. As an improved model organism with functional components of the melatonin synthesis pathway and melatonin-dependent circadian regulations, the new line will be useful to researchers studying melatonin physiological functions in a variety of fields including, but not limited to, circadian biology and neuroscience. In particular, the congenic line will be useful to speed up introduction of melatonin production capacity into genetically-modified mouse lines of interest such as knockout lines, many of which are on B6 or mixed B6 backgrounds. The melatonin-proficient B6 congenic line will be widely distributed. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Effect of glial cell line-derived neurotrophic factor on behavior and key members of the brain serotonin system in mouse strains genetically predisposed to behavioral disorders.

PubMed

Naumenko, Vladimir S; Bazovkina, Daria V; Semenova, Alina A; Tsybko, Anton S; Il'chibaeva, Tatyana V; Kondaurova, Elena M; Popova, Nina K

2013-12-01

The effect of glial cell line-derived neurotrophic factor (GDNF) on behavior and on the serotonin (5-HT) system of a mouse strain predisposed to depressive-like behavior, ASC/Icg (Antidepressant Sensitive Cataleptics), in comparison with the parental "nondepressive" CBA/Lac mice was studied. Within 7 days after acute administration, GDNF (800 ng, i.c.v.) decreased cataleptic immobility but increased depressive-like behavioral traits in both investigated mouse strains and produced anxiolytic effects in ASC mice. The expression of the gene encoding the key enzyme for 5-HT biosynthesis in the brain, tryptophan hydroxylase-2 (Tph-2), and 5-HT1A receptor gene in the midbrain as well as 5-HT2A receptor gene in the frontal cortex were increased in GDNF-treated ASC mice. At the same time, GDNF decreased 5-HT1A and 5-HT2A receptor gene expression in the hippocampus of ASC mice. GDNF failed to change Tph2, 5-HT1A , or 5-HT2A receptor mRNA levels in CBA mice as well as 5-HT transporter gene expression and 5-HT1A and 5-HT2A receptor functional activity in both investigated mouse strains. The results show 1) a GDNF-induced increase in the expression of key genes of the brain 5-HT system, Tph2, 5-HT1A , and 5-HT2A receptors, and 2) significant genotype-dependent differences in the 5-HT system response to GDNF treatment. The data suggest that genetically defined cross-talk between neurotrophic factors and the brain 5-HT system underlies the variability in behavioral response to GDNF. Copyright © 2013 Wiley Periodicals, Inc.

Chemopreventive effect of Korean Angelica root extract on TRAMP carcinogenesis and integrative “omic” profiling of affected neuroendocrine carcinomas

PubMed Central

Zhang, Jinhui; Wang, Lei; Zhang, Yong; Li, Li; Tang, Suni; Xing, Chengguo; Kim, Sung-Hoon; Jiang, Cheng; Lü, Junxuan

2016-01-01

Angelica gigas Nakai (AGN) root ethanol extract exerts anti-cancer activity in several allograft and xenograft models. Here we examined its chemopreventive efficacy through gavage administration against primary carcinogenesis in the transgenic adenocarcinoma of mouse prostate (TRAMP) model. Male C57BL/6 TRAMP mice and wild type littermates were given a daily gavage (5 mg/mouse, Monday-Friday) of AGN or vehicle, beginning at 8 weeks of age (WOA). All mice were terminated at 24 WOA, unless earlier euthanasia was necessitated by large tumors. Whereas AGN-treated TRAMP mice decreased dorsolateral prostate lesion growth by 30% (P = 0.009), they developed fewer and smaller neuroendocrine-carcinomas (NE-Ca) (0.12 g/mouse) than vehicle-treated counterparts (0.81g/mouse, P = 0.037). We analyzed the proteome and transcriptome of banked NE-Ca to gain molecular insights. Angiogenesis-antibody array detected a substantial reduction in AGN-treated NE-Ca of basic fibroblast growth factor (FGF2), an angiogenesis stimulator. iTRAQ proteomics plus data mining suggested changes of genes upstream and downstream of FGF2 functionally consistent with AGN inhibiting FGF2/FGFR1 signaling at different levels of the transduction cascade. Moreover, AGN upregulated mRNA of genes related to immune responses, restored expression of many tumor suppressor genes, and prostate function and muscle differentiation genes. On the other hand, AGN down-regulated mRNA of genes related to neuron signaling, oncofetal antigens, inflammation and mast cells, Wnt signaling, embryonic morphogenesis, biosynthesis, cell adhesion, motility, invasion and angiogenesis. These changes suggest not only multiple cancer cell targeting actions of AGN but also impact on the tumor microenvironments such as angiogenesis, inflammation and immune surveillance. PMID:25307620

Few Differences in Metabolic Network Use Found Between Salmonella enterica Colonization of Plants and Typhoidal Mice.

PubMed

Kwan, Grace; Plagenz, Brett; Cowles, Kimberly; Pisithkul, Tippapha; Amador-Noguez, Daniel; Barak, Jeri D

2018-01-01

The human enteric pathogen Salmonella enterica leads a cross-kingdom lifestyle, actively colonizing and persisting on plants in between animal hosts. One of the questions that arises from this dual lifestyle is how S. enterica is able to adapt to such divergent hosts. Metabolic pathways required for S. enterica animal colonization and virulence have been previously identified, but the metabolism of this bacterium on plants is poorly understood. To determine the requirements for plant colonization by S. enterica , we first screened a library of metabolic mutants, previously examined in a systemic mouse typhoidal model, for competitive plant colonization fitness on alfalfa seedlings. By comparing our results to those reported in S. enterica -infected murine spleens, we found that the presence of individual nutrients differed between the two host niches. Yet, similar metabolic pathways contributed to S. enterica colonization of both plants and animals, such as the biosynthesis of amino acids, purines, and vitamins and the catabolism of glycerol and glucose. However, utilization of at least three metabolic networks differed during the bacterium's plant- and animal-associated lifestyles. Whereas both fatty acid biosynthesis and degradation contributed to S. enterica animal colonization, only fatty acid biosynthesis was required during plant colonization. Though serine biosynthesis was required in both hosts, S. enterica used different pathways within the serine metabolic network to achieve this outcome. Lastly, the metabolic network surrounding manA played different roles during colonization of each host. In animal models of infection, O-antigen production downstream of manA facilitates immune evasion. We discovered that manA contributed to S. enterica attachment, to seeds and germinated seedlings, and was essential for growth in early seedling exudates, when mannose is limited. However, only seedling attachment was linked to O-antigen production, indicating that manA played additional roles critical for plant colonization that were independent of surface polysaccharide production. The integrated view of S. enterica metabolism throughout its life cycle presented here provides insight on how metabolic versatility and adaption of known physiological pathways for alternate functions enable a zoonotic pathogen to thrive in niches spanning across multiple kingdoms of life.

Ablation of Dihydroceramide Desaturase Confers Resistance to Etoposide-Induced Apoptosis In Vitro

PubMed Central

Siddique, Monowarul M.; Bikman, Benjamin T.; Wang, Liping; Ying, Li; Reinhardt, Erin; Shui, Guanghou; Wenk, Markus R.; Summers, Scott A.

2012-01-01

Sphingolipid biosynthesis is potently upregulated by factors associated with cellular stress, including numerous chemotherapeutics, inflammatory cytokines, and glucocorticoids. Dihydroceramide desaturase 1 (Des1), the third enzyme in the highly conserved pathway driving sphingolipid biosynthesis, introduces the 4,5-trans-double bond that typifies most higher-order sphingolipids. Surprisingly, recent studies have shown that certain chemotherapeutics and other drugs inhibit Des1, giving rise to a number of sphingolipids that lack the characteristic double bond. In order to assess the effect of an altered sphingolipid profile (via Des1 inhibition) on cell function, we generated isogenic mouse embryonic fibroblasts lacking both Des1 alleles. Lipidomic profiling revealed that these cells contained higher levels of dihydroceramide than wild-type fibroblasts and that complex sphingolipids were comprised predominantly of the saturated backbone (e.g. sphinganine vs. sphingosine, dihydrosphingomyelin vs. sphingomyelin, etc.). Des1 ablation activated pro-survival and anabolic signaling intermediates (e.g. Akt/PKB, mTOR, MAPK, etc.) and provided protection from apoptosis caused by etoposide, a chemotherapeutic that induces sphingolipid synthesis by upregulating several sphingolipid biosynthesizing enzymes. These data reveal that the double bond present in most sphingolipids has a profound impact on cell survival pathways, and that the manipulation of Des1 could have important effects on apoptosis. PMID:22984457

Inhibition of protease-resistant prion protein formation by porphyrins and phthalocyanines

PubMed Central

Caughey, Winslow S.; Raymond, Lynne D.; Horiuchi, Motohiro; Caughey, Byron

1998-01-01

A central aspect of pathogenesis in the transmissible spongiform encephalopathies or prion diseases is the conversion of normal protease-sensitive prion protein (PrP-sen) to the abnormal protease-resistant form, PrP-res. Here we identify porphyrins and phthalocyanines as inhibitors of PrP-res accumulation. The most potent of these tetrapyrroles had IC50 values of 0.5–1 μM in scrapie-infected mouse neuroblastoma (ScNB) cell cultures. Inhibition was observed without effects on protein biosynthesis in general or PrP-sen biosynthesis in particular. Tetrapyrroles also inhibited PrP-res formation in a cell-free reaction composed predominantly of hamster PrP-res and PrP-sen. Inhibitors were found among phthalocyanines, deuteroporphyrins IX, and meso-substituted porphines; examples included compounds containing anionic, neutral protic, and cationic peripheral substituents and various metals. We conclude that certain tetrapyrroles specifically inhibit the conversion of PrP-sen to PrP-res without apparent cytotoxic effects. The inhibition observed in the cell-free conversion reaction suggests that the mechanism involved direct interactions of the tetrapyrrole with PrP-res and/or PrP-sen. These findings introduce a new class of inhibitors of PrP-res formation that represents a potential source of therapeutic agents for transmissible spongiform encephalopathies. PMID:9770449

The expression and regulation of enzymes mediating the biosynthesis of triglycerides and phospholipids in keratinocytes/epidermis

PubMed Central

Feingold, Kenneth R

2011-01-01

Triglycerides and phospholipids play an important role in epidermal permability barrier formation and function. They are synthesized de novo in the epidermis via the glycerol-3-phosphate pathway, catalyzed sequentially by a group of enzymes that have multiple isoforms including glycerol-3-phosphate acyltransferase (GPAT), 1-acylglycerol-3-phosphate acyltransferase (AGPAT), Lipin and diacylglycerol acyltransferase (DGAT). Here we review the current knowledge of GPAT, AGPAT, Lipin and DGAT enzymes in keratinocytes/epidermis focusing on the expression levels of the various isoforms and their localization in mouse epidermis. Additionally, the factors regulating their gene expression, including calcium induced differentiation, PPAR and LXR activators, and the effect of acute permeability barrier disruption will be discussed. PMID:21695015

Antimicrobial Treatment Improves Mycobacterial Survival in Nonpermissive Growth Conditions

PubMed Central

Turapov, Obolbek; Waddell, Simon J.; Burke, Bernard; Glenn, Sarah; Sarybaeva, Asel A.; Tudo, Griselda; Labesse, Gilles; Young, Danielle I.; Young, Michael; Andrew, Peter W.; Butcher, Philip D.; Cohen-Gonsaud, Martin

2014-01-01

Antimicrobials targeting cell wall biosynthesis are generally considered inactive against nonreplicating bacteria. Paradoxically, we found that under nonpermissive growth conditions, exposure of Mycobacterium bovis BCG bacilli to such antimicrobials enhanced their survival. We identified a transcriptional regulator, RaaS (for regulator of antimicrobial-assisted survival), encoded by bcg1279 (rv1219c) as being responsible for the observed phenomenon. Induction of this transcriptional regulator resulted in reduced expression of specific ATP-dependent efflux pumps and promoted long-term survival of mycobacteria, while its deletion accelerated bacterial death under nonpermissive growth conditions in vitro and during macrophage or mouse infection. These findings have implications for the design of antimicrobial drug combination therapies for persistent infectious diseases, such as tuberculosis. PMID:24590482

Effect of diphenyl ether herbicides and oxadiazon on porphyrin biosynthesis in mouse liver, rat primary hepatocyte culture and HepG2 cells.

PubMed

Krijt, J; van Holsteijn, I; Hassing, I; Vokurka, M; Blaauboer, B J

1993-01-01

The effects of the herbicides fomesafen, oxyfluorfen, oxadiazon and fluazifop-butyl on porphyrin accumulation in mouse liver, rat primary hepatocyte culture and HepG2 cells were investigated. Ten days of herbicide feeding (0.25% in the diet) increased the liver porphyrins in male C57B1/6J mice from 1.4 +/- 0.6 to 4.8 +/- 2.1 (fomesafen) 16.9 +2- 2.9 (oxyfluorfen) and 25.9 +/- 3.1 (oxadiazon) nmol/g wet weight, respectively. Fluazifop-butyl had no effect on liver porphyrin metabolism. Fomesafen, oxyfluorfen and oxadiazon increased the cellular porphyrin content of rat hepatocytes after 24 h of incubation (control, 3.2 pmol/mg protein, fomesafen, oxyfluorfen and oxadiazon at 0.125 mM concentration 51.5, 54.3 and 44.0 pmol/mg protein, respectively). The porphyrin content of HepG2 cells increased from 1.6 to 18.2, 10.6 and 9.2 pmol/mg protein after 24 h incubation with the three herbicides. Fluazifop-butyl increased hepatic cytochrome P450 levels and ethoxy- and pentoxyresorufin O-dealkylase (EROD and PROD) activity, oxyfluorfen increased PROD activity. Peroxisomal palmitoyl CoA oxidation increased after fomesafen and fluazifop treatment to about 500% of control values both in mouse liver and rat hepatocytes. Both rat hepatocytes and HepG2 cells can be used as a test system for the porphyrogenic potential of photobleaching herbicides.

Photosystem II Component Lifetimes in the Cyanobacterium Synechocystis sp. Strain PCC 6803

PubMed Central

Yao, Danny C. I.; Brune, Daniel C.; Vavilin, Dmitri; Vermaas, Wim F. J.

2012-01-01

To gain insight in the lifetimes of photosystem II (PSII) chlorophyll and proteins, a combined stable isotope labeling (15N)/mass spectrometry method was used to follow both old and new pigments and proteins. Photosystem I-less Synechocystis cells were grown to exponential or post-exponential phase and then diluted in BG-11 medium with [15N]ammonium and [15N]nitrate. PSII was isolated, and the masses of PSII protein fragments and chlorophyll were determined. Lifetimes of PSII components ranged from 1.5 to 40 h, implying that at least some of the proteins and chlorophyll turned over independently from each other. Also, a significant amount of nascent PSII components accumulated in thylakoids when cells were in post-exponential growth phase. In a mutant lacking small Cab-like proteins (SCPs), most PSII protein lifetimes were unaffected, but the lifetime of chlorophyll and the amount of nascent PSII components that accumulated were decreased. In the absence of SCPs, one of the PSII biosynthesis intermediates, the monomeric PSII complex without CP43, was missing. Therefore, SCPs may stabilize nascent PSII protein complexes. Moreover, upon SCP deletion, the rate of chlorophyll synthesis and the accumulation of early tetrapyrrole precursors were drastically reduced. When [14N]aminolevulinic acid (ALA) was supplemented to 15N-BG-11 cultures, the mutant lacking SCPs incorporated much more exogenous ALA into chlorophyll than the control demonstrating that ALA biosynthesis was impaired in the absence of SCPs. This illustrates the major effects that nonstoichiometric PSII components such as SCPs have on intermediates and assembly but not on the lifetime of PSII proteins. PMID:22090028

Fatty acids of pulmonary surfactant phosphatidylcholine from fetal rabbit lung tissue in culture. Biosynthesis of n-10 monoenoic fatty acids.

PubMed

Longmuir, K J; Resele-Tiden, C; Rossi, M E

1988-08-01

We have previously reported that fetal rabbit lung tissue in organ culture produces a lamellar body material (pulmonary surfactant) with a lower percentage of disaturated phosphatidylcholine than is typically found in rabbit lung in vivo (Longmuir, K.J., C. Resele-Tiden, and L. Sykes. 1985. Biochim. Biophys. Acta. 833: 135-143). This investigation was conducted to identify all fatty acids present in the lamellar body phosphatidylcholine, and to determine whether the low level of disaturated phosphatidylcholine is due to excessive unsaturated fatty acid at position sn-1, sn-2, or both. Fetal rabbit lung tissue, 23 days gestation, was maintained in culture for 7 days in defined (serum-free) medium. Phospholipids were labeled in culture with [1-14C]acetate or [U-14C]glycerol (to follow de novo fatty acid biosynthesis), or with [1-14C]palmitic acid (to follow incorporation of exogenously supplied fatty acid). Radiolabeled fatty acid methyl esters obtained from lamellar body phosphatidylcholine were first separated by reverse-phase thin-layer chromatography (TLC) into two fractions of 1) 14:0 + 16:1 and 2) 16:0 + 18:1. Complete separation of the individual saturated and monoenoic fatty acids was achieved by silver nitrate TLC of the two fractions. Monoenoic fatty acid double bond position was determined by permanganate-periodate oxidation followed by HPLC of the carboxylic acid phenacyl esters. Lamellar body phosphatidylcholine contained four monoenoic fatty acids: 1) palmitoleic acid, 16:1 cis-9; 2) oleic acid, 18:1 cis-9; 3) cis-vaccenic acid, 18:1 cis-11; and 4) 6-hexadecenoic acid, 16:1 cis-6. In addition, 8-octadecenoic acid, 18:1 cis-8, was found in the fatty acids of the tissue homogenate. The abnormally low disaturated phosphatidylcholine content in lamellar body material was the result of abnormally high levels of monoenoic fatty acid (principally 16:1 cis-9) found at position sn-2. Position sn-1 contained normal levels of saturated fatty acid. The biosynthesis of the unusual n-10 fatty acids was observed from the start of culture throughout the entire 7-day culture period, and was observed in incubations of tissue slices of day 23 fetal rabbit lung. This is the first report of the biosynthesis of n-10 fatty acids (16:1 cis-6 and 18:1 cis-8) in a mammalian tissue other than skin, where these fatty acids are found in the secretory product (sebum) of sebaceous glands.

FnrL and Three Dnr Regulators Are Used for the Metabolic Adaptation to Low Oxygen Tension in Dinoroseobacter shibae

PubMed Central

Ebert, Matthias; Laaß, Sebastian; Thürmer, Andrea; Roselius, Louisa; Eckweiler, Denitsa; Daniel, Rolf; Härtig, Elisabeth; Jahn, Dieter

2017-01-01

The heterotrophic marine bacterium Dinoroseobacter shibae utilizes aerobic respiration and anaerobic denitrification supplemented with aerobic anoxygenic photosynthesis for energy generation. The aerobic to anaerobic transition is controlled by four Fnr/Crp family regulators in a unique cascade-type regulatory network. FnrL is utilizing an oxygen-sensitive Fe-S cluster for oxygen sensing. Active FnrL is inducing most operons encoding the denitrification machinery and the corresponding heme biosynthesis. Activation of gene expression of the high oxygen affinity cbb3-type and repression of the low affinity aa3-type cytochrome c oxidase is mediated by FnrL. Five regulator genes including dnrE and dnrF are directly controlled by FnrL. Multiple genes of the universal stress protein (USP) and cold shock response are further FnrL targets. DnrD, most likely sensing NO via a heme cofactor, co-induces genes of denitrification, heme biosynthesis, and the regulator genes dnrE and dnrF. DnrE is controlling genes for a putative Na+/H+ antiporter, indicating a potential role of a Na+ gradient under anaerobic conditions. The formation of the electron donating primary dehydrogenases is coordinated by FnrL and DnrE. Many plasmid encoded genes were DnrE regulated. DnrF is controlling directly two regulator genes including the Fe-S cluster biosynthesis regulator iscR, genes of the electron transport chain and the glutathione metabolism. The genes for nitrate reductase and CO dehydrogenase are repressed by DnrD and DnrF. Both regulators in concert with FnrL are inducing the photosynthesis genes. One of the major denitrification operon control regions, the intergenic region between nirS and nosR2, contains one Fnr/Dnr binding site. Using regulator gene mutant strains, lacZ-reporter gene fusions in combination with promoter mutagenesis, the function of the single Fnr/Dnr binding site for FnrL-, DnrD-, and partly DnrF-dependent nirS and nosR2 transcriptional activation was shown. Overall, the unique regulatory network of the marine bacterium D. shibae for the transition from aerobic to anaerobic growth composed of four Crp/Fnr family regulators was elucidated. PMID:28473807

The biosynthesis of nitrous oxide in the green alga Chlamydomonas reinhardtii.

PubMed

Plouviez, Maxence; Wheeler, David; Shilton, Andy; Packer, Michael A; McLenachan, Patricia A; Sanz-Luque, Emanuel; Ocaña-Calahorro, Francisco; Fernández, Emilio; Guieysse, Benoit

2017-07-01

Over the last decades, several studies have reported emissions of nitrous oxide (N 2 O) from microalgal cultures and aquatic ecosystems characterized by a high level of algal activity (e.g. eutrophic lakes). As N 2 O is a potent greenhouse gas and an ozone-depleting pollutant, these findings suggest that large-scale cultivation of microalgae (and possibly, natural eutrophic ecosystems) could have a significant environmental impact. Using the model unicellular microalga Chlamydomonas reinhardtii, this study was conducted to investigate the molecular basis of microalgal N 2 O synthesis. We report that C. reinhardtii supplied with nitrite (NO 2 - ) under aerobic conditions can reduce NO 2 - into nitric oxide (NO) using either a mitochondrial cytochrome c oxidase (COX) or a dual enzymatic system of nitrate reductase (NR) and amidoxime-reducing component, and that NO is subsequently reduced into N 2 O by the enzyme NO reductase (NOR). Based on experimental evidence and published literature, we hypothesize that when nitrate (NO 3 - ) is the main Nitrogen source and the intracellular concentration of NO 2 - is low (i.e. under physiological conditions), microalgal N 2 O synthesis involves the reduction of NO 3 - to NO 2 - by NR followed by the reduction of NO 2 - to NO by the dual system involving NR. This microalgal N 2 O pathway has broad implications for environmental science and algal biology because the pathway of NO 3 - assimilation is conserved among microalgae, and because its regulation may involve NO. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

ABA, GA(3), and nitrate may control seed germination of Crithmum maritimum (Apiaceae) under saline conditions.

PubMed

Atia, Abdallah; Debez, Ahmed; Barhoumi, Zouhaier; Smaoui, Abderrazak; Abdelly, Chedly

2009-08-01

Impaired germination is common among halophyte seeds exposed to salt stress, partly resulting from the salt-induced reduction of the growth regulator contents in seeds. Thus, the understanding of hormonal regulation during the germination process is a main key: (i) to overcome the mechanisms by which NaCl-salinity inhibit germination; and (ii) to improve the germination of these species when challenged with NaCl. In the present investigation, the effects of ABA, GA(3), NO(-)(3), and NH(+)(4) on the germination of the oilseed halophyte Crithmum maritimum (Apiaceae) were assessed under NaCl-salinity (up to 200 mM NaCl). Seeds were collected from Tabarka rocky coasts (N-W of Tunisia). The exogenous application of GA(3), nitrate (either as NaNO(3) or KNO(3)), and NH(4)Cl enhanced germination under NaCl salinity. The beneficial impact of KNO(3) on germination upon seed exposure to NaCl salinity was rather due to NO(-)(3) than to K(+), since KCl failed to significantly stimulate germination. Under optimal conditions for germination (0 mM NaCl), ABA inhibited germination over time in a dose dependent manner, but KNO(3) completely restored the germination parameters. Under NaCl salinity, the application of fluridone (FLU) an inhibitor of ABA biosynthesis, stimulated substantially seed germination. Taken together, our results point out that NO(-)(3) and GA(3) mitigate the NaCl-induced reduction of seed germination, and that NO(-)(3) counteracts the inhibitory effect of ABA on germination of C. maritimum.

Metagenomic Insights Into the Microbial Community and Nutrient Cycling in the Western Subarctic Pacific Ocean.

PubMed

Li, Yingdong; Jing, Hongmei; Xia, Xiaomin; Cheung, Shunyan; Suzuki, Koji; Liu, Hongbin

2018-01-01

The composition and metabolic functions of prokaryotic communities in the western subarctic Pacific (WSP), where strong mixing of waters from the Sea of Okhotsk and the East Kamchatka Current result in transfer to the Oyashio Current, were investigated using a shotgun metagenome sequencing approach. Functional metabolic genes related to nutrient cycling of nitrogen, sulfur, carbohydrates, iron and amino acids were differently distributed between the surface and deep waters of the WSP. Genes related to nitrogen metabolism were mainly found in deep waters, where Thaumarchaeaota, Sphingomonadales , and Pseudomonadales were closely associated and performing important roles in ammonia oxidation, assimilatory nitrate reduction, and dissimilatory nitrate reduction processes, respectively. In addition, orders affiliated to Spingobacteria and Alphaproteobacteria were crucial for sulfate reduction and abundant at 3000 m, whereas orders affiliated to Gammaproteobacteria , which harbored the most sulfate reduction genes, were abundant at 1000 m. Additionally, when compared with the East Kamchatka Current, the prokaryotes in the Oyashio Current were likely to consume more energy for synthesizing cellular components. Also, genes encoding iron transport and siderophore biosynthesis proteins were in low abundance, indicating that the iron was not a limiting factor in the Oyashio current. In contrast, in the East Kamchatka Current, prokaryotes were more likely to directly utilize the amino acids and absorb iron from the environment. Overall, our data indicated that the transformation from the East Kamchatka Current to the Oyashio Current reshapes not only the composition of microbial community, but also the function of the metabolic processes. These results extended our knowledge of the microbial composition and potential metabolism in the WSP.

Metagenomic Insights Into the Microbial Community and Nutrient Cycling in the Western Subarctic Pacific Ocean

PubMed Central

Li, Yingdong; Jing, Hongmei; Xia, Xiaomin; Cheung, Shunyan; Suzuki, Koji; Liu, Hongbin

2018-01-01

The composition and metabolic functions of prokaryotic communities in the western subarctic Pacific (WSP), where strong mixing of waters from the Sea of Okhotsk and the East Kamchatka Current result in transfer to the Oyashio Current, were investigated using a shotgun metagenome sequencing approach. Functional metabolic genes related to nutrient cycling of nitrogen, sulfur, carbohydrates, iron and amino acids were differently distributed between the surface and deep waters of the WSP. Genes related to nitrogen metabolism were mainly found in deep waters, where Thaumarchaeaota, Sphingomonadales, and Pseudomonadales were closely associated and performing important roles in ammonia oxidation, assimilatory nitrate reduction, and dissimilatory nitrate reduction processes, respectively. In addition, orders affiliated to Spingobacteria and Alphaproteobacteria were crucial for sulfate reduction and abundant at 3000 m, whereas orders affiliated to Gammaproteobacteria, which harbored the most sulfate reduction genes, were abundant at 1000 m. Additionally, when compared with the East Kamchatka Current, the prokaryotes in the Oyashio Current were likely to consume more energy for synthesizing cellular components. Also, genes encoding iron transport and siderophore biosynthesis proteins were in low abundance, indicating that the iron was not a limiting factor in the Oyashio current. In contrast, in the East Kamchatka Current, prokaryotes were more likely to directly utilize the amino acids and absorb iron from the environment. Overall, our data indicated that the transformation from the East Kamchatka Current to the Oyashio Current reshapes not only the composition of microbial community, but also the function of the metabolic processes. These results extended our knowledge of the microbial composition and potential metabolism in the WSP. PMID:29670596

MiR-106a Associated with Diabetic Peripheral Neuropathy Through the Regulation of 12/15-LOX-meidiated Oxidative/Nitrative Stress.

PubMed

Wu, Yuanbo; Xu, Dandan; Zhu, Xiang; Yang, Guangwei; Ren, Mingshan

2017-01-01

Diabetic peripheral neuropathy (DPN) is a common complication of diabetes with a complex pathogenesis. Study has reported that oxidative stress and nitrative stress are all involved in the DPN. However, the mechanism between them is still unclear. In the present study, we investigated whether miR-106a regulated 12/15-Lipoxygenase (12/15-LOX) of oxidative/nitrative stress (OS/NS) in DPN. Dorsal root ganglion (DRG) was isolated from 10 healthy mice and 10 DPN mice. DPN mouse model was established by the injection of streptozotocin (STZ), and high glucose was used for inducing the in vitro model of DPN. In DPN mice, the levels of fasting blood-glucose (FBG) level, Methane Dicarboxylic Aldehyde (MDA) and the Inducible Nitric Oxide Synthase (iNOS) were increased, while the levels of motor nerve conduction velocity (MNCV), sensory nerve conduction velocity (SNCV) and superoxide dismutase (SOD) were decreased. MiR-106a level in DRG cells of DPN was decreased. The 12/15-LOX expression and cell apoptosis were increased. DRG cells subjected to high glucose resulted in the same effects as above. MiR-106a was observed to target 12/15-LOX and regulated its expression. MiR-106a overexpression reversed the effect that was induced by high glucose, however, overexpression of 12/15-LOX reversed the effect that was induced by miR-106a overexpression. MiR-106 may be associated with 12/15-LOX mediated OS/NS in DPN and may be considered as a potential therapeutic target. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Cannabidiol Protects against Doxorubicin-Induced Cardiomyopathy by Modulating Mitochondrial Function and Biogenesis.

PubMed

Hao, Enkui; Mukhopadhyay, Partha; Cao, Zongxian; Erdélyi, Katalin; Holovac, Eileen; Liaudet, Lucas; Lee, Wen-Shin; Haskó, György; Mechoulam, Raphael; Pacher, Pál

2015-01-06

Doxorubicin (DOX) is a widely used, potent chemotherapeutic agent; however, its clinical application is limited because of its dose-dependent cardiotoxicity. DOX's cardiotoxicity involves increased oxidative/nitrative stress, impaired mitochondrial function in cardiomyocytes/endothelial cells and cell death. Cannabidiol (CBD) is a nonpsychotropic constituent of marijuana, which is well tolerated in humans, with antioxidant, antiinflammatory and recently discovered antitumor properties. We aimed to explore the effects of CBD in a well-established mouse model of DOX-induced cardiomyopathy. DOX-induced cardiomyopathy was characterized by increased myocardial injury (elevated serum creatine kinase and lactate dehydrogenase levels), myocardial oxidative and nitrative stress (decreased total glutathione content and glutathione peroxidase 1 activity, increased lipid peroxidation, 3-nitrotyrosine formation and expression of inducible nitric oxide synthase mRNA), myocardial cell death (apoptotic and poly[ADP]-ribose polymerase 1 [PARP]-dependent) and cardiac dysfunction (decline in ejection fraction and left ventricular fractional shortening). DOX also impaired myocardial mitochondrial biogenesis (decreased mitochondrial copy number, mRNA expression of peroxisome proliferator-activated receptor γ coactivator 1-alpha, peroxisome proliferator-activated receptor alpha, estrogen-related receptor alpha), reduced mitochondrial function (attenuated complex I and II activities) and decreased myocardial expression of uncoupling protein 2 and 3 and medium-chain acyl-CoA dehydrogenase mRNA. Treatment with CBD markedly improved DOX-induced cardiac dysfunction, oxidative/nitrative stress and cell death. CBD also enhanced the DOX-induced impaired cardiac mitochondrial function and biogenesis. These data suggest that CBD may represent a novel cardioprotective strategy against DOX-induced cardiotoxicity, and the above-described effects on mitochondrial function and biogenesis may contribute to its beneficial properties described in numerous other models of tissue injury.

Cannabidiol Protects against Doxorubicin-Induced Cardiomyopathy by Modulating Mitochondrial Function and Biogenesis

PubMed Central

Hao, Enkui; Mukhopadhyay, Partha; Cao, Zongxian; Erdélyi, Katalin; Holovac, Eileen; Liaudet, Lucas; Lee, Wen-Shin; Haskó, György; Mechoulam, Raphael; Pacher, Pál

2015-01-01

Doxorubicin (DOX) is a widely used, potent chemotherapeutic agent; however, its clinical application is limited because of its dose-dependent cardiotoxicity. DOX’s cardiotoxicity involves increased oxidative/nitrative stress, impaired mitochondrial function in cardiomyocytes/endothelial cells and cell death. Cannabidiol (CBD) is a nonpsychotropic constituent of marijuana, which is well tolerated in humans, with antioxidant, antiinflammatory and recently discovered antitumor properties. We aimed to explore the effects of CBD in a well-established mouse model of DOX-induced cardiomyopathy. DOX-induced cardiomyopathy was characterized by increased myocardial injury (elevated serum creatine kinase and lactate dehydrogenase levels), myocardial oxidative and nitrative stress (decreased total glutathione content and glutathione peroxidase 1 activity, increased lipid peroxidation, 3-nitrotyrosine formation and expression of inducible nitric oxide synthase mRNA), myocardial cell death (apoptotic and poly[ADP]-ribose polymerase 1 [PARP]-dependent) and cardiac dysfunction (decline in ejection fraction and left ventricular fractional shortening). DOX also impaired myocardial mitochondrial biogenesis (decreased mitochondrial copy number, mRNA expression of peroxisome proliferator-activated receptor γ coactivator 1-alpha, peroxisome proliferator-activated receptor alpha, estrogen-related receptor alpha), reduced mitochondrial function (attenuated complex I and II activities) and decreased myocardial expression of uncoupling protein 2 and 3 and medium-chain acyl-CoA dehydrogenase mRNA. Treatment with CBD markedly improved DOX-induced cardiac dysfunction, oxidative/nitrative stress and cell death. CBD also enhanced the DOX-induced impaired cardiac mitochondrial function and biogenesis. These data suggest that CBD may represent a novel cardioprotective strategy against DOX-induced cardiotoxicity, and the above-described effects on mitochondrial function and biogenesis may contribute to its beneficial properties described in numerous other models of tissue injury. PMID:25569804

Age-dependent α-synuclein aggregation in the Microcebus murinus lemur primate

PubMed Central

Canron, Marie-Hélène; Perret, Martine; Vital, Anne; Bézard, Erwan; Dehay, Benjamin

2012-01-01

Since age-dependent deposition of Aβ-amyloid has been reported in the Microcebus murinus, we posited that this animal could as well be a model of age-related synucleinopathy. We characterized the distribution of Aβ-amyloid, α-synuclein and two of its modified forms in the brain of Microcebus murinus aged from 1.5 to 10 years. Intracytoplasmic α-synuclein aggregates were observed only in aged animals in different brain regions, which were also phospho-Ser129 and nitrated α-synuclein immunoreactive. Age-dependent α-synuclein aggregation occurs spontaneously in mouse lemur primates. Microcebus murinus may provide a model to study age-associated α-synucleinopathy and for testing putative therapeutic interventions for both Alzheimer's and Parkinson's diseases. PMID:23205271

Sterol intermediates of cholesterol biosynthesis inhibit hair growth and trigger an innate immune response in cicatricial alopecia.

PubMed

Panicker, Sreejith P; Ganguly, Taneeta; Consolo, Mary; Price, Vera; Mirmirani, Paradi; Honda, Kord; Karnik, Pratima

2012-01-01

Primary cicatricial alopecia (PCA) is a group of inflammatory hair disorders that cause scarring and permanent hair loss. Previous studies have implicated PPARγ, a transcription factor that integrates lipogenic and inflammatory signals, in the pathogenesis of PCA. However, it is unknown what triggers the inflammatory response in these disorders, whether the inflammation is a primary or secondary event in disease pathogenesis, and whether the inflammatory reaction reflects an autoimmune process. In this paper, we show that the cholesterol biosynthetic pathway is impaired in the skin and hair follicles of PCA patients. Treatment of hair follicle cells with BM15766, a cholesterol biosynthesis inhibitor, or 7-dehydrocholesterol (7-DHC), a sterol precursor, stimulates the expression of pro-inflammatory chemokine genes. Painting of mouse skin with 7-DHC or BM15766 inhibits hair growth, causes follicular plugging and induces the infiltration of inflammatory cells into the interfollicular dermis. Our results demonstrate that cholesterologenic changes within hair follicle cells trigger an innate immune response that is associated with the induction of toll-like receptor (TLR) and interferon (IFN) gene expression, and the recruitment of macrophages that surround the hair follicles and initiate their destruction. These findings reveal a previously unsuspected role for cholesterol precursors in PCA pathogenesis and identify a novel link between sterols and inflammation that may prove transformative in the diagnosis and treatment of these disorders.

An extremely high dietary iodide supply forestalls severe hypothyroidism in Na+/I- symporter (NIS) knockout mice.

PubMed

Ferrandino, Giuseppe; Kaspari, Rachel R; Reyna-Neyra, Andrea; Boutagy, Nabil E; Sinusas, Albert J; Carrasco, Nancy

2017-07-13

The sodium/iodide symporter (NIS) mediates active iodide (I - ) accumulation in the thyroid, the first step in thyroid hormone (TH) biosynthesis. Mutations in the SLC5A5 gene encoding NIS that result in a non-functional protein lead to congenital hypothyroidism due to I - transport defect (ITD). ITD is a rare autosomal disorder that, if not treated promptly in infancy, can cause mental retardation, as the TH decrease results in improper development of the nervous system. However, in some patients, hypothyroidism has been ameliorated by unusually large amounts of dietary I - . Here we report the first NIS knockout (KO) mouse model, obtained by targeting exons 6 and 7 of the Slc5a5 gene. In NIS KO mice, in the thyroid, stomach, and salivary gland, NIS is absent, and hence there is no active accumulation of the NIS substrate pertechnetate ( 99m TcO 4 - ). NIS KO mice showed undetectable serum T 4 and very low serum T 3 levels when fed a diet supplying the minimum I - requirement for rodents. These hypothyroid mice displayed oxidative stress in the thyroid, but not in the brown adipose tissue or liver. Feeding the mice a high-I - diet partially rescued TH biosynthesis, demonstrating that, at high I - concentrations, I - enters the thyroid through routes other than NIS.

The clinical heterogeneity of coenzyme Q10 deficiency results from genotypic differences in the Coq9 gene

PubMed Central

Luna-Sánchez, Marta; Díaz-Casado, Elena; Barca, Emanuele; Tejada, Miguel Ángel; Montilla-García, Ángeles; Cobos, Enrique Javier; Escames, Germaine; Acuña-Castroviejo, Dario; Quinzii, Catarina M; López, Luis Carlos

2015-01-01

Primary coenzyme Q10 (CoQ10) deficiency is due to mutations in genes involved in CoQ biosynthesis. The disease has been associated with five major phenotypes, but a genotype–phenotype correlation is unclear. Here, we compare two mouse models with a genetic modification in Coq9 gene (Coq9Q95X and Coq9R239X), and their responses to 2,4-dihydroxybenzoic acid (2,4-diHB). Coq9R239X mice manifest severe widespread CoQ deficiency associated with fatal encephalomyopathy and respond to 2,4-diHB increasing CoQ levels. In contrast, Coq9Q95X mice exhibit mild CoQ deficiency manifesting with reduction in CI+III activity and mitochondrial respiration in skeletal muscle, and late-onset mild mitochondrial myopathy, which does not respond to 2,4-diHB. We show that these differences are due to the levels of COQ biosynthetic proteins, suggesting that the presence of a truncated version of COQ9 protein in Coq9R239X mice destabilizes the CoQ multiprotein complex. Our study points out the importance of the multiprotein complex for CoQ biosynthesis in mammals, which may provide new insights to understand the genotype–phenotype heterogeneity associated with human CoQ deficiency and may have a potential impact on the treatment of this mitochondrial disorder. PMID:25802402

Methamphetamine Accelerates Cellular Senescence through Stimulation of De Novo Ceramide Biosynthesis

PubMed Central

Astarita, Giuseppe; Avanesian, Agnesa; Grimaldi, Benedetto; Realini, Natalia; Justinova, Zuzana; Panlilio, Leight V.; Basit, Abdul; Piomelli, Daniele

2015-01-01

Methamphetamine is a highly addictive psychostimulant that causes profound damage to the brain and other body organs. Post mortem studies of human tissues have linked the use of this drug to diseases associated with aging, such as coronary atherosclerosis and pulmonary fibrosis, but the molecular mechanism underlying these findings remains unknown. Here we used functional lipidomics and transcriptomics experiments to study abnormalities in lipid metabolism in select regions of the brain and, to a greater extent, peripheral organs and tissues of rats that self-administered methamphetamine. Experiments in various cellular models (primary mouse fibroblasts and myotubes) allowed us to investigate the molecular mechanisms of systemic inflammation and cellular aging related to methamphetamine abuse. We report now that methamphetamine accelerates cellular senescence and activates transcription of genes involved in cell-cycle control and inflammation by stimulating production of the sphingolipid messenger ceramide. This pathogenic cascade is triggered by reactive oxygen species, likely generated through methamphetamine metabolism via cytochrome P450, and involves the recruitment of nuclear factor-κB (NF-κB) to induce expression of enzymes in the de novo pathway of ceramide biosynthesis. Inhibitors of NF-κB signaling and ceramide formation prevent methamphetamine-induced senescence and systemic inflammation in rats self-administering the drug, attenuating their health deterioration. The results suggest new therapeutic strategies to reduce the adverse consequences of methamphetamine abuse and improve effectiveness of abstinence treatments. PMID:25671639

Metabolite identification in fecal microbiota transplantation mouse livers and combined proteomics with chronic unpredictive mild stress mouse livers.

PubMed

Li, Bo; Guo, Kenan; Zeng, Li; Zeng, Benhua; Huo, Ran; Luo, Yuanyuan; Wang, Haiyang; Dong, Meixue; Zheng, Peng; Zhou, Chanjuan; Chen, Jianjun; Liu, Yiyun; Liu, Zhao; Fang, Liang; Wei, Hong; Xie, Peng

2018-01-31

Major depressive disorder (MDD) is a common mood disorder. Gut microbiota may be involved in the pathogenesis of depression via the microbe-gut-brain axis. Liver is vulnerable to exposure of bacterial products translocated from the gut via the portal vein and may be involved in the axis. In this study, germ-free mice underwent fecal microbiota transplantation from MDD patients and healthy controls. Behavioral tests verified the depression model. Metabolomics using gas chromatography-mass spectrometry, nuclear magnetic resonance, and liquid chromatography-mass spectrometry determined the influence of microbes on liver metabolism. With multivariate statistical analysis, 191 metabolites were distinguishable in MDD mice from control (CON) mice. Compared with CON mice, MDD mice showed lower levels for 106 metabolites and higher levels for 85 metabolites. These metabolites are associated with lipid and energy metabolism and oxidative stress. Combined analyses of significantly changed proteins in livers from another depression model induced by chronic unpredictive mild stress returned a high score for the Lipid Metabolism, Free Radical Scavenging, and Molecule Transports network, and canonical pathways were involved in energy metabolism and tryptophan degradation. The two mouse models of depression suggest that changes in liver metabolism might be involved in the pathogenesis of MDD. Conjoint analyses of fecal, serum, liver, and hippocampal metabolites from fecal microbiota transplantation mice suggested that aminoacyl-tRNA biosynthesis significantly changed and fecal metabolites showed a close relationship with the liver. These findings may help determine the biological mechanisms of depression and provide evidence about "depression microbes" impacting on liver metabolism.

Human Fetal Testis Xenografts Are Resistant to Phthalate-Induced Endocrine Disruption

PubMed Central

Heger, Nicholas E; Hall, Susan J; Sandrof, Moses A; McDonnell, Elizabeth V; Hensley, Janan B; McDowell, Erin N; Martin, Kayla A; Gaido, Kevin W; Johnson, Kamin J

2012-01-01

Background: In utero exposure to endocrine-disrupting chemicals may contribute to testicular dysgenesis syndrome (TDS), a proposed constellation of increasingly common male reproductive tract abnormalities (including hypospadias, cryptorchidism, hypospermatogenesis, and testicular cancer). Male rats exposed in utero to certain phthalate plasticizers exhibit multinucleated germ cell (MNG) induction and suppressed steroidogenic gene expression and testosterone production in the fetal testis, causing TDS-consistent effects of hypospadias and cryptorchidism. Mice exposed to phthalates in utero exhibit MNG induction only. This disparity in response demonstrates a species-specific sensitivity to phthalate-induced suppression of fetal Leydig cell steroidogenesis. Importantly, ex vivo phthalate exposure of the fetal testis does not recapitulate the species-specific endocrine disruption, demonstrating the need for a new bioassay to assess the human response to phthalates. Objectives: In this study, we aimed to develop and validate a rat and mouse testis xenograft bioassay of phthalate exposure and examine the human fetal testis response. Methods: Fetal rat, mouse, and human testes were xenografted into immunodeficient rodent hosts, and hosts were gavaged with a range of phthalate doses over multiple days. Xenografts were harvested and assessed for histopathology and steroidogenic end points. Results: Consistent with the in utero response, phthalate exposure induced MNG formation in rat and mouse xenografts, but only rats exhibited suppressed steroidogenesis. Across a range of doses, human fetal testis xenografts exhibited MNG induction but were resistant to suppression of steroidogenic gene expression. Conclusions: Phthalate exposure of grafted human fetal testis altered fetal germ cells but did not reduce expression of genes that regulate fetal testosterone biosynthesis. PMID:22511013

A transgenic approach to study argininosuccinate synthetase gene expression

PubMed Central

2014-01-01

Background Argininosuccinate synthetase (ASS) participates in urea, nitric oxide and arginine production. Besides transcriptional regulation, a post-transcriptional regulation affecting nuclear precursor RNA stability has been reported. To study whether such post-transcriptional regulation underlines particular temporal and spatial ASS expression, and to investigate how human ASS gene behaves in a mouse background, a transgenic mouse system using a modified bacterial artificial chromosome carrying the human ASS gene tagged with EGFP was employed. Results Two lines of ASS-EGFP transgenic mice were generated: one with EGFP under transcriptional control similar to that of the endogenous ASS gene, another with EGFP under both transcriptional and post-transcriptional regulation as that of the endogenous ASS mRNA. EGFP expression in the liver, the organ for urea production, and in the intestine and kidney that are responsible for arginine biosynthesis, was examined. Organs taken from embryos E14.5 stage to young adult were examined under a fluorescence microscope either directly or after cryosectioning. The levels of EGFP and endogenous mouse Ass mRNAs were also quantified by S1 nuclease mapping. EGFP fluorescence and EGFP mRNA levels in both the liver and kidney were found to increase progressively from embryonic stage toward birth. In contrast, EGFP expression in the intestine was higher in neonates and started to decline at about 3 weeks after birth. Comparison between the EGFP profiles of the two transgenic lines indicated the developmental and tissue-specific regulation was mainly controlled at the transcriptional level. The ASS transgene was of human origin. EGFP expression in the liver followed essentially the mouse Ass pattern as evidenced by zonation distribution of fluorescence and the level of EGFP mRNA at birth. However, in the small intestine, Ass mRNA level declined sharply at 3 week of age, and yet substantial EGFP mRNA was still detectable at this stage. Thus, the time course of EGFP expression in the transgenic mice resembled that of the human ASS gene. Conclusions We demonstrate that the transgenic mouse system reported here has the merit of sensitivity and direct visualization advantage, and is ideal for annotating temporal and spatial expression profiles and the regulation mode of the ASS gene. PMID:24884799

Nitric oxide-releasing flurbiprofen reduces formation of proinflammatory hydrogen sulfide in lipopolysaccharide-treated rat

PubMed Central

Anuar, Farhana; Whiteman, Matthew; Siau, Jia Ling; Kwong, Shing Erl; Bhatia, Madhav; Moore, Philip K

2006-01-01

The biosynthesis of both nitric oxide (NO) and hydrogen sulfide (H2S) is increased in lipopolysaccharide (LPS)-injected mice and rats but their interaction in these models is not known. In this study we examined the effect of the NO donor, nitroflurbiprofen (and the parent molecule flurbiprofen) on NO and H2S metabolism in tissues from LPS-pretreated rats. Administration of LPS (10 mg kg−1, i.p.; 6 h) resulted in an increase (P<0.05) in plasma TNF-α, IL-1β and nitrate/nitrite (NOx) concentrations, liver H2S synthesis (from added cysteine), CSE mRNA, inducible nitric oxide synthase (iNOS), myeloperoxidase (MPO) activity (marker for neutrophil infiltration) and nuclear factor-kappa B (NF-κB) activation. Nitroflurbiprofen (3–30 mg kg−1, i.p.) administration resulted in a dose-dependent inhibition of the LPS-mediated increase in plasma TNF-α, IL-1β and NOx concentration, liver H2S synthesis (55.00±0.95 nmole mg protein−1, c.f. 62.38±0.47 nmole mg protein−1, n=5, P<0.05), CSE mRNA, iNOS, MPO activity and NF-κB activation. Flurbiprofen (21 mg kg−1, i.p.) was without effect. These results show for the first time that nitroflurbiprofen downregulates the biosynthesis of proinflammatory H2S and suggest that such an effect may contribute to the augmented anti-inflammatory activity of this compound. These data also highlight the existence of ‘crosstalk' between NO and H2S in this model of endotoxic shock. PMID:16491094

Synergistic Activation of Steroidogenic Acute Regulatory Protein Expression and Steroid Biosynthesis by Retinoids: Involvement of cAMP/PKA Signaling

PubMed Central

Manna, Pulak R.; Slominski, Andrzej T.; King, Steven R.; Stetson, Cloyce L.

2014-01-01

Both retinoic acid receptors (RARs) and retinoid X receptors (RXRs) mediate the action of retinoids that play important roles in reproductive development and function, as well as steroidogenesis. Regulation of steroid biosynthesis is principally mediated by the steroidogenic acute regulatory protein (StAR); however, the modes of action of retinoids in the regulation of steroidogenesis remain obscure. In this study we demonstrate that all-trans retinoic acid (atRA) enhances StAR expression, but not its phosphorylation (P-StAR), and progesterone production in MA-10 mouse Leydig cells. Activation of the protein kinase A (PKA) cascade, by dibutyrl-cAMP or type I/II PKA analogs, markedly increased retinoid-responsive StAR, P-StAR, and steroid levels. Targeted silencing of endogenous RARα and RXRα, with small interfering RNAs, resulted in decreases in 9-cis RA-stimulated StAR and progesterone levels. Truncation of and mutational alterations in the 5′-flanking region of the StAR gene demonstrated the importance of the −254/−1-bp region in retinoid responsiveness. An oligonucleotide probe encompassing an RXR/liver X receptor recognition motif, located within the −254/−1-bp region, specifically bound MA-10 nuclear proteins and in vitro transcribed/translated RXRα and RARα in EMSAs. Transcription of the StAR gene in response to atRA and dibutyrl-cAMP was influenced by several factors, its up-regulation being dependent on phosphorylation of cAMP response-element binding protein (CREB). Chromatin immunoprecipitation studies revealed the association of phosphorylation of CREB, CREB binding protein, RXRα, and RARα to the StAR promoter. Further studies elucidated that hormone-sensitive lipase plays an important role in atRA-mediated regulation of the steroidogenic response that involves liver X receptor signaling. These findings delineate the molecular events by which retinoids influence cAMP/PKA signaling and provide additional and novel insight into the regulation of StAR expression and steroidogenesis in mouse Leydig cells. PMID:24265455

Transcription of the Streptococcus pyogenes Hyaluronic Acid Capsule Biosynthesis Operon Is Regulated by Previously Unknown Upstream Elements

PubMed Central

Falaleeva, Marina; Zurek, Oliwia W.; Watkins, Robert L.; Reed, Robert W.; Ali, Hadeel; Sumby, Paul; Voyich, Jovanka M.

2014-01-01

The important human pathogen Streptococcus pyogenes (group A Streptococcus [GAS]) produces a hyaluronic acid (HA) capsule that plays critical roles in immune evasion. Previous studies showed that the hasABC operon encoding the capsule biosynthesis enzymes is under the control of a single promoter, P1, which is negatively regulated by the two-component regulatory system CovR/S. In this work, we characterize the sequence upstream of P1 and identify a novel regulatory region controlling transcription of the capsule biosynthesis operon in the M1 serotype strain MGAS2221. This region consists of a promoter, P2, which initiates transcription of a novel small RNA, HasS, an intrinsic transcriptional terminator that inefficiently terminates HasS, permitting read-through transcription of hasABC, and a putative promoter which lies upstream of P2. Electrophoretic mobility shift assays, quantitative reverse transcription-PCR, and transcriptional reporter data identified CovR as a negative regulator of P2. We found that the P1 and P2 promoters are completely repressed by CovR, and capsule expression is regulated by the putative promoter upstream of P2. Deletion of hasS or of the terminator eliminates CovR-binding sequences, relieving repression and increasing read-through, hasA transcription, and capsule production. Sequence analysis of 44 GAS genomes revealed a high level of polymorphism in the HasS sequence region. Most of the HasS variations were located in the terminator sequences, suggesting that this region is under strong selective pressure. We discovered that the terminator deletion mutant is highly resistant to neutrophil-mediated killing and is significantly more virulent in a mouse model of GAS invasive disease than the wild-type strain. Together, these results are consistent with the naturally occurring mutations in this region modulating GAS virulence. PMID:25287924

Polycyclic Aromatic Hydrocarbons (PAHs) Mediate Transcriptional Activation of the ATP Binding Cassette Transporter ABCB6 Gene via the Aryl Hydrocarbon Receptor (AhR)*

PubMed Central

Chavan, Hemantkumar; Krishnamurthy, Partha

2012-01-01

Liver is endowed with a mechanism to induce hepatic cytochromes P450 (CYP450s) in response to therapeutic drugs and environmental contaminants, leading to increased detoxification and elimination of the xenobiotics. Each CYP450 is composed of an apoprotein moiety and a heme prosthetic group, which is required for CYP450 activity. Thus, under conditions of CYP450 induction, there is a coordinate increase in heme biosynthesis to compensate for the increased expression of CYP450s. ABCB6, a mitochondrial ATP binding cassette transporter, which regulates coproporphyrinogen transport from the cytoplasm into the mitochondria to complete heme biosynthesis, represents a previously unrecognized rate-limiting step in heme biosynthesis. However, it is not known if exposure to drugs and environmental contaminants induces ABCB6 expression, to assure an adequate and apparently coordinated supply of heme for the generation of functional cytochrome holoprotein. In the present study, we demonstrate that polycyclic aromatic hydrocarbons (PAHs), the widely distributed environmental toxicants shown to induce porphyrin accumulation causing hepatic porphyria, up-regulate ABCB6 expression in both mice and humans. Using siRNA technology and Abcb6 knock-out mice, we demonstrate that PAH-mediated increase in hepatic porphyrins is compromised in the absence of ABCB6. Moreover, in vivo studies in aryl hydrocarbon receptor (AhR) knock-out mice demonstrate that PAH induction of ABCB6 is mediated by AhR. Promoter activation studies combined with electrophoretic mobility shift assay and chromatin immunoprecipitation assay demonstrate direct interactions between the AhR binding sites in the ABCB6 promoter and the AhR receptor, implicating drug activation mechanisms for ABCB6 similar to those found in inducible cytochrome P450s. These studies are the first to describe direct transcriptional activation of both mouse and human ABCB6 by xenobiotics. PMID:22761424

Osthole, a coumadin analog from Cnidium monnieri (L.) Cusson, stimulates corticosterone secretion by increasing steroidogenic enzyme expression in mouse Y1 adrenocortical tumor cells.

PubMed

Pan, Zhiqiang; Fang, Zhaoqin; Lu, Wenli; Liu, Xiaomei; Zhang, Yuanyuan

2015-12-04

Osthole is an O-methylated coumadin, which was isolated and purified from the seeds of Cnidium monnieri (L.) Cusson. Osthole is a commonly used traditional Chinese medicine to treat patients with Kidney-Yang deficiency patients, who exhibit clinical signs similar to those of glucocorticoid withdrawal. However, the mechanism of action of osthole is not fully understood. This study was designed to reveal the effects of osthole on corticosterone production in mouse Y1 cell. Mouse Y1 adrenocortical cells were used to evaluate corticosterone production, which was quantified by enzyme-linked immunosorbent assay (ELISA) kits. Cell viability was tested using the MTT assay, and the mRNA and protein expression of genes encoding steroidogenic enzymes and transcription factors was monitored by quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) and western blotting, respectively. Osthole stimulated corticosterone secretion from mouse Y1 cells in a dose- and time-dependent manner, and osthole enhanced the effect of dibutyryl-cAMP (Bu2cAMP) on corticosterone production. Further, osthole also increased StAR and CYP11B1 mRNA expression in a dose-dependent manner and enhanced the expression of transcription factors such as HSD3B1, FDX1, POR and RXRα as well as immediate early genes such as NR4A1. Moreover, osthole significantly increased SCARB1(SRB1) mRNA and StAR protein expression in the presence or absence of Bu2cAMP; these proteins are an important for the transport of the corticosteroid precursor cholesterol transport into mitochondria. Our results show that the promotion of corticosterone biosynthesis and secretion is a novel effect of osthole, suggesting that this agent can be utilized for the prevention and treatment of Kidney-Yang deficiency syndrome. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Biosynthesis of ribosomal RNA in nucleoli regulates pluripotency and differentiation ability of pluripotent stem cells.

PubMed

Watanabe-Susaki, Kanako; Takada, Hitomi; Enomoto, Kei; Miwata, Kyoko; Ishimine, Hisako; Intoh, Atsushi; Ohtaka, Manami; Nakanishi, Mahito; Sugino, Hiromu; Asashima, Makoto; Kurisaki, Akira

2014-12-01

Pluripotent stem cells have been shown to have unique nuclear properties, for example, hyperdynamic chromatin and large, condensed nucleoli. However, the contribution of the latter unique nucleolar character to pluripotency has not been well understood. Here, we show that fibrillarin (FBL), a critical methyltransferase for ribosomal RNA (rRNA) processing in nucleoli, is one of the proteins highly expressed in pluripotent embryonic stem (ES) cells. Stable expression of FBL in ES cells prolonged the pluripotent state of mouse ES cells cultured in the absence of leukemia inhibitory factor (LIF). Analyses using deletion mutants and a point mutant revealed that the methyltransferase activity of FBL regulates stem cell pluripotency. Knockdown of this gene led to significant delays in rRNA processing, growth inhibition, and apoptosis in mouse ES cells. Interestingly, both partial knockdown of FBL and treatment with actinomycin D, an inhibitor of rRNA synthesis, induced the expression of differentiation markers in the presence of LIF and promoted stem cell differentiation into neuronal lineages. Moreover, we identified p53 signaling as the regulatory pathway for pluripotency and differentiation of ES cells. These results suggest that proper activity of rRNA production in nucleoli is a novel factor for the regulation of pluripotency and differentiation ability of ES cells. © 2014 AlphaMed Press.

Transgenic expression of cyclooxygenase-2 (COX2) causes premature aging phenotypes in mice.

PubMed

Kim, Joohwee; Vaish, Vivek; Feng, Mingxiao; Field, Kevin; Chatzistamou, Ioulia; Shim, Minsub

2016-10-07

Cyclooxygenase (COX) is a key enzyme in the biosynthesis of prostanoids, lipid signaling molecules that regulate various physiological processes. COX2, one of the isoforms of COX, is highly inducible in response to a wide variety of cellular and environmental stresses. Increased COX2 expression is thought to play a role in the pathogenesis of many age-related diseases. COX2 expression is also reported to be increased in the tissues of aged humans and mice, which suggests the involvement of COX2 in the aging process. However, it is not clear whether the increased COX2 expression is causal to or a result of aging. We have now addressed this question by creating an inducible COX2 transgenic mouse model. Here we show that post-natal expression of COX2 led to a panel of aging-related phenotypes. The expression of p16, p53, and phospho-H2AX was increased in the tissues of COX2 transgenic mice. Additionally, adult mouse lung fibroblasts from COX2 transgenic mice exhibited increased expression of the senescence-associated β-galactosidase. Our study reveals that the increased COX2 expression has an impact on the aging process and suggests that modulation of COX2 and its downstream signaling may be an approach for intervention of age-related disorders.

coq7/clk-1 regulates mitochondrial respiration and the generation of reactive oxygen species via coenzyme Q.

PubMed

Nakai, Daisuke; Shimizu, Takahiko; Nojiri, Hidetoshi; Uchiyama, Satoshi; Koike, Hideo; Takahashi, Mayumi; Hirokawa, Katsuiku; Shirasawa, Takuji

2004-10-01

coq7/clk-1 was isolated from a long-lived mutant of Caenorhabditis elegans, and shows sluggish behaviours and an extended lifespan. In C. elegans and Saccharomyces cerevisiae, coq7/clk-1 is required for the biosynthesis of coenzyme Q (CoQ), an essential co-factor in mitochondrial respiration. The clk-1 mutant contains dietary CoQ(8) from Escherichia coli and demethoxyubiquinone 9 (DMQ9) instead of CoQ(9). In a previous study, we generated COQ7-deficient mice by targeted disruption of the coq7 gene and reported that mouse coq7/clk-1 is also essential for CoQ synthesis, maintenance of mitochondrial integrity and neurogenesis. In the present study, we rescued COQ7-deficient mice from embryonic lethality and established a mouse model with decreased CoQ level by transgene expression of COQ7/CLK-1. A biochemical analysis showed a concomitant decrease in CoQ(9), mitochondrial respiratory enzyme activity and the generation of reactive oxygen species (ROS) in the mitochondria of CoQ-insufficient mice. This implied that the depressed activity of respiratory enzymes and the depressed production of ROS may play a physiological role in the control of lifespan in mammalian species and of C. elegans.

STMN1 Promotes Progesterone Production Via StAR Up-regulation in Mouse Granulosa Cells.

PubMed

Dou, Yun-De; Zhao, Han; Huang, Tao; Zhao, Shi-Gang; Liu, Xiao-Man; Yu, Xiao-Chen; Ma, Zeng-Xiang; Zhang, Yu-Chao; Liu, Tao; Gao, Xuan; Li, Lei; Lu, Gang; Chan, Wai-Yee; Gao, Fei; Liu, Hong-Bin; Chen, Zi-Jiang

2016-06-08

Stathmin 1 (STMN1) is a biomarker in several types of neoplasms. It plays an important role in cell cycle progression, mitosis, signal transduction and cell migration. In ovaries, STMN1 is predominantly expressed in granulosa cells (GCs). However, little is known about the role of STMN1 in ovary. In this study, we demonstrated that STMN1 is overexpressed in GCs in patients with polycystic ovary syndrome (PCOS). In mouse primary GCs, the overexpression of STMN1 stimulated progesterone production, whereas knockdown of STMN1 decreased progesterone production. We also found that STMN1 positively regulates the expression of Star (steroidogenic acute regulatory protein) and Cyp11a1 (cytochrome P450 family 11 subfamily A member 1). Promoter and ChIP assays indicated that STMN1 increased the transcriptional activity of Star and Cyp11a1 by binding to their promoter regions. The data suggest that STMN1 mediates the progesterone production by modulating the promoter activity of Star and Cyp11a1. Together, our findings provide novel insights into the molecular mechanisms of STMN1 in ovary GC steroidogenesis. A better understanding of this potential interaction between STMN1 and Star in progesterone biosynthesis in GCs will facilitate the discovery of new therapeutic targets in PCOS.

Interaction of human, rat, and mouse immunoglobulin A (IgA) with Staphylococcal superantigen-like 7 (SSL7) decoy protein and leukocyte IgA receptor.

PubMed

Wines, Bruce D; Ramsland, Paul A; Trist, Halina M; Gardam, Sandra; Brink, Robert; Fraser, John D; Hogarth, P Mark

2011-09-23

Host survival depends on an effective immune system and pathogen survival on the effectiveness of immune evasion mechanisms. Staphylococcus aureus utilizes a number of molecules to modulate host immunity, including the SSL family of which SSL7 binds IgA and inhibits Fcα receptor I (FcαRI)-mediated function. Other Gram-positive bacterial pathogens produce IgA binding proteins, which, similar to SSL7, also bind the Fc at the CH2/CH3 interface (the junction between constant domains 2 and 3 of the heavy chain). The opposing activities of the host FcαRI-IgA receptor ligand pair and the pathogen decoy proteins select for host and pathogen variants, which exert stronger protection or evasion, respectively. Curiously, mouse but not rat IgA contains a putative N-linked glycosylation site in the center of this host receptor and pathogen-binding site. Here, we demonstrate that this site is glycosylated and that the effect of amino acid changes and glycosylation of the CH2/CH3 interface inhibits interaction with the pathogen IgA binding protein SSL7, while maintaining binding of pIgR, essential to the biosynthesis and transport of SIgA.

Hepatic expression of spermatogenic genes and their transiently remarkable downregulations in Wistar-Kyoto rats in response to lead-nitrate administration: strain-difference in the gene expression patterns.

PubMed

Nemoto, Kiyomitsu; Ito, Sei; Yoshida, Chiaki; Miyata, Misaki; Kojima, Misaki; Degawa, Masakuni

2011-06-01

Administration of lead ion (Pb) to rats and mice affects hepatic functions such as the induction of hepatic cell proliferation and upregulation of cholesterol biosynthesis. To identify the genes for which expression changes in response to Pb-administration, we analyzed hepatic gene expression patterns in stroke-prone spontaneously hypertensive rat (SHRSP), its normotensive control, Wistar-Kyoto rat (WKY), and Spraque-Dawley (SD) rat strains, 3, 6, and 12 hr later after single i.v. injection of lead nitrate (LN) at a dose of 100 µmol using a DNA microarray technique. The data analysis demonstrated that the expression of a great number of genes was transiently and remarkably downregulated 3 hr after LN-injection, and then recovered to control levels only in LN-injected WKY. These normal hepatic expression levels in WKY and SHRSP were much higher than those in SD rats. Furthermore, most of these genes were ones thought to be expressed specifically in the spermatids and/or testes; i.e. genes encoding protamin 1, transition protein 1, and transition protein 2. These findings suggest that the regulation system common to expression of all of these genes could be a target site of Pb-toxic action, at least, in the liver of WKY, and that this system might be similar to the system essential for spermatogenesis, especially spermiogenesis, in the testis. In addition, it appears that clarifying the cause of the difference between the systems of WKY and SHRSP might aid in identifying the pathologic genes in SHRSP. Finally, it will be an important to clarify how the products of the genes related to spermatogenesis, including spermiogenesis, are functional in the livers of WKY and SHRSP.

Staphylococcus aureus golden pigment impairs neutrophil killing and promotes virulence through its antioxidant activity.

PubMed

Liu, George Y; Essex, Anthony; Buchanan, John T; Datta, Vivekanand; Hoffman, Hal M; Bastian, John F; Fierer, Joshua; Nizet, Victor

2005-07-18

Golden color imparted by carotenoid pigments is the eponymous feature of the human pathogen Staphylococcus aureus. Here we demonstrate a role of this hallmark phenotype in virulence. Compared with the wild-type (WT) bacterium, a S. aureus mutant with disrupted carotenoid biosynthesis is more susceptible to oxidant killing, has impaired neutrophil survival, and is less pathogenic in a mouse subcutaneous abscess model. The survival advantage of WT S. aureus over the carotenoid-deficient mutant is lost upon inhibition of neutrophil oxidative burst or in human or murine nicotinamide adenine dinucleotide phosphate oxidase-deficient hosts. Conversely, heterologous expression of the S. aureus carotenoid in the nonpigmented Streptococcus pyogenes confers enhanced oxidant and neutrophil resistance and increased animal virulence. Blocking S. aureus carotenogenesis increases oxidant sensitivity and decreases whole-blood survival, suggesting a novel target for antibiotic therapy.

33 CFR 126.28 - Ammonium nitrate, ammonium nitrate fertilizers, fertilizer mixtures, or nitro carbo nitrate...

Code of Federal Regulations, 2010 CFR

2010-07-01

... nitrate fertilizers, fertilizer mixtures, or nitro carbo nitrate; general provisions. 126.28 Section 126..., ammonium nitrate fertilizers, fertilizer mixtures, or nitro carbo nitrate; general provisions. (a) When any item of ammonium nitrate, ammonium nitrate fertilizers, fertilizer mixtures, or nitro carbo nitrate...

33 CFR 126.28 - Ammonium nitrate, ammonium nitrate fertilizers, fertilizer mixtures, or nitro carbo nitrate...

Code of Federal Regulations, 2014 CFR

2014-07-01

... nitrate fertilizers, fertilizer mixtures, or nitro carbo nitrate; general provisions. 126.28 Section 126..., ammonium nitrate fertilizers, fertilizer mixtures, or nitro carbo nitrate; general provisions. (a) When any item of ammonium nitrate, ammonium nitrate fertilizers, fertilizer mixtures, or nitro carbo nitrate...

33 CFR 126.28 - Ammonium nitrate, ammonium nitrate fertilizers, fertilizer mixtures, or nitro carbo nitrate...

Code of Federal Regulations, 2013 CFR

2013-07-01

... nitrate fertilizers, fertilizer mixtures, or nitro carbo nitrate; general provisions. 126.28 Section 126..., ammonium nitrate fertilizers, fertilizer mixtures, or nitro carbo nitrate; general provisions. (a) When any item of ammonium nitrate, ammonium nitrate fertilizers, fertilizer mixtures, or nitro carbo nitrate...

33 CFR 126.28 - Ammonium nitrate, ammonium nitrate fertilizers, fertilizer mixtures, or nitro carbo nitrate...

Code of Federal Regulations, 2011 CFR

2011-07-01

... nitrate fertilizers, fertilizer mixtures, or nitro carbo nitrate; general provisions. 126.28 Section 126..., ammonium nitrate fertilizers, fertilizer mixtures, or nitro carbo nitrate; general provisions. (a) When any item of ammonium nitrate, ammonium nitrate fertilizers, fertilizer mixtures, or nitro carbo nitrate...

33 CFR 126.28 - Ammonium nitrate, ammonium nitrate fertilizers, fertilizer mixtures, or nitro carbo nitrate...

Code of Federal Regulations, 2012 CFR

2012-07-01

... nitrate fertilizers, fertilizer mixtures, or nitro carbo nitrate; general provisions. 126.28 Section 126..., ammonium nitrate fertilizers, fertilizer mixtures, or nitro carbo nitrate; general provisions. (a) When any item of ammonium nitrate, ammonium nitrate fertilizers, fertilizer mixtures, or nitro carbo nitrate...

Enhanced production of astaxanthin by Chromochloris zofingiensis in a microplate-based culture system under high light irradiation.

PubMed

Chen, Jun-Hui; Liu, Lu; Wei, Dong

2017-12-01

The green microalga Chromochloris zofingiensis is a promising producer of natural astaxanthin. In the present study, C. zofingiensis was first cultivated in shake flasks under low light irradiation and then subjected to continuous high light irradiation, which effectively promoted astaxanthin production. In addition, a microplate-based culture system in concert with high light irradiation from blue light and white light above 150μmolm -2 s -1 was constructed and applied to improve astaxanthin production. Blue light exerted more positive influences on astaxanthin accumulation, but when the light intensity was increased to 300μmolm -2 s -1 , astaxanthin biosynthesis was substantially inhibited. Conversely, in a nitrogen-deprived culture under white light, the highest astaxanthin content for C. zofingiensis, 7.1mg/g, was obtained. The highest astaxanthin yield achieved was 38.9mg/L in a culture with 0.1g/L nitrate under the same culture conditions. This study demonstrates that C. zofingiensis has great potential for natural astaxanthin production. Copyright © 2017 Elsevier Ltd. All rights reserved.

Geobacter metallireducens gen. nov. sp. nov., a microorganism capable of coupling the complete oxidation of organic compounds to the reduction of iron and other metals

USGS Publications Warehouse

Lovley, D.R.; Giovannoni, S.J.; White, D.C.; Champine, J.E.; Phillips, E.J.P.; Gorby, Y.A.; Goodwin, S.

1993-01-01

The gram-negative metal-reducing microorganism, previously known as strain GS-15, was further characterized. This strict anaerobe oxidizes several short-chain fatty acids, alcohols, and monoaromatic compounds with Fe(III) as the sole electron acceptor. Furthermore, acetate is also oxidized with the reduction of Mn(IV), U(VI), and nitrate. In whole cell suspensions, the c-type cytochrome(s) of this organism was oxidized by physiological electron acceptors and also by gold, silver, mercury, and chromate. Menaquinone was recovered in concentrations comparable to those previously found in gram-negative sulfate reducers. Profiles of the phospholipid ester-linked fatty acids indicated that both the anaerobic desaturase and the branched pathways for fatty acid biosynthesis were operative. The organism contained three lipopolysaccharide hydroxy fatty acids which have not been previously reported in microorganisms, but have been observed in anaerobic freshwater sediments. The 16S rRNA sequence indicated that this organism belongs in the delta proteobacteria. Its closest known relative is Desulfuromonas acetoxidans. The name Geobacter metallireducens is proposed.

Production of novel antibiotics zeamines through optimizing Dickeya zeae fermentation conditions.

PubMed

Liao, Lisheng; Cheng, Yingying; Liu, Shiyin; Zhou, Jianuan; An, Shuwen; Lv, Mingfa; Chen, Yufan; Gu, Yanfang; Chen, Shaohua; Zhang, Lian-Hui

2014-01-01

Dickeya zeae strain EC1 was recently shown to produce a new type of phytotoxins designated as zeamine and zeamine II, which are potent wide-spectrum antibiotics against Gram-positive and Gram-negative bacterial pathogens, suggesting their promising potential as clinical medicines. In this study, the optimized medium composition and culture conditions for biosynthesis of novel antibiotics zeamines have been established by using response surface methodology, largely increasing the yield of zeamines from original about 7.35 µg · mL(-1) in minimal medium to about 150 µg · mL(-1) in LS5 medium. The study identified the major factors contributing to zeamines production, which include nitrate, sucrose, asparaginate, mineral elements Mg2+ and K+, and optimized amount of phosphate. In addition, the results showed that overexpression of zmsK in D. zeae strain EC1 could further increase zeamines yield to about 180 µg · mL(-1) in LS5 medium. The findings from this study could facilitate further characterization and utilization of these two novel antibiotics, and also provide useful clues for understanding the regulatory mechanisms that govern D. zeae virulence.

The Modular Organization of Protein Interactions in Escherichia coli

PubMed Central

Peregrín-Alvarez, José M.; Xiong, Xuejian; Su, Chong; Parkinson, John

2009-01-01

Escherichia coli serves as an excellent model for the study of fundamental cellular processes such as metabolism, signalling and gene expression. Understanding the function and organization of proteins within these processes is an important step towards a ‘systems’ view of E. coli. Integrating experimental and computational interaction data, we present a reliable network of 3,989 functional interactions between 1,941 E. coli proteins (∼45% of its proteome). These were combined with a recently generated set of 3,888 high-quality physical interactions between 918 proteins and clustered to reveal 316 discrete modules. In addition to known protein complexes (e.g., RNA and DNA polymerases), we identified modules that represent biochemical pathways (e.g., nitrate regulation and cell wall biosynthesis) as well as batteries of functionally and evolutionarily related processes. To aid the interpretation of modular relationships, several case examples are presented, including both well characterized and novel biochemical systems. Together these data provide a global view of the modular organization of the E. coli proteome and yield unique insights into structural and evolutionary relationships in bacterial networks. PMID:19798435

Denitrifying SUP05 Require Exogenous Nitrogen

NASA Astrophysics Data System (ADS)

Shah, V.; Chang, B. X.; Morris, R. M.

2016-02-01

Members of the SUP05 clade of gamma-proteobacteria are among the most abundant chemoautotrophs in the ocean. Environmental sequencing studies suggest that they have critical roles in mediating carbon fixation, denitrification, and sulfur oxidation in oxygen minimum zones (OMZs). They have evaded cultivation and little is known about the specific growth requirements or substrate ranges that determine their abundance, distribution and impact on marine biogeochemical cycles. We evaluated the genetic potential of an isolate from the SUP05 clade "Ca. Thioglobus autotrophica strain EF1" to fix carbon, reduce nitrogen and oxidize sulfur under anaerobic growth conditions. Growth experiments support genomic predictions, indicating that strain EF1 is a facultatively anaerobic sulfur-oxidizing chemolithoautotroph that reduces nitrate to nitrite and nitric oxide to nitrous oxide. These experiments also revealed that strain EF1 is limited for growth by ammonium, which indicates that it requires an exogenous source of nitrogen for biosynthesis. Evidence that SUP05 cells produce nitrite and nitrous oxide and require exogenous nitrogen suggests that they have important roles in nitrogen cycling and that their growth is ultimately limited by the degradation of sinking organic matter.

Blood plasma response and urinary excretion of nitrite and nitrate in milk-fed calves after oral nitrite and nitrate administration.

PubMed

Hüsler, B R.; Blum, J W.

2001-05-01

There is marked endogenous production of nitrate in young calves. Here we have studied the contribution of exogenous nitrate and nitrite to plasma concentrations and urinary excretion of nitrite and nitrate in milk-fed calves. In experiment 1, calves were fed 0 or 200 &mgr;mol nitrate or nitrite/kg(0.75) or 100 &mgr;mol nitrite plus 100 &mgr;mol nitrate/kg(0.75) with milk for 3 d. In experiment 2, calves were fed 400 &mgr;mol nitrate or nitrite/kg(0.75) with milk for 1 d. Plasma nitrate rapidly and comparably increased after feeding nitrite, nitrate or nitrite plus nitrate. The rise of plasma nitrate was greater if 400 than 200 &mgr;mol nitrate or nitrite/kg(0.75) were fed. Plasma nitrate decreased slowly after the 3-d administration of 200 &mgr;mol nitrate or nitrite/kg(0.75) and reached pre-experimental concentrations 4 d later. Urinary nitrate excretions nearly identically increased if nitrate, nitrite or nitrite plus nitrate were administered and excreted amounts were greater if 400 than 200 &mgr;mol nitrate or nitrite/kg(0.75) were fed. After nitrite ingestion plasma nitrite only transiently increased after 2 and 4 h and urinary excretion rates remained unchanged. Plasma nitrate concentration remained unchanged if milk was not supplemented with nitrite or nitrate. Nitrate concentrations were stable for 24 h after addition of nitrite to full blood in vitro, whereas nitrite concentrations decreased within 2 h. In conclusion, plasma nitrate concentrations and urinary nitrate excretions are enhanced dose-dependently by feeding low amounts of nitrate and nitrite, whereas after ingested nitrite only a transient and small rise of plasma nitrite is observed because of rapid conversion to nitrate.

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

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.

Mouse fat storage-inducing transmembrane protein 2 (FIT2) promotes lipid droplet accumulation in plants.

PubMed

Cai, Yingqi; McClinchie, Elizabeth; Price, Ann; Nguyen, Thuy N; Gidda, Satinder K; Watt, Samantha C; Yurchenko, Olga; Park, Sunjung; Sturtevant, Drew; Mullen, Robert T; Dyer, John M; Chapman, Kent D

2017-07-01

Fat storage-inducing transmembrane protein 2 (FIT2) is an endoplasmic reticulum (ER)-localized protein that plays an important role in lipid droplet (LD) formation in animal cells. However, no obvious homologue of FIT2 is found in plants. Here, we tested the function of FIT2 in plant cells by ectopically expressing mouse (Mus musculus) FIT2 in Nicotiana tabacum suspension-cultured cells, Nicotiana benthamiana leaves and Arabidopsis thaliana plants. Confocal microscopy indicated that the expression of FIT2 dramatically increased the number and size of LDs in leaves of N. benthamiana and Arabidopsis, and lipidomics analysis and mass spectrometry imaging confirmed the accumulation of neutral lipids in leaves. FIT2 also increased seed oil content by ~13% in some stable, overexpressing lines of Arabidopsis. When expressed transiently in leaves of N. benthamiana or suspension cells of N. tabacum, FIT2 localized specifically to the ER and was often concentrated at certain regions of the ER that resembled ER-LD junction sites. FIT2 also colocalized at the ER with other proteins known to be involved in triacylglycerol biosynthesis or LD formation in plants, but not with ER resident proteins involved in electron transfer or ER-vesicle exit sites. Collectively, these results demonstrate that mouse FIT2 promotes LD accumulation in plants, a surprising functional conservation in the context of a plant cell given the apparent lack of FIT2 homologues in higher plants. These results suggest also that FIT2 expression represents an effective synthetic biology strategy for elaborating neutral lipid compartments in plant tissues for potential biofuel or bioproduct purposes. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Vitamin B5 and N-acetylcysteine in nonalcoholic steatohepatitis: a pre-clinical study in a dietary mouse model

PubMed Central

Machado, Mariana Verdelho; Kruger, Leandi; Jewell, Mark L.; Michelotti, Gregory Alexander; de Almeida Pereira, Thiago; Xie, Guanhua; Moylan, Cynthia A.; Diehl, Anna Mae

2015-01-01

Background Nonalcoholic fatty liver disease (NAFLD) is the number one cause of chronic liver disease and second indication for liver transplantation in the Western world. Effective therapy is still not available. Previously we showed a critical role for caspase-2 in the pathogenesis of nonalcoholic steatohepatitis (NASH), the potentially progressive form of NAFLD. An imbalance between free Coenzyme A (CoA) and acyl-CoA ratio is known to induce caspase-2 activation. Objectives We aimed to evaluate CoA metabolism and the effects of supplementation with CoA precursors, pantothenate and cysteine, in mouse models of NASH. Methods CoA metabolism was evaluated in methionine-choline deficient (MCD) and Western diet mouse models of NASH. MCD-diet fed mice were treated with pantothenate and N-acetylcysteine or placebo to determine effects on NASH. Results Liver free CoA content was reduced, pantothenate kinase (PANK), the rate-limiting enzyme in the CoA biosynthesis pathway, was down-regulated, and CoA degrading enzymes were increased in mice with NASH. Decreased hepatic free CoA content was associated with increased caspase-2 activity, and correlated with worse liver cell apoptosis, inflammation and fibrosis. Treatment with pantothenate and N-acetylcysteine did not inhibit caspase-2 activation, improve NASH, normalize PANK expression, or restore free CoA levels in MCD diet-fed mice. Conclusion In mice with NASH, hepatic CoA metabolism is impaired, leading to decreased free CoA content, activation of caspase-2, and increased liver cell apoptosis. Dietary supplementation with CoA precursors did not restore CoA levels or improve NASH, suggesting that alternative approaches are necessary to normalize free CoA during NASH. PMID:26403427

Mouse fat storage-inducing transmembrane protein 2 (FIT2) promotes lipid droplet accumulation in plants

DOE PAGES

Cai, Yingqi; McClinchie, Elizabeth; Price, Ann; ...

2017-01-18

Fat storage-inducing transmembrane protein 2 (FIT2) is an endoplasmic reticulum (ER)-localized protein that plays an important role in lipid droplet (LD) formation in animal cells. However, no obvious homologue of FIT2 is found in plants. We tested the function of FIT2 in plant cells by ectopically expressing mouse (Mus musculus) FIT2 in Nicotiana tabacum suspension-cultured cells, Nicotiana benthamiana leaves and Arabidopsis thaliana plants. Confocal microscopy indicated that the expression of FIT2 dramatically increased the number and size of LDs in leaves of N. benthamiana and Arabidopsis, and lipidomics analysis and mass spectrometry imaging confirmed the accumulation of neutral lipids inmore » leaves. FIT2 also increased seed oil content by ~13% in some stable, overexpressing lines of Arabidopsis. Furthermore, when expressed transiently in leaves of N. benthamiana or suspension cells of N. tabacum, FIT2 localized specifically to the ER and was often concentrated at certain regions of the ER that resembled ER-LD junction sites. FIT2 also colocalized at the ER with other proteins known to be involved in triacylglycerol biosynthesis or LD formation in plants, but not with ER resident proteins involved in electron transfer or ERvesicle exit sites. Collectively, these results demonstrate that mouse FIT2 promotes LD accumulation in plants, a surprising functional conservation in the context of a plant cell given the apparent lack of FIT2 homologues in higher plants. Our results suggest also that FIT2 expression represents an effective synthetic biology strategy for elaborating neutral lipid compartments in plant tissues for potential biofuel or bioproduct purposes.« less

Translational analysis of mouse and human placental protein and mRNA reveals distinct molecular pathologies in human preeclampsia.

PubMed

Cox, Brian; Sharma, Parveen; Evangelou, Andreas I; Whiteley, Kathie; Ignatchenko, Vladimir; Ignatchenko, Alex; Baczyk, Dora; Czikk, Marie; Kingdom, John; Rossant, Janet; Gramolini, Anthony O; Adamson, S Lee; Kislinger, Thomas

2011-12-01

Preeclampsia (PE) adversely impacts ~5% of pregnancies. Despite extensive research, no consistent biomarkers or cures have emerged, suggesting that different molecular mechanisms may cause clinically similar disease. To address this, we undertook a proteomics study with three main goals: (1) to identify a panel of cell surface markers that distinguish the trophoblast and endothelial cells of the placenta in the mouse; (2) to translate this marker set to human via the Human Protein Atlas database; and (3) to utilize the validated human trophoblast markers to identify subgroups of human preeclampsia. To achieve these goals, plasma membrane proteins at the blood tissue interfaces were extracted from placentas using intravascular silica-bead perfusion, and then identified using shotgun proteomics. We identified 1181 plasma membrane proteins, of which 171 were enriched at the maternal blood-trophoblast interface and 192 at the fetal endothelial interface with a 70% conservation of expression in humans. Three distinct molecular subgroups of human preeclampsia were identified in existing human microarray data by using expression patterns of trophoblast-enriched proteins. Analysis of all misexpressed genes revealed divergent dysfunctions including angiogenesis (subgroup 1), MAPK signaling (subgroup 2), and hormone biosynthesis and metabolism (subgroup 3). Subgroup 2 lacked expected changes in known preeclampsia markers (sFLT1, sENG) and uniquely overexpressed GNA12. In an independent set of 40 banked placental specimens, GNA12 was overexpressed during preeclampsia when co-incident with chronic hypertension. In the current study we used a novel translational analysis to integrate mouse and human trophoblast protein expression with human microarray data. This strategy identified distinct molecular pathologies in human preeclampsia. We conclude that clinically similar preeclampsia patients exhibit divergent placental gene expression profiles thus implicating divergent molecular mechanisms in the origins of this disease.

Mouse Conjunctival Forniceal Gene Expression during Postnatal Development and Its Regulation by Krüppel-like Factor 4

PubMed Central

Gupta, Divya; Harvey, Stephen A. K.; Kaminski, Naftali

2011-01-01

Purpose. To identify the changes in postnatal mouse conjunctival forniceal gene expression and their regulation by Klf4 during the eye-opening stage when the goblet cells first appear. Methods. Laser microdissection (LMD) was used to collect conjunctival forniceal cells from postnatal (PN) day 9, PN14 and PN20 wild-type (WT), and PN14 Klf4-conditional null (Klf4CN) mice, in which goblet cells are absent, developing, present, and missing, respectively. Microarrays were used to compare gene expression among these groups. Expression of selected genes was validated by quantitative RT-PCR, and spatiotemporal expression was assessed by in situ hybridization. Results. This study identified 668, 251, 1160, and 139 transcripts that were increased and 492, 377, 1419, and 57 transcripts that were decreased between PN9 and PN14, PN14 and PN20, PN9 and PN20, and PN14 WT and Klf4CN conjunctiva, respectively. Transcripts encoding transcription factors Spdef, FoxA1, and FoxA3 that regulate goblet cell development in other mucosal epithelia, and epithelium-specific Ets (ESE) transcription factor family members were increased during conjunctival development. Components of pathways related to the mesenchymal–epithelial transition, glycoprotein biosynthesis, mucosal immunity, signaling, and endocytic and neural regulation were increased during conjunctival development. Conjunctival Klf4 target genes differed significantly from the previously identified corneal Klf4 target genes, implying tissue-dependent regulatory targets for Klf4. Conclusions. The changes in gene expression accompanying mouse conjunctival development were identified, and the role of Klf4 in this process was determined. This study provides new probes for examining conjunctival development and function and reveals that the gene regulatory network necessary for goblet cell development is conserved across different mucosal epithelia. PMID:21398290

Mouse fat storage-inducing transmembrane protein 2 (FIT2) promotes lipid droplet accumulation in plants

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

Cai, Yingqi; McClinchie, Elizabeth; Price, Ann

Fat storage-inducing transmembrane protein 2 (FIT2) is an endoplasmic reticulum (ER)-localized protein that plays an important role in lipid droplet (LD) formation in animal cells. However, no obvious homologue of FIT2 is found in plants. We tested the function of FIT2 in plant cells by ectopically expressing mouse (Mus musculus) FIT2 in Nicotiana tabacum suspension-cultured cells, Nicotiana benthamiana leaves and Arabidopsis thaliana plants. Confocal microscopy indicated that the expression of FIT2 dramatically increased the number and size of LDs in leaves of N. benthamiana and Arabidopsis, and lipidomics analysis and mass spectrometry imaging confirmed the accumulation of neutral lipids inmore » leaves. FIT2 also increased seed oil content by ~13% in some stable, overexpressing lines of Arabidopsis. Furthermore, when expressed transiently in leaves of N. benthamiana or suspension cells of N. tabacum, FIT2 localized specifically to the ER and was often concentrated at certain regions of the ER that resembled ER-LD junction sites. FIT2 also colocalized at the ER with other proteins known to be involved in triacylglycerol biosynthesis or LD formation in plants, but not with ER resident proteins involved in electron transfer or ERvesicle exit sites. Collectively, these results demonstrate that mouse FIT2 promotes LD accumulation in plants, a surprising functional conservation in the context of a plant cell given the apparent lack of FIT2 homologues in higher plants. Our results suggest also that FIT2 expression represents an effective synthetic biology strategy for elaborating neutral lipid compartments in plant tissues for potential biofuel or bioproduct purposes.« less

Widespread cerebellar transcriptome changes in Ts65Dn Down syndrome mouse model after lifelong running.

PubMed

Walus, Marius; Kida, Elizabeth; Rabe, Ausma; Albertini, Giorgio; Golabek, Adam A

2016-01-01

Our previous study showed an improvement in locomotor deficits after voluntary lifelong running in Ts65Dn mice, an animal model for Down syndrome (DS). In the present study, we employed mouse microarrays printed with 55,681 probes in an attempt to identify molecular changes in the cerebellar transcriptome that might contribute to the observed behavioral benefits of voluntary long-term running in Ts65Dn mice. Euploid mice were processed in parallel for comparative purposes in some analyses. We found that running significantly changed the expression of 4,315 genes in the cerebellum of Ts65Dn mice, over five times more than in euploid animals, up-regulating 1,991 and down-regulating 2,324 genes. Functional analysis of these genes revealed a significant enrichment of 92 terms in the biological process category, including regulation of biosynthesis and metabolism, protein modification, phosphate metabolism, synaptic transmission, development, regulation of cell death/apoptosis, protein transport, development, neurogenesis and neuron differentiation. The KEGG pathway database identified 18 pathways that are up-regulated and two that are down-regulated by running that were associated with learning, memory, cell signaling, proteolysis, regeneration, cell cycle, proliferation, growth, migration, and survival. Of six mRNA protein products we tested by immunoblotting, four showed significant running-associated changes in their levels, the most prominent in glutaminergic receptor metabotropic 1, and two showed changes that were close to significant. Thus, unexpectedly, our data point to the high molecular plasticity of Ts65Dn mouse cerebellum, which translated into humans with DS, suggests that the motor deficits of individuals with DS could markedly benefit from prolonged exercise. Copyright © 2015 Elsevier B.V. All rights reserved.

Circulating exosomes potentiate tumor malignant properties in a mouse model of chronic sleep fragmentation

PubMed Central

Khalyfa, Abdelnaby; Almendros, Isaac; Gileles-Hillel, Alex; Akbarpour, Mahzad; Trzepizur, Wojciech; Mokhlesi, Babak; Huang, Lei; Andrade, Jorge; Farré, Ramon; Gozal, David

2016-01-01

Background Chronic sleep fragmentation (SF) increases cancer aggressiveness in mice. Exosomes exhibit pleiotropic biological functions, including immune regulatory functions, antigen presentation, intracellular communication and inter-cellular transfer of RNA and proteins. We hypothesized that SF-induced alterations in biosynthesis and cargo of plasma exosomes may affect tumor cell properties. Results SF-derived exosomes increased tumor cell proliferation (~13%), migration (~2.3-fold) and extravasation (~10%) when compared to exosomes from SC-exposed mice. Similarly, Pre exosomes from OSA patients significantly enhanced proliferation and migration of human adenocarcinoma cells compared to Post. SF-exosomal cargo revealed 3 discrete differentially expressed miRNAs, and exploration of potential mRNA targets in TC1 tumor cells uncovered 132 differentially expressed genes that encode for multiple cancer-related pathways. Methods Plasma-derived exosomes from C57/B6 mice exposed to 6 wks of SF or sleep control (SC), and from adult human patients with obstructive sleep apnea (OSA) before (Pre) and after adherent treatment for 6 wks (Post) were co-cultured with mouse lung TC1 or human adenocarcinoma tumor cell lines, respectively. Proliferation, migration, invasion, endothelial barrier integrity and extravasation assays of tumor cells were performed. Plasma mouse exosomal miRNAs were profiled with arrays, and transcriptomic assessments of TC1 cells exposed to SF or SC exosomes were conducted to identify gene targets. Conclusions Chronic SF induces alterations in exosomal miRNA cargo that alter the biological properties of TC1 lung tumor cells to enhance their proliferative, migratory and extravasation properties, and similar findings occur in OSA patients, in whom SF is a constitutive component of their sleep disorder. Thus, exosomes could participate, at least in part, in the adverse cancer outcomes observed in OSA. PMID:27419627

N-Glycolylneuraminic acid deficiency worsens cardiac and skeletal muscle pathophysiology in α-sarcoglycan-deficient mice

PubMed Central

Martin, Paul T; Camboni, Marybeth; Xu, Rui; Golden, Bethannie; Chandrasekharan, Kumaran; Wang, Chiou-Miin; Varki, Ajit; Janssen, Paul M L

2013-01-01

Roughly 3 million years ago, an inactivating deletion occurred in CMAH, the human gene encoding CMP-Neu5Ac (cytidine-5′-monophospho-N-acetylneuraminic acid) hydroxylase (Chou HH, Takematsu H, Diaz S, Iber J, Nickerson E, Wright KL, Muchmore EA, Nelson DL, Warren ST, Varki A. 1998. A mutation in human CMP-sialic acid hydroxylase occurred after the Homo-Pan divergence. Proc Natl Acad Sci USA. 95:11751–11756). This inactivating deletion is now homozygous in all humans, causing the loss of N-glycolylneuraminic acid (Neu5Gc) biosynthesis in all human cells and tissues. The CMAH enzyme is active in other mammals, including mice, where Neu5Gc is an abundant form of sialic acid on cellular membranes, including those in cardiac and skeletal muscle. We recently demonstrated that the deletion of mouse Cmah worsened the severity of pathophysiology measures related to muscular dystrophy in mdx mice, a model for Duchenne muscular dystrophy (Chandrasekharan K, Yoon JH, Xu Y, deVries S, Camboni M, Janssen PM, Varki A, Martin PT. 2010. A human-specific deletion in mouse Cmah increases disease severity in the mdx model of Duchenne muscular dystrophy. Sci Transl Med. 2:42–54). Here, we demonstrate similar changes in cardiac and skeletal muscle pathology and physiology resulting from Cmah deletion in α-sarcoglycan-deficient (Sgca−/−) mice, a model for limb girdle muscular dystrophy 2D. These experiments demonstrate that loss of mouse Cmah can worsen disease severity in more than one form of muscular dystrophy and suggest that Cmah may be a general genetic modifier of muscle disease. PMID:23514716

Development of accelerated net nitrate uptake. [Zea mays L

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

MacKown, C.T.; McClure, P.R.

1988-05-01

Upon initial nitrate exposure, net nitrate uptake rates in roots of a wide variety of plants accelerate within 6 to 8 hours to substantially greater rates. Effects of solution nitrate concentrations and short pulses of nitrate ({le}1 hour) upon nitrate-induced acceleration of nitrate uptake in maize (Zea mays L.) were determined. Root cultures of dark-grown seedlings, grown without nitrate, were exposed to 250 micromolar nitrate for 0.25 to 1 hour or to various solution nitrate concentration (10-250 micromolar) for 1 hour before returning them to a nitrate-free solution. Net nitrate uptake rates were assayed at various periods following nitrate exposuremore » and compared to rates of roots grown either in the absence of nitrate (CaSO{sub 4}-grown) or with continuous nitrate for at least 20 hours. Three hours after initial nitrate exposure, nitrate pulse treatments increased nitrate uptake rates three- to four-fold compared to the rates of CaSO{sub 4}-grown roots. When cycloheximide (5 micrograms per milliliter) was included during a 1-hour pulse with 250 micromolar nitrate, development of the accelerated nitrate uptake state was delayed. Otherwise, nitrate uptake rates reached maximum values within 6 hours before declining. Maximum rates, however, were significantly less than those of roots exposed continuously for 20, 32, or 44 hours. Pulsing for only 0.25 hour with 250 micromolar nitrate and for 1 hour with 10 micromolar caused acceleration of nitrate uptake, but the rates attained were either less than or not sustained for a duration comparable to those of roots pulsed for 1 hour with 250 micromolar nitrate. These results indicate that substantial development of nitrate-induced accelerated nitrate uptake state can be achieved by small endogenous accumulations of nitrate, which appear to moderate the activity or level of root nitrate uptake.« less

Acceleration of atherogenesis by COX-1-dependent prostanoid formation in low density lipoprotein receptor knockout mice.

PubMed

Praticò, D; Tillmann, C; Zhang, Z B; Li, H; FitzGerald, G A

2001-03-13

The cyclooxygenase (COX) product, prostacyclin (PGI(2)), inhibits platelet activation and vascular smooth-muscle cell migration and proliferation. Biochemically selective inhibition of COX-2 reduces PGI(2) biosynthesis substantially in humans. Because deletion of the PGI(2) receptor accelerates atherogenesis in the fat-fed low density lipoprotein receptor knockout mouse, we wished to determine whether selective inhibition of COX-2 would accelerate atherogenesis in this model. To address this hypothesis, we used dosing with nimesulide, which inhibited COX-2 ex vivo, depressed urinary 2,3 dinor 6-keto PGF(1alpha) by approximately 60% but had no effect on thromboxane formation by platelets, which only express COX-1. By contrast, the isoform nonspecific inhibitor, indomethacin, suppressed platelet function and thromboxane formation ex vivo and in vivo, coincident with effects on PGI(2) biosynthesis indistinguishable from nimesulide. Indomethacin reduced the extent of atherosclerosis by 55 +/- 4%, whereas nimesulide failed to increase the rate of atherogenesis. Despite their divergent effects on atherogenesis, both drugs depressed two indices of systemic inflammation, soluble intracellular adhesion molecule-1, and monocyte chemoattractant protein-1 to a similar but incomplete degree. Neither drug altered serum lipids and the marked increase in vascular expression of COX-2 during atherogenesis. Accelerated progression of atherosclerosis is unlikely during chronic intake of specific COX-2 inhibitors. Furthermore, evidence that COX-1-derived prostanoids contribute to atherogenesis suggests that controlled evaluation of the effects of nonsteroidal anti-inflammatory drugs and/or aspirin on plaque progression in humans is timely.

Pyridoxal phosphate synthases PdxS/PdxT are required for Actinobacillus pleuropneumoniae viability, stress tolerance and virulence.

PubMed

Xie, Fang; Li, Gang; Wang, Yalei; Zhang, Yanhe; Zhou, Long; Wang, Chengcheng; Liu, Shuanghong; Liu, Siguo; Wang, Chunlai

2017-01-01

Pyridoxal 5'-phosphate (PLP) is an essential cofactor for numerous enzymes involved in a diversity of cellular processes in living organisms. Previous analysis of the Actinobacillus pleuropneumoniae S-8 genome sequence revealed the presence of pdxS and pdxT genes, which are implicated in deoxyxylulose 5-phosphate (DXP)-independent pathway of PLP biosynthesis; however, little is known about their roles in A. pleuropneumoniae pathogenicity. Our data demonstrated that A. pleuropneumoniae could synthesize PLP by PdxS and PdxT enzymes. Disruption of the pdxS and pdxT genes rendered the pathogen auxotrophic for PLP, and the defective growth as a result of these mutants was chemically compensated by the addition of PLP, suggesting the importance of PLP production for A. pleuropneumoniae growth and viability. Additionally, the pdxS and pdxT deletion mutants displayed morphological defects as indicated by irregular and aberrant shapes in the absence of PLP. The reduced growth of the pdxS and pdxT deletion mutants under osmotic and oxidative stress conditions suggests that the PLP synthases PdxS/PdxT are associated with the stress tolerance of A. pleuropneumoniae. Furthermore, disruption of the PLP biosynthesis pathway led to reduced colonization and attenuated virulence of A. pleuropneumoniae in the BALB/c mouse model. The data presented in this study reveal the critical role of PLP synthases PdxS/PdxT in viability, stress tolerance, and virulence of A. pleuropneumoniae.

Pyruvate carboxylase is critical for non–small-cell lung cancer proliferation

PubMed Central

Sellers, Katherine; Fox, Matthew P.; Bousamra, Michael; Slone, Stephen P.; Higashi, Richard M.; Miller, Donald M.; Wang, Yali; Yan, Jun; Yuneva, Mariia O.; Deshpande, Rahul; Lane, Andrew N.; Fan, Teresa W.-M.

2015-01-01

Anabolic biosynthesis requires precursors supplied by the Krebs cycle, which in turn requires anaplerosis to replenish precursor intermediates. The major anaplerotic sources are pyruvate and glutamine, which require the activity of pyruvate carboxylase (PC) and glutaminase 1 (GLS1), respectively. Due to their rapid proliferation, cancer cells have increased anabolic and energy demands; however, different cancer cell types exhibit differential requirements for PC- and GLS-mediated pathways for anaplerosis and cell proliferation. Here, we infused patients with early-stage non–small-cell lung cancer (NSCLC) with uniformly 13C-labeled glucose before tissue resection and determined that the cancerous tissues in these patients had enhanced PC activity. Freshly resected paired lung tissue slices cultured in 13C6-glucose or 13C5,15N2-glutamine tracers confirmed selective activation of PC over GLS in NSCLC. Compared with noncancerous tissues, PC expression was greatly enhanced in cancerous tissues, whereas GLS1 expression showed no trend. Moreover, immunohistochemical analysis of paired lung tissues showed PC overexpression in cancer cells rather than in stromal cells of tumor tissues. PC knockdown induced multinucleation, decreased cell proliferation and colony formation in human NSCLC cells, and reduced tumor growth in a mouse xenograft model. Growth inhibition was accompanied by perturbed Krebs cycle activity, inhibition of lipid and nucleotide biosynthesis, and altered glutathione homeostasis. These findings indicate that PC-mediated anaplerosis in early-stage NSCLC is required for tumor survival and proliferation. PMID:25607840

Novel assay for simultaneous measurement of pyridine mononucleotides synthesizing activities allows dissection of the NAD(+) biosynthetic machinery in mammalian cells.

PubMed

Zamporlini, Federica; Ruggieri, Silverio; Mazzola, Francesca; Amici, Adolfo; Orsomando, Giuseppe; Raffaelli, Nadia

2014-11-01

The redox coenzyme NAD(+) is also a rate-limiting co-substrate for several enzymes that consume the molecule, thus rendering its continuous re-synthesis indispensable. NAD(+) biosynthesis has emerged as a therapeutic target due to the relevance of NAD(+) -consuming reactions in complex intracellular signaling networks whose alteration leads to many neurologic and metabolic disorders. Distinct metabolic routes, starting from various precursors, are known to support NAD(+) biosynthesis with tissue/cell-specific efficiencies, probably reflecting differential expression of the corresponding rate-limiting enzymes, i.e. nicotinamide phosphoribosyltransferase, quinolinate phosphoribosyltransferase, nicotinate phosphoribosyltransferase and nicotinamide riboside kinase. Understanding the contribution of these enzymes to NAD(+) levels depending on the tissue/cell type and metabolic status is necessary for the rational design of therapeutic strategies aimed at modulating NAD(+) availability. Here we report a simple, fast and sensitive coupled fluorometric assay that enables simultaneous determination of the four activities in whole-cell extracts and biological fluids. Its application to extracts from various mouse tissues, human cell lines and plasma yielded for the first time an overall picture of the tissue/cell-specific distribution of the activities of the various enzymes. The screening enabled us to gather novel findings, including (a) the presence of quinolinate phosphoribosyltransferase and nicotinamide riboside kinase in all examined tissues/cell lines, indicating that quinolinate and nicotinamide riboside are relevant NAD(+) precursors, and (b) the unexpected occurrence of nicotinate phosphoribosyltransferase in human plasma. © 2014 FEBS.

Analgesic Effects of Toad Cake and Toad-cake-containing Herbal Drugs

PubMed Central

Inoue, Eiji; Shimizu, Yasuharu; Masui, Ryo; Usui, Tomomi; Sudoh, Keiichi

2014-01-01

Objectives: This study was conducted to clarify the analgesic effect of toad cake and toad-cake-containing herbal drugs. Methods: We counted the writhing response of mice after the intraperitoneal administration of acetic acid as a nociceptive pain model and the withdrawal response after the plantar surface stimulation of the hind paw induced by partial sciatic nerve ligation of the mice as a neuropathic pain model to investigate the analgesic effect of toad cake and toad-cake-containing herbal drugs. A co-treatment study with serotonin biosynthesis inhibitory drug 4-chloro- DL-phenylalanine methyl ester hydrochloride (PCPA), the catecholamine biosynthesis inhibitory drug α-methyl- DL-tyrosine methyl ester hydrochloride (AMPT) or the opioid receptor antagonist naloxone hydrochloride was also conducted. Results: Analgesic effects in a mouse model of nociceptive pain and neuropathic pain were shown by oral administration of toad cake and toad-cake-containing herbal drugs. The effects of toad cake and toad-cake-containing herbal drugs disappeared upon co-treatment with PCPA, but not with AMPT or naloxone in the nociceptive pain model; the analgesic effect of toad-cake-containing herbal drugs also disappeared upon co-treatment with PCPA in the neuropathic pain model. Conclusion: Toad cake and toad-cake-containing herbal drugs have potential for the treatments of nociceptive pain and of neuropathic pain, such as post-herpetic neuralgia, trigeminal neuralgia, diabetic neuralgia, and postoperative or posttraumatic pain, by activation of the central serotonin nervous system. PMID:25780693

Analgesic Effects of Toad Cake and Toad-cake-containing Herbal Drugs: Analgesic effects of toad cake.

PubMed

Inoue, Eiji; Shimizu, Yasuharu; Masui, Ryo; Usui, Tomomi; Sudoh, Keiichi

2014-03-01

This study was conducted to clarify the analgesic effect of toad cake and toad-cake-containing herbal drugs. We counted the writhing response of mice after the intraperitoneal administration of acetic acid as a nociceptive pain model and the withdrawal response after the plantar surface stimulation of the hind paw induced by partial sciatic nerve ligation of the mice as a neuropathic pain model to investigate the analgesic effect of toad cake and toad-cake-containing herbal drugs. A co-treatment study with serotonin biosynthesis inhibitory drug 4-chloro- DL-phenylalanine methyl ester hydrochloride (PCPA), the catecholamine biosynthesis inhibitory drug α-methyl- DL-tyrosine methyl ester hydrochloride (AMPT) or the opioid receptor antagonist naloxone hydrochloride was also conducted. Analgesic effects in a mouse model of nociceptive pain and neuropathic pain were shown by oral administration of toad cake and toad-cake-containing herbal drugs. The effects of toad cake and toad-cake-containing herbal drugs disappeared upon co-treatment with PCPA, but not with AMPT or naloxone in the nociceptive pain model; the analgesic effect of toad-cake-containing herbal drugs also disappeared upon co-treatment with PCPA in the neuropathic pain model. Toad cake and toad-cake-containing herbal drugs have potential for the treatments of nociceptive pain and of neuropathic pain, such as post-herpetic neuralgia, trigeminal neuralgia, diabetic neuralgia, and postoperative or posttraumatic pain, by activation of the central serotonin nervous system.

Development of a Highly Biocompatible Antituberculosis Nanodelivery Formulation Based on Para-Aminosalicylic Acid—Zinc Layered Hydroxide Nanocomposites

PubMed Central

Arulselvan, Palanisamy; El Zowalaty, Mohamed Ezzat; Fakurazi, Sharida; Webster, Thomas J.; Geilich, Benjamin; Hussein, Mohd Zobir

2014-01-01

Tuberculosis is a lethal epidemic, difficult to control disease, claiming thousands of lives every year. We have developed a nanodelivery formulation based on para-aminosalicylic acid (PAS) and zinc layered hydroxide using zinc nitrate salt as a precursor. The developed formulation has a fourfold higher efficacy of PAS against mycobacterium tuberculosis with a minimum inhibitory concentration (MIC) found to be at 1.40 μg/mL compared to the free drug PAS with a MIC of 5.0 μg/mL. The newly developed formulation was also found active against Gram-positive bacteria, Gram-negative bacteria, and Candida albicans. The formulation was also found to be biocompatible with human normal lung cells MRC-5 and mouse fibroblast cells-3T3. The in vitro release of PAS from the formulation was found to be sustained in a human body simulated phosphate buffer saline (PBS) solution at pH values of 7.4 and 4.8. Most importantly the nanocomposite prepared using zinc nitrate salt was advantageous in terms of yield and free from toxic zinc oxide contamination and had higher biocompatibility compared to one prepared using a zinc oxide precursor. In summary, these promising in vitro results are highly encouraging for the continued investigation of para-aminosalicylic acid and zinc layered hydroxide nanocomposites in vivo and eventual preclinical studies. PMID:25050392

Short-term effects of a high nitrate diet on nitrate metabolism in healthy individuals.

PubMed

Bondonno, Catherine P; Liu, Alex H; Croft, Kevin D; Ward, Natalie C; Puddey, Ian B; Woodman, Richard J; Hodgson, Jonathan M

2015-03-12

Dietary nitrate, through the enterosalivary nitrate-nitrite-NO pathway, can improve blood pressure and arterial stiffness. How long systemic nitrate and nitrite remain elevated following cessation of high nitrate intake is unknown. In 19 healthy men and women, the time for salivary and plasma nitrate and nitrite to return to baseline after 7 days increased nitrate intake from green leafy vegetables was determined. Salivary and plasma nitrate and nitrite was measured at baseline [D0], end of high nitrate diet [D7], day 9 [+2D], day 14 [+7D] and day 21 [+14D]. Urinary nitrite and nitrate was assessed at D7 and +14D. Increased dietary nitrate for 7 days resulted in a more than fourfold increase in saliva and plasma nitrate and nitrite (p < 0.001) measured at [D7]. At [+2D] plasma nitrite and nitrate had returned to baseline while saliva nitrate and nitrite were more than 1.5 times higher than at baseline levels. By [+7D] all metabolites had returned to baseline levels. The pattern of response was similar between men and women. Urinary nitrate and nitrate was sevenfold higher at D7 compared to +14D. These results suggest that daily ingestion of nitrate may be required to maintain the physiological changes associated with high nitrate intake.

Possibility of Salicornia europaea use for the human liquid wastes inclusion into BLSS intrasystem mass exchange

NASA Astrophysics Data System (ADS)

Tikhomirova, Natalia A.; Ushakova, Sofya A.; Tikhomirov, Alexander A.; Kalacheva, Galina S.; Gros, Jean-Bernard

One of the ways of solving the problem of the human liquid wastes utilization in bioregenerative life support systems (BLSS) can be the use of halophytic vegetable plant Salicornia europaea capable of accumulating sodium chloride in rather high concentrations. Since the most specific higher plant function in BLSS, which at present cannot be substituted by physicochemical processes, appears to be the biosynthesis of a wide spectrum of nutritive substances necessary for a human, the object of the given work was the investigation of the S. europaea productivity, biochemical and mineral composition when grown under close to optimal BLSS vegetative component conditions. As the use of human urine after its preliminary physicochemical processing is supposed to be the mineral solution basis for the S. europaea cultivation, it is necessary to clear up the effect of reduced nitrogen on plants growth. Ground research was carried out. Biochemical composition of the S. europaea edible part showed that crude protein was contained in the highest degree. At that the content of crude protein (24% per dry weight) and cellulose (4.7% per dry weight) was higher in the plants grown on solutions containing amide nitrogen in comparison with the plants grown on solutions with nitrate nitrogen (15.4%—3.1% correspondingly). The water-soluble sugar contents were not high in the S. europaea edible part and depending on the nitrogen nutrition form they amounted to 1.1% (amide nitrogen) and 1.5% (nitrate nitrogen). The polysaccharide number (except cellulose) was rather higher and varied from 7.7% to 8.2%. Although the lipid content in the S. europaea plants was relatively low (7% per dry weight), it was shown that the plant lipids are characterized by a high nonsaturation degree mainly due to alpha linolenic and linoleic acids. Nitrogen nutrition form did not significantly affect the S. europaea productivity, and dry edible biomass of one plant was 8.6 g. Sodium and its concentrations predominated in the plant mineral composition and amounted in average to 9% per dry weight. Thus the S. europaea being the vegetable plant it can be the source of several biochemical substances and essential fatty acids. The present work also considers the influence of nitrate and amide forms of nitrogen on S. europaea biochemical and mineral composition.

21 CFR 181.33 - Sodium nitrate and potassium nitrate.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Sodium nitrate and potassium nitrate. 181.33...-Sanctioned Food Ingredients § 181.33 Sodium nitrate and potassium nitrate. Sodium nitrate and potassium nitrate are subject to prior sanctions issued by the U.S. Department of Agriculture for use as sources of...

21 CFR 181.33 - Sodium nitrate and potassium nitrate.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Sodium nitrate and potassium nitrate. 181.33...-Sanctioned Food Ingredients § 181.33 Sodium nitrate and potassium nitrate. Sodium nitrate and potassium nitrate are subject to prior sanctions issued by the U.S. Department of Agriculture for use as sources of...

21 CFR 181.33 - Sodium nitrate and potassium nitrate.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Sodium nitrate and potassium nitrate. 181.33... nitrate and potassium nitrate. Sodium nitrate and potassium nitrate are subject to prior sanctions issued... potassium nitrite, in the production of cured red meat products and cured poultry products. [48 FR 1705, Jan...

49 CFR 176.415 - Permit requirements for Division 1.5, ammonium nitrates, and certain ammonium nitrate fertilizers.

Code of Federal Regulations, 2011 CFR

2011-10-01

... nitrates, and certain ammonium nitrate fertilizers. 176.415 Section 176.415 Transportation Other... requirements for Division 1.5, ammonium nitrates, and certain ammonium nitrate fertilizers. (a) Except as... Captain of the Port (COTP). (1) Ammonium nitrate UN1942, ammonium nitrate fertilizers containing more than...

49 CFR 176.415 - Permit requirements for Division 1.5, ammonium nitrates, and certain ammonium nitrate fertilizers.

Code of Federal Regulations, 2013 CFR

2013-10-01

... nitrates, and certain ammonium nitrate fertilizers. 176.415 Section 176.415 Transportation Other... requirements for Division 1.5, ammonium nitrates, and certain ammonium nitrate fertilizers. (a) Except as... Captain of the Port (COTP). (1) Ammonium nitrate UN1942, ammonium nitrate fertilizers containing more than...

49 CFR 176.415 - Permit requirements for Division 1.5, ammonium nitrates, and certain ammonium nitrate fertilizers.

Code of Federal Regulations, 2014 CFR

2014-10-01

... nitrates, and certain ammonium nitrate fertilizers. 176.415 Section 176.415 Transportation Other... requirements for Division 1.5, ammonium nitrates, and certain ammonium nitrate fertilizers. (a) Except as... Captain of the Port (COTP). (1) Ammonium nitrate UN1942, ammonium nitrate fertilizers containing more than...

49 CFR 176.415 - Permit requirements for Division 1.5, ammonium nitrates, and certain ammonium nitrate fertilizers.

Code of Federal Regulations, 2012 CFR

2012-10-01

... nitrates, and certain ammonium nitrate fertilizers. 176.415 Section 176.415 Transportation Other... requirements for Division 1.5, ammonium nitrates, and certain ammonium nitrate fertilizers. (a) Except as... Captain of the Port (COTP). (1) Ammonium nitrate UN1942, ammonium nitrate fertilizers containing more than...

Low levels of glutathione are sufficient for survival of keratinocytes after UV irradiation and for healing of mouse skin wounds.

PubMed

Telorack, Michèle; Abplanalp, Jeannette; Werner, Sabine

2016-08-01

Reduced levels of the cellular antioxidant glutathione are associated with premature skin aging, cancer and impaired wound healing, but the in vivo functions of glutathione in the skin remain largely unknown. Therefore, we analyzed mice lacking the modifier subunit of the glutamate cysteine ligase (Gclm), the enzyme that catalyzes the rate-limiting step of glutathione biosynthesis. Glutathione levels in the skin of these mice were reduced by 70 %. However, neither skin development and homeostasis, nor UVA- or UVB-induced apoptosis in the epidermis were affected. Histomorphometric analysis of excisional wounds did not reveal wound healing abnormalities in young Gclm-deficient mice, while the area of hyperproliferative epithelium as well as keratinocyte proliferation were affected in aged mice. These findings suggest that low levels of glutathione are sufficient for wound repair in young mice, but become rate-limiting upon aging.

Phosphatidylcholine and the CDP-Choline Cycle

PubMed Central

Fagone, Paolo; Jackowski, Suzanne

2012-01-01

The CDP-choline pathway of phosphatidylcholine (PtdCho) biosynthesis was first described more than 50 years ago. Investigation of the CDP-choline pathway in yeast provides a basis for understanding the CDP-choline pathway in mammals. PtdCho is considered as an intermediate in a cycle of synthesis and degradation, and the activity of a CDP-choline cycle is linked to subcellular membrane lipid movement. The components of the mammalian CDP-choline pathway include choline transport, choline kinase, phosphocholine cytidylyltransferase, and choline phosphotransferase activities. The protein isoforms and biochemical mechanisms of regulation of the pathway enzymes are related to their cell and tissue-specific functions. Regulated PtdCho turnover mediated by phospholipases or neuropathy target esterase participates in the mammalian CDP-choline cycle. Knockout mouse models define the biological functions of the CDP-choline cycle in mammalian cells and tissues. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism. PMID:23010477

Transmissible microbial and metabolomic remodeling by soluble dietary fiber improves metabolic homeostasis

PubMed Central

He, Baokun; Nohara, Kazunari; Ajami, Nadim J.; Michalek, Ryan D.; Tian, Xiangjun; Wong, Matthew; Losee-Olson, Susan H.; Petrosino, Joseph F.; Yoo, Seung-Hee; Shimomura, Kazuhiro; Chen, Zheng

2015-01-01

Dietary fibers are increasingly appreciated as beneficial nutritional components. However, a requisite role of gut microbiota in fiber function and the overall impact of fibers on metabolomic flux remain unclear. We herein showed enhancing effects of a soluble resistant maltodextrin (RM) on glucose homeostasis in mouse metabolic disease models. Remarkably, fecal microbiota transplantation (FMT) caused pronounced and time-dependent improvement in glucose tolerance in RM recipient mice, indicating a causal relationship between microbial remodeling and metabolic efficacy. Microbial 16S sequencing revealed transmissible taxonomic changes correlated with improved metabolism, notably enrichment of probiotics and reduction of Alistipes and Bacteroides known to associate with high fat/protein diets. Metabolomic profiling further illustrated broad changes, including enrichment of phenylpropionates and decreases in key intermediates of glucose utilization, cholesterol biosynthesis and amino acid fermentation. These studies elucidate beneficial roles of RM-dependent microbial remodeling in metabolic homeostasis, and showcase prevalent health-promoting potentials of dietary fibers. PMID:26040234

Selective targeting of mutant adenomatous polyposis coli (APC) in colorectal cancer.

PubMed

Zhang, Lu; Theodoropoulos, Panayotis C; Eskiocak, Ugur; Wang, Wentian; Moon, Young-Ah; Posner, Bruce; Williams, Noelle S; Wright, Woodring E; Kim, Sang Bum; Nijhawan, Deepak; De Brabander, Jef K; Shay, Jerry W

2016-10-19

Mutations in the adenomatous polyposis coli (APC) gene are common in colorectal cancer (CRC), and more than 90% of those mutations generate stable truncated gene products. We describe a chemical screen using normal human colonic epithelial cells (HCECs) and a series of oncogenically progressed HCECs containing a truncated APC protein. With this screen, we identified a small molecule, TASIN-1 (truncated APC selective inhibitor-1), that specifically kills cells with APC truncations but spares normal and cancer cells with wild-type APC. TASIN-1 exerts its cytotoxic effects through inhibition of cholesterol biosynthesis. In vivo administration of TASIN-1 inhibits tumor growth of CRC cells with truncated APC but not APC wild-type CRC cells in xenograft models and in a genetically engineered CRC mouse model with minimal toxicity. TASIN-1 represents a potential therapeutic strategy for prevention and intervention in CRC with mutant APC. Copyright © 2016, American Association for the Advancement of Science.

Short-Term Effects of a High Nitrate Diet on Nitrate Metabolism in Healthy Individuals

PubMed Central

Bondonno, Catherine P.; Liu, Alex H.; Croft, Kevin D.; Ward, Natalie C.; Puddey, Ian B.; Woodman, Richard J.; Hodgson, Jonathan M.

2015-01-01

Dietary nitrate, through the enterosalivary nitrate-nitrite-NO pathway, can improve blood pressure and arterial stiffness. How long systemic nitrate and nitrite remain elevated following cessation of high nitrate intake is unknown. In 19 healthy men and women, the time for salivary and plasma nitrate and nitrite to return to baseline after 7 days increased nitrate intake from green leafy vegetables was determined. Salivary and plasma nitrate and nitrite was measured at baseline [D0], end of high nitrate diet [D7], day 9 [+2D], day 14 [+7D] and day 21 [+14D]. Urinary nitrite and nitrate was assessed at D7 and +14D. Increased dietary nitrate for 7 days resulted in a more than fourfold increase in saliva and plasma nitrate and nitrite (p < 0.001) measured at [D7]. At [+2D] plasma nitrite and nitrate had returned to baseline while saliva nitrate and nitrite were more than 1.5 times higher than at baseline levels. By [+7D] all metabolites had returned to baseline levels. The pattern of response was similar between men and women. Urinary nitrate and nitrate was sevenfold higher at D7 compared to +14D. These results suggest that daily ingestion of nitrate may be required to maintain the physiological changes associated with high nitrate intake. PMID:25774606

Adverse hepatic and cardiac responses to rosiglitazone in a new mouse model of type 2 diabetes: relation to dysregulated phosphatidylcholine metabolism.

PubMed

Pan, Huei-Ju; Lin, Yiming; Chen, Yuqing E; Vance, Dennis E; Leiter, Edward H

2006-07-01

Given the heterogeneous nature of metabolic dysfunctions associated with insulin resistance and type 2 diabetes (T2D), a single pharmaceutical cannot be expected to provide complication-free therapy in all patients. Thiazolidinediones (TZD) increase insulin sensitivity, reduce blood glucose and improve cardiovascular parameters. However, in addition to increasing fat mass, TZD have the potential in certain individuals to exacerbate underlying hepatosteatosis and diabetic cardiomyopathy. Pharmacogenetics should allow patient selection to maximize therapy and minimize risk. To this end, we have combined two genetically diverse inbred strains, NON/Lt and NZO/Lt, to produce a "negative heterosis" increasing the frequency of T2D in F1 males. As in humans with T2D, treatment of diabetic and hyperlipemic F1 males with rosiglitazone (Rosi), an agonist of peroxisome proliferator-activated gamma receptor (PPARgamma), reverses these disease phenotypes. However, the hybrid genome perturbed both major pathways for phosphatidylcholine (PC) biosynthesis in the liver, and effected remarkable alterations in the composition of cardiolipin in heart mitochondria. These metabolic defects severely exacerbated an underlying hepatosteatosis and increased levels of the adipokine, plasminogen activator inhibitor-1 (PAI-1), a risk factor for cardiovascular events. This model system demonstrates how the power of mouse genetics can be used to identify the metabolic signatures of individuals who may be prone to drug side effects.

Specificity and rate of human and mouse liver and plasma phosphatidylcholine synthesis analyzed in vivo[S

PubMed Central

Pynn, Christopher J.; Henderson, Neil G.; Clark, Howard; Koster, Grielof; Bernhard, Wolfgang; Postle, Anthony D.

2011-01-01

Phosphatidylcholine (PC) synthesis by the direct cytidine diphosphate choline (CDP-choline) pathway in rat liver generates predominantly mono- and di-unsaturated molecular species, while polyunsaturated PC species are synthesized largely by the phosphatidylethanolamine-N-methyltransferase (PEMT) pathway. Although altered PC synthesis has been suggested to contribute to development of hepatocarcinoma and nonalcoholic steatohepatitis, analysis of the specificity of hepatic PC metabolism in human patients has been limited by the lack of sensitive and safe methodologies. Here we incorporated a deuterated methyl-d9-labled choline chloride, to quantify biosynthesis fluxes through both of the PC synthetic pathways in vivo in human volunteers and compared these fluxes with those in mice. Rates and molecular specificities of label incorporated into mouse liver and plasma PC were very similar and strongly suggest that label incorporation into human plasma PC can provide a direct measure of hepatic PC synthesis in human subjects. Importantly, we demonstrate for the first time that the PEMT pathway in human liver is selective for polyunsaturated PC species, especially those containing docosahexaenoic acid. Finally, we present a multiple isotopomer distribution analysis approach, based on transfer of deuterated methyl groups to S-adenosylmethionine and subsequent sequential methylations of PE, to quantify absolute flux rates through the PEMT pathway that are applicable to studies of liver dysfunction in clinical studies. PMID:21068006

Inhibition of ultraviolet-B epidermal ornithine decarboxylase induction and skin carcinogenesis in hairless mice by topical indomethacin and triamcinolone acetonide

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

Lowe, N.J.; Connor, M.J.; Breeding, J.

1982-10-01

Modulation of ultraviolet-B (UVB) skin carcinogenesis by topical treatment with two antiinflammatory drugs expected to have different mechanisms of action has been studied in the hairless mouse. Indomethacin is a nonsteroidal antiinflammatory agent which may act by inhibiting prostaglandin biosynthesis. Triamcinolone acetonide is a steroidal antiinflammatory agent. Both of these drugs inhibited the induction of epidermal ornithine decarboxylase by UVB when applied topically in a acetone vehicle. A UVB skin tumor study was designed. Groups of mice were irradiated daily with UVB for 20 days, each mouse receiving a total of 17.1 kJ UVB per sq m. Group 1 wasmore » treated with acetone immediately after each irradiation; Group 2 received 700 nmol indomethacin in acetone immediately after each irradiation; Group 3 received 14.4 nmol triamcinolone acetonide in acetone immediately after each irradiation. Mice were killed after 52 weeks, and the tumors were excised and examined histologically. Both topical indomethacin and topical triamcinolone acetonide were effective in reducing the incidence and size of the skin tumors induced by UVB. This evidence supports the hypothesis that the induction of ornithine decarboxylase may be a critical component of UVB skin carcinogenesis and that inhibition of ornithine decarboxylase induction can be used as a screen for agents which will inhibit UVB skin carcinogenesis.« less

Novel Inhibitors of Plasmodium falciparum Dihydroorotate Dehydrogenase with Anti-malarial Activity in the Mouse Model

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

Booker, Michael L.; Bastos, Cecilia M.; Kramer, Martin L.

Plasmodium falciparum, the causative agent of the most deadly form of human malaria, is unable to salvage pyrimidines and must rely on de novo biosynthesis for survival. Dihydroorotate dehydrogenase (DHODH) catalyzes the rate-limiting step in the pyrimidine biosynthetic pathway and represents a potential target for anti-malarial therapy. A high throughput screen and subsequent medicinal chemistry program identified a series of N-alkyl-5-(1H-benzimidazol-1-yl)thiophene-2-carboxamides with low nanomolar in vitro potency against DHODH from P. falciparum, P. vivax, and P. berghei. The compounds were selective for the parasite enzymes over human DHODH, and x-ray structural data on the analog Genz-667348, demonstrated that species selectivitymore » could be attributed to amino acid differences in the inhibitor-binding site. Compounds from this series demonstrated in vitro potency against the 3D7 and Dd2 strains of P. falciparum, good tolerability and oral exposure in the mouse, and ED{sub 50} values in the 4-day murine P. berghei efficacy model of 13-21 mg/kg/day with oral twice-daily dosing. In particular, treatment with Genz-667348 at 100 mg/kg/day resulted in sterile cure. Two recent analogs of Genz-667348 are currently undergoing pilot toxicity testing to determine suitability as clinical development candidates.« less

Expression of resolvin D1 biosynthetic pathways in salivary epithelium.

PubMed

Leigh, N J; Nelson, J W; Mellas, R E; Aguirre, A; Baker, O J

2014-03-01

Resolvins are potent anti-inflammatory mediators derived from ω-3 fatty acids. Results from our previous studies indicated that resolvin D1 (RvD1) blocks pro-inflammatory responses in salivary glands. Furthermore, RvD1 enhances salivary epithelial integrity, demonstrating its potential use for the restoration of salivary gland function in Sjögren's syndrome (SS). We investigated whether the RvD1 biosynthetic machinery (e.g., cytosolic phospholipase A2, calcium-independent phospholipase A2, 12/15 and 5-lipoxygenase) is expressed in mouse submandibular glands (mSMG), using qPCR and Western blot analyses. Additionally, we determined the localization of RvD1 biosynthetic machinery in mSMG and human minor salivary glands (hMSG), with and without SS, using confocal microscopy. Finally, we measured RvD1 levels in cell supernatants from mSMG cell cultures and freshly isolated mSMG cells, with and without SS, using ELISA. Our results indicate that: (1) RvD1 machinery is expressed in mouse and human salivary glands; (2) polar distribution of RvD1 biosynthetic machinery is lost in hMSG with SS; (3) RvD1 levels in mSMG cell culture supernatants increased with time; and (4) RvD1 levels in mSMG cell supernatants, with and without SS, were similar. These studies demonstrate that the RvD1 biosynthesis machinery is expressed and functional in salivary glands with and without SS.

Standing Genetic Variation in Contingency Loci Drives the Rapid Adaptation of Campylobacter jejuni to a Novel Host

PubMed Central

Jerome, John P.; Bell, Julia A.; Plovanich-Jones, Anne E.; Barrick, Jeffrey E.; Brown, C. Titus; Mansfield, Linda S.

2011-01-01

The genome of the food-borne pathogen Campylobacter jejuni contains multiple highly mutable sites, or contingency loci. It has been suggested that standing variation at these loci is a mechanism for rapid adaptation to a novel environment, but this phenomenon has not been shown experimentally. In previous work we showed that the virulence of C. jejuni NCTC11168 increased after serial passage through a C57BL/6 IL-10-/- mouse model of campylobacteriosis. Here we sought to determine the genetic basis of this adaptation during passage. Re-sequencing of the 1.64Mb genome to 200-500X coverage allowed us to define variation in 23 contingency loci to an unprecedented depth both before and after in vivo adaptation. Mutations in the mouse-adapted C. jejuni were largely restricted to the homopolymeric tracts of thirteen contingency loci. These changes cause significant alterations in open reading frames of genes in surface structure biosynthesis loci and in genes with only putative functions. Several loci with open reading frame changes also had altered transcript abundance. The increase in specific phases of contingency loci during in vivo passage of C. jejuni, coupled with the observed virulence increase and the lack of other types of genetic changes, is the first experimental evidence that these variable regions play a significant role in C. jejuni adaptation and virulence in a novel host. PMID:21283682

Green synthesis of silver nanoparticles by Ricinus communis var. carmencita leaf extract and its antibacterial study

NASA Astrophysics Data System (ADS)

Ojha, Sunita; Sett, Arghya; Bora, Utpal

2017-09-01

In this study, we report synthesis of silver nanoparticles (RcAgNPs) from silver nitrate solution using methanolic leaf extract of Ricinus communis var. carmencita. The polyphenols present in the leaves reduce Ag++ ions to Ag0 followed by a color change. Silver nanoparticle formation was ensured by surface plasmon resonance between 400 nm to 500 nm. Crystallinity of the synthesized nanoparticles was confirmed by UHRTEM, SAED and XRD analysis. The capping of phytochemicals and thermal stability of RcAgNPs were assessed by FTIR spectra and TGA analysis, respectively. It also showed antibacterial activity against both gram positive and gram negative strains. RcAgNPs were non-toxic against normal cell line (mouse fibroblast cell line L929) at lower concentrations (80 µg ml-1).

46 CFR 148.205 - Ammonium nitrate and ammonium nitrate fertilizers.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 46 Shipping 5 2011-10-01 2011-10-01 false Ammonium nitrate and ammonium nitrate fertilizers. 148... Materials § 148.205 Ammonium nitrate and ammonium nitrate fertilizers. (a) This section applies to the stowage and transportation in bulk of ammonium nitrate and the following fertilizers composed of uniform...

46 CFR 148.205 - Ammonium nitrate and ammonium nitrate fertilizers.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 46 Shipping 5 2014-10-01 2014-10-01 false Ammonium nitrate and ammonium nitrate fertilizers. 148... Materials § 148.205 Ammonium nitrate and ammonium nitrate fertilizers. (a) This section applies to the stowage and transportation in bulk of ammonium nitrate and the following fertilizers composed of uniform...

46 CFR 148.205 - Ammonium nitrate and ammonium nitrate fertilizers.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 46 Shipping 5 2012-10-01 2012-10-01 false Ammonium nitrate and ammonium nitrate fertilizers. 148... Materials § 148.205 Ammonium nitrate and ammonium nitrate fertilizers. (a) This section applies to the stowage and transportation in bulk of ammonium nitrate and the following fertilizers composed of uniform...

46 CFR 148.205 - Ammonium nitrate and ammonium nitrate fertilizers.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 46 Shipping 5 2013-10-01 2013-10-01 false Ammonium nitrate and ammonium nitrate fertilizers. 148... Materials § 148.205 Ammonium nitrate and ammonium nitrate fertilizers. (a) This section applies to the stowage and transportation in bulk of ammonium nitrate and the following fertilizers composed of uniform...

Transgenic mice overexpressing tyrosine-to-cysteine mutant human alpha-synuclein: a progressive neurodegenerative model of diffuse Lewy body disease.

PubMed

Zhou, Wenbo; Milder, Julie B; Freed, Curt R

2008-04-11

Abnormal aggregation of human alpha-synuclein in Lewy bodies and Lewy neurites is a pathological hallmark of Parkinson disease and dementia with Lewy bodies. Studies have shown that oxidation and nitration of alpha-synuclein lead to the formation of stable dimers and oligomers through dityrosine cross-linking. Previously we have reported that tyrosine-to-cysteine mutations, particularly at the tyrosine 39 residue (Y39C), significantly enhanced alpha-synuclein fibril formation and neurotoxicity. In the current study, we have generated transgenic mice expressing the Y39C mutant human alpha-synuclein gene controlled by the mouse Thy1 promoter. Mutant human alpha-synuclein was widely expressed in transgenic mouse brain, resulting in 150% overexpression relative to endogenous mouse alpha-synuclein. At age 9-12 months, transgenic mice began to display motor dysfunction in rotarod testing. Older animals aged 15-18 months showed progressive accumulation of human alpha-synuclein oligomers, associated with worse motor function and cognitive impairment in the Morris water maze. By age 21-24 months, alpha-synuclein aggregates were further increased, accompanied by severe behavioral deficits. At this age, transgenic mice developed neuropathology, such as Lewy body-like alpha-synuclein and ubiquitin-positive inclusions, phosphorylation at Ser(129) of human alpha-synuclein, and increased apoptotic cell death. In summary, Y39C human alpha-synuclein transgenic mice show age-dependent, progressive neuronal degeneration with motor and cognitive deficits similar to diffuse Lewy body disease. The time course of alpha-synuclein oligomer accumulation coincided with behavioral and pathological changes, indicating that these oligomers may initiate protein aggregation, disrupt cellular function, and eventually lead to neuronal death.

Paralytic shellfish toxin biosynthesis in cyanobacteria and dinoflagellates: A molecular overview.

PubMed

Wang, Da-Zhi; Zhang, Shu-Fei; Zhang, Yong; Lin, Lin

2016-03-01

Paralytic shellfish toxins (PSTs) are a group of water soluble neurotoxic alkaloids produced by two different kingdoms of life, prokaryotic cyanobacteria and eukaryotic dinoflagellates. Owing to the wide distribution of these organisms, these toxic secondary metabolites account for paralytic shellfish poisonings around the world. On the other hand, their specific binding to voltage-gated sodium channels makes these toxins potentially useful in pharmacological and toxicological applications. Much effort has been devoted to the biosynthetic mechanism of PSTs, and gene clusters encoding 26 proteins involved in PST biosynthesis have been unveiled in several cyanobacterial species. Functional analysis of toxin genes indicates that PST biosynthesis in cyanobacteria is a complex process including biosynthesis, regulation, modification and export. However, less is known about the toxin biosynthesis in dinoflagellates owing to our poor understanding of the massive genome and unique chromosomal characteristics [1]. So far, few genes involved in PST biosynthesis have been identified from dinoflagellates. Moreover, the proteins involved in PST production are far from being totally explored. Thus, the origin and evolution of PST biosynthesis in these two kingdoms are still controversial. In this review, we summarize the recent progress on the characterization of genes and proteins involved in PST biosynthesis in cyanobacteria and dinoflagellates, and discuss the standing evolutionary hypotheses concerning the origin of toxin biosynthesis as well as future perspectives in PST biosynthesis. Paralytic shellfish toxins (PSTs) are a group of potent neurotoxins which specifically block voltage-gated sodium channels in excitable cells and result in paralytic shellfish poisonings (PSPs) around the world. Two different kingdoms of life, cyanobacteria and dinoflagellates are able to produce PSTs. However, in contrast with cyanobacteria, our understanding of PST biosynthesis in dinoflagellates is extremely limited owing to their unique features. The origin and evolution of PST biosynthesis in these two kingdoms are still controversial. High-throughput omics technologies, such as genomics, transcriptomics and proteomics provide powerful tools for the study of PST biosynthesis in cyanobacteria and dinoflagellates, and have shown their powerful potential with regard to revealing genes and proteins involved in PST biosynthesis in two kingdoms. This review summarizes the recent progress in PST biosynthesis in cyanobacteria and dinoflagellates with focusing on the novel insights from omics technologies, and discusses the evolutionary relationship of toxin biosynthesis genes between these two kingdoms. Copyright © 2015 Elsevier B.V. All rights reserved.

Molecular Components of Nitrate and Nitrite Efflux in Yeast

PubMed Central

Cabrera, Elisa; González-Montelongo, Rafaela; Giraldez, Teresa; de la Rosa, Diego Alvarez

2014-01-01

Some eukaryotes, such as plant and fungi, are capable of utilizing nitrate as the sole nitrogen source. Once transported into the cell, nitrate is reduced to ammonium by the consecutive action of nitrate and nitrite reductase. How nitrate assimilation is balanced with nitrate and nitrite efflux is unknown, as are the proteins involved. The nitrate assimilatory yeast Hansenula polymorpha was used as a model to dissect these efflux systems. We identified the sulfite transporters Ssu1 and Ssu2 as effective nitrate exporters, Ssu2 being quantitatively more important, and we characterize the Nar1 protein as a nitrate/nitrite exporter. The use of strains lacking either SSU2 or NAR1 along with the nitrate reductase gene YNR1 showed that nitrate reductase activity is not required for net nitrate uptake. Growth test experiments indicated that Ssu2 and Nar1 exporters allow yeast to cope with nitrite toxicity. We also have shown that the well-known Saccharomyces cerevisiae sulfite efflux permease Ssu1 is also able to excrete nitrite and nitrate. These results characterize for the first time essential components of the nitrate/nitrite efflux system and their impact on net nitrate uptake and its regulation. PMID:24363367

Nitrate and periplasmic nitrate reductases

PubMed Central

Sparacino-Watkins, Courtney; Stolz, John F.; Basu, Partha

2014-01-01

The nitrate anion is a simple, abundant and relatively stable species, yet plays a significant role in global cycling of nitrogen, global climate change, and human health. Although it has been known for quite some time that nitrate is an important species environmentally, recent studies have identified potential medical applications. In this respect the nitrate anion remains an enigmatic species that promises to offer exciting science in years to come. Many bacteria readily reduce nitrate to nitrite via nitrate reductases. Classified into three distinct types – periplasmic nitrate reductase (Nap), respiratory nitrate reductase (Nar) and assimilatory nitrate reductase (Nas), they are defined by their cellular location, operon organization and active site structure. Of these, Nap proteins are the focus of this review. Despite similarities in the catalytic and spectroscopic properties Nap from different Proteobacteria are phylogenetically distinct. This review has two major sections: in the first section, nitrate in the nitrogen cycle and human health, taxonomy of nitrate reductases, assimilatory and dissimilatory nitrate reduction, cellular locations of nitrate reductases, structural and redox chemistry are discussed. The second section focuses on the features of periplasmic nitrate reductase where the catalytic subunit of the Nap and its kinetic properties, auxiliary Nap proteins, operon structure and phylogenetic relationships are discussed. PMID:24141308

21 CFR 181.33 - Sodium nitrate and potassium nitrate.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Sodium nitrate and potassium nitrate. 181.33...-Sanctioned Food Ingredients § 181.33 Sodium nitrate and potassium nitrate. Sodium nitrate and potassium... nitrite, with or without sodium or potassium nitrite, in the production of cured red meat products and...

21 CFR 181.33 - Sodium nitrate and potassium nitrate.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Sodium nitrate and potassium nitrate. 181.33...-Sanctioned Food Ingredients § 181.33 Sodium nitrate and potassium nitrate. Sodium nitrate and potassium... nitrite, with or without sodium or potassium nitrite, in the production of cured red meat products and...

Arabidopsis Nitrate Transporter NRT1.9 Is Important in Phloem Nitrate Transport[W][OA

PubMed Central

Wang, Ya-Yun; Tsay, Yi-Fang

2011-01-01

This study of the Arabidopsis thaliana nitrate transporter NRT1.9 reveals an important function for a NRT1 family member in phloem nitrate transport. Functional analysis in Xenopus laevis oocytes showed that NRT1.9 is a low-affinity nitrate transporter. Green fluorescent protein and β-glucuronidase reporter analyses indicated that NRT1.9 is a plasma membrane transporter expressed in the companion cells of root phloem. In nrt1.9 mutants, nitrate content in root phloem exudates was decreased, and downward nitrate transport was reduced, suggesting that NRT1.9 may facilitate loading of nitrate into the root phloem and enhance downward nitrate transport in roots. Under high nitrate conditions, the nrt1.9 mutant showed enhanced root-to-shoot nitrate transport and plant growth. We conclude that phloem nitrate transport is facilitated by expression of NRT1.9 in root companion cells. In addition, enhanced root-to-shoot xylem transport of nitrate in nrt1.9 mutants points to a negative correlation between xylem and phloem nitrate transport. PMID:21571952

Nitrate analogs as attractants for soybean cyst nematode.

PubMed

Hosoi, Akito; Katsuyama, Tsutomu; Sasaki, Yasuyuki; Kondo, Tatsuhiko; Yajima, Shunsuke; Ito, Shinsaku

2017-08-01

Soybean cyst nematode (SCN) Heterodera glycines Ichinohe, a plant parasite, is one of the most serious pests of soybean. In this paper, we report that SCN is attracted to nitrate and its analogs. We performed attraction assays to screen for novel attractants for SCN and found that nitrates were attractants for SCN and SCN recognized nitrate gradients. However, attraction of SCN to nitrates was not observed on agar containing nitrate. To further elucidate the attraction mechanism in SCN, we performed attraction assays using nitrate analogs ([Formula: see text], [Formula: see text], [Formula: see text]). SCN was attracted to all nitrate analogs; however, attraction of SCN to nitrate analogs was not observed on agar containing nitrate. In contrast, SCN was attracted to azuki root, irrespective of presence or absence of nitrate in agar media. Our results suggest that the attraction mechanisms differ between plant-derived attractant and nitrate.

Inhibition of bile salt transport by drugs associated with liver injury in primary hepatocytes from human, monkey, dog, rat, and mouse

PubMed Central

Zhang, Jie; He, Kan; Cai, Lining; Chen, Yu-Chuan; Yang, Yifan; Shi, Qin; Woolf, Thomas F.; Ge, Weigong; Guo, Lei; Borlak, Jürgen; Tong, Weida

2018-01-01

Interference of bile salt transport is one of the underlying mechanisms for drug-induced liver injury (DILI). We developed a novel bile salt transport activity assay involving in situ biosynthesis of bile salts from their precursors in primary human, monkey, dog, rat, and mouse hepatocytes in suspension as well as LC-MS/MS determination of extracellular bile salts transported out of hepatocytes. Glycine- and taurine-conjugated bile acids were rapidly formed in hepatocytes and effectively transported into the extracellular medium. The bile salt formation and transport activities were time– and bile-acid-concentration–dependent in primary human hepatocytes. The transport activity was inhibited by the bile salt export pump (BSEP) inhibitors ketoconazole, saquinavir, cyclosporine, and troglitazone. The assay was used to test 86 drugs for their potential to inhibit bile salt transport activity in human hepatocytes, which included 35 drugs associated with severe DILI (sDILI) and 51 with non-severe DILI (non-sDILI). Approximately 60% of the sDILI drugs showed potent inhibition (with IC50 values <50 μM), but only about 20% of the non-sDILI drugs showed this strength of inhibition in primary human hepatocytes and these drugs are associated only with cholestatic and mixed hepatocellular cholestatic (mixed) injuries. The sDILI drugs, which did not show substantial inhibition of bile salt transport activity, are likely to be associated with immune-mediated liver injury. Twenty-four drugs were also tested in monkey, dog, rat and mouse hepatocytes. Species differences in potency were observed with mouse being less sensitive than other species to inhibition of bile salt transport. In summary, a novel assay has been developed using hepatocytes in suspension from human and animal species that can be used to assess the potential for drugs and/or drug-derived metabolites to inhibit bile salt transport and/or formation activity. Drugs causing sDILI, except those by immune-mediated mechanism, are highly associated with potent inhibition of bile salt transport. PMID:27000539

Inhibition of bile salt transport by drugs associated with liver injury in primary hepatocytes from human, monkey, dog, rat, and mouse.

PubMed

Zhang, Jie; He, Kan; Cai, Lining; Chen, Yu-Chuan; Yang, Yifan; Shi, Qin; Woolf, Thomas F; Ge, Weigong; Guo, Lei; Borlak, Jürgen; Tong, Weida

2016-08-05

Interference of bile salt transport is one of the underlying mechanisms for drug-induced liver injury (DILI). We developed a novel bile salt transport activity assay involving in situ biosynthesis of bile salts from their precursors in primary human, monkey, dog, rat, and mouse hepatocytes in suspension as well as LC-MS/MS determination of extracellular bile salts transported out of hepatocytes. Glycine- and taurine-conjugated bile acids were rapidly formed in hepatocytes and effectively transported into the extracellular medium. The bile salt formation and transport activities were time‒ and bile-acid-concentration‒dependent in primary human hepatocytes. The transport activity was inhibited by the bile salt export pump (BSEP) inhibitors ketoconazole, saquinavir, cyclosporine, and troglitazone. The assay was used to test 86 drugs for their potential to inhibit bile salt transport activity in human hepatocytes, which included 35 drugs associated with severe DILI (sDILI) and 51 with non-severe DILI (non-sDILI). Approximately 60% of the sDILI drugs showed potent inhibition (with IC50 values <50 μM), but only about 20% of the non-sDILI drugs showed this strength of inhibition in primary human hepatocytes and these drugs are associated only with cholestatic and mixed hepatocellular cholestatic (mixed) injuries. The sDILI drugs, which did not show substantial inhibition of bile salt transport activity, are likely to be associated with immune-mediated liver injury. Twenty-four drugs were also tested in monkey, dog, rat and mouse hepatocytes. Species differences in potency were observed with mouse being less sensitive than other species to inhibition of bile salt transport. In summary, a novel assay has been developed using hepatocytes in suspension from human and animal species that can be used to assess the potential for drugs and/or drug-derived metabolites to inhibit bile salt transport and/or formation activity. Drugs causing sDILI, except those by immune-mediated mechanism, are highly associated with potent inhibition of bile salt transport. Published by Elsevier Ireland Ltd.

Low Testosterone Alters the Activity of Mouse Prostate Stem Cells.

PubMed

Zhou, Ye; Copeland, Ben; Otto-Duessel, Maya; He, Miaoling; Markel, Susan; Synold, Tim W; Jones, Jeremy O

2017-04-01

Low serum testosterone (low T) has been repeatedly linked to worse outcomes in men with newly diagnosed prostate cancer (PC). How low T contributes to these outcomes is unknown. Here we demonstrate that exposure to low T causes significant changes in the mouse prostate and prostate stem cells. Mice were castrated and implanted with capsules to achieve castrate, normal, or sub-physiological levels of T. After 6 weeks of treatment, LC-MS/MS was used to quantify the levels of T and dihydrotestosterone (DHT) in serum and prostate tissue. FACS was used to quantify the percentages of purported prostate stem and transit amplifying (TA) cells in mouse prostates. Prostate tissues were also stained for the presence of CD68+ cells and RNA was extracted from prostate tissue or specific cell populations to measure changes in transcript levels with low T treatment. Despite having significantly different levels of T and DHT in the serum, T and DHT concentrations in prostate tissue from different T treatment groups were similar. Low T treatment resulted in significant alterations in the expression of androgen biosynthesis genes, which may be related to maintaining prostate androgen levels. Furthermore, the expression of androgen-regulated genes in the prostate was similar among all T treatment groups, demonstrating that the mouse prostate can maintain functional levels of androgens despite low serum T levels. Low T increased the frequency of prostate stem and TA cells in adult prostate tissue and caused major transcriptional changes in those cells. Gene ontology analysis suggested that low T caused inflammatory responses and immunofluorescent staining indicated that low T treatment led to the increased presence of CD68+ macrophages in prostate tissue. Low T alters the AR signaling axis which likely leads to maintenance of functional levels of prostate androgens. Low T also induces quantitative and qualitative changes in prostate stem cells which appear to lead to inflammatory macrophage infiltration. These changes are proposed to lead to an aggressive phenotype once cancers develop and may contribute to the poor outcomes in men with low T. Prostate 77:530-541, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Bioactivation of organic nitrates and the mechanism of nitrate tolerance.

PubMed

Klemenska, Emila; Beresewicz, Andrzej

2009-01-01

Organic nitrates, such as nitroglycerin, are commonly used in the therapy of cardiovascular disease. Long-term therapy with these drugs, however, results in the rapid development of nitrate tolerance, limiting their hemodynamic and anti-ischemic efficacy. In addition, nitrate tolerance is associated with the expression of potentially deleterious modifications such as increased oxidative stress, endothelial dysfunction, and sympathetic activation. In this review we discuss current concepts regarding the mechanisms of organic nitrate bioactivation, nitrate tolerance, and nitrate-mediated oxidative stress and endothelial dysfunction. We also examine how hydralazine may prevent nitrate tolerance and related endothelial dysfunction.

Respiratory Nitrate Ammonification by Shewanella oneidensis MR-1▿

PubMed Central

Cruz-García, Claribel; Murray, Alison E.; Klappenbach, Joel A.; Stewart, Valley; Tiedje, James M.

2007-01-01

Anaerobic cultures of Shewanella oneidensis MR-1 grown with nitrate as the sole electron acceptor exhibited sequential reduction of nitrate to nitrite and then to ammonium. Little dinitrogen and nitrous oxide were detected, and no growth occurred on nitrous oxide. A mutant with the napA gene encoding periplasmic nitrate reductase deleted could not respire or assimilate nitrate and did not express nitrate reductase activity, confirming that the NapA enzyme is the sole nitrate reductase. Hence, S. oneidensis MR-1 conducts respiratory nitrate ammonification, also termed dissimilatory nitrate reduction to ammonium, but not respiratory denitrification. PMID:17098906

Nitrate transport in the cyanobacterium Anacystis nidulans R2. Kinetic and energetic aspects.

PubMed Central

Rodríguez, R; Lara, C; Guerrero, M G

1992-01-01

Nitrate transport has been studied in the cyanobacterium Anacystis nidulans R2 by monitoring intracellular nitrate accumulation in intact cells of the mutant strain FM6, which lacks nitrate reductase activity and is therefore unable to reduce the transported nitrate. Kinetic analysis of nitrate transport as a function of external nitrate concentration revealed apparent substrate inhibition, with a peak velocity at 20-25 microM-nitrate. A Ks (NO3-) of 1 microM was calculated. Nitrate transport exhibited a stringent requirement for Na+. Neither Li+ nor K+ could substitute for Na+. Monensin depressed nitrate transport in a concentration-dependent manner, inhibition being more than 60% at 2 microM, indicating that the Na(+)-dependence of active nitrate transport relies on the maintenance of a Na+ electrochemical gradient. The operation of an Na+/NO3- symport system is suggested. Nitrite behaved as an effective competitive inhibitor of nitrate transport, with a Ki (NO2-) of 3 microM. The time course of nitrite inhibition of nitrate transport was consistent with competitive inhibition by mixed alternative substrates. Nitrate and nitrite might be transported by the same carrier. PMID:1554347

Relation of nitrate concentrations to baseflow in the Raccoon River, Iowa

USGS Publications Warehouse

Schilling, K.E.; Lutz, D.S.

2004-01-01

Excessive nitrate-nitrogen (nitrate) export from the Raccoon River in west central Iowa is an environmental concern to downstream receptors. The 1972 to 2000 record of daily streamflow and the results from 981 nitrate measurements were examined to describe the relation of nitrate to streamflow in the Raccoon River. No long term trends in streamflow and nitrate concentrations were noted in the 28-year record. Strong seasonal patterns were evident in nitrate concentrations, with higher concentrations occurring in spring and fall. Nitrate concentrations were linearly related to streamflow at daily, monthly, seasonal, and annual time scales. At all time scales evaluated, the relation was improved when baseflow was used as the discharge variable instead of total streamflow. Nitrate concentrations were found to be highly stratified according to flow, but there was little relation of nitrate to streamflow within each flow range. Simple linear regression models developed to predict monthly mean nitrate concentrations explained as much as 76 percent of the variability in the monthly nitrate concentration data for 2001. Extrapolation of current nitrate baseflow relations to historical conditions in the Raccoon River revealed that increasing baseflow over the 20th century could account for a measurable increase in nitrate concentrations.

Efficient syntheses of climate relevant isoprene nitrates and (1R,5S)-(-)-myrtenol nitrate.

PubMed

Bew, Sean P; Hiatt-Gipson, Glyn D; Mills, Graham P; Reeves, Claire E

2016-01-01

Here we report the chemoselective synthesis of several important, climate relevant isoprene nitrates using silver nitrate to mediate a 'halide for nitrate' substitution. Employing readily available starting materials, reagents and Horner-Wadsworth-Emmons chemistry the synthesis of easily separable, synthetically versatile 'key building blocks' (E)- and (Z)-3-methyl-4-chlorobut-2-en-1-ol as well as (E)- and (Z)-1-((2-methyl-4-bromobut-2-enyloxy)methyl)-4-methoxybenzene has been achieved using cheap, 'off the shelf' materials. Exploiting their reactivity we have studied their ability to undergo an 'allylic halide for allylic nitrate' substitution reaction which we demonstrate generates (E)- and (Z)-3-methyl-4-hydroxybut-2-enyl nitrate, and (E)- and (Z)-2-methyl-4-hydroxybut-2-enyl nitrates ('isoprene nitrates') in 66-80% overall yields. Using NOESY experiments the elucidation of the carbon-carbon double bond configuration within the purified isoprene nitrates has been established. Further exemplifying our 'halide for nitrate' substitution chemistry we outline the straightforward transformation of (1R,2S)-(-)-myrtenol bromide into the previously unknown monoterpene nitrate (1R,2S)-(-)-myrtenol nitrate.

Plasma nitrate and nitrite are increased by a high nitrate supplement, but not by high nitrate foods in older adults

PubMed Central

Miller, Gary D.; Marsh, Anthony P.; Dove, Robin W.; Beavers, Daniel; Presley, Tennille; Helms, Christine; Bechtold, Erika; King, S. Bruce; Kim-Shapiro, Daniel

2012-01-01

Little is known about the effect of dietary nitrate on the nitrate/nitrite/NO (nitric oxide) cycle in older adults. We examined the effect of a 3-day control diet vs. high nitrate diet, with and without a high nitrate supplement (beetroot juice), on plasma nitrate and nitrite kinetics, and blood pressure using a randomized four period cross-over controlled design. We hypothesized that the high nitrate diet would show higher levels of plasma nitrate/nitrite and blood pressure compared to the control diet, which would be potentiated by the supplement. Participants were eight normotensive older men and women (5 female, 3 male, 72.5±4.7 yrs) with no overt disease or medications that affect NO metabolism. Plasma nitrate and nitrite levels and blood pressure were measured prior to and hourly for 3 hours after each meal. The mean daily changes in plasma nitrate and nitrite were significantly different from baseline for both control diet+supplement (p<0.001 and =0.017 for nitrate and nitrite, respectively) and high nitrate diet+supplement (p=0.001 and 0.002), but not for control diet (p=0.713 and 0.741) or high nitrate diet (p=0.852 and 0.500). Blood pressure decreased from the morning baseline measure to the three 2 hr post-meal follow-up time-points for all treatments, but there was no main effect for treatment. In healthy older adults, a high nitrate supplement consumed at breakfast elevated plasma nitrate and nitrite levels throughout the day. This observation may have practical utility for the timing of intake of a nitrate supplement with physical activity for older adults with vascular dysfunction. PMID:22464802

The changing trend in nitrate concentrations in major aquifers due to historical nitrate loading from agricultural land across England and Wales from 1925 to 2150.

PubMed

Wang, L; Stuart, M E; Lewis, M A; Ward, R S; Skirvin, D; Naden, P S; Collins, A L; Ascott, M J

2016-01-15

Nitrate is necessary for agricultural productivity, but can cause considerable problems if released into aquatic systems. Agricultural land is the major source of nitrates in UK groundwater. Due to the long time-lag in the groundwater system, it could take decades for leached nitrate from the soil to discharge into freshwaters. However, this nitrate time-lag has rarely been considered in environmental water management. Against this background, this paper presents an approach to modelling groundwater nitrate at the national scale, to simulate the impacts of historical nitrate loading from agricultural land on the evolution of groundwater nitrate concentrations. An additional process-based component was constructed for the saturated zone of significant aquifers in England and Wales. This uses a simple flow model which requires modelled recharge values, together with published aquifer properties and thickness data. A spatially distributed and temporally variable nitrate input function was also introduced. The sensitivity of parameters was analysed using Monte Carlo simulations. The model was calibrated using national nitrate monitoring data. Time series of annual average nitrate concentrations along with annual spatially distributed nitrate concentration maps from 1925 to 2150 were generated for 28 selected aquifer zones. The results show that 16 aquifer zones have an increasing trend in nitrate concentration, while average nitrate concentrations in the remaining 12 are declining. The results are also indicative of the trend in the flux of groundwater nitrate entering rivers through baseflow. The model thus enables the magnitude and timescale of groundwater nitrate response to be factored into source apportionment tools and to be taken into account alongside current planning of land-management options for reducing nitrate losses. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Serotonergic system antagonists target breast tumor initiating cells and synergize with chemotherapy to shrink human breast tumor xenografts

PubMed Central

Gwynne, William D; Hallett, Robin M; Girgis-Gabardo, Adele; Bojovic, Bojana; Dvorkin-Gheva, Anna; Aarts, Craig; Dias, Kay; Bane, Anita; Hassell, John A

2017-01-01

Breast tumors comprise an infrequent tumor cell population, termed breast tumor initiating cells (BTIC), which sustain tumor growth, seed metastases and resist cytotoxic therapies. Hence therapies are needed to target BTIC to provide more durable breast cancer remissions than are currently achieved. We previously reported that serotonergic system antagonists abrogated the activity of mouse BTIC resident in the mammary tumors of a HER2-overexpressing model of breast cancer. Here we report that antagonists of serotonin (5-hydroxytryptamine; 5-HT) biosynthesis and activity, including US Federal Food and Drug Administration (FDA)-approved antidepressants, targeted BTIC resident in numerous breast tumor cell lines regardless of their clinical or molecular subtype. Notably, inhibitors of tryptophan hydroxylase 1 (TPH1), required for 5-HT biosynthesis in select non-neuronal cells, the serotonin reuptake transporter (SERT) and several 5-HT receptors compromised BTIC activity as assessed by functional sphere-forming assays. Consistent with these findings, human breast tumor cells express TPH1, 5-HT and SERT independent of their molecular or clinical subtype. Exposure of breast tumor cells ex vivo to sertraline (Zoloft), a selective serotonin reuptake inhibitor (SSRI), reduced BTIC frequency as determined by transplanting drug-treated tumor cells into immune-compromised mice. Moreover, another SSRI (vilazodone; Viibryd) synergized with chemotherapy to shrink breast tumor xenografts in immune-compromised mice by inhibiting tumor cell proliferation and inducing their apoptosis. Collectively our data suggest that antidepressants in combination with cytotoxic anticancer therapies may be an appropriate treatment regimen for testing in clinical trials. PMID:28404880

Serotonergic system antagonists target breast tumor initiating cells and synergize with chemotherapy to shrink human breast tumor xenografts.

PubMed

Gwynne, William D; Hallett, Robin M; Girgis-Gabardo, Adele; Bojovic, Bojana; Dvorkin-Gheva, Anna; Aarts, Craig; Dias, Kay; Bane, Anita; Hassell, John A

2017-05-09

Breast tumors comprise an infrequent tumor cell population, termed breast tumor initiating cells (BTIC), which sustain tumor growth, seed metastases and resist cytotoxic therapies. Hence therapies are needed to target BTIC to provide more durable breast cancer remissions than are currently achieved. We previously reported that serotonergic system antagonists abrogated the activity of mouse BTIC resident in the mammary tumors of a HER2-overexpressing model of breast cancer. Here we report that antagonists of serotonin (5-hydroxytryptamine; 5-HT) biosynthesis and activity, including US Federal Food and Drug Administration (FDA)-approved antidepressants, targeted BTIC resident in numerous breast tumor cell lines regardless of their clinical or molecular subtype. Notably, inhibitors of tryptophan hydroxylase 1 (TPH1), required for 5-HT biosynthesis in select non-neuronal cells, the serotonin reuptake transporter (SERT) and several 5-HT receptors compromised BTIC activity as assessed by functional sphere-forming assays. Consistent with these findings, human breast tumor cells express TPH1, 5-HT and SERT independent of their molecular or clinical subtype. Exposure of breast tumor cells ex vivo to sertraline (Zoloft), a selective serotonin reuptake inhibitor (SSRI), reduced BTIC frequency as determined by transplanting drug-treated tumor cells into immune-compromised mice. Moreover, another SSRI (vilazodone; Viibryd) synergized with chemotherapy to shrink breast tumor xenografts in immune-compromised mice by inhibiting tumor cell proliferation and inducing their apoptosis. Collectively our data suggest that antidepressants in combination with cytotoxic anticancer therapies may be an appropriate treatment regimen for testing in clinical trials.

Mitochondrial COQ9 is a lipid-binding protein that associates with COQ7 to enable coenzyme Q biosynthesis

PubMed Central

Lohman, Danielle C.; Forouhar, Farhad; Beebe, Emily T.; Stefely, Matthew S.; Minogue, Catherine E.; Ulbrich, Arne; Stefely, Jonathan A.; Sukumar, Shravan; Luna-Sánchez, Marta; Jochem, Adam; Lew, Scott; Seetharaman, Jayaraman; Xiao, Rong; Wang, Huang; Westphall, Michael S.; Wrobel, Russell L.; Everett, John K.; Mitchell, Julie C.; López, Luis C.; Coon, Joshua J.; Tong, Liang; Pagliarini, David J.

2014-01-01

Coenzyme Q (CoQ) is an isoprenylated quinone that is essential for cellular respiration and is synthesized in mitochondria by the combined action of at least nine proteins (COQ1–9). Although most COQ proteins are known to catalyze modifications to CoQ precursors, the biochemical role of COQ9 remains unclear. Here, we report that a disease-related COQ9 mutation leads to extensive disruption of the CoQ protein biosynthetic complex in a mouse model, and that COQ9 specifically interacts with COQ7 through a series of conserved residues. Toward understanding how COQ9 can perform these functions, we solved the crystal structure of Homo sapiens COQ9 at 2.4 Å. Unexpectedly, our structure reveals that COQ9 has structural homology to the TFR family of bacterial transcriptional regulators, but that it adopts an atypical TFR dimer orientation and is not predicted to bind DNA. Our structure also reveals a lipid-binding site, and mass spectrometry-based analyses of purified COQ9 demonstrate that it associates with multiple lipid species, including CoQ itself. The conserved COQ9 residues necessary for its interaction with COQ7 comprise a surface patch around the lipid-binding site, suggesting that COQ9 might serve to present its bound lipid to COQ7. Collectively, our data define COQ9 as the first, to our knowledge, mammalian TFR structural homolog and suggest that its lipid-binding capacity and association with COQ7 are key features for enabling CoQ biosynthesis. PMID:25339443

Role of Acinetobactin-Mediated Iron Acquisition Functions in the Interaction of Acinetobacter baumannii Strain ATCC 19606T with Human Lung Epithelial Cells, Galleria mellonella Caterpillars, and Mice

PubMed Central

Gaddy, Jennifer A.; Arivett, Brock A.; McConnell, Michael J.; López-Rojas, Rafael; Pachón, Jerónimo

2012-01-01

Acinetobacter baumannii, which causes serious infections in immunocompromised patients, expresses high-affinity iron acquisition functions needed for growth under iron-limiting laboratory conditions. In this study, we determined that the initial interaction of the ATCC 19606T type strain with A549 human alveolar epithelial cells is independent of the production of BasD and BauA, proteins needed for acinetobactin biosynthesis and transport, respectively. In contrast, these proteins are required for this strain to persist within epithelial cells and cause their apoptotic death. Infection assays using Galleria mellonella larvae showed that impairment of acinetobactin biosynthesis and transport functions significantly reduces the ability of ATCC 19606T cells to persist and kill this host, a defect that was corrected by adding inorganic iron to the inocula. The results obtained with these ex vivo and in vivo approaches were validated using a mouse sepsis model, which showed that expression of the acinetobactin-mediated iron acquisition system is critical for ATCC 19606T to establish an infection and kill this vertebrate host. These observations demonstrate that the virulence of the ATCC 19606T strain depends on the expression of a fully active acinetobactin-mediated system. Interestingly, the three models also showed that impairment of BasD production results in an intermediate virulence phenotype compared to those of the parental strain and the BauA mutant. This observation suggests that acinetobactin intermediates or precursors play a virulence role, although their contribution to iron acquisition is less relevant than that of mature acinetobactin. PMID:22232188

Source Areas of Water and Nitrate in a Peatland Catchment, Minnesota, USA

NASA Astrophysics Data System (ADS)

Sebestyen, S. D.

2017-12-01

In nitrogen polluted forests, stream nitrate concentrations increase and some unprocessed atmospheric nitrate may be transported to streams during stormflow events. This understanding has emerged from forests with upland mineral soils. In contrast, catchments with northern peatlands may have both upland soils and lowlands with deep organic soils, each with unique effects on nitrate transport and processing. While annual budgets show nitrate yields to be relatively lower from peatland than upland-dominated catchments, little is known about particular runoff events when stream nitrate concentrations have been higher (despite long periods with little or no nitrate in outlet streams) or the reasons why. I used site knowledge and expansive/extensive monitoring at the Marcell Experimental Forest in Minnesota, along with a targeted 2-year study to determine landscape areas, water sources, and nitrate sources that affected stream nitrate variation in a peatland catchment. I combined streamflow, upland runoff, snow amount, and frost depth data from long-term monitoring with nitrate concentration, yield, and isotopic data to show that up to 65% of stream nitrate during snowmelt of 2009 and 2010 was unprocessed atmospheric nitrate. Up to 46% of subsurface runoff from upland soils during 2009 was unprocessed atmospheric nitrate, which shows the uplands to be a stream nitrate source during 2009, but not during 2010 when upland runoff concentrations were below the detection limit. Differences are attributable to variations in water and nitrate sources. Little snow (a nitrate source), less upland runoff relative to peatland runoff, and deeper soil frost in the peatland caused a relatively larger input of nitrate from the uplands to the stream during 2009 and the peatland to the stream during 2010. Despite the near-absence of stream nitrate during much of rest of the year, these findings show an important time when nitrate transport affected downstream aquatic ecosystems, reasons why nitrate was transported, and that atmospheric nitrate pollution had a direct effect on a stream in a peatland catchment. Furthermore, this work illustrates how long-term monitoring when coupled with shorter-duration studies allows contemporary questions to be addressed within legacy catchment studies.

Pseudo-constitutivity of nitrate-responsive genes in nitrate reductase mutants

PubMed Central

Schinko, Thorsten; Gallmetzer, Andreas; Amillis, Sotiris; Strauss, Joseph

2013-01-01

In fungi, transcriptional activation of genes involved in NO3- assimilation requires the presence of an inducer (nitrate or nitrite) and low intracellular concentrations of the pathway products ammonium or glutamine. In Aspergillus nidulans, the two transcription factors NirA and AreA act synergistically to mediate nitrate/nitrite induction and nitrogen metabolite derepression, respectively. In all studied fungi and in plants, mutants lacking nitrate reductase (NR) activity express nitrate-metabolizing enzymes constitutively without the addition of inducer molecules. Based on their work in A. nidulans, Cove and Pateman proposed an “autoregulation control” model for the synthesis of nitrate metabolizing enzymes in which the functional nitrate reductase molecule would act as co-repressor in the absence and as co-inducer in the presence of nitrate. However, NR mutants could simply show “pseudo-constitutivity” due to induction by nitrate which accumulates over time in NR-deficient strains. Here we examined this possibility using strains which lack flavohemoglobins (fhbs), and are thus unable to generate nitrate internally, in combination with nitrate transporter mutations (nrtA, nrtB) and a GFP-labeled NirA protein. Using different combinations of genotypes we demonstrate that nitrate transporters are functional also in NR null mutants and show that the constitutive phenotype of NR mutants is not due to nitrate accumulation from intracellular sources but depends on the activity of nitrate transporters. However, these transporters are not required for nitrate signaling because addition of external nitrate (10 mM) leads to standard induction of nitrate assimilatory genes in the nitrate transporter double mutants. We finally show that NR does not regulate NirA localization and activity, and thus the autoregulation model, in which NR would act as a co-repressor of NirA in the absence of nitrate, is unlikely to be correct. Results from this study instead suggest that transporter-mediated NO3- accumulation in NR deficient mutants, originating from traces of nitrate in the media, is responsible for the constitutive expression of NirA-regulated genes, and the associated phenotype is thus termed “pseudo-constitutive”. PMID:23454548

Serum nitrate/nitrite concentration correlates with gastric juice nitrate/nitrite: a possible marker for mutagenesis of the proximal stomach.

PubMed

Kishikawa, Hiroshi; Nishida, Jiro; Ichikawa, Hitoshi; Kaida, Shogo; Matsukubo, Takashi; Miura, Soichiro; Morishita, Tetsuo; Hibi, Toshifumi

2011-01-01

In the normal acid-secreting stomach, luminally generated nitric oxide, which contributes to carcinogenesis in the proximal stomach, is associated with the concentration of nitrate plus nitrite (nitrate/nitrite) in gastric juice. We investigated whether the serum nitrate/nitrite concentration is associated with that of gastric juice and whether it can be used as a serum marker. Serum and gastric juice nitrate/nitrite concentration, Helicobacter pylori antibody, and gastric pH were measured in 176 patients undergoing upper endoscopy. Multiple regression analysis revealed that serum nitrate/nitrite concentration was the best independent predictor of gastric juice nitrate/nitrite concentration. On single regression analysis, serum and gastric juice nitrate/nitrite concentration were significantly correlated, according to the following equation: gastric juice nitrate/nitrite concentration (μmol/l) = 3.93 - 0.54 × serum nitrate/nitrite concentration (μmol/l; correlation coefficient = 0.429, p < 0.001). In analyses confined to subjects with gastric pH less than 2.0, and in those with serum markers suggesting normal acid secretion (pepsinogen-I >30 ng/ml and negative H. pylori antibody), the serum nitrate/nitrite concentration was an independent predictor of the gastric juice nitrate/nitrite concentration (p < 0.001). Measuring the serum nitrate/nitrite concentration has potential in estimating the gastric juice nitrate/nitrite concentration. The serum nitrate/nitrite concentration could be useful as a marker for mutagenesis in the proximal stomach. Copyright © 2011 S. Karger AG, Basel.

Carrageenan-induced mouse paw oedema is biphasic, age-weight dependent and displays differential nitric oxide cyclooxygenase-2 expression

PubMed Central

Posadas, Inmaculada; Bucci, Mariarosaria; Roviezzo, Fiorentina; Rossi, Antonietta; Parente, Luca; Sautebin, Lidia; Cirino, Giuseppe

2004-01-01

Injection of carrageenan 1% (50 μl) in the mouse paw causes a biphasic response: an early inflammatory response that lasts 6 h and a second late response that peaks at 72 h, declining at 96 h. Only mice 7- or 8-week old, weighing 32–34 g, displayed a consistent response in both phases. In 8-week-old mice, myeloperoxidase (MPO) levels are significantly elevated in the early phase at 6 h and reach their maximum at 24 h to decline to basal value at 48 h. Nitrate+nitrite (NOx) levels in the paw are maximal after 2 h and slowly decline thereafter in contrast to prostaglandin E2 levels that peak in the second phase at the 72 h point. Western blot analysis showed that inducible nitric oxide synthase (iNOS) is detectable at 6 h and cyclooxygenase 2 (COX-2) at 24 h point, respectively. Analysis of endothelial nitric oxide synthase (eNOS), iNOS and COX-2 expression at 6 and 24 h in 3–8-week-old mice demonstrated that both eNOS and iNOS expressions are dependent upon the age–weight of mice, as opposite to COX-2 that is present only in the second phase of the oedema and is not linked to mouse age–weight. Subplantar injection of carrageenan to C57BL/6J causes a biphasic oedema that is significantly reduced by about 20% when compared to CD1 mice. Interestingly, in these mice, iNOS expression is absent up to 6 h, as opposite to CD1, and becomes detectable at the 24 h point. Cyclooxygenase (COX-1) expression is upregulated between 4 and 24 h after carrageenan injection, whereas in CD1 mice COX-1 remains unchanged after irritant agent injection. MPO levels are maximal at the 24 h point and they are significantly lower, at 6 h point, than MPO levels detected in CD1 mice. In conclusion, mouse paw oedema is biphasic and age-weight dependent. The present results are the first report on the differential expressions of eNOS, iNOS, COX-1 and COX-2 in response to carrageenan injection in the two phases of the mouse paw oedema. PMID:15155540

Fungus mediated synthesis of biomedically important cerium oxide nanoparticles

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

Khan, Shadab Ali; Ahmad, Absar, E-mail: a.ahmad@ncl.res.in

2013-10-15

Graphical abstract: - Highlights: • First time biological synthesis of cerium oxide oxide nanoparticles using fungus Humicola sp. • Complete characterization of cerium oxide nanoparticles. • Biosynthesis of naturally protein capped, luminescent and water dispersible CeO{sub 2} nanoparticles. • Biosynthesized CeO{sub 2} nanoparticles can be used for many biomedical applications. - Abstract: Nanomaterials can be synthesized by chemical, physical and the more recently discovered biological routes. The biological routes are advantageous over the chemical and physical ones as unlike these, the biological synthesis protocols occur at ambient conditions, are cheap, non-toxic and eco-friendly. Although purely biological and bioinspired methods formore » the synthesis of nanomaterials are environmentally benign and energy conserving processes, their true potential has not been explored yet and attempts are being made to extend the formation of technologically important nanoparticles using microorganisms like fungi. Though there have been reports on the biosynthesis of oxide nanoparticles by our group in the past, no attempts have been made to employ fungi for the synthesis of nanoparticles of rare earth metals or lanthanides. Here we report for the first time, the bio-inspired synthesis of biomedically important cerium oxide (CeO{sub 2}) nanoparticles using the thermophilic fungus Humicola sp. The fungus Humicola sp. when exposed to aqueous solutions of oxide precursor cerium (III) nitrate hexahydrate (CeN{sub 3}O{sub 9}·6H{sub 2}O) results in the extracellular formation of CeO{sub 2} nanoparticles containing Ce (III) and Ce (IV) mixed oxidation states, confirmed by X-ray Photoemission Spectroscopy (XPS). The formed nanoparticles are naturally capped by proteins secreted by the fungus and thus do not agglomerate, are highly stable, water dispersible and are highly fluorescent as well. The biosynthesized nanoparticles were characterized by UV–vis spectroscopy, Photoluminescence spectroscopy (PL), Transmission Electron Microscopy (TEM), Fourier Transform Infrared Spectroscopy (FTIR), X-Ray Diffraction (XRD) and X-ray Photoemission Spectroscopy (XPS)« less

Biofabrication of zinc oxide nanoparticles using fruit extract of Rosa canina and their toxic potential against bacteria: A mechanistic approach.

PubMed

Jafarirad, Saeed; Mehrabi, Meysam; Divband, Baharak; Kosari-Nasab, Morteza

2016-02-01

The use of plant extract in the biosynthesis of nanoparticles (NPs) can be an eco-friendly approach and have been suggested as a possible alternative to classic methods namely physical and chemical procedures. In this study, the biosynthesis of zinc oxide (ZnO) NPs by both "conventional heating" (CH) and "microwave irradiation" (MI) methods has been reported. Stable and spherical ZnONPs were produced using zinc nitrate and flesh extract of Rosa canina fruit (rosehip) which was used as a precursor. The flesh extract acts as a reducing and capping agent for generation of ZnONPs. The structural, morphological and colloidal properties of the as-synthesized NPs have been confirmed by X-ray Diffraction (XRD), Scanning Electron Microscopy (SEM), Energy Dispersive X-ray (EDX), Fourier transform Infrared (FT-IR) and Dynamic Light Scattering (DLS). In comparison with the CH method, the MI method has some advantages such as significantly short reaction time (within 8min) owing to the high heating rate and thus the accelerated reaction rate. Both methods led to the synthesis of nearly identical NPs with respect to shape and size according to the results of DLS, XRD and SEM techniques. The possible mechanism for synthesis pathway has been proposed based on FT IR results, XRD patterns, potentiometric data and antioxidant activity. In addition, the antibacterial activity of as-prepared ZnONPs was investigated against several bacteria such as Listeria monocytogenes, Escherichia coli, Salmonella typhimurium. Moreover, the efficacy of ZnONPs to treat cancer cell lines were measured by means of cell viability test via MTT assay in which concentrations of 0.05 and 0.1mg/mL of ZnONPs induced a very low toxicity. Thus, the present investigation reveals that ZnONPs have the potential for various medical and industrial applications. Copyright © 2015 Elsevier B.V. All rights reserved.

Aminoethyl nitrate – the novel super nitrate?

PubMed Central

Bauersachs, Johann

2009-01-01

Long-term use of most organic nitrates is limited by development of tolerance, induction of oxidative stress and endothelial dysfunction. In this issue of the BJP, Schuhmacher et al. characterized a novel class of organic nitrates with amino moieties (aminoalkyl nitrates). Aminoethyl nitrate was identified as a novel organic mononitrate with high potency but devoid of induction of mitochondrial oxidative stress. Cross-tolerance to nitroglycerin or the endothelium-dependent agonist acetylcholine after in vivo treatment was not observed. Like all nitrates, aminoethyl nitrate induced vasorelaxation by activation of soluble guanylate cyclase. Thus, in contrast to the prevailing view, high potency in an organic nitrate is not necessarily accompanied by induction of oxidative stress or endothelial dysfunction. This work from Daiber's group is an important step forward in the understanding of nitrate bioactivation, tolerance phenomena and towards the development of better organic nitrates for clinical use. PMID:19732062

The nitrate time bomb: a numerical way to investigate nitrate storage and lag time in the unsaturated zone.

PubMed

Wang, L; Butcher, A S; Stuart, M E; Gooddy, D C; Bloomfield, J P

2013-10-01

Nitrate pollution in groundwater, which is mainly from agricultural activities, remains an international problem. It threatens the environment, economics and human health. There is a rising trend in nitrate concentrations in many UK groundwater bodies. Research has shown it can take decades for leached nitrate from the soil to discharge into groundwater and surface water due to the 'store' of nitrate and its potentially long travel time in the unsaturated and saturated zones. However, this time lag is rarely considered in current water nitrate management and policy development. The aim of this study was to develop a catchment-scale integrated numerical method to investigate the nitrate lag time in the groundwater system, and the Eden Valley, UK, was selected as a case study area. The method involves three models, namely the nitrate time bomb-a process-based model to simulate the nitrate transport in the unsaturated zone (USZ), GISGroundwater--a GISGroundwater flow model, and N-FM--a model to simulate the nitrate transport in the saturated zone. This study answers the scientific questions of when the nitrate currently in the groundwater was loaded into the unsaturated zones and eventually reached the water table; is the rising groundwater nitrate concentration in the study area caused by historic nitrate load; what caused the uneven distribution of groundwater nitrate concentration in the study area; and whether the historic peak nitrate loading has reached the water table in the area. The groundwater nitrate in the area was mainly from the 1980s to 2000s, whilst the groundwater nitrate in most of the source protection zones leached into the system during 1940s-1970s; the large and spatially variable thickness of the USZ is one of the major reasons for unevenly distributed groundwater nitrate concentrations in the study area; the peak nitrate loading around 1983 has affected most of the study area. For areas around the Bowscar, Beacon Edge, Low Plains, Nord Vue, Dale Springs, Gamblesby, Bankwood Springs, and Cliburn, the peak nitrate loading will arrive at the water table in the next 34 years; statistical analysis shows that 8.7 % of the Penrith Sandstone and 7.3 % of the St Bees Sandstone have not been affected by peak nitrate. This research can improve the scientific understanding of nitrate processes in the groundwater system and support the effective management of groundwater nitrate pollution for the study area. With a limited number of parameters, the method and models developed in this study are readily transferable to other areas.

Light-Dark Changes in Cytosolic Nitrate Pools Depend on Nitrate Reductase Activity in Arabidopsis Leaf Cells1[w

PubMed Central

Cookson, Sarah J.; Williams, Lorraine E.; Miller, Anthony J.

2005-01-01

Several different cellular processes determine the size of the metabolically available nitrate pool in the cytoplasm. These processes include not only ion fluxes across the plasma membrane and tonoplast but also assimilation by the activity of nitrate reductase (NR). In roots, the maintenance of cytosolic nitrate activity during periods of nitrate starvation and resupply (M. van der Leij, S.J. Smith, A.J. Miller [1998] Planta 205: 64–72; R.-G. Zhen, H.-W. Koyro, R.A. Leigh, A.D. Tomos, A.J. Miller [1991] Planta 185: 356–361) suggests that this pool is regulated. Under nitrate-replete conditions vacuolar nitrate is a membrane-bound store that can release nitrate to the cytoplasm; after depletion of cytosolic nitrate, tonoplast transporters would serve to restore this pool. To study the role of assimilation, specifically the activity of NR in regulating the size of the cytosolic nitrate pool, we have compared wild-type and mutant plants. In leaf mesophyll cells, light-to-dark transitions increase cytosolic nitrate activity (1.5–2.8 mm), and these changes were reversed by dark-to-light transitions. Such changes were not observed in nia1nia2 NR-deficient plants indicating that this change in cytosolic nitrate activity was dependent on the presence of functional NR. Furthermore, in the dark, the steady-state cytosolic nitrate activities were not statistically different between the two types of plant, indicating that NR has little role in determining resting levels of nitrate. Epidermal cells of both wild type and NR mutants had cytosolic nitrate activities that were not significantly different from mesophyll cells in the dark and were unaltered by dark-to-light transitions. We propose that the NR-dependent changes in cytosolic nitrate provide a cellular mechanism for the diurnal changes in vacuolar nitrate storage, and the results are discussed in terms of the possible signaling role of cytosolic nitrate. PMID:15908593

Identifying source and formation altitudes of nitrates in drinking water from Réunion Island, France, using a multi-isotopic approach.

PubMed

Rogers, Karyne M; Nicolini, Eric; Gauthier, Virginie

2012-09-01

Nitrate concentrations, water isotopes (δ(2)H and δ(18)O(water)) and associated nitrate isotopes (δ(15)N(nitrate) and δ(18)O(nitrate)) from 10 drinking water wells, 5 fresh water springs and the discharge from 3 wastewater treatment stations in Réunion Island, located in the Indian Ocean, were analysed. We used a multi isotopic approach to investigate the extent of nitrate contamination, nitrate formation altitude and source of nitrates in Réunion Island's principal aquifer. Water from these study sites contained between 0.1 and 85.3 mg/L nitrate. δ(15)N(nitrate) values between +6 and +14‰ suggested the main sources of contamination were animal and/or human waste, rather than inorganic (synthetic) fertilisers, infiltrating through the subsurface into the saturated zone, due to rainfall leaching of the unsaturated zone at various altitudes of precipitation. Based on δ(15)N(nitrate) values alone, it was not possible to distinguish between animal and human activities responsible for the contamination of each specific catchment. However, using a multi isotope approach (δ(18)O(water) and δ(15)N(nitrate)), it was possible to relate the average altitude of rainfall infiltration (δ(18)O(water)) associated with the nitrate contamination (δ(18)O(nitrate)). This relationship between land use, rainfall recharge altitude and isotopic composition (δ(15)N(nitrate) and δ(18)O(water)) discriminated between the influences of human waste at lower (below 600 m elevation) or animal derived contamination (at elevations between 600 and 1300 m). By further comparing the theoretical altitude of nitrate formation calculated by the δ(18)O(nitrate), it was possible to determine that only 5 out of 15 fresh water wells and springs followed the conservative nitrate formation mechanism of 2/3δ(18)O(water)+1/3δ(18)O(air), to give nitrate formation altitudes which corresponded to land use activities. Copyright © 2012 Elsevier B.V. All rights reserved.

49 CFR 176.410 - Division 1.5 materials, ammonium nitrate and ammonium nitrate mixtures.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 49 Transportation 2 2011-10-01 2011-10-01 false Division 1.5 materials, ammonium nitrate and ammonium nitrate mixtures. 176.410 Section 176.410 Transportation Other Regulations Relating to... nitrate and ammonium nitrate mixtures. (a) This section prescribes requirements to be observed with...

49 CFR 176.410 - Division 1.5 materials, ammonium nitrate and ammonium nitrate mixtures.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 49 Transportation 2 2012-10-01 2012-10-01 false Division 1.5 materials, ammonium nitrate and ammonium nitrate mixtures. 176.410 Section 176.410 Transportation Other Regulations Relating to... nitrate and ammonium nitrate mixtures. (a) This section prescribes requirements to be observed with...

49 CFR 176.410 - Division 1.5 materials, ammonium nitrate and ammonium nitrate mixtures.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 49 Transportation 2 2013-10-01 2013-10-01 false Division 1.5 materials, ammonium nitrate and ammonium nitrate mixtures. 176.410 Section 176.410 Transportation Other Regulations Relating to... nitrate and ammonium nitrate mixtures. (a) This section prescribes requirements to be observed with...

49 CFR 176.410 - Division 1.5 materials, ammonium nitrate and ammonium nitrate mixtures.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 49 Transportation 2 2014-10-01 2014-10-01 false Division 1.5 materials, ammonium nitrate and ammonium nitrate mixtures. 176.410 Section 176.410 Transportation Other Regulations Relating to... nitrate and ammonium nitrate mixtures. (a) This section prescribes requirements to be observed with...

California GAMA Special Study: An isotopic and dissolved gas investigation of nitrate source and transport to a public supply well in California's Central Valley

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

Singleton, M J; Moran, J E; Esser, B K

2010-04-14

This study investigates nitrate contamination of a deep municipal drinking water production well in Ripon, CA to demonstrate the utility of natural groundwater tracers in constraining the sources and transport of nitrate to deep aquifers in the Central Valley. The goal of the study was to investigate the origin (source) of elevated nitrate and the potential for the deep aquifer to attenuate anthropogenic nitrate. The site is ideal for such an investigation. The production well is screened from 165-325 feet below ground surface and a number of nearby shallow and deep monitoring wells were available for sampling. Furthermore, potential sourcesmore » of nitrate contamination to the well had been identified, including a fertilizer supply plant located approximately 1000 feet to the east and local almond groves. A variety of natural isotopic and dissolved gas tracers including {sup 3}H-{sup 3}He groundwater age and the isotopic composition of nitrate are applied to identify nitrate sources and to characterize nitrate transport. An advanced method for sampling production wells is employed to help identify contaminant contributions from specific screen intervals. Nitrate transport: Groundwater nitrate at this field site is not being actively denitrified. Groundwater parameters indicate oxic conditions, the dissolved gas data shows no evidence for excess nitrogen as the result of denitrification, and nitrate-N and -O isotope compositions do not display patterns typical of denitrification. Contaminant nitrate source: The ambient nitrate concentration in shallow groundwater at the Ripon site ({approx}12 mg/L as nitrate) is typical of shallow groundwaters affected by recharge from agricultural and urban areas. Nitrate concentrations in Ripon City Well 12 (50-58 mg/L as nitrate) are significantly higher than these ambient concentrations, indicating an additional source of anthropogenic nitrate is affecting groundwater in the capture zone of this municipal drinking water well. This study provides two new pieces of evidence that the Ripon Farm Services Plant is the source of elevated nitrate in Ripon City Well 12. (1) Chemical mass balance calculations using nitrate concentration, nitrate isotopic composition, and initial tritium activity all indicate that that the source water for elevated nitrate to Ripon City Well 12 is a very small component of the water produced by City Well 12 and thus must have extremely high nitrate concentration. The high source water nitrate concentration ({approx}1500 mg/L as nitrate) required by these mass balance calculations precludes common sources of nitrate such as irrigated agriculture, dairy wastewater, and septic discharge. Shallow groundwater under the Ripon Farm Services RFS plant does contain extremely high concentrations of nitrate (>1700 mg/L as nitrate). (2) Nitrogen and oxygen isotope compositions of nitrate indicate that the additional anthropogenic nitrate source to Ripon City Well 12 is significantly enriched in {delta}{sup 18}O-NO{sub 3}, an isotopic signature consistent with synthetic nitrate fertilizer, and not with human or animal wastewater discharge (i.e. dairy operations, septic system discharge, or municipal wastewater discharge), or with organic fertilizer. Monitoring wells on and near the RFS plant also have high {delta}{sup 18}O-NO{sub 3}, and the plant has handled and stored synthetic nitrate fertilizer that will have this isotopic signature. The results described here highlight the complexity of attributing nitrate found in long screened, high capacity wells to specific sources. In this case, the presence of a very high concentration source near the well site combined with sampling using multiple isotopic tracer techniques and specialized depth-specific techniques allowed fingerprinting of the source in the mixed-age samples drawn from the production well.« less

Synthesis and standardization of biologically synthesized silver nanoparticles

NASA Astrophysics Data System (ADS)

Roy, Swarup; Das, Tapan Kumar

2013-06-01

The biological silver nanoparticle was synthesized extracellularly by using a fungi Aspergillus foetidus. The live cell filtrate of fungi has been used as reducing agent in the process of nanoparticles synthesis. In 50 ml cell filtrate a volume of AgNO3 stock solution was added to make finally the concentration as 1 mM of AgNO3 and allowed to shake in an incubator for several hrs in dark. The changed color was considered as the primary indication of nanoparticles formation and studies of UV-VIS, DLS, FTIR, AFM, TEM, EDS, Zeta pot. and nitrate reductase assay confirmed the same. It was indicated that stable & 20-40 nm roughly spherical shaped silver nanoparticles was formed. To standardize the nanoparticles biosynthesis different physical parameters like Substrate cone. (0-8 mM), PH-(5-12), Temp.-(5-50°C), incubation time (0-120) hrs and salinity (0.1-1.0 %) were investigated and it was observed that 4 mM AgNO3 conc., PH-9, Temp. -30°C, incubation time 72h and 0.2 % salinity were found to be optimum for the synthesis & stability of the silver nanoparticles.

Bacterial cellulose synthesis mechanism of facultative anaerobe Enterobacter sp. FY-07.

PubMed

Ji, Kaihua; Wang, Wei; Zeng, Bing; Chen, Sibin; Zhao, Qianqian; Chen, Yueqing; Li, Guoqiang; Ma, Ting

2016-02-25

Enterobacter sp. FY-07 can produce bacterial cellulose (BC) under aerobic and anaerobic conditions. Three potential BC synthesis gene clusters (bcsI, bcsII and bcsIII) of Enterobacter sp. FY-07 have been predicted using genome sequencing and comparative genome analysis, in which bcsIII was confirmed as the main contributor to BC synthesis by gene knockout and functional reconstitution methods. Protein homology, gene arrangement and gene constitution analysis indicated that bcsIII had high identity to the bcsI operon of Enterobacter sp. 638; however, its arrangement and composition were same as those of BC synthesizing operon of G. xylinum ATCC53582 except for the flanking sequences. According to the BC biosynthesizing process, oxygen is not directly involved in the reactions of BC synthesis, however, energy is required to activate intermediate metabolites and synthesize the activator, c-di-GMP. Comparative transcriptome and metabolite quantitative analysis demonstrated that under anaerobic conditions genes involved in the TCA cycle were downregulated, however, genes in the nitrate reduction and gluconeogenesis pathways were upregulated, especially, genes in three pyruvate metabolism pathways. These results suggested that Enterobacter sp. FY-07 could produce energy efficiently under anaerobic conditions to meet the requirement of BC biosynthesis.

Bacterial cellulose synthesis mechanism of facultative anaerobe Enterobacter sp. FY-07

PubMed Central

Ji, Kaihua; Wang, Wei; Zeng, Bing; Chen, Sibin; Zhao, Qianqian; Chen, Yueqing; Li, Guoqiang; Ma, Ting

2016-01-01

Enterobacter sp. FY-07 can produce bacterial cellulose (BC) under aerobic and anaerobic conditions. Three potential BC synthesis gene clusters (bcsI, bcsII and bcsIII) of Enterobacter sp. FY-07 have been predicted using genome sequencing and comparative genome analysis, in which bcsIII was confirmed as the main contributor to BC synthesis by gene knockout and functional reconstitution methods. Protein homology, gene arrangement and gene constitution analysis indicated that bcsIII had high identity to the bcsI operon of Enterobacter sp. 638; however, its arrangement and composition were same as those of BC synthesizing operon of G. xylinum ATCC53582 except for the flanking sequences. According to the BC biosynthesizing process, oxygen is not directly involved in the reactions of BC synthesis, however, energy is required to activate intermediate metabolites and synthesize the activator, c-di-GMP. Comparative transcriptome and metabolite quantitative analysis demonstrated that under anaerobic conditions genes involved in the TCA cycle were downregulated, however, genes in the nitrate reduction and gluconeogenesis pathways were upregulated, especially, genes in three pyruvate metabolism pathways. These results suggested that Enterobacter sp. FY-07 could produce energy efficiently under anaerobic conditions to meet the requirement of BC biosynthesis. PMID:26911736

Production of Novel Antibiotics Zeamines through Optimizing Dickeya zeae Fermentation Conditions

PubMed Central

Liao, Lisheng; Cheng, Yingying; Liu, Shiyin; Zhou, Jianuan; An, Shuwen; Lv, Mingfa; Chen, Yufan; Gu, Yanfang; Chen, Shaohua; Zhang, Lian-Hui

2014-01-01

Dickeya zeae strain EC1 was recently shown to produce a new type of phytotoxins designated as zeamine and zeamine II, which are potent wide-spectrum antibiotics against Gram-positive and Gram-negative bacterial pathogens, suggesting their promising potential as clinical medicines. In this study, the optimized medium composition and culture conditions for biosynthesis of novel antibiotics zeamines have been established by using response surface methodology, largely increasing the yield of zeamines from original about 7.35 µg·mL−1 in minimal medium to about 150 µg·mL−1 in LS5 medium. The study identified the major factors contributing to zeamines production, which include nitrate, sucrose, asparaginate, mineral elements Mg2+ and K+, and optimized amount of phosphate. In addition, the results showed that overexpression of zmsK in D. zeae strain EC1 could further increase zeamines yield to about 180 µg·mL−1 in LS5 medium. The findings from this study could facilitate further characterization and utilization of these two novel antibiotics, and also provide useful clues for understanding the regulatory mechanisms that govern D. zeae virulence. PMID:25541733

Formation of friable embryogenic callus in cassava is enhanced under conditions of reduced nitrate, potassium and phosphate

PubMed Central

Utsumi, Yoshinori; Utsumi, Chikako; Tanaka, Maho; Ha, Vu The; Matsui, Akihiro; Takahashi, Satoshi; Seki, Motoaki

2017-01-01

Agrobacterium-mediated transformation is an important research tool for the genetic improvement of cassava. The induction of friable embryogenic callus (FEC) is considered as a key step in cassava transformation. In the present study, the media composition was optimized for enhancing the FEC induction, and the effect of the optimized medium on gene expression was evaluated. In relative comparison to MS medium, results demonstrated that using a medium with reducing nutrition (a 10-fold less concentration of nitrogen, potassium, and phosphate), the increased amount of vitamin B1 (10 mg/L) and the use of picrolam led to reprogram non-FEC to FEC. Gene expression analyses revealed that FEC on modified media increased the expression of genes related to the regulation of polysaccharide biosynthesis and breakdown of cell wall components in comparison to FEC on normal CIM media, whereas the gene expression associated with energy flux was not dramatically altered. It is hypothesized that we reprogram non-FEC to FEC under low nitrogen, potassium and phosphate and high vitamin B1. These findings were more effective in inducing FEC formation than the previous protocol. It might contribute to development of an efficient transformation strategy in cassava. PMID:28806727

Oxaloacetate Transport into Plant Mitochondria1

PubMed Central

Hanning, Iris; Baumgarten, Katharina; Schott, Karin; Heldt, Hans W.

1999-01-01

The properties of oxaloacetate (OA) transport into mitochondria from potato (Solanum tuberosum) tuber and pea (Pisum sativum) leaves were studied by measuring the uptake of 14C-labeled OA into liposomes with incorporated mitochondrial membrane proteins preloaded with various dicarboxylates or citrate. OA was found to be transported in an obligatory counterexchange with malate, 2-oxoglutarate, succinate, citrate, or aspartate. Phtalonate inhibited all of these countertransports. OA-malate countertransport was inhibited by 4,4′-dithiocyanostilbene-2,2′-disulfonate and pyridoxal phosphate, and also by p-chloromercuribenzene sulfonate and mersalyl, indicating that a lysine and a cysteine residue of the translocator protein are involved in the transport. From these and other inhibition studies, we concluded that plant mitochondria contain an OA translocator that differs from all other known mitochondrial translocators. Major functions of this translocator are the export of reducing equivalents from the mitochondria via the malate-OA shuttle and the export of citrate via the citrate-OA shuttle. In the cytosol, citrate can then be converted either into 2-oxoglutarate for use as a carbon skeleton for nitrate assimilation or into acetyl-coenzyme A for use as a precursor for fatty acid elongation or isoprenoid biosynthesis. PMID:10069840

Proteomics of the organohalide-respiring Epsilonproteobacterium Sulfurospirillum multivorans adapted to tetrachloroethene and other energy substrates

PubMed Central

Goris, Tobias; Schiffmann, Christian L.; Gadkari, Jennifer; Schubert, Torsten; Seifert, Jana; Jehmlich, Nico; von Bergen, Martin; Diekert, Gabriele

2015-01-01

Organohalide respiration is an environmentally important but poorly characterized type of anaerobic respiration. We compared the global proteome of the versatile organohalide-respiring Epsilonproteobacterium Sulfurospirillum multivorans grown with different electron acceptors (fumarate, nitrate, or tetrachloroethene [PCE]). The most significant differences in protein abundance were found for gene products of the organohalide respiration region. This genomic region encodes the corrinoid and FeS cluster containing PCE reductive dehalogenase PceA and other proteins putatively involved in PCE metabolism such as those involved in corrinoid biosynthesis. The latter gene products as well as PceA and a putative quinol dehydrogenase were almost exclusively detected in cells grown with PCE. This finding suggests an electron flow from the electron donor such as formate or pyruvate via the quinone pool and a quinol dehydrogenase to PceA and the terminal electron acceptor PCE. Two putative accessory proteins, an IscU-like protein and a peroxidase-like protein, were detected with PCE only and might be involved in PceA maturation. The proteome of cells grown with pyruvate instead of formate as electron donor indicates a route of electrons from reduced ferredoxin via an Epsilonproteobacterial complex I and the quinone pool to PCE. PMID:26387727

Simple biosynthesis of zinc oxide nanoparticles using nature's source, and it's in vitro bio-activity

NASA Astrophysics Data System (ADS)

Zare, Elham; Pourseyedi, Shahram; Khatami, Mehrdad; Darezereshki, Esmaeel

2017-10-01

Nanoparticles with antimicrobial activity, especially as a new class of biomedical materials for use in increasing the level of public health in daily life have emerged. In this study, green synthesis of zinc oxide) ZnO(nanoparticles was studied by Cuminum cyminum (cumin) as novel natural source and zinc nitrate [Zn(NO3)2] as Zn2+ source. The results showed that parameters such as concentration, time, temperature and pH have a direct impact on the synthesis of zinc nanoparticles and change in any of the factors causing the change in the process of synthesis. The properties of synthesized nanoparticles were examined by UV-visible Spectrophotometer, X-ray diffraction spectroscopy and transmission electron microscopy (TEM). The UV-visible spectroscopy presented the absorption peak in the range of 370 nm. Transmission electron microscopy images of synthesized nanoparticles are mainly spherical or oval with an average size of about 7 nm. The effect of antimicrobial nanoparticles calculated using disk diffusion method and broth MIC and MBC in different strains of bacteria, which showed that gram positive and negative were sensitive to zinc oxide nanoparticles. The sensitivity of gram-negative bacteria was more.

Biosynthesis and characterization of ZnO nanoparticles using the aqueous leaf extract of Imperata cylindrica L.

NASA Astrophysics Data System (ADS)

Saputra, I. S.; Yulizar, Y.

2017-04-01

ZnO nanoparticles (ZnO NPs) were biosynthesized.The growth was observed by a sol-gel method. ZnO were successfully formed through the reaction of zinc nitrate tetrahydrate Zn(NO3)2.4H2O precursor with aqueous leaf extract of Imperata cylindrica L (ICL). The structural and optical properties of ZnO were investigated. The as-synthesized products were characterized by UV-Visible (UV-Vis), UV diffuse reflectance spectroscopy (UV-DRS), Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), scanning electron microscopy (SEM), and energy-dispersive X-ray spectroscopy (EDS). UV-Vis absorption data showed hydrolysis and characteristic of absorption peak at 300 nm of Zn(OH)2. UV-DRS confirmed that ZnO NPs has the indirect band gap at 3.13 eV. FTIR spectrum revealed the functional groups and indicated the presence of protein as the capping and stabilizing agent on the ZnO surface. Powder XRD studies indicated the formation of pure wurtzite hexagonal structure with particle size of 11.9 nm. The detailed morphological and structural characterizations revealed that the synthesized products were hexagonal nanochip.

Effects of stress ethylene inhibitors on sweet clover (Melilotus Alba L.) seedling growth in microgravity

NASA Technical Reports Server (NTRS)

Gallegos, Gregory L.; Peterson, Barbara V.; Brown, Christopher S.; Guikema, James A.

1995-01-01

Previous experiments from our lab have shown that seeds of sweet clover, when germinated and grown within the Fluid Processing Apparatus (FPA) on a slow rotating clinostat produce significantly greater levels of the volatile stress hormone, ethylene, when compared to seeds treated the same but without clinorotation. In both conditions, carbon dioxide levels reached high levels and seedling growth was inhibited. However, clinorotation inhibited growth to a greater extent. To help determine to what extent microgravity influences stress ethylene production and to what extent ethylene inhibits seedling growth, we have extended the above experiments by growing sweet clover in the presence of aminooxyacetic acid (AOA) and silver nitrate (AgNO3), inhibitors of stress ethylene biosynthesis and action, respectively. Seeds of sweet clover were germinated and grown for five days in the FPA under two gravity conditions: under stationary conditions on Earth and in microgravity onboard the space shuttle, Discovery (STS-63), which launched Feb. 3, 1995. Upon recovery, gas samples were aspirated from the growth chambers and carbon dioxide and ethylene concentrations were measured using a gas chromatograph. Then the tissue was weighed, photographed and fixed, and is current undergoing further morphological and microscopic characterization.

Green biosynthesis of silver nanoparticles using Curcuma longa tuber powder

PubMed Central

Shameli, Kamyar; Ahmad, Mansor Bin; Zamanian, Ali; Sangpour, Parvanh; Shabanzadeh, Parvaneh; Abdollahi, Yadollah; Zargar, Mohsen

2012-01-01

Green synthesis of noble metal nanoparticles is a vastly developing area of research. Metallic nanoparticles have received great attention from chemists, physicists, biologists, and engineers who wish to use them for the development of a new-generation of nanodevices. In this study, silver nanoparticles were biosynthesized from aqueous silver nitrate through a simple and eco-friendly route using Curcuma longa tuber-powder extracts, which acted as a reductant and stabilizer simultaneously. Characterizations of nanoparticles were done using different methods, which included ultraviolet-visible spectroscopy, powder X-ray diffraction, transmission electron microscopy, scanning electron microscopy, energy-dispersive X-ray fluorescence spectrometry, and Fourier-transform infrared spectroscopy. The ultraviolet-visible spectrum of the aqueous medium containing silver nanoparticles showed an absorption peak at around 415 nm. Transmission electron microscopy showed that mean diameter and standard deviation for the formation of silver nanoparticles was 6.30 ± 2.64 nm. Powder X-ray diffraction showed that the particles are crystalline in nature, with a face-centered cubic structure. The most needed outcome of this work will be the development of value-added products from C. longa for biomedical and nanotechnology-based industries. PMID:23341739

Food sources of nitrates and nitrites: the physiologic context for potential health benefits.

PubMed

Hord, Norman G; Tang, Yaoping; Bryan, Nathan S

2009-07-01

The presence of nitrates and nitrites in food is associated with an increased risk of gastrointestinal cancer and, in infants, methemoglobinemia. Despite the physiologic roles for nitrate and nitrite in vascular and immune function, consideration of food sources of nitrates and nitrites as healthful dietary components has received little attention. Approximately 80% of dietary nitrates are derived from vegetable consumption; sources of nitrites include vegetables, fruit, and processed meats. Nitrites are produced endogenously through the oxidation of nitric oxide and through a reduction of nitrate by commensal bacteria in the mouth and gastrointestinal tract. As such, the dietary provision of nitrates and nitrites from vegetables and fruit may contribute to the blood pressure-lowering effects of the Dietary Approaches to Stop Hypertension (DASH) diet. We quantified nitrate and nitrite concentrations by HPLC in a convenience sample of foods. Incorporating these values into 2 hypothetical dietary patterns that emphasize high-nitrate or low-nitrate vegetable and fruit choices based on the DASH diet, we found that nitrate concentrations in these 2 patterns vary from 174 to 1222 mg. The hypothetical high-nitrate DASH diet pattern exceeds the World Health Organization's Acceptable Daily Intake for nitrate by 550% for a 60-kg adult. These data call into question the rationale for recommendations to limit nitrate and nitrite consumption from plant foods; a comprehensive reevaluation of the health effects of food sources of nitrates and nitrites is appropriate. The strength of the evidence linking the consumption of nitrate- and nitrite-containing plant foods to beneficial health effects supports the consideration of these compounds as nutrients.

Safety in the Chemical Laboratory: Nitric Acid, Nitrates, and Nitro Compounds.

ERIC Educational Resources Information Center

Bretherick, Leslie

1989-01-01

Discussed are the potential hazards associated with nitric acid, inorganic and organic nitrate salts, alkyl nitrates, acyl nitrates, aliphatic nitro compounds, aromatic nitro compounds, and nitration reactions. (CW)

49 CFR 176.415 - Permit requirements for Division 1.5, ammonium nitrates, and certain ammonium nitrate fertilizers.

Code of Federal Regulations, 2010 CFR

2010-10-01

... nitrates, and certain ammonium nitrate fertilizers. 176.415 Section 176.415 Transportation Other... Solids), Class 5 (Oxidizers and Organic Peroxides), and Division 1.5 Materials § 176.415 Permit requirements for Division 1.5, ammonium nitrates, and certain ammonium nitrate fertilizers. (a) Except as...

The Arabidopsis NRG2 Protein Mediates Nitrate Signaling and Interacts with and Regulates Key Nitrate Regulators[OPEN

PubMed Central

Zhao, Lufei; Zhang, Chengfei; Li, Zehui; Lei, Zhao; Liu, Fei; Guan, Peizhu; Crawford, Nigel M.

2016-01-01

We show that NITRATE REGULATORY GENE2 (NRG2), which we identified using forward genetics, mediates nitrate signaling in Arabidopsis thaliana. A mutation in NRG2 disrupted the induction of nitrate-responsive genes after nitrate treatment by an ammonium-independent mechanism. The nitrate content in roots was lower in the mutants than in the wild type, which may have resulted from reduced expression of NRT1.1 (also called NPF6.3, encoding a nitrate transporter/receptor) and upregulation of NRT1.8 (also called NPF7.2, encoding a xylem nitrate transporter). Genetic and molecular data suggest that NRG2 functions upstream of NRT1.1 in nitrate signaling. Furthermore, NRG2 directly interacts with the nitrate regulator NLP7 in the nucleus, but nuclear retention of NLP7 in response to nitrate is not dependent on NRG2. Transcriptomic analysis revealed that genes involved in four nitrogen-related clusters including nitrate transport and response to nitrate were differentially expressed in the nrg2 mutants. A nitrogen compound transport cluster containing some members of the NRT/PTR family was regulated by both NRG2 and NRT1.1, while no nitrogen-related clusters showed regulation by both NRG2 and NLP7. Thus, NRG2 plays a key role in nitrate regulation in part through modulating NRT1.1 expression and may function with NLP7 via their physical interaction. PMID:26744214

Disruption of the rice nitrate transporter OsNPF2.2 hinders root-to-shoot nitrate transport and vascular development

PubMed Central

Li, Yuge; Ouyang, Jie; Wang, Ya-Yun; Hu, Rui; Xia, Kuaifei; Duan, Jun; Wang, Yaqin; Tsay, Yi-Fang; Zhang, Mingyong

2015-01-01

Plants have evolved to express some members of the nitrate transporter 1/peptide transporter family (NPF) to uptake and transport nitrate. However, little is known of the physiological and functional roles of this family in rice (Oryza sativa L.). Here, we characterized the vascular specific transporter OsNPF2.2. Functional analysis using cDNA-injected Xenopus laevis oocytes revealed that OsNPF2.2 is a low-affinity, pH-dependent nitrate transporter. Use of a green fluorescent protein tagged OsNPF2.2 showed that the transporter is located in the plasma membrane in the rice protoplast. Expression analysis showed that OsNPF2.2 is nitrate inducible and is mainly expressed in parenchyma cells around the xylem. Disruption of OsNPF2.2 increased nitrate concentration in the shoot xylem exudate when nitrate was supplied after a deprivation period; this result suggests that OsNPF2.2 may participate in unloading nitrate from the xylem. Under steady-state nitrate supply, the osnpf2.2 mutants maintained high levels of nitrate in the roots and low shoot:root nitrate ratios; this observation suggests that OsNPF2.2 is involved in root-to-shoot nitrate transport. Mutation of OsNPF2.2 also caused abnormal vasculature and retarded plant growth and development. Our findings demonstrate that OsNPF2.2 can unload nitrate from the xylem to affect the root-to-shoot nitrate transport and plant development. PMID:25923512

Profound differences between humans and rodents in the ability to concentrate salivary nitrate: Implications for translational research.

PubMed

Montenegro, Marcelo F; Sundqvist, Michaela L; Nihlén, Carina; Hezel, Michael; Carlström, Mattias; Weitzberg, Eddie; Lundberg, Jon O

2016-12-01

In humans dietary circulating nitrate accumulates rapidly in saliva through active transport in the salivary glands. By this mechanism resulting salivary nitrate concentrations are 10-20 times higher than in plasma. In the oral cavity nitrate is reduced by commensal bacteria to nitrite, which is subsequently swallowed and further metabolized to nitric oxide (NO) and other bioactive nitrogen oxides in blood and tissues. This entero-salivary circulation of nitrate is central in the various NO-like effects observed after ingestion of inorganic nitrate. The very same system has also been the focus of toxicologists studying potential carcinogenic effects of nitrite-dependent nitrosamine formation. Whether active transport of nitrate and accumulation in saliva occurs also in rodents is not entirely clear. Here we measured salivary and plasma levels of nitrate and nitrite in humans, rats and mice after administration of a standardized dose of nitrate. After oral (humans) or intraperitoneal (rodents) sodium nitrate administration (0.1mmol/kg), plasma nitrate levels increased markedly reaching ~300µM in all three species. In humans ingestion of nitrate was followed by a rapid increase in salivary nitrate to >6000µM, ie 20 times higher than those found in plasma. In contrast, in rats and mice salivary nitrate concentrations never exceeded the levels in plasma. Nitrite levels in saliva and plasma followed a similar pattern, ie marked increases in humans but modest elevations in rodents. In mice there was also no accumulation of nitrate in the salivary glands as measured directly in whole glands obtained after acute administration of nitrate. This study suggests that in contrast to humans, rats and mice do not actively concentrate circulating nitrate in saliva. These apparent species differences should be taken into consideration when studying the nitrate-nitrite-nitric oxide pathway in rodents, when calculating doses, exploring physiological, therapeutic and toxicological effects and comparing with human data. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Novel pathway of 3-hydroxyanthranilic acid formation in limazepine biosynthesis reveals evolutionary relation between phenazines and pyrrolobenzodiazepines.

PubMed

Pavlikova, Magdalena; Kamenik, Zdenek; Janata, Jiri; Kadlcik, Stanislav; Kuzma, Marek; Najmanova, Lucie

2018-05-17

Natural pyrrolobenzodiazepines (PBDs) form a large and structurally diverse group of antitumour microbial metabolites produced through complex pathways, which are encoded within biosynthetic gene clusters. We sequenced the gene cluster of limazepines and proposed their biosynthetic pathway based on comparison with five available gene clusters for the biosynthesis of other PBDs. Furthermore, we tested two recombinant proteins from limazepine biosynthesis, Lim5 and Lim6, with the expected substrates in vitro. The reactions monitored by LC-MS revealed that limazepine biosynthesis involves a new way of 3-hydroxyanthranilic acid formation, which we refer to as the chorismate/DHHA pathway and which represents an alternative to the kynurenine pathway employed for the formation of the same precursor in the biosynthesis of other PBDs. The chorismate/DHHA pathway is presumably also involved in the biosynthesis of PBD tilivalline, several natural products unrelated to PBDs, and its part is shared also with phenazine biosynthesis. The similarities between limazepine and phenazine biosynthesis indicate tight evolutionary links between these groups of compounds.

Alcoholic Liver Disease: A Mouse Model Reveals Protection by Lactobacillus fermentum

PubMed Central

Barone, Rosario; Rappa, Francesca; Macaluso, Filippo; Caruso Bavisotto, Celeste; Sangiorgi, Claudia; Di Paola, Gaia; Tomasello, Giovanni; Di Felice, Valentina; Marcianò, Vito; Farina, Felicia; Zummo, Giovanni; Conway de Macario, Everly; J.L. Macario, Alberto; Cocchi, Massimo; Cappello, MD, Francesco; Marino Gammazza, Antonella

2016-01-01

Objectives: Alcoholism is one of the most devastating diseases with high incidence, but knowledge of its pathology and treatment is still plagued with gaps mostly because of the inherent limitations of research with patients. We developed an animal model for studying liver histopathology, Hsp (heat-shock protein)-chaperones involvement, and response to treatment. Methods: The system was standardized using mice to which ethanol was orally administered alone or in combination with Lactobacillus fermentum following a precise schedule over time and applying, at predetermined intervals, a battery of techniques (histology, immunohistochemistry, western blotting, real-time PCR, immunoprecipitation, 3-nitrotyrosine labeling) to assess liver pathology (e.g., steatosis, fibrosis), and Hsp60 and iNOS (inducible form of nitric oxide synthase) gene expression and protein levels, and post-translational modifications. Results: Typical ethanol-induced liver pathology occurred and the effect of the probiotic could be reliably monitored. Steatosis score, iNOS levels, and nitrated proteins (e.g., Hsp60) decreased after probiotic intake. Conclusions: We describe a mouse model useful for studying liver disease induced by chronic ethanol intake and for testing pertinent therapeutic agents, e.g., probiotics. We tested L. fermentum, which reduced considerably ethanol-induced tissue damage and deleterious post-translational modifications of the chaperone Hsp60. The model is available to test other agents and probiotics with therapeutic potential in alcoholic liver disease. PMID:26795070

An Abnormal Nitric Oxide Metabolism Contributes to Brain Oxidative Stress in the Mouse Model for the Fragile X Syndrome, a Possible Role in Intellectual Disability

PubMed Central

Lima-Cabello, Elena; Garcia-Guirado, Francisco; Calvo-Medina, Rocio; el Bekay, Rajaa; Perez-Costillas, Lucia; Quintero-Navarro, Carolina; Sanchez-Salido, Lourdes

2016-01-01

Background. Fragile X syndrome is the most common genetic cause of mental disability. Although many research has been performed, the mechanism underlying the pathogenesis is unclear and needs further investigation. Oxidative stress played major roles in the syndrome. The aim was to investigate the nitric oxide metabolism, protein nitration level, the expression of NOS isoforms, and furthermore the activation of the nuclear factor NF-κB-p65 subunit in different brain areas on the fragile X mouse model. Methods. This study involved adult male Fmr1-knockout and wild-type mice as controls. We detected nitric oxide metabolism and the activation of the nuclear factor NF-κBp65 subunit, comparing the mRNA expression and protein content of the three NOS isoforms in different brain areas. Results. Fmr1-KO mice showed an abnormal nitric oxide metabolism and increased levels of protein tyrosine nitrosylation. Besides that, nuclear factor NF-κB-p65 and inducible nitric oxide synthase appeared significantly increased in the Fmr1-knockout mice. mRNA and protein levels of the neuronal nitric oxide synthase appeared significantly decreased in the knockout mice. However, the epithelial nitric oxide synthase isoform displayed no significant changes. Conclusions. These data suggest the potential involvement of an abnormal nitric oxide metabolism in the pathogenesis of the fragile X syndrome. PMID:26788253

Ant Trail Pheromone Biosynthesis Is Triggered by a Neuropeptide Hormone

PubMed Central

Choi, Man-Yeon; Vander Meer, Robert K.

2012-01-01

Our understanding of insect chemical communication including pheromone identification, synthesis, and their role in behavior has advanced tremendously over the last half-century. However, endocrine regulation of pheromone biosynthesis has progressed slowly due to the complexity of direct and/or indirect hormonal activation of the biosynthetic cascades resulting in insect pheromones. Over 20 years ago, a neurohormone, pheromone biosynthesis activating neuropeptide (PBAN) was identified that stimulated sex pheromone biosynthesis in a lepidopteran moth. Since then, the physiological role, target site, and signal transduction of PBAN has become well understood for sex pheromone biosynthesis in moths. Despite that PBAN-like peptides (∼200) have been identified from various insect Orders, their role in pheromone regulation had not expanded to the other insect groups except for Lepidoptera. Here, we report that trail pheromone biosynthesis in the Dufour's gland (DG) of the fire ant, Solenopsis invicta, is regulated by PBAN. RNAi knock down of PBAN gene (in subesophageal ganglia) or PBAN receptor gene (in DG) expression inhibited trail pheromone biosynthesis. Reduced trail pheromone was documented analytically and through a behavioral bioassay. Extension of PBAN's role in pheromone biosynthesis to a new target insect, mode of action, and behavioral function will renew research efforts on the involvement of PBAN in pheromone biosynthesis in Insecta. PMID:23226278

New insights into bioactivation of organic nitrates, nitrate tolerance and cross-tolerance.

PubMed

Daiber, A; Wenzel, P; Oelze, M; Münzel, T

2008-01-01

Organic nitrates still represent a group of very effective anti-ischemic drugs used for the treatment of patients with stable angina, acute myocardial infarction and chronic congestive heart failure. Long-term therapy with organic nitrates, however, results in a rapid development of nitrate tolerance blunting their hemodynamic and antiischemic efficacy. Recent studies revealed that mitochondrial reactive oxygen species (ROS) formation and a subsequent oxidative inactivation of nitrate reductase, the mitochondrial aldehyde dehydrogenase (ALDH-2), play an important role for the development of nitrate and crosstolerance. The present review focuses firstly on the role of ALDH-2 for organic nitrate bioactivation and secondly on the role of oxidative stress in the development of tolerance and cross-tolerance (endothelial dysfunction) in response to various organic nitrates. Finally, we would like to draw the reader's attention to the protective properties of the organic nitrate pentaerithrityl tetranitrate (PETN), which, in contrast to all other organic nitrates, is able to upregulate enzymes with a strong antioxidative capacity thereby preventing tolerance and the development of endothelial dysfunction.

Substrate binding site for nitrate reductase of Escherichia coli is on the inner aspect of the membrane.

PubMed Central

Kristjansson, J K; Hollocher, T C

1979-01-01

Escherichia coli grown anaerobically on nitrate exhibited the same transport barrier to reduction of chlorate, relative to nitrate, as that exhibited by Paracoccus denitrificans. This establishes that the nitrate binding site of nitrate reductase (EC 1.7.99.4) in E. coli must also lie on the cell side of the nitrate transporter which is associated with the plasma membrane. Because nitrate reductase is membrane bound, the nitrate binding site is thus located on the inner aspect of the membrane. Nitrate pulse studies on E. coli in the absence of valinomycin showed a small transient alkalinization (leads to H+/NO3- congruent to --0.07) which did not occur with oxygen pulses. By analogy with P. denitrificans, the alkaline transient is interpreted to arise from proton-linked nitrate uptake which is closely followed by nitrite efflux. The result is consistent with internal reduction of nitrate, whereas external reduction would be expected to give leads to H+/NO3-ratios approaching --2. PMID:374343

Nitrate biosensors and biological methods for nitrate determination.

PubMed

Sohail, Manzar; Adeloju, Samuel B

2016-06-01

The inorganic nitrate (NO3‾) anion is present under a variety of both natural and artificial environmental conditions. Nitrate is ubiquitous within the environment, food, industrial and physiological systems and is mostly present as hydrated anion of a corresponding dissolved salt. Due to the significant environmental and toxicological effects of nitrate, its determination and monitoring in environmental and industrial waters are often necessary. A wide range of analytical techniques are available for nitrate determination in various sample matrices. This review discusses biosensors available for nitrate determination using the enzyme nitrate reductase (NaR). We conclude that nitrate determination using biosensors is an excellent non-toxic alternative to all other available analytical methods. Over the last fifteen years biosensing technology for nitrate analysis has progressed very well, however, there is a need to expedite the development of nitrate biosensors as a suitable alternative to non-enzymatic techniques through the use of different polymers, nanostructures, mediators and strategies to overcome oxygen interference. Copyright © 2016 Elsevier B.V. All rights reserved.

Differential Effect of Irradiance and Nutrient Nitrate on the Relationship of in Vivo and in Vitro Nitrate Reductase Assay in Chlorophyllous Tissues 1

PubMed Central

Jones, Richard Wyn; Sheard, Robert W.

1977-01-01

Growth at increasing continuous irradiance (at high nutrient nitrate) and nutrient nitrate concentrations (at high continuous irradiance) furnished increases in the in vivo and in vitro nitrate reductase activities of corn (Zea mays L.), field peas (Pisum arvense L.), wheat (Triticum aestivum L.), barley (Hordeum vulgare L.), and globe amaranth (Gomphrena globosa L.) leaves and of marrow (Cucurbita pepo L.) cotyledons. Ratios of in vivo to in vitro activity declined exponentially in all species with increasing nitrate reductase levels promoted by nutrient nitrate. The ratios were more nearly independent of nitrate reductase levels generated by adjusting the irradiance; major exceptions were marrow and wheat at low (1.5 klux and less) irradiances and peas throughout the irradiance range, where decreases in the ratio were accompanied by increases in in situ nitrate concentration. The ratio also increased at the highest irradiance (39.2 klux) in wheat and barley, associated with a decline of in vitro nitrate reductase. These differences in response to irradiance and nutrient nitrate indicate that the in vivo assay does not provide a simple measure of nitrate reductase but rather yields a more composite measure of nitrate reduction, possibly related both to nitrate reductase level and to the supply of reductant for in vivo activity. PMID:16659888

Observations of fine and coarse particle nitrate at several rural locations in the United States

NASA Astrophysics Data System (ADS)

Lee, Taehyoung; Yu, Xiao-Ying; Ayres, Benjamin; Kreidenweis, Sonia M.; Malm, William C.; Collett, Jeffrey L.

Nitrate comprises an important part of aerosol mass at many non-urban locations during some times of the year. Little is known, however, about the chemical form and size distribution of particulate nitrate in these environments. While submicron ammonium nitrate is often assumed to be the dominant species, this assumption is rarely tested. Properties of aerosol nitrate were characterized at several IMPROVE monitoring sites during a series of field studies. Study sites included Bondville, Illinois (February 2003), San Gorgonio Wilderness Area, California (April and July 2003), Grand Canyon National Park, Arizona (May 2003), Brigantine National Wildlife Refuge, New Jersey (November 2003), and Great Smoky Mountains National Park, Tennessee (July/August 2004). Nitrate was found predominantly in submicron ammonium nitrate particles during the Bondville and San Gorgonio (April) campaigns. Coarse mode nitrate particles, resulting from reactions of nitric acid or its precursors with sea salt or soil dust, were more important at Grand Canyon and Great Smoky Mountains. Both fine and coarse mode nitrate were important during the studies at Brigantine and San Gorgonio (July). These results, which complement earlier findings about the importance of coarse particle nitrate at Yosemite and Big Bend National Parks, suggest a need to more closely examine common assumptions regarding the importance of ammonium nitrate at non-urban sites, to include pathways for coarse mode nitrate formation in regional models, and to consider impacts of coarse particle nitrate on visibility. Because coarse particle nitrate modes often extend well below 2.5 μm aerodynamic diameter, measurements of PM 2.5 nitrate in these environments should not automatically be assumed to contain only ammonium nitrate.

Ground-water movement and nitrate in ground water, East Erda area, Tooele County, Utah, 1997-2000

USGS Publications Warehouse

Susong, D.D.

2005-01-01

Nitrate was discovered in ground water in the east Erda area of Tooele County, Utah, in 1994. The U.S. Geological Survey, in cooperation with Tooele County, investigated the ground-water flow system and water quality in the eastern part of Tooele Valley to determine (1) the vertical and horizontal distribution of nitrate, (2) the direction of movement of the nitrate contamination, and (3) the source of the nitrate. The potentiometric surface of the upper part of the basin-fill aquifer indicates that the general direction of ground-water flow is to the northwest, the flow system is complex, and there is a ground-water mound probably associated with springs. The spatial distribution of nitrate reflects the flow system with the nitrate contamination split into a north and south part by the ground-water mound. The distribution of dissolved solids and sulfate in ground water varies spatially. Vertical profiles of nitrate in water from selected wells indicate that nitrate contamination generally is in the upper part of the saturated zone and in some wells has moved downward. Septic systems, mining and smelting, agriculture, and natural sources were considered to be possible sources of nitrate contamination in the east Erda area. Septic systems are not the source of nitrate because water from wells drilled upgradient of all septic systems in the area had elevated nitrate concentrations. Mining and smelting activity are a possible source of nitrate contamination but few data are available to link nitrate contamination with mining sites. Natural and agricultural sources of nitrate are present east of the Erda area but few data are available about these sources. The source(s) of nitrate in the east Erda area could not be clearly delineated in spite of considerable effort and expenditure of resources.

Geochemical controls on microbial nitrate-dependent U(IV) oxidation

USGS Publications Warehouse

Senko, John M.; Suflita, Joseph M.; Krumholz, Lee R.

2005-01-01

After reductive immobilization of uranium, the element may be oxidized and remobilized in the presence of nitrate by the activity of dissimilatory nitrate-reducing bacteria. We examined controls on microbially mediated nitrate-dependent U(IV) oxidation in landfill leachate-impacted subsurface sediments. Nitrate-dependent U(IV)-oxidizing bacteria were at least two orders of magnitude less numerous in these sediments than glucose- or Fe(II)-oxidizing nitrate-reducing bacteria and grew more slowly than the latter organisms, suggesting that U(IV) is ultimately oxidized by Fe(III) produced by nitrate-dependent Fe(II)-oxidizing bacteria or by oxidation of Fe(II) by nitrite that accumulates during organotrophic dissimilatory nitrate reduction. We examined the effect of nitrate and reductant concentration on nitrate-dependent U(IV) oxidation in sediment incubations and used the initial reductive capacity (RDC = [reducing equivalents] - [oxidizing equivalents]) of the incubations as a unified measurement of the nitrate or reductant concentration. When we lowered the RDC with progressively higher nitrate concentrations, we observed a corresponding increase in the extent of U(IV) oxidation, but did not observe this relationship between RDC and U(IV) oxidation rate, especially when RDC > 0, suggesting that nitrate concentration strongly controls the extent, but not the rate of nitrate-dependent U(IV) oxidation. On the other hand, when we raised the RDC in sediment incubations with progressively higher reductant (acetate, sulfide, soluble Fe(II), or FeS) concentrations, we observed progressively lower extents and rates of nitrate-dependent U(IV) oxidation. Acetate was a relatively poor inhibitor of nitrate-dependent U(IV) oxidation, while Fe(II) was the most effective inhibitor. Based on these results, we propose that it may be possible to predict the stability of U(IV) in a bioremediated aquifer based on the geochemical characteristics of that aquifer.

Post-anthesis nitrate uptake is critical to yield and grain protein content in Sorghum bicolor.

PubMed

Worland, Belinda; Robinson, Nicole; Jordan, David; Schmidt, Susanne; Godwin, Ian

2017-09-01

Crops only use ∼50% of applied nitrogen (N) fertilizer creating N losses and pollution. Plants need to efficiently uptake and utilize N to meet growing global food demands. Here we investigate how the supply and timing of nitrate affects N status and yield in Sorghum bicolor (sorghum). Sorghum was grown in pots with either 10mM (High) or 1mM (Low) nitrate supply. Shortly before anthesis the nitrate supply was either maintained, increased 10-fold or eliminated. Leaf sheaths of sorghum grown with High nitrate accumulated nitrate in concentrations >3-times higher than leaves. Removal of nitrate supply pre-anthesis resulted in the rapid reduction of stored nitrate in all organs. Plants receiving a 10-fold increase in nitrate supply pre-anthesis achieved similar grain yield and protein content and 29% larger grains than those maintained on High nitrate, despite receiving 24% less nitrate over the whole growth period. In sorghum, plant available N is important throughout development, particularly anthesis and grain filling, for grain yield and grain protein content. Nitrate accumulation in leaf sheaths presents opportunities for the genetic analysis of mechanisms behind nitrate storage and remobilization in sorghum to improve N use efficiency. Copyright © 2017 Elsevier GmbH. All rights reserved.

Dietary nitrate as modulator of physical performance and cardiovascular health.

PubMed

Kerley, Conor P

2017-11-01

Early interventional trials reported improvements in cardiac and exercise outcomes with inorganic nitrate ingestion. The current review aims to provide a brief update of recent evidence regarding ergogenic and cardiovascular effects of dietary nitrate and practical recommendations. Recent evidence has been inconsistent and questions remain regarding effective dose, duration, and source of nitrate and cohorts likely to benefit. Dietary nitrate may be most relevant to those with vascular/metabolic impairments, those engaging in short-term, intense exercise, deconditioned individuals, and those with a low dietary nitrate intake. The evidence for cardiovascular/exercise benefit is plausible but inconsistent. However, dietary nitrate, in contrast to pharmacological nitrate, has a high benefit-risk ratio. Although nitrate supplementation has grown in popularity, it is suggested that increased green vegetables consumption may provide similar/superior benefits to nitrate supplementation in a cheaper, safer, and potentially tastier context.

Simultaneous reduction of nitrate and selenate by cell suspensions of selenium-respiring bacteria

USGS Publications Warehouse

Oremland, R.S.; Blum, J.S.; Bindi, A.B.; Dowdle, P.R.; Herbel, M.; Stolz, J.F.

1999-01-01

Washed-cell suspensions of Sulfurospirillum barnesii reduced selenate [Se(VI)] when cells were cultured with nitrate, thiosulfate, arsenate, or fumarate as the electron acceptor. When the concentration of the electron donor was limiting, Se(VI) reduction in whole cells was approximately fourfold greater in Se(VI)-grown cells than was observed in nitrate-grown cells; correspondingly, nitrate reduction was ~11-fold higher in nitrate-grown cells than in Se(VI)-grown cells. However, a simultaneous reduction of nitrate and Se(VI) was observed in both cases. At nonlimiting electron donor concentrations, nitrate- grown cells suspended with equimolar nitrate and selenate achieved a complete reductive removal of nitrogen and selenium oxyanions, with the bulk of nitrate reduction preceding that of selenate reduction. Chloramphenicol did not inhibit these reductions. The Se(VI)-respiring haloalkaliphile Bacillus arsenicoselenatis gave similar results, but its Se(VI) reductase was not constitutive in nitrate-grown cells. No reduction of Se(VI) was noted for Bacillus selenitireducens, which respires selenite. The results of kinetic experiments with cell membrane preparations of S. barnesii suggest the presence of constitutive selenate and nitrate reduction, as well as an inducible, high- affinity nitrate reductase in nitrate-grown cells which also has a low affinity for selenate. The simultaneous reduction of micromolar Se(VI) in the presence of millimolar nitrate indicates that these organisms may have a functional use in bioremediating nitrate-rich, seleniferous agricultural wastewaters. Results with 75Se-selenate tracer show that these organisms can lower ambient Se(VI) concentrations to levels in compliance with new regulations proposed for release of selenium oxyanions into the environment.

Study on the influence of the decoking agent on the activity of limestone in wet flue gas desulfurization

NASA Astrophysics Data System (ADS)

Li, Qianjun; Xu, Dongyang; Wu, Yunxia; Yu, Jin

2017-01-01

Influence of the main components of decoking agent (magnesium nitrate, aluminum nitrate, copper nitrate, ammonium nitrate and actual decoking agent) on the activity of limestone is studied in laboratory by MET method. Results show that magnesium nitrate, ammonium nitrate and copper nitrate almost has no effect on the activity of limestone. With the concentration increasing, aluminum nitrate has an increasing inhibition on the dissolution of limestone. Fly ash has inhibition on dissolution of limestone due to the blockage of limestone pore by fly ash. The actual decoking agent has almost no effect on the limestone.

Organic Nitrates: A Complex Family of Atmospheric Trace Constituents

NASA Astrophysics Data System (ADS)

Ballschmiter, K.; Fischer, R. G.; Grünert, A.; Kastler, J.; Schneider, M.; Woidich, S.

2003-04-01

Biogenic and geogenic hydrocarbons are the precursors of organic nitrates that are formed as tropospheric photo-oxidation products in the presence of NOx. Air chemistry leads to a very complex pattern of nitric acid esters: alkyl nitrates, aryl-alkyl nitrates, and bifunctional nitrates like alkyl dinitrates, hydroxy alkyl nitrates and carbonyl alkyl nitrates. We have analyzed the pattern of organic nitrates in air samples after adsorption/thermal desorption (low volume sampling-LVS) or adsorption/solvent desorption (high volume sampling-HVS) by capillary gas chromatography with electron capture (ECD) and mass spectrometric detection (MSD) using air aliquotes of 100 up to 3000 liters on column. The complexity of the organic nitrates found in air requires a group pre-separation by normal phase liquid chromatography. A detection limit per compound of 0.005 ppt(v) is achieved by our approach. We have synthesized a broad spectrum of organic nitrates as reference compounds. Air samples were taken from central Europe, the US West (Utah, Nevada, California), and the North- and South Atlantic including Antarctica. Levels and patterns of the regional and global occurrence of the various groups of C1-C12 organic nitrates including dinitrates and hydroxy nitrates and nitrates of isoprene (2-methylbutadiene) are presented. Werner G., J. Kastler, R. Looser, K. Ballschmiter: "Organic nitrates of isoprene as atmospheric trace compounds" Angewandte Chemie - International Edition (1999) 38: 1634-1637. Woidich S., O. Froescheis, O. Luxenhofer, K. Ballschmiter: "EI- and NCI-mass spectrometry of arylalkyl nitrates and their occurrence in urban air" Fresenius J. Anal. Chem. (1999) 364 : 91-99. Kastler, J; Jarman, W; Ballschmiter, K.: "Multifunctional organic nitrates as constituents in European and US urban photo-smog" Fresenius J. Anal. Chem. (2000) 368:244-249. Schneider M., K. Ballschmiter: "C3-C14 alkyl nitrates in remote South Atlantic air" Chemosphere (1999) 38: 233-244. Fischer, R G; Kastler, J; Ballschmiter, K.: "Levels and pattern of alkyl nitrates, multifunctional alkyl nitrates, and halocarbons in the air over the Atlantic Ocean" J. Geophys. Research (2000) 105:14473-14494. Fischer R.G. , R. Weller, H.-W. Jacobi, K. Ballschmiter: "Levels and patterns of volatile organic nitrates and halocarbons in the air at Neumayer Station (70°), Antarctic" Chemosphere (2002) 48:981-992

Dietary nitrate protects submandibular gland from hyposalivation in ovariectomized rats via suppressing cell apoptosis.

PubMed

Xu, Yipu; Pang, Baoxing; Hu, Liang; Feng, Xiaoyu; Hu, Lei; Wang, Jingsong; Zhang, Chunmei; Wang, Songlin

2018-02-26

Xerostomia, a major oral symptom of menopause, is a subjective feeling of dry mouth associated with oral pain and difficulties in deglutition and speech, which significantly reduces patient's quality of life. Dietary nitrate, which can be converted to nitric oxide, has multiple physiological functions in the body, including antioxidant activity and vasodilatation; however, its protective effect against xerostomia remains poorly understood. The present study aimed to evaluate the effects of dietary nitrate on estrogen deficiency-induced xerostomia. We established an ovariectomized (OVX) rat model, which included five groups: sham-operated, OVX, OVX + 0.4 mM nitrate, OVX + 2 mM nitrate, and OVX + 4 mM nitrate (n = 6). After ovariectomy, animals in the nitrate treatment groups received appropriate amounts of sodium nitrate dissolved in distilled water for 3 months. The results showed that nitrate treatment reduced body weight and water intake, and increased serum nitrate and nitrite levels. Furthermore, nitrate uptake increased saliva secretion as evidenced by saliva flow rates and aquaporin 5 expression, and alleviated histological lesions as evidenced by reduction of the fibrotic area and cell atrophy in the salivary glands. Although protective effects of nitrate against estrogen deficiency-induced xerostomia were observed at all doses, treatment with 2 mM nitrate was more effective than that with 0.4 mM and 4 mM nitrate. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and caspase-3 expression analyses showed that nitrate also protected cells from apoptosis, possibly through upregulation of Cu-Zn superoxide dismutase (Cu-Zn SOD) known to inhibit oxidative stress-related apoptosis. Our findings indicate that nitrate could improve functional activity of the salivary glands in OVX rats by suppressing apoptosis and upregulating Cu-Zn SOD expression, suggesting that dietary nitrate may potentially prevent hyposalivation in menopausal women. Copyright © 2018. Published by Elsevier Inc.

Nitrate Reduction Functional Genes and Nitrate Reduction Potentials Persist in Deeper Estuarine Sediments. Why?

PubMed Central

Papaspyrou, Sokratis; Smith, Cindy J.; Dong, Liang F.; Whitby, Corinne; Dumbrell, Alex J.; Nedwell, David B.

2014-01-01

Denitrification and dissimilatory nitrate reduction to ammonium (DNRA) are processes occurring simultaneously under oxygen-limited or anaerobic conditions, where both compete for nitrate and organic carbon. Despite their ecological importance, there has been little investigation of how denitrification and DNRA potentials and related functional genes vary vertically with sediment depth. Nitrate reduction potentials measured in sediment depth profiles along the Colne estuary were in the upper range of nitrate reduction rates reported from other sediments and showed the existence of strong decreasing trends both with increasing depth and along the estuary. Denitrification potential decreased along the estuary, decreasing more rapidly with depth towards the estuary mouth. In contrast, DNRA potential increased along the estuary. Significant decreases in copy numbers of 16S rRNA and nitrate reducing genes were observed along the estuary and from surface to deeper sediments. Both metabolic potentials and functional genes persisted at sediment depths where porewater nitrate was absent. Transport of nitrate by bioturbation, based on macrofauna distributions, could only account for the upper 10 cm depth of sediment. A several fold higher combined freeze-lysable KCl-extractable nitrate pool compared to porewater nitrate was detected. We hypothesised that his could be attributed to intracellular nitrate pools from nitrate accumulating microorganisms like Thioploca or Beggiatoa. However, pyrosequencing analysis did not detect any such organisms, leaving other bacteria, microbenthic algae, or foraminiferans which have also been shown to accumulate nitrate, as possible candidates. The importance and bioavailability of a KCl-extractable nitrate sediment pool remains to be tested. The significant variation in the vertical pattern and abundance of the various nitrate reducing genes phylotypes reasonably suggests differences in their activity throughout the sediment column. This raises interesting questions as to what the alternative metabolic roles for the various nitrate reductases could be, analogous to the alternative metabolic roles found for nitrite reductases. PMID:24728381

Oxygen and nitrogen isotopic composition of nitrate in commercial fertilizers, nitric acid, and reagent salts.

PubMed

Michalski, Greg; Kolanowski, Michelle; Riha, Krystin M

2015-01-01

Nitrate is a key component of synthetic fertilizers that can be beneficial to crop production in agro-ecosystems, but can also cause damage to natural ecosystems if it is exported in large amounts. Stable isotopes, both oxygen and nitrogen, have been used to trace the sources and fate of nitrate in various ecosystems. However, the oxygen isotope composition of synthetic and organic nitrates is poorly constrained. Here, we present a study on the N and O isotope composition of nitrate-based fertilizers. The δ(15)N values of synthetic and natural nitrates were 0 ± 2 ‰ similar to the air N2 from which they are derived. The δ(18)O values of synthetic nitrates were 23 ± 3 ‰, similar to air O2, and natural nitrate fertilizer δ(18)O values (55 ± 5 ‰) were similar to those observed in atmospheric nitrate. The Δ(17)O values of synthetic fertilizer nitrate were approximately zero following a mass-dependent isotope relationship, while natural nitrate fertilizers had Δ(17)O values of 18 ± 2 ‰ similar to nitrate produced photochemically in the atmosphere. These narrow ranges of values can be used to assess the amount of nitrate arising from fertilizers in mixed systems where more than one nitrate source exists (soil, rivers, and lakes) using simple isotope mixing models.

Challenges with nitrate therapy and nitrate tolerance: prevalence, prevention, and clinical relevance.

PubMed

Thadani, Udho

2014-08-01

Nitrate therapy has been an effective treatment for ischemic heart disease for over 100 years. The anti-ischemic and exercise-promoting benefits of sublingually administered nitrates are well established. Nitroglycerin is indicated for the relief of an established attack of angina and for prophylactic use, but its effects are short lived. In an effort to increase the duration of beneficial effects, long-acting orally administered and topical applications of nitrates have been developed; however, following their continued or frequent daily use, patients soon develop tolerance to these long-acting nitrate preparations. Once tolerance develops, patients begin losing the protective effects of the long-acting nitrate therapy. By providing a nitrate-free interval, or declining nitrate levels at night, one can overcome or reduce the development of tolerance, but cannot provide 24-h anti-anginal and anti-ischemic protection. In addition, patients may be vulnerable to occurrence of rebound angina and myocardial ischemia during periods of absent nitrate levels at night and early hours of the morning, and worsening of exercise capacity prior to the morning dose of the medication. This has been a concern with nitroglycerin patches but not with oral formulations of isosorbide-5 mononitrates, and has not been adequately studied with isosorbide dinitrate. This paper describes problems associated with nitrate tolerance, reviews mechanisms by which nitrate tolerance and loss of efficacy develop, and presents strategies to avoid nitrate tolerance and maintain efficacy when using long-acting nitrate formulations.

The Gne M712T mouse as a model for human glomerulopathy.

PubMed

Kakani, Sravan; Yardeni, Tal; Poling, Justin; Ciccone, Carla; Niethamer, Terren; Klootwijk, Enriko D; Manoli, Irini; Darvish, Daniel; Hoogstraten-Miller, Shelley; Zerfas, Patricia; Tian, E; Ten Hagen, Kelly G; Kopp, Jeffrey B; Gahl, William A; Huizing, Marjan

2012-04-01

Pathological glomerular hyposialylation has been implicated in certain unexplained glomerulopathies, including minimal change nephrosis, membranous glomerulonephritis, and IgA nephropathy. We studied our previously established mouse model carrying a homozygous mutation in the key enzyme of sialic acid biosynthesis, N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase. Mutant mice died before postnatal day 3 (P3) from severe glomerulopathy with podocyte effacement and segmental glomerular basement membrane splitting due to hyposialylation. Administration of the sialic acid precursor N-acetylmannosamine (ManNAc) led to improved sialylation and survival of mutant pups beyond P3. We determined the onset of the glomerulopathy in the embryonic stage. A lectin panel, distinguishing normally sialylated from hyposialylated glycans, used WGA, SNA, PNA, Jacalin, HPA, and VVA, indicating glomerular hyposialylation of predominantly O-linked glycoproteins in mutant mice. The glomerular glycoproteins nephrin and podocalyxin were hyposialylated in this unique murine model. ManNAc treatment appeared to ameliorate the hyposialylation status of mutant mice, indicated by a lectin histochemistry pattern similar to that of wild-type mice, with improved sialylation of both nephrin and podocalyxin, as well as reduced albuminuria compared with untreated mutant mice. These findings suggest application of our lectin panel for categorizing human kidney specimens based on glomerular sialylation status. Moreover, the partial restoration of glomerular architecture in ManNAc-treated mice highlights ManNAc as a potential treatment for humans affected with disorders of glomerular hyposialylation. Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

FolC2-mediated folate metabolism contributes to suppression of inflammation by probiotic Lactobacillus reuteri.

PubMed

Thomas, Carissa M; Saulnier, Delphine M A; Spinler, Jennifer K; Hemarajata, Peera; Gao, Chunxu; Jones, Sara E; Grimm, Ashley; Balderas, Miriam A; Burstein, Matthew D; Morra, Christina; Roeth, Daniel; Kalkum, Markus; Versalovic, James

2016-10-01

Bacterial-derived compounds from the intestinal microbiome modulate host mucosal immunity. Identification and mechanistic studies of these compounds provide insights into host-microbial mutualism. Specific Lactobacillus reuteri strains suppress production of the proinflammatory cytokine, tumor necrosis factor (TNF), and are protective in a mouse model of colitis. Human-derived L. reuteri strain ATCC PTA 6475 suppresses intestinal inflammation and produces 5,10-methenyltetrahydrofolic acid polyglutamates. Insertional mutagenesis identified the bifunctional dihydrofolate synthase/folylpolyglutamate synthase type 2 (folC2) gene as essential for 5,10-methenyltetrahydrofolic acid polyglutamate biosynthesis, as well as for suppression of TNF production by activated human monocytes, and for the anti-inflammatory effect of L. reuteri 6475 in a trinitrobenzene sulfonic acid-induced mouse model of acute colitis. In contrast, folC encodes the enzyme responsible for folate polyglutamylation but does not impact TNF suppression by L. reuteri. Comparative transcriptomics between wild-type and mutant L. reuteri strains revealed additional genes involved in immunomodulation, including previously identified hdc genes involved in histidine to histamine conversion. The folC2 mutant yielded diminished hdc gene cluster expression and diminished histamine production, suggesting a link between folate and histadine/histamine metabolism. The identification of genes and gene networks regulating production of bacterial-derived immunoregulatory molecules may lead to improved anti-inflammatory strategies for digestive diseases. © 2016 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

Antitumor activity of a novel and orally available inhibitor of serine palmitoyltransferase

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

Yaguchi, Masahiro; Shibata, Sachio; Satomi, Yoshinori

Metabolic reprogramming is an essential hallmark of neoplasia. Therefore, targeting cancer metabolism, including lipid synthesis, has attracted much interest in recent years. Serine palmitoyltransferase (SPT) plays a key role in the initial and rate-limiting step of de novo sphingolipid biosynthesis, and inhibiting SPT activity prevents the proliferation of certain cancer cells. Here, we identified a novel and orally available SPT inhibitor, compound-2. Compound-2 showed an anti-proliferative effect in several cancer cell models, reducing the levels of the sphingolipids ceramide and sphingomyelin. In the presence of compound-2, exogenously added S1P partially compensated the intracellular sphingolipid levels through the salvage pathway bymore » partially rescuing compound-2-induced cytotoxicity. This suggested that the mechanism underlying the anti-proliferative effect of compound-2 involved the reduction of sphingolipid levels. Indeed, compound-2 promoted multinuclear formation with reduced endogenous sphingomyelin levels specifically in a compound-2-sensitive cell line, indicating that the effect was induced by sphingolipid reduction. Furthermore, compound-2 showed potent antitumor activity without causing significant body weight loss in the PL-21 acute myeloid leukemia mouse xenograft model. Therefore, SPT may be an attractive therapeutic anti-cancer drug target for which compound-2 may be a promising new drug. - Highlights: • We discovered compound-2, a novel and orally available SPT inhibitor. • Compound-2 was cytotoxic against PL-21 acute myeloid leukemia cells. • Compound-2 showed antitumor activity in the PL-21 mouse xenograft model.« less

Nicotinamide Forestalls Pathology and Cognitive Decline in Alzheimer Mice: Evidence for Improved Neuronal Bioenergetics and Autophagy Procession

PubMed Central

Liu, Dong; Pitta, Michael; Jiang, Haiyang; Lee, Jong-Hwan; Zhang, Guofeng; Chen, Xinzhi; Kawamoto, Elisa M.; Mattson, Mark P.

2012-01-01

Impaired brain energy metabolism and oxidative stress are implicated in cognitive decline and the pathological accumulations of amyloid β-peptide (Aβ) and hyperphosphorylated Tau (p-Tau) in Alzheimer's disease (AD). To determine whether improving brain energy metabolism will forestall disease progress in AD, the impact of the NAD+ precursor nicotinamide on brain cell mitochondrial function and macroautophagy, bioenergetics-related signaling and cognitive performance were studied in cultured neurons and in a mouse model of AD. Oxidative stress resulted in decreased mitochondrial mass, mitochondrial degeneration and autophagosome accumulation in neurons. Nicotinamide preserved mitochondrial integrity and autophagy function, and reduced neuronal vulnerability to oxidative/metabolic insults and Aβ toxicity. NAD+ biosynthesis, autophagy and PI3K signaling were required for the neuroprotective action of nicotinamide. Treatment of 3xTgAD mice with nicotinamide for 8 months resulted in improved cognitive performance, and reduced Aβ and p-Tau pathologies in hippocampus and cerebral cortex. Nicotinamide treatment preserved mitochondrial integrity, and improved autophagy-lysosome procession by enhancing lysosome/autolysosome acidification to reduce autophagosome accumulation. Treatment of 3xTgAD mice with nicotinamide resulted in elevated levels of activated neuroplasticity-related kinases (Akt and ERKs) and the transcription factor cyclic AMP response element-binding protein in the hippocampus and cerebral cortex. Thus, nicotinamide suppresses AD pathology and cognitive decline in a mouse model of AD by a mechanism involving improved brain bioenergetics with preserved functionality of mitochondria and the autophagy system. PMID:23273573

IDH1 deficiency attenuates gluconeogenesis in mouse liver by impairing amino acid utilization.

PubMed

Ye, Jing; Gu, Yu; Zhang, Feng; Zhao, Yuanlin; Yuan, Yuan; Hao, Zhenyue; Sheng, Yi; Li, Wanda Y; Wakeham, Andrew; Cairns, Rob A; Mak, Tak W

2017-01-10

Although the enzymatic activity of isocitrate dehydrogenase 1 (IDH1) was defined decades ago, its functions in vivo are not yet fully understood. Cytosolic IDH1 converts isocitrate to α-ketoglutarate (α-KG), a key metabolite regulating nitrogen homeostasis in catabolic pathways. It was thought that IDH1 might enhance lipid biosynthesis in liver or adipose tissue by generating NADPH, but we show here that lipid contents are relatively unchanged in both IDH1-null mouse liver and IDH1-deficient HepG2 cells generated using the CRISPR-Cas9 system. Instead, we found that IDH1 is critical for liver amino acid (AA) utilization. Body weights of IDH1-null mice fed a high-protein diet (HPD) were abnormally low. After prolonged fasting, IDH1-null mice exhibited decreased blood glucose but elevated blood alanine and glycine compared with wild-type (WT) controls. Similarly, in IDH1-deficient HepG2 cells, glucose consumption was increased, but alanine utilization and levels of intracellular α-KG and glutamate were reduced. In IDH1-deficient primary hepatocytes, gluconeogenesis as well as production of ammonia and urea were decreased. In IDH1-deficient whole livers, expression levels of genes involved in AA metabolism were reduced, whereas those involved in gluconeogenesis were up-regulated. Thus, IDH1 is critical for AA utilization in vivo and its deficiency attenuates gluconeogenesis primarily by impairing α-KG-dependent transamination of glucogenic AAs such as alanine.

Disrupted SOX10 function causes spongiform neurodegeneration in gray tremor mice

PubMed Central

Anderson, Sarah R.; Lee, Inyoul; Ebeling, Christine; Stephenson, Dennis A.; Schweitzer, Kelsey M.; Baxter, David; Moon, Tara M.; LaPierre, Sarah; Jaques, Benjamin; Silvius, Derek; Wegner, Michael; Hood, Leroy E.; Carlson, George; Gunn, Teresa M.

2014-01-01

Mice homozygous for the gray tremor (gt) mutation have a pleiotropic phenotype that includes pigmentation defects, megacolon, whole body tremors, sporadic seizures, hypo- and dysmyelination of the CNS and PNS, vacuolation of the CNS, and early death. Vacuolation similar to that caused by prions was originally reported to be transmissible, but subsequent studies showed the inherited disease was not infectious. The gt mutation mapped to distal mouse chromosome 15, to the same region as Sox10, which encodes a transcription factor with essential roles in neural crest survival and differentiation. As dominant mutations in mouse or human SOX10 cause white spotting and intestinal aganglionosis, we screened the Sox10 coding region for mutations in gt/gt DNA. An adenosine to guanine transversion was identified in exon 2 that changes a highly conserved glutamic acid residue in the SOX10 DNA binding domain to glycine. This mutant allele was not seen in wildtype mice, including the related GT/Le strain, and failed to complement a Sox10 null allele. Gene expression analysis revealed significant down-regulation of genes involved in myelin lipid biosynthesis pathways in gt/gt brains. Knockout mice for some of these genes develop CNS vacuolation and/or myelination defects, suggesting that their down-regulation may contribute to these phenotypes in gt mutants and could underlie the neurological phenotypes associated with Peripheral demyelinating neuropathy-Central dysmyelinating leukodystrophy-Waardenburg syndrome-Hirschsprung (PCWH) disease, caused by mutations in human SOX10. PMID:25399070

Presence of nitrate NO 3 a ects animal production, photocalysis is a possible solution

NASA Astrophysics Data System (ADS)

Barba-Molina, Heli; Barba-Ortega, J.; Joya, M. R.

2016-02-01

Farmers and ranchers depend on the successful combination of livestock and crops. However, they have lost in the production by nitrate pollution. Nitrate poisoning in cattle is caused by the consumption of an excessive amount of nitrate or nitrite from grazing or water. Both humans and livestock can be affected. It would appear that well fertilised pasture seems to take up nitrogen from the soil and store it as nitrate in the leaf. Climatic conditions, favour the uptake of nitrate. Nitrate poisoning is a noninfectious disease condition that affects domestic ruminants. It is a serious problem, often resulting in the death of many animals. When nitrogen fertilizers are used to enrich soils, nitrates may be carried by rain, irrigation and other surface waters through the soil into ground water. Human and animal wastes can also contribute to nitrate contamination of ground water. A possible method to decontaminate polluted water by nitrates is with methods of fabrication of zero valent iron nanoparticles (FeNps) are found to affect their efficiency in nitrate removal from water.

Total salivary nitrates and nitrites in oral health and periodontal disease.

PubMed

Sánchez, Gabriel A; Miozza, Valeria A; Delgado, Alejandra; Busch, Lucila

2014-01-30

It is well known that nitrites are increased in saliva from patients with periodontal disease. In the oral cavity, nitrites may derive partly from the reduction of nitrates by oral bacteria. Nitrates have been reported as a defence-related mechanism. Thus, the aim of the present study was to determine the salivary levels of total nitrate and nitrite and their relationship, in unstimulated and stimulated saliva from periodontal healthy subjects, and from patients with chronic periodontal disease. Nitrates and nitrites were determined in saliva from thirty healthy subjects and forty-four patients with periodontal disease. A significant increase in salivary nitrates and nitrites was observed. Nitrates and nitrites concentration was related to clinical attachment level (CAL). A positive and significant Pearson's correlation was found between salivary total nitrates and nitrites. Periodontal treatment induced clinical improvement and decreased nitrates and nitrites. It is concluded that salivary nitrates and nitrites increase, in patients with periodontal disease, could be related to defence mechanisms. The possibility that the salivary glands respond to oral infectious diseases by increasing nitrate secretion should be explored further. Copyright © 2013 Elsevier Inc. All rights reserved.

Assessing the Role of Sewers and Atmospheric Deposition as Nitrate Contamination Sources to Urban Surface Waters using Stable Nitrate Isotopes

NASA Astrophysics Data System (ADS)

Sikora, M. T.; Elliott, E. M.

2009-12-01

Excess nitrate (NO3-) contributes to the overall degraded quality of streams in many urban areas. These systems are often dominated by impervious surfaces and storm sewers that can route atmospherically deposited nitrogen, from both wet and dry deposition, to waterways. Moreover, in densely populated watersheds there is the potential for interaction between urban waterways and sewer systems. The affects of accumulated nitrate in riverine and estuary systems include low dissolved oxygen, loss of species diversity, increased mortality of aquatic species, and general eutrophication of the waterbody. However, the dynamics of nitrate pollution from each source and it’s affect on urban waterways is poorly constrained. The isotopes of nitrogen and oxygen in nitrate have been proven effective in helping to distinguish contamination sources to ground and surface waters. In order to improve our understanding of urban nitrate pollution sources and dynamics, we examined nitrate isotopes (δ15N and δ18O) in base- and stormflow samples collected over a two-year period from a restored urban stream in Pittsburgh, Pennsylvania (USA). Nine Mile Run drains a 1,600 hectare urban watershed characterized by 38% impervious surface cover. Prior work has documented high nitrate export from the watershed (~19 kg NO3- ha-1 yr-1). Potential nitrate sources to the watershed include observed sewer overflows draining directly to the stream, as well as atmospheric deposition (~23 kg NO3- ha-1 yr-1). In this and other urban systems with high percentages of impervious surfaces, there is likely minimal input from nitrate derived from soil or fertilizer. In this presentation, we examine spatial and temporal patterns in nitrate isotopic composition collected at five locations along Nine Mile Run characterized by both sanitary and combined-sewer cross-connections. Preliminary isotopic analysis of low-flow winter streamwater samples suggest nitrate export from Nine Mile Run is primarily influenced by inputs of human waste despite high rates of atmospheric nitrate deposition. Further isotopic analysis of nitrate will examine seasonal variations in nitrate sources; compare nitrate dynamics and sources during low- versus high-flows, and the influence of interannual climatic variability on nitrate export.

AaEIN3 Mediates the Downregulation of Artemisinin Biosynthesis by Ethylene Signaling Through Promoting Leaf Senescence in Artemisia annua.

PubMed

Tang, Yueli; Li, Ling; Yan, Tingxiang; Fu, Xueqing; Shi, Pu; Shen, Qian; Sun, Xiaofen; Tang, Kexuan

2018-01-01

Artemisinin is an important drug for malaria treatment, which is exclusively produced in Artemisia annua . It's important to dissect the regulatory mechanism of artemisinin biosynthesis by diverse plant hormones and transcription factors. Our study shows ethylene, a plant hormone which accelerates flower and leaf senescence and fruit ripening, suppressed the expression of genes encoding three key enzymes ADS, DBR2, CYP71AV1, and a positive regulator AaORA involved in artemisinin biosynthesis. Then we isolated the gene encoding ETHYLENE-INSENSITIVE3 (EIN3), a key transcription factor in ethylene signaling pathway, by screening the transcriptome and genome database from Artemisia annua , named AaEIN3 . Overexpressing AaEIN3 suppressed artemisinin biosynthesis, while repressing its expression with RNAi enhanced artemisinin biosynthesis in Artemisia annua , indicating AaEIN3 negatively regulates artemisinin biosynthesis. Further study showed the downregulation of artemisinin biosynthesis by ethylene required the mediation of AaEIN3. AaEIN3 could accelerate leaf senescence, and leaf senescence attenuated the expression of ADS, DBR2, CYP71AV1 , and AaORA that are involved in artemisinin biosynthesis. Collectively, our study demonstrated a negative correlation between ethylene signaling and artemisinin biosynthesis, which is ascribed to AaEIN3-induced senescence process of leaves. Our work provided novel knowledge on the regulatory network of plant hormones for artemisinin metabolic pathway.

AaEIN3 Mediates the Downregulation of Artemisinin Biosynthesis by Ethylene Signaling Through Promoting Leaf Senescence in Artemisia annua

PubMed Central

Tang, Yueli; Li, Ling; Yan, Tingxiang; Fu, Xueqing; Shi, Pu; Shen, Qian; Sun, Xiaofen; Tang, Kexuan

2018-01-01

Artemisinin is an important drug for malaria treatment, which is exclusively produced in Artemisia annua. It’s important to dissect the regulatory mechanism of artemisinin biosynthesis by diverse plant hormones and transcription factors. Our study shows ethylene, a plant hormone which accelerates flower and leaf senescence and fruit ripening, suppressed the expression of genes encoding three key enzymes ADS, DBR2, CYP71AV1, and a positive regulator AaORA involved in artemisinin biosynthesis. Then we isolated the gene encoding ETHYLENE-INSENSITIVE3 (EIN3), a key transcription factor in ethylene signaling pathway, by screening the transcriptome and genome database from Artemisia annua, named AaEIN3. Overexpressing AaEIN3 suppressed artemisinin biosynthesis, while repressing its expression with RNAi enhanced artemisinin biosynthesis in Artemisia annua, indicating AaEIN3 negatively regulates artemisinin biosynthesis. Further study showed the downregulation of artemisinin biosynthesis by ethylene required the mediation of AaEIN3. AaEIN3 could accelerate leaf senescence, and leaf senescence attenuated the expression of ADS, DBR2, CYP71AV1, and AaORA that are involved in artemisinin biosynthesis. Collectively, our study demonstrated a negative correlation between ethylene signaling and artemisinin biosynthesis, which is ascribed to AaEIN3-induced senescence process of leaves. Our work provided novel knowledge on the regulatory network of plant hormones for artemisinin metabolic pathway. PMID:29675029

Effects of nitrate addition to a diet on fermentation and microbial populations in the rumen of goats, with special reference to Selenomonas ruminantium having the ability to reduce nitrate and nitrite.

PubMed

Asanuma, Narito; Yokoyama, Shota; Hino, Tsuneo

2015-04-01

This study investigated the effects of dietary nitrate addition on ruminal fermentation characteristics and microbial populations in goats. The involvement of Selenomonas ruminantium in nitrate and nitrite reduction in the rumen was also examined. As the result of nitrate feeding, the total concentration of ruminal volatile fatty acids decreased, whereas the acetate : propionate ratio and the concentrations of ammonia and lactate increased. Populations of methanogens, protozoa and fungi, as estimated by real-time PCR, were greatly decreased as a result of nitrate inclusion in the diet. There was modest or little impact of nitrate on the populations of prevailing species or genus of bacteria in the rumen, whereas Streptococcus bovis and S. ruminantium significantly increased. Both the activities of nitrate reductase (NaR) and nitrite reductase (NiR) per total mass of ruminal bacteria were increased by nitrate feeding. Quantification of the genes encoding NaR and NiR by real-time PCR with primers specific for S. ruminantium showed that these genes were increased by feeding nitrate, suggesting that the growth of nitrate- and nitrite-reducing S. ruminantium is stimulated by nitrate addition. Thus, S. ruminantium is likely to play a major role in nitrate and nitrite reduction in the rumen. © 2014 Japanese Society of Animal Science.

Test/QA Plan for Verification of Nitrate Sensors for Groundwater Remediation Monitoring

EPA Science Inventory

A submersible nitrate sensor is capable of collecting in-situ measurements of dissolved nitrate concentrations in groundwater. Although several types of nitrate sensors currently exist, this verification test will focus on submersible sensors equipped with a nitrate-specific ion...

Ipso Nitration. Regiospecific Nitration via Ipso Nitration Products.

DTIC Science & Technology

1985-05-20

products of nitration of alkylbenzenes and alkylphenol derivatives. The general pattern envisioned is shown in Scheme 1. In order to realize this...we have also explored solid state nitration of various alkylphenols . This procedure involves adsorbing concentrated nitric acid on alumina, followed

Cytosolic invertase contributes to the supply of substrate for cellulose biosynthesis in developing wood.

PubMed

Rende, Umut; Wang, Wei; Gandla, Madhavi Latha; Jönsson, Leif J; Niittylä, Totte

2017-04-01

Carbon for cellulose biosynthesis is derived from sucrose. Cellulose is synthesized from uridine 5'-diphosphoglucose (UDP-glucose), but the enzyme(s) responsible for the initial sucrose cleavage and the source of UDP-glucose for cellulose biosynthesis in developing wood have not been defined. We investigated the role of CYTOSOLIC INVERTASEs (CINs) during wood formation in hybrid aspen (Populus tremula × tremuloides) and characterized transgenic lines with reduced CIN activity during secondary cell wall biosynthesis. Suppression of CIN activity by 38-55% led to a 9-13% reduction in crystalline cellulose. The changes in cellulose were reflected in reduced diameter of acid-insoluble cellulose microfibrils and increased glucose release from wood upon enzymatic digestion of cellulose. Reduced CIN activity decreased the amount of the cellulose biosynthesis precursor UDP-glucose in developing wood, pointing to the likely cause of the cellulose phenotype. The findings suggest that CIN activity has an important role in the cellulose biosynthesis of trees, and indicate that cellulose biosynthesis in wood relies on a quantifiable UDP-glucose pool. The results also introduce a concept of altering cellulose microfibril properties by modifying substrate supply to cellulose biosynthesis. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

Habitual Dietary Nitrate Intake in Highly Trained Athletes.

PubMed

Jonvik, Kristin L; Nyakayiru, Jean; van Dijk, Jan-Willem; Wardenaar, Floris C; van Loon, Luc J C; Verdijk, Lex B

2017-04-01

Although beetroot juice, as a nitrate carrier, is a popular ergogenic supplement among athletes, nitrate is consumed through the regular diet as well. We aimed to assess the habitual dietary nitrate intake and identify the main contributing food sources in a large group of highly trained athletes. Dutch highly trained athletes (226 women and 327 men) completed 2-4 web-based 24-hr dietary recalls and questionnaires within a 2- to 4-week period. The nitrate content of food products and food groups was determined systematically based on values found in regulatory reports and scientific literature. These were then used to calculate each athlete's dietary nitrate intake from the web-based recalls. The median[IQR] habitual nitrate intake was 106[75-170] mg/d (range 19-525 mg/d). Nitrate intake correlated with energy intake (ρ = 0.28, p < .001), and strongly correlated with vegetable intake (ρ = 0.78, p < .001). In accordance, most of the dietary nitrate was consumed through vegetables, potatoes and fruit, accounting for 74% of total nitrate intake, with lettuce and spinach contributing most. When corrected for energy intake, nitrate intake was substantially higher in female vs male athletes (12.8[9.2-20.0] vs 9.4[6.2-13.8] mg/MJ; p < .001). This difference was attributed to the higher vegetable intake in female vs male athletes (150[88-236] vs 114[61-183] g/d; p < .001). In conclusion, median daily intake of dietary nitrate in highly trained athletes was 106 mg, with large interindividual variation. Dietary nitrate intake was strongly associated with the intake of vegetables. Increasing the intake of nitrate-rich vegetables in the diet might serve as an alternative strategy for nitrate supplementation.

In low transpiring conditions, uncoupling the BnNrt2.1 and BnNrt1.1 NO3- transporters by glutamate treatment reveals the essential role of BnNRT2.1 for nitrate uptake and the nitrate-signaling cascade during growth

PubMed Central

Leblanc, Antonin; Segura, Raphaël; Deleu, Carole; Le Deunff, Erwan

2013-01-01

In plants, the nitrate transporters, NRT1.1 and NRT2.1, are mainly responsible for nitrate uptake. Intriguingly, both nitrate transporters are located in a complementary manner in different cells layers of the mature root suggesting that their coordination should occur during nitrate uptake and plant growth. This hypothesis was examined on 5-d-old rape seedlings grown on agar medium supplemented with 1 or 5mM nitrate. Seedlings were treated with increasing potassium glutamate concentrations in order to uncouple the two nitrate transporters by inhibiting BnNRT2.1 expression and activity specifically. In both nitrate treatments, increasing the glutamate concentrations from 0.5 to 10mM induced a reduction in 15NO3- uptake and an inhibition of N assimilation. The decrease in 15NO3- uptake was caused by downregulation of BnNRT2.1 expression but surprisingly it was not compensated by the upregulation of BnNRT1.1. This created an unprecedented physiological situation where the effects of the nitrate signal on shoot growth were solely modulated by nitrate absorption. In these conditions, the osmotic water flow for volumetric shoot growth was mainly dependent on active nitrate transport and nitrate signaling. This behavior was confirmed by the allometric relationships found between changes in the root length with 15N and water accumulation in the shoot. These findings demonstrate that the BnNRT2.1 transporter is essential for nitrate uptake and growth, and renew the question of the respective roles of the NRT2.1 and NRT1.1 transporters in nitrate uptake and sensing at the whole plant level. PMID:23299418

Distinguishing summertime atmospheric production of nitrate across the East Antarctic Ice Sheet

NASA Astrophysics Data System (ADS)

Shi, G.; Buffen, A. M.; Ma, H.; Hu, Z.; Sun, B.; Li, C.; Yu, J.; Ma, T.; An, C.; Jiang, S.; Li, Y.; Hastings, M. G.

2018-06-01

Surface snow and atmospheric samples collected along a traverse from the coast to the ice sheet summit (Dome A) are used to investigate summertime atmospheric production of nitrate (NO3-) across East Antarctica. The strong relationship observed between δ15N and δ18O of nitrate in the surface snow suggests a large (lesser) extent of nitrate photolysis in the interior (coastal) region. A linear correlation between the oxygen isotopes of nitrate (δ18O and Δ17O) indicates mixing of various oxidants that react with NOx (NOx = NO + NO2) to produce atmospheric nitrate. On the plateau, the isotopes of snow nitrate are best explained by local reoxidation chemistry of NOx, possibly occurring in both condensed and gas phases. Nitrate photolysis results in redistribution of snow nitrate, and the plateau snow is a net exporter of nitrate and its precursors. Our results suggest that while snow-sourced NOx from the plateau due to photolysis is a significant input to the nitrate budget in coastal snow (up to ∼35%), tropospheric transport from mid-low latitudes dominates (∼65%) coastal snow nitrate. The linear relationship of δ18O vs. Δ17O of the snow nitrate suggests a predominant role of hydroxyl radical (OH) and ozone (O3) in nitrate production, although a high Δ17O(O3) is required to explain the observations. Across Antarctica the oxygen isotope composition of OH appears to be dominated by exchange with water vapor, despite the very dry environment. One of the largest uncertainties in quantifying nitrate production pathways is the limited knowledge of atmospheric oxidant isotopic compositions.

Nitrate Utilization by the Diatom Skeletonema costatum

PubMed Central

Serra, Juan L.; Llama, Maria J.; Cadenas, Eduardo

1978-01-01

Nitrate uptake has been studied in nitrogen-deficient cells of the marine diatom Skeletonema costatum. When these cells are incubated in the presence of nitrate, this ion is quickly taken up from the medium, and nitrite is excreted by the cells. Nitrite is excreted following classical saturation kinetics, its rate being independent of nitrate concentration in the incubation medium for nitrate concentration values higher than 3 micromolar. Nitrate uptake shows mixed-transfer kinetics, which can be attributed to the simultaneous contributions of mediated and diffusion transfer. Cycloheximide and p-hydroxymercuribenzoate inhibit the carrier-mediated contribution to nitrate uptake, without affecting the diffusion component. When cells are preincubated with nitrate, the net nitrogen uptake is increased. PMID:16660652

Mechanisms of nitrate capture in biochar: Are they related to biochar properties, post-treatment and soil environment?

NASA Astrophysics Data System (ADS)

Cimo, Giulia; Haller, Andreas; Spokas, Kurt; Novak, Jeff; Ippolito, Jim; Löhnertz, Otmar; Kammann, Claudia

2017-04-01

Biochar use in soils is assumed to increase soil fertility and the efficiency of nutrient use, particularly nitrogen. It was demonstrated recently that biochar is able to capture considerable amounts of the mobile anion nitrate which was observed in co-composted as well as field aged biochar1,2. Moreover the nitrate was not sufficiently extractable with standard methods from biochar particles; extractions had to be repeated to effectively remove the nitrate1. Subsequently the co-composted nitrate-enriched biochar stimulated plant growth due to N supply to the plants2. However, in a field study in sandy soil in Germany, a different biochar also captured nitrate, increasing the topsoil nitrate concentration and likely reducing nitrate leaching to subsoils1. This was particularly seen after a dry year in the re-picked and analysed particles. However, in the field experiment this aged, nitrate-enriched biochar did not improve crop yields3. To better understand the way biochar interacts with nitrate we undertook several laboratory experiments with 13 well characterized biochars produced from cypress, pine and grapewood at 350, 500, 700 and 900 °C including one Kon-Tiki produced grapewood biochar (600-700°C). Our results showed that (1) pure, pristine (not post-treated) biochar captured more nitrate when they were air-moist and not totally dry; that (2) letting biochar particles dry in nitrate solution forces more nitrate into biochar particles than incubating them in the solution, but (3) that shaking during drying nevertheless caused a higher nitrate uptake into biochar particles; that(4) the counter ion K+ in nitrate solution was more effective than Na+ for N-loading of biochar; (5)that drying a soil-biochar mix in nitrate solution produced a higher nitrate loading of the mixture (i.e. the biochar) than drying both components separately in the same solution; (6)that a higher biochar production temperature caused higher nitrate capture up to 700-900°C. Furthermore we found (7)that this captured nitrate was well protected against leaching, (8)that repeated drying-wetting cycles increased nitrate capture, with the amount protected against leaching remaining more or less constant; and (9) that an organic "coating" (or application of the nitrate in an organic solution, here: black tea) increased biochars' capability of nitrate capture. Our results thus underline that the phenomenon of nitrate capture is not purely due to ionic mechanisms but may partly rely on physical interactions and the pore structure of the biochar. Acknowledgement: JC acknowledges funding by the COST action TD1107 (short term scientific mission), CK acknowledges the financial support of DFG grant no. Ka3442/1-1 and of the HMWK Hessia funded OptiChar4EcoVin project. 1-Haider, G., Steffens, D., Müller, C. & Kammann, C. I. Standard extraction methods may underestimate nitrate stocks captured by field aged biochar. J. Environ. Qual. 45, 1196-1204 (2016). 2-Kammann, C. I. et al. Plant growth improvement mediated by nitrate capture in co-composted biochar. Scientific Reports 5, doi: 10.1038/srep11080 (2015). 3-Haider, G., Steffens, D., Moser, G., Müller, C. & Kammann, C. I. Biochar reduced nitrate leaching and improved soil moisture content without yield improvements in a four-year field study. Agri. Ecosys. Environ. 237, 80-94 (2017).

The preservation of long-range transported nitrate in snow at Summit, Greenland (Invited)

NASA Astrophysics Data System (ADS)

Hastings, M. G.

2013-12-01

Nitrate is one of the major anions found in polar and alpine snow, both today and in the past. Deposition of nitrate to snow surfaces results from reactions of nitrogen oxides (NOx) with oxidants in the atmosphere, resulting in the production of HNO3 that is incorporated into precipitation or reacts on the surface of particles. Several factors motivate studying nitrate concentration in ice cores including reconstructing past levels of NOx, tropospheric oxidant concentrations and natural variability in NOx sources. The link between the atmospheric concentration of NOx and nitrate concentration in ice core records is problematic because post-depositional processing, such as photolysis and evaporation, can impact the concentration of nitrate in snow. Recent work has shown that the isotopic ratios of nitrate (15N/14N, 18O/16O, 17O/16O) can be a powerful tool for tracing post-depositional loss of nitrate from surface snow. The isotopic composition of nitrate has been shown to contain information about the source of the nitrate (i.e, NOx sources) and the oxidation processes that convert NOx to nitrate in the atmosphere prior to deposition. Results from a number of studies at Summit, Greenland reveal limited loss of nitrate from surface snow during highly photoactive periods, and the oxygen isotopic signatures in snow nitrate appear to be representative of atmospheric deposition of nitrate from outside of Summit. Higher than expected oxygen isotope ratios (18O/16O, 17O/16O) found in Summit summertime nitrate were expected to be dependent upon local photochemistry in which nitrate in the snow is photolyzed to NOx that is then oxidized above the snow by BrO to reform nitrate (i.e., BrONO2). However, the oxygen isotopic composition of nitrate collected at high time resolution in surface snow does not show any link to local gas phase concentrations of a number of species, including BrO. Furthermore, the combination of nitrogen and oxygen isotope data reveals interesting insights into the contributions of nitrate sources to Summit. There are several important implications of this work including that nitrate at Summit appears to be largely preserved in surface snow during photoactive periods, and that nitrate in snow at Summit also appears to be representative of long-range transported nitrate/NOx. The surface snow work is further substantiated by relationships found between and among seasonally-resolved ice core measurements of the isotopic composition of nitrate, nitrate concentration and a suite of chemical and elemental tracers. The seasonality observed in 15N/14N ratios in an ice core representing accumulation since 1760 C.E. cannot be explained by diffusion or other processes occurring in the firn over time. A marked negative trend in 15N/14N since industrialization, parallels a nearly three-fold increase in nitrate concentration as well as pronounced increases in tracers such as excess lead and non-sea-salt sulfur. This, along with independent estimates of oil burning and transport studies, indicate that North American oil combustion is the primary driver of the modern negative trend in 15N/14N of nitrate. The high, positive 15N/14N ratios found in pre-industrial ice link to biomass burning based upon concentrations of black carbon and ammonium.

Genistein exposure inhibits growth and alters steroidogenesis in adult mouse antral follicles

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

Patel, Shreya, E-mail: Shreya.patel214@gmail.com; Peretz, Jackye, E-mail: Jackye.peretz@gmail.com; Pan, Yuan-Xiang, E-mail: yxpan@illinois.edu

Genistein is a naturally occurring isoflavone phytoestrogen commonly found in plant products such as soybeans, lentils, and chickpeas. Genistein, like other phytoestrogens, has the potential to mimic, enhance, or impair the estradiol biosynthesis pathway, thereby potentially altering ovarian follicle growth. Previous studies have inconsistently indicated that genistein exposure may alter granulosa cell proliferation and hormone production, but no studies have examined the effects of genistein on intact antral follicles. Thus, this study was designed to test the hypothesis that genistein exposure inhibits follicle growth and steroidogenesis in intact antral follicles. To test this hypothesis, antral follicles isolated from CD-1 micemore » were cultured with vehicle (dimethyl sulfoxide; DMSO) or genistein (6.0 and 36 μM) for 18–96 h. Every 24 h, follicle diameters were measured to assess growth. At the end of each culture period, the media were pooled to measure hormone levels, and the cultured follicles were collected to measure expression of cell cycle regulators and steroidogenic enzymes. The results indicate that genistein (36 μM) inhibits growth of mouse antral follicles. Additionally, genistein (6.0 and 36 μM) increases progesterone, testosterone, and dehydroepiandrosterone (DHEA) levels, but decreases estrone and estradiol levels. The results also indicate that genistein alters the expression of steroidogenic enzymes at 24, 72 and 96 h, and the expression of cell cycle regulators at 18 h. These data indicate that genistein exposure inhibits antral follicle growth by inhibiting the cell cycle, alters sex steroid hormone levels, and dysregulates steroidogenic enzymes in cultured mouse antral follicles. - Highlights: • Genistein exposure inhibits antral follicle growth. • Genistein exposure alters expression of cell cycle regulators. • Genistein exposure alters sex steroid hormones. • Genistein exposure alters expression of steroidogenic enzymes. • Genistein exposure alters Esr1 and Esr2 expression.« less

MEF2 Cooperates With Forskolin/cAMP and GATA4 to Regulate Star Gene Expression in Mouse MA-10 Leydig Cells.

PubMed

Daems, Caroline; Di-Luoffo, Mickaël; Paradis, Élise; Tremblay, Jacques J

2015-07-01

In Leydig cells, steroidogenic acute regulatory protein (STAR) participates in cholesterol shuttling from the outer to the inner mitochondrial membrane, the rate-limiting step in steroidogenesis. Steroid hormone biosynthesis and steroidogenic gene expression are regulated by LH, which activates various signaling pathways and transcription factors, including cAMP/Ca(2+)/CAMK (Ca(2+)/calmodulin-dependent kinase)-myocyte enhancer factor 2 (MEF2). The 4 MEF2 transcription factors are essential regulators of cell differentiation and organogenesis in numerous tissues. Recently, MEF2 was identified in Sertoli and Leydig cells of the testis. Here, we report that MEF2 regulates steroidogenesis in mouse MA-10 Leydig cells by acting on the Star gene. In MA-10 cells depleted of MEF2 using siRNAs (small interfering RNAs), STAR protein levels, Star mRNA levels, and promoter activity were significantly decreased. On its own, MEF2 did not activate the mouse Star promoter but was found to cooperate with forskolin/cAMP. By chromatin immunoprecipitation and DNA precipitation assays, we confirmed MEF2 binding to a consensus element located at -232 bp of the Star promoter. Mutation or deletion of the MEF2 element reduced but did not abrogate the MEF2/cAMP cooperation, indicating that MEF2 cooperates with other DNA-bound transcription factor(s). We identified GATA4 (GATA binding protein 4) as a partner for MEF2 in Leydig cells, because mutation of the GATA element abrogated the MEF2/cAMP cooperation on a reporter lacking a MEF2 element. MEF2 and GATA4 interact as revealed by coimmunoprecipitation, and MEF2 and GATA4 transcriptionally cooperate on the Star promoter. Altogether, our results define MEF2 as a novel regulator of steroidogenesis and Star transcription in Leydig cells and identify GATA4 as a key partner for MEF2-mediated action.

Distribution of histaminergic neuronal cluster in the rat and mouse hypothalamus.

PubMed

Moriwaki, Chinatsu; Chiba, Seiichi; Wei, Huixing; Aosa, Taishi; Kitamura, Hirokazu; Ina, Keisuke; Shibata, Hirotaka; Fujikura, Yoshihisa

2015-10-01

Histidine decarboxylase (HDC) catalyzes the biosynthesis of histamine from L-histidine and is expressed throughout the mammalian nervous system by histaminergic neurons. Histaminergic neurons arise in the posterior mesencephalon during the early embryonic period and gradually develop into two histaminergic substreams around the lateral area of the posterior hypothalamus and the more anterior peri-cerebral aqueduct area before finally forming an adult-like pattern comprising five neuronal clusters, E1, E2, E3, E4, and E5, at the postnatal stage. This distribution of histaminergic neuronal clusters in the rat hypothalamus appears to be a consequence of neuronal development and reflects the functional differentiation within each neuronal cluster. However, the close linkage between the locations of histaminergic neuronal clusters and their physiological functions has yet to be fully elucidated because of the sparse information regarding the location and orientation of each histaminergic neuronal clusters in the hypothalamus of rats and mice. To clarify the distribution of the five-histaminergic neuronal clusters more clearly, we performed an immunohistochemical study using the anti-HDC antibody on serial sections of the rat hypothalamus according to the brain maps of rat and mouse. Our results confirmed that the HDC-immunoreactive (HDCi) neuronal clusters in the hypothalamus of rats and mice are observed in the ventrolateral part of the most posterior hypothalamus (E1), ventrolateral part of the posterior hypothalamus (E2), ventromedial part from the medial to the posterior hypothalamus (E3), periventricular part from the anterior to the medial hypothalamus (E4), and diffusely extended part of the more dorsal and almost entire hypothalamus (E5). The stereological estimation of the total number of HDCi neurons of each clusters revealed the larger amount of the rat than the mouse. The characterization of histaminergic neuronal clusters in the hypothalamus of rats and mice may provide useful information for further investigations. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Sodium phenylbutyrate controls neuroinflammatory and antioxidant activities and protects dopaminergic neurons in mouse models of Parkinson's disease.

PubMed

Roy, Avik; Ghosh, Anamitra; Jana, Arundhati; Liu, Xiaojuan; Brahmachari, Saurav; Gendelman, Howard E; Pahan, Kalipada

2012-01-01

Neuroinflammation and oxidative stress underlie the pathogenesis of various neurodegenerative disorders. Here we demonstrate that sodium phenylbutyrate (NaPB), an FDA-approved therapy for reducing plasma ammonia and glutamine in urea cycle disorders, can suppress both proinflammatory molecules and reactive oxygen species (ROS) in activated glial cells. Interestingly, NaPB also decreased the level of cholesterol but involved only intermediates, not the end product of cholesterol biosynthesis pathway for these functions. While inhibitors of both geranylgeranyl transferase (GGTI) and farnesyl transferase (FTI) inhibited the activation of NF-κB, inhibitor of GGTI, but not FTI, suppressed the production of ROS. Accordingly, a dominant-negative mutant of p21(rac), but not p21(ras), attenuated the production of ROS from activated microglia. Inhibition of both p21(ras) and p21(rac) activation by NaPB in microglial cells suggests that NaPB exerts anti-inflammatory and antioxidative effects via inhibition of these small G proteins. Consistently, we found activation of both p21(ras) and p21(rac)in vivo in the substantia nigra of acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease. Oral administration of NaPB reduced nigral activation of p21(ras) and p21(rac), protected nigral reduced glutathione, attenuated nigral activation of NF-κB, inhibited nigral expression of proinflammatory molecules, and suppressed nigral activation of glial cells. These findings paralleled dopaminergic neuronal protection, normalized striatal neurotransmitters, and improved motor functions in MPTP-intoxicated mice. Consistently, FTI and GGTI also protected nigrostriata in MPTP-intoxicated mice. Furthermore, NaPB also halted the disease progression in a chronic MPTP mouse model. These results identify novel mode of action of NaPB and suggest that NaPB may be of therapeutic benefit for neurodegenerative disorders.

Sodium Phenylbutyrate Controls Neuroinflammatory and Antioxidant Activities and Protects Dopaminergic Neurons in Mouse Models of Parkinson’s Disease

PubMed Central

Jana, Arundhati; Liu, Xiaojuan; Brahmachari, Saurav; Gendelman, Howard E.; Pahan, Kalipada

2012-01-01

Neuroinflammation and oxidative stress underlie the pathogenesis of various neurodegenerative disorders. Here we demonstrate that sodium phenylbutyrate (NaPB), an FDA-approved therapy for reducing plasma ammonia and glutamine in urea cycle disorders, can suppress both proinflammatory molecules and reactive oxygen species (ROS) in activated glial cells. Interestingly, NaPB also decreased the level of cholesterol but involved only intermediates, not the end product of cholesterol biosynthesis pathway for these functions. While inhibitors of both geranylgeranyl transferase (GGTI) and farnesyl transferase (FTI) inhibited the activation of NF-κB, inhibitor of GGTI, but not FTI, suppressed the production of ROS. Accordingly, a dominant-negative mutant of p21rac, but not p21ras, attenuated the production of ROS from activated microglia. Inhibition of both p21ras and p21rac activation by NaPB in microglial cells suggests that NaPB exerts anti-inflammatory and antioxidative effects via inhibition of these small G proteins. Consistently, we found activation of both p21ras and p21rac in vivo in the substantia nigra of acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson’s disease. Oral administration of NaPB reduced nigral activation of p21ras and p21rac, protected nigral reduced glutathione, attenuated nigral activation of NF-κB, inhibited nigral expression of proinflammatory molecules, and suppressed nigral activation of glial cells. These findings paralleled dopaminergic neuronal protection, normalized striatal neurotransmitters, and improved motor functions in MPTP-intoxicated mice. Consistently, FTI and GGTI also protected nigrostriata in MPTP-intoxicated mice. Furthermore, NaPB also halted the disease progression in a chronic MPTP mouse model. These results identify novel mode of action of NaPB and suggest that NaPB may be of therapeutic benefit for neurodegenerative disorders. PMID:22723850

40 CFR 418.40 - Applicability; description of the ammonium nitrate subcategory.

Code of Federal Regulations, 2012 CFR

2012-07-01

... ammonium nitrate subcategory. 418.40 Section 418.40 Protection of Environment ENVIRONMENTAL PROTECTION... Ammonium Nitrate Subcategory § 418.40 Applicability; description of the ammonium nitrate subcategory. The provisions of this subpart are applicable to discharges resulting from the manufacture of ammonium nitrate...

40 CFR 418.40 - Applicability; description of the ammonium nitrate subcategory.

Code of Federal Regulations, 2011 CFR

2011-07-01

... ammonium nitrate subcategory. 418.40 Section 418.40 Protection of Environment ENVIRONMENTAL PROTECTION... Ammonium Nitrate Subcategory § 418.40 Applicability; description of the ammonium nitrate subcategory. The provisions of this subpart are applicable to discharges resulting from the manufacture of ammonium nitrate...

40 CFR 418.40 - Applicability; description of the ammonium nitrate subcategory.

Code of Federal Regulations, 2013 CFR

2013-07-01

... ammonium nitrate subcategory. 418.40 Section 418.40 Protection of Environment ENVIRONMENTAL PROTECTION... Ammonium Nitrate Subcategory § 418.40 Applicability; description of the ammonium nitrate subcategory. The provisions of this subpart are applicable to discharges resulting from the manufacture of ammonium nitrate...

40 CFR 418.40 - Applicability; description of the ammonium nitrate subcategory.

Code of Federal Regulations, 2014 CFR

2014-07-01

... ammonium nitrate subcategory. 418.40 Section 418.40 Protection of Environment ENVIRONMENTAL PROTECTION... Ammonium Nitrate Subcategory § 418.40 Applicability; description of the ammonium nitrate subcategory. The provisions of this subpart are applicable to discharges resulting from the manufacture of ammonium nitrate...

Histological characteristics following a long-term nitrate-rich diet in miniature pigs with parotid atrophy

PubMed Central

Xia, Dengsheng; Qu, Xingmin; Tran, Simon D; Schmidt, Laura L; Qin, Lizheng; Zhang, Chunmei; Cui, Xiuyu; Deng, Dajun; Wang, Songlin

2015-01-01

The aim of this study was to investigate the histological characteristics following a 2-year nitrate-rich diet in miniature pigs with parotid atrophy. Using averages collected data from three time points at 6, 12, and 24 months following the induction of parotid gland atrophy, salivary nitrate levels of the nitrate-diet parotid-atrophied group (17.3±3.9 ng/µl) were close to those of the control group (19.6±5.1 ng/µl). Compared to the control group, the nitrate-diet group had significantly higher nitrate levels in blood (P < 0.05) and urine (P < 0.001). Histological and electron microscopy analyses showed no abnormalities in the organs of experimental or control animals. No significant differences on apoptosis rate were found in liver and kidney tissues between the standard- and nitrate-diet groups. Therefore, dietary nitrate supplementation could restore salivary nitrate levels. High-dose nitrate loading for 2 years had no observed systemic toxicity in miniature pigs with parotid atrophy. PMID:26261499

High temperature two component explosive

DOEpatents

Mars, James E.; Poole, Donald R.; Schmidt, Eckart W.; Wang, Charles

1981-01-01

A two component, high temperature, thermally stable explosive composition comprises a liquid or low melting oxidizer and a liquid or low melting organic fuel. The oxidizer and fuel in admixture are incapable of substantial spontaneous exothermic reaction at temperatures on the order of 475.degree. K. At temperatures on the order of 475.degree. K., the oxidizer and fuel in admixture have an activation energy of at least about 40 kcal/mol. As a result of the high activation energy, the preferred explosive compositions are nondetonable as solids at ambient temperature, and become detonable only when heated beyond the melting point. Preferable oxidizers are selected from alkali or alkaline earth metal nitrates, nitrites, perchlorates, and/or mixtures thereof. Preferred fuels are organic compounds having polar hydrophilic groups. The most preferred fuels are guanidinium nitrate, acetamide and mixtures of the two. Most preferred oxidizers are eutectic mixtures of lithium nitrate, potassium nitrate and sodium nitrate, of sodium nitrite, sodium nitrate and potassium nitrate, and of potassium nitrate, calcium nitrate and sodium nitrate.

Evidence for the Existence of Autotrophic Nitrate-Reducing Fe(II)-Oxidizing Bacteria in Marine Coastal Sediment

PubMed Central

Laufer, Katja; Røy, Hans; Jørgensen, Bo Barker

2016-01-01

ABSTRACT Nitrate-reducing Fe(II)-oxidizing microorganisms were described for the first time ca. 20 years ago. Most pure cultures of nitrate-reducing Fe(II) oxidizers can oxidize Fe(II) only under mixotrophic conditions, i.e., when an organic cosubstrate is provided. A small number of nitrate-reducing Fe(II)-oxidizing cultures have been proposed to grow autotrophically, but unambiguous evidence for autotrophy has not always been provided. Thus, it is still unclear whether or to what extent Fe(II) oxidation coupled to nitrate reduction is an enzymatically catalyzed and energy-yielding autotrophic process or whether Fe(II) is abiotically oxidized by nitrite from heterotrophic nitrate reduction. The aim of the present study was to find evidence for the existence of autotrophic nitrate-reducing Fe(II) oxidizers in coastal marine sediments. Microcosm incubations showed that with increasing incubation times, the stoichiometric ratio of reduced nitrate/oxidized Fe(II) [NO3−reduced/Fe(II)oxidized] decreased, indicating a decreasing contribution of heterotrophic denitrification and/or an increasing contribution of autotrophic nitrate-reducing Fe(II) oxidation over time. After incubations of sediment slurries for >10 weeks, nitrate-reducing activity ceased, although nitrate was still present. This suggests that heterotrophic nitrate reduction had ceased due to the depletion of readily available organic carbon. However, after the addition of Fe(II) to these batch incubation mixtures, the nitrate-reducing activity resumed, and Fe(II) was oxidized, indicating the activity of autotrophic nitrate-reducing Fe(II) oxidizers. The concurrent reduction of 14C-labeled bicarbonate concentrations unambiguously proved that autotrophic C fixation occurred during Fe(II) oxidation and nitrate reduction. Our results clearly demonstrated that autotrophic nitrate-reducing Fe(II)-oxidizing bacteria were present in the investigated coastal marine sediments. IMPORTANCE Twenty years after the discovery of nitrate-reducing Fe(II) oxidizers, it is still controversially discussed whether autotrophic nitrate-reducing Fe(II)-oxidizing microorganisms exist and to what extent Fe(II) oxidation in this reduction/oxidation process is enzymatically catalyzed or which role abiotic side reactions of Fe(II) with reactive N species play. Most pure cultures of nitrate-reducing Fe(II) oxidizers are mixotrophic; i.e., they need an organic cosubstrate to maintain their activity over several cultural transfers. For the few existing autotrophic isolates and enrichment cultures, either the mechanism of nitrate-reducing Fe(II) oxidation is not known or evidence for their autotrophic lifestyle is controversial. In the present study, we provide evidence for the existence of autotrophic nitrate-reducing Fe(II) oxidizers in coastal marine sediments. The evidence is based on stoichiometries of nitrate reduction and Fe(II) oxidation determined in microcosm incubations and the incorporation of carbon from CO2 under conditions that favor the activity of nitrate-reducing Fe(II) oxidizers. PMID:27496777

Factors influencing protein tyrosine nitration – structure-based predictive models

PubMed Central

Bayden, Alexander S.; Yakovlev, Vasily A.; Graves, Paul R.; Mikkelsen, Ross B.; Kellogg, Glen E.

2010-01-01

Models for exploring tyrosine nitration in proteins have been created based on 3D structural features of 20 proteins for which high resolution X-ray crystallographic or NMR data are available and for which nitration of 35 total tyrosines has been experimentally proven under oxidative stress. Factors suggested in previous work to enhance nitration were examined with quantitative structural descriptors. The role of neighboring acidic and basic residues is complex: for the majority of tyrosines that are nitrated the distance to the heteroatom of the closest charged sidechain corresponds to the distance needed for suspected nitrating species to form hydrogen bond bridges between the tyrosine and that charged amino acid. This suggests that such bridges play a very important role in tyrosine nitration. Nitration is generally hindered for tyrosines that are buried and for those tyrosines where there is insufficient space for the nitro group. For in vitro nitration, closed environments with nearby heteroatoms or unsaturated centers that can stabilize radicals are somewhat favored. Four quantitative structure-based models, depending on the conditions of nitration, have been developed for predicting site-specific tyrosine nitration. The best model, relevant for both in vitro and in vivo cases predicts 30 of 35 tyrosine nitrations (positive predictive value) and has a sensitivity of 60/71 (11 false positives). PMID:21172423

Open-Source Photometric System for Enzymatic Nitrate Quantification

PubMed Central

Wittbrodt, B. T.; Squires, D. A.; Walbeck, J.; Campbell, E.; Campbell, W. H.; Pearce, J. M.

2015-01-01

Nitrate, the most oxidized form of nitrogen, is regulated to protect people and animals from harmful levels as there is a large over abundance due to anthropogenic factors. Widespread field testing for nitrate could begin to address the nitrate pollution problem, however, the Cadmium Reduction Method, the leading certified method to detect and quantify nitrate, demands the use of a toxic heavy metal. An alternative, the recently proposed Environmental Protection Agency Nitrate Reductase Nitrate-Nitrogen Analysis Method, eliminates this problem but requires an expensive proprietary spectrophotometer. The development of an inexpensive portable, handheld photometer will greatly expedite field nitrate analysis to combat pollution. To accomplish this goal, a methodology for the design, development, and technical validation of an improved open-source water testing platform capable of performing Nitrate Reductase Nitrate-Nitrogen Analysis Method. This approach is evaluated for its potential to i) eliminate the need for toxic chemicals in water testing for nitrate and nitrite, ii) reduce the cost of equipment to perform this method for measurement for water quality, and iii) make the method easier to carryout in the field. The device is able to perform as well as commercial proprietary systems for less than 15% of the cost for materials. This allows for greater access to the technology and the new, safer nitrate testing technique. PMID:26244342

Dietary intake of nitrate relative to antioxidant vitamin in relation to breast cancer risk: a case-control study.

PubMed

Yang, Yoon Jung; Hwang, Se Hee; Kim, Hyun Ja; Nam, Seok-Jin; Kong, Gu; Kim, Mi Kyung

2010-01-01

Nitrate is a precursor in the endogenous formation of N-nitroso compounds, which are potent animal carcinogens, whereas antioxidant vitamins have been suggested to protect against carcinogenesis. Interestingly, nitrate and antioxidant vitamins stem from the same dietary sources. We investigated whether the intake of nitrate relative to antioxidant vitamins is associated with the risk of breast cancer. A total of 362 breast cancer cases were matched to the 362 controls by age and menopausal status. Dietary intake was assessed using a quantitative food frequency questionnaire with 121 food items by trained interviewers. The nitrate to antioxidant vitamin consumption ratio was then calculated. Conditional logistic regression analysis was used to obtain odds ratios (ORs) and corresponding 95% confidence intervals (CI). Mean intakes of nitrate for cases and controls were 421 mg/day and 424 mg/day, respectively. Intakes of nitrate, nitrate/beta-carotene, nitrate/vitamin C, and nitrate/vitamin E were not associated with breast cancer risk. However, higher breast cancer risk was observed with higher intake of nitrate/folate (OR = 2.03, 95% CI = 1.16-3.54, P for trend = 0.052). Our results suggest that lowering the ratio of nitrate to folate intake may be effective in reducing breast cancer risk.

Open-Source Photometric System for Enzymatic Nitrate Quantification.

PubMed

Wittbrodt, B T; Squires, D A; Walbeck, J; Campbell, E; Campbell, W H; Pearce, J M

2015-01-01

Nitrate, the most oxidized form of nitrogen, is regulated to protect people and animals from harmful levels as there is a large over abundance due to anthropogenic factors. Widespread field testing for nitrate could begin to address the nitrate pollution problem, however, the Cadmium Reduction Method, the leading certified method to detect and quantify nitrate, demands the use of a toxic heavy metal. An alternative, the recently proposed Environmental Protection Agency Nitrate Reductase Nitrate-Nitrogen Analysis Method, eliminates this problem but requires an expensive proprietary spectrophotometer. The development of an inexpensive portable, handheld photometer will greatly expedite field nitrate analysis to combat pollution. To accomplish this goal, a methodology for the design, development, and technical validation of an improved open-source water testing platform capable of performing Nitrate Reductase Nitrate-Nitrogen Analysis Method. This approach is evaluated for its potential to i) eliminate the need for toxic chemicals in water testing for nitrate and nitrite, ii) reduce the cost of equipment to perform this method for measurement for water quality, and iii) make the method easier to carryout in the field. The device is able to perform as well as commercial proprietary systems for less than 15% of the cost for materials. This allows for greater access to the technology and the new, safer nitrate testing technique.

Absence of an effect of high nitrate intake from beetroot juice on blood pressure in treated hypertensive individuals: a randomized controlled trial.

PubMed

Bondonno, Catherine P; Liu, Alex H; Croft, Kevin D; Ward, Natalie C; Shinde, Sujata; Moodley, Yuben; Lundberg, Jon O; Puddey, Ian B; Woodman, Richard J; Hodgson, Jonathan M

2015-08-01

Dietary nitrate, which is in green leafy vegetables and beetroot, decreases blood pressure through the enterosalivary nitrate-nitrite-nitric oxide pathway in healthy individuals. Whether similar effects would occur in individuals with treated hypertension and, therefore, at increased risk of cardiovascular disease is unclear. We assessed whether increased dietary nitrate intake by using beetroot juice for 1 wk lowers blood pressure in treated hypertensive men and women. Participants (n = 27) were recruited to a randomized, placebo-controlled, double-blind crossover trial. The effect of 1-wk intake of nitrate-rich beetroot juice was compared with 1-wk intake of nitrate-depleted beetroot juice (placebo). The primary outcome was blood pressure assessed by measuring home blood pressure during the intervention and 24-h ambulatory blood pressure on day 7 of the intervention. Other outcomes included nitrate metabolism assessed by measuring nitrate and nitrite in plasma, saliva, and urine. Relative to the placebo, 1-wk intake of nitrate-rich beetroot juice resulted in a 3-fold increase in plasma nitrite and nitrate, a 7-fold increase in salivary nitrite, an 8-fold higher salivary nitrate, and a 4-fold increase in both urinary nitrite and nitrate (P < 0.001). However, no differences in home blood pressure and 24-h ambulatory blood pressure were observed with 1-wk intake of nitrate-rich beetroot juice in comparison with the placebo. An increase in dietary nitrate intake may not be an effective short-term approach to further lower blood pressure in treated hypertensive subjects. © 2015 American Society for Nutrition.

Physiological responses of intertidal marine brown algae to nitrogen deprivation and resupply of nitrate and ammonium.

PubMed

Young, Erica B; Berges, John A; Dring, Matthew J

2009-04-01

Intertidal macroalgae Fucus and Laminaria experience seasonally fluctuating inorganic N supply. This study examined the effects of long-term N deprivation, recovery following N resupply, and effects of elevated ammonium and nitrate exposure on N acquisition in intertidal algae using manipulations of N supply in tank culture. Over 15 weeks of N deprivation, internal N and nitrate reductase activity (NRA) declined, but maximum quantum yield of PSII was unaffected in Fucus serratus and Fucus vesiculosus. Low NRA was maintained despite no external nitrate availability and depletion of internal pools, suggesting a constitutive NRA, insensitive to N supply. Nitrate resupplied to N-starved thalli was rapidly taken up and internal nitrate pools and NRA increased. Exposure to elevated (50 microM) nitrate over 4 days stimulated nitrate uptake and NRA in Laminaria digitata and F. serratus. Exposure to elevated ammonium suppressed NRA in L. digitata but not in F. serratus. This novel insensitivity of NRA to ammonium in Fucus contrasts with regulation of NRA in other algae and higher plants. Ammonium suppression of NRA in L. digitata was not via inhibition of nitrate uptake and was independent of nitrate availability. L. digitata showed a higher capacity for internal nitrate storage when exposed to elevated ambient nitrate, but NRA was lower than in Fucus. All species maintained nitrate assimilation capacity in excess of nitrate uptake capacity. N uptake and storage strategies of these intertidal macroalgae are adaptive to life in fluctuating N supply, and distinct regulation of N metabolism in Fucus vs Laminaria may relate to position in the intertidal zone.

Presence of Nitrate-Accumulating Sulfur Bacteria and Their Influence on Nitrogen Cycling in a Shallow Coastal Marine Sediment

PubMed Central

Sayama, Mikio

2001-01-01

Nitrate flux between sediment and water, nitrate concentration profile at the sediment-water interface, and in situ sediment denitrification activity were measured seasonally at the innermost part of Tokyo Bay, Japan. For the determination of sediment nitrate concentration, undisturbed sediment cores were sectioned into 5-mm depth intervals and each segment was stored frozen at −30°C. The nitrate concentration was determined for the supernatants after centrifuging the frozen and thawed sediments. Nitrate in the uppermost sediment showed a remarkable seasonal change, and its seasonal maximum of up to 400 μM was found in October. The directions of the diffusive nitrate fluxes predicted from the interfacial concentration gradients were out of the sediment throughout the year. In contrast, the directions of the total nitrate fluxes measured by the whole-core incubation were into the sediment at all seasons. This contradiction between directions indicates that a large part of the nitrate pool extracted from the frozen surface sediments is not a pore water constituent, and preliminary examinations demonstrated that the nitrate was contained in the intracellular vacuoles of filamentous sulfur bacteria dwelling on or in the surface sediment. Based on the comparison between in situ sediment denitrification activity and total nitrate flux, it is suggested that intracellular nitrate cannot be directly utilized by sediment denitrification, and the probable fate of the intracellular nitrate is hypothesized to be dissimilatory reduction to ammonium. The presence of nitrate-accumulating sulfur bacteria therefore may lower nature's self-purification capacity (denitrification) and exacerbate eutrophication in shallow coastal marine environments. PMID:11472923

A comparison of organic and inorganic nitrates/nitrites.

PubMed

Omar, Sami A; Artime, Esther; Webb, Andrew J

2012-05-15

Although both organic and inorganic nitrates/nitrites mediate their principal effects via nitric oxide, there are many important differences. Inorganic nitrate and nitrite have simple ionic structures and are produced endogenously and are present in the diet, whereas their organic counterparts are far more complex, and, with the exception of ethyl nitrite, are all medicinally synthesised products. These chemical differences underlie the differences in pharmacokinetic properties allowing for different modalities of administration, particularly of organic nitrates, due to the differences in their bioavailability and metabolic profiles. Whilst the enterosalivary circulation is a key pathway for orally ingested inorganic nitrate, preventing an abrupt effect or toxic levels of nitrite and prolonging the effects, this is not used by organic nitrates. The pharmacodynamic differences are even greater; while organic nitrates have potent acute effects causing vasodilation, inorganic nitrite's effects are more subtle and dependent on certain conditions. However, in chronic use, organic nitrates are considerably limited by the development of tolerance and endothelial dysfunction, whereas inorganic nitrate/nitrite may compensate for diminished endothelial function, and tolerance has not been reported. Also, while inorganic nitrate/nitrite has important cytoprotective effects against ischaemia-reperfusion injury, continuous use of organic nitrates may increase injury. While there are concerns that inorganic nitrate/nitrite may induce carcinogenesis, direct evidence of this in humans is lacking. While organic nitrates may continue to dominate the therapeutic arena, this may well change with the increasing recognition of their limitations, and ongoing discovery of beneficial effects and specific advantages of inorganic nitrate/nitrite. Copyright © 2012 Elsevier Inc. All rights reserved.

Role of nitrite, urate and pepsin in the gastroprotective effects of saliva

PubMed Central

Rocha, Bárbara S.; Lundberg, Jon O; Radi, Rafael; Laranjinha, João

2016-01-01

Dietary nitrate is now recognized as an alternative substrate for nitric oxide (•NO) production in the gut. This novel pathway implies the sequential reduction of nitrate to nitrite, •NO and other bioactive nitrogen oxides but the physiological relevance of these oxidants has remained elusive. We have previously shown that dietary nitrite fuels an hitherto unrecognized nitrating pathway at acidic gastric pH, through which pepsinogen is nitrated in the gastric mucosa, yielding a less active form of pepsin in vitro. Here, we demonstrate that pepsin is nitrated in vivo and explore the functional impact of protein nitration by means of peptic ulcer development. Upon administration of pentagastrin and human nitrite-rich saliva or sodium nitrite to rats, nitrated pepsin was detected in the animal's stomach by immunoprecipitation. •NO was measured in the gastric headspace before and after nitrite instillation by chemiluminescence. At the end of each procedure, the stomach's lesions, ranging from gastric erosions to haemorrhagic ulcers, were scored. Nitrite increased gastric •NO by 200-fold (p<0.05) and nitrated pepsin was detected both in the gastric juice and the mucosa (p<0.05). Exogenous urate, a scavenger of nitrogen dioxide radical, blunted •NO detection and inhibited pepsin nitration, suggesting an underlining free radical-dependent mechanism for nitration. Functionally, pepsin nitration prevented the development of gastric ulcers, as the lesions were only apparent when pepsin nitration was inhibited by urate. In sum, this work unravels a novel dietary-dependent nitrating pathway in which pepsin is nitrated and inactivated in the stomach, preventing the progression of gastric ulcers. PMID:27156250

REDUCTION OF NITRATE THROUGH THE USE OF NITRATE REDUCTASE FOR THE SMARTCHEM AUTOANALYZER

EPA Science Inventory

The standard method for the determination of nitrate in drinking water, USEPA Method 353.2 “Determination of Nitrate-Nitrite by Automated Colorimetry,” employs cadmium as the reductant for the conversion of nitrate to nitrite. The nitrite is then analyzed colorimetrically by way ...

EXTRACTION OF URANYL NITRATE FROM AQUEOUS SOLUTIONS

DOEpatents

Furman, N.H.; Mundy, R.J.

1957-12-10

An improvement in the process is described for extracting aqueous uranyl nitrate solutions with an organic solvent such as ether. It has been found that the organic phase will extract a larger quantity of uranyl nitrate if the aqueous phase contains in addition to the uranyl nitrate, a quantity of some other soluble nitrate to act as a salting out agent. Mentioned as suitable are the nitrates of lithium, calcium, zinc, bivalent copper, and trivalent iron.

Alkali metal nitrate purification

DOEpatents

Fiorucci, Louis C.; Morgan, Michael J.

1986-02-04

A process is disclosed for removing contaminants from impure alkali metal nitrates containing them. The process comprises heating the impure alkali metal nitrates in solution form or molten form at a temperature and for a time sufficient to effect precipitation of solid impurities and separating the solid impurities from the resulting purified alkali metal nitrates. The resulting purified alkali metal nitrates in solution form may be heated to evaporate water therefrom to produce purified molten alkali metal nitrates suitable for use as a heat transfer medium. If desired, the purified molten form may be granulated and cooled to form discrete solid particles of purified alkali metal nitrates.

Enhanced removal of nitrate from water using nZVI@MWCNTs composite: synthesis, kinetics and mechanism of reduction.

PubMed

Babaei, Ali Akbar; Azari, Ali; Kalantary, Roshanak Rezaei; Kakavandi, Babak

2015-01-01

Herein, multi-wall carbon nanotubes (MWCNTs) were used as the carrier of nano-zero valent iron (nZVI) particles to fabricate a composite known as nZVI@MWCNTs. The composite was then characterized and applied in the nitrate removal process in a batch system under anoxic conditions. The influential parameters such as pH, various concentrations of nitrate and composite were investigated within 240 min of the reaction. The mechanism, kinetics and end-products of nitrate reduction were also evaluated. Results revealed that the removal nitrate percentage for nZVI@MWCNTs composite was higher than that of nZVI and MWCNTs alone. Experimental data from nitrate reduction were fitted to the Langmuir-Hinshelwood kinetic model. The values of observed rate constant (kobs) decreased with increasing the initial concentration of nitrate. Our experiments proved that the nitrate removal efficiency was favorable once both high amounts of nZVI@MWCNTs and low concentrations of nitrate were applied. The predominant end-products of the nitrate reduction were ammonium (84%) and nitrogen gas (15%). Our findings also revealed that ZVI@MWCNTs is potentially a good composite for removal/reduction of nitrate from aqueous solutions.

Knock-Down of a Tonoplast Localized Low-Affinity Nitrate Transporter OsNPF7.2 Affects Rice Growth under High Nitrate Supply

PubMed Central

Hu, Rui; Qiu, Diyang; Chen, Yi; Miller, Anthony J.; Fan, Xiaorong; Pan, Xiaoping; Zhang, Mingyong

2016-01-01

The large nitrate transporter 1/peptide transporter family (NPF) has been shown to transport diverse substrates, including nitrate, amino acids, peptides, phytohormones, and glucosinolates. However, the rice (Oryza sativa) root-specific family member OsNPF7.2 has not been functionally characterized. Here, our data show that OsNPF7.2 is a tonoplast localized low-affinity nitrate transporter, that affects rice growth under high nitrate supply. Expression analysis showed that OsNPF7.2 was mainly expressed in the elongation and maturation zones of roots, especially in the root sclerenchyma, cortex and stele. It was also induced by high concentrations of nitrate. Subcellular localization analysis showed that OsNPF7.2 was localized on the tonoplast of large and small vacuoles. Heterologous expression in Xenopus laevis oocytes suggested that OsNPF7.2 was a low-affinity nitrate transporter. Knock-down of OsNPF7.2 retarded rice growth under high concentrations of nitrate. Therefore, we deduce that OsNPF7.2 plays a role in intracellular allocation of nitrate in roots, and thus influences rice growth under high nitrate supply. PMID:27826301

Genetic identification of a gene involved in constitutive, high-affinity nitrate transport in higher plants.

PubMed Central

Wang, R; Crawford, N M

1996-01-01

Two mutations have been found in a gene (NRT2) of Arabidopsis thaliana that specifically impair constitutive, high-affinity nitrate uptake. These mutants were selected for resistance to 0.1 mM chlorate in the absence of nitrate. Progency from one of the backcrossed mutants showed no constitutive uptake of nitrate below 0.5 mM at pH 7.0 in liquid culture (that is, within 30 min of initial exposure to nitrate). All other uptake activities measured (high-affinity phosphate and sulfate uptake, inducible high-affinity nitrate uptake, and constitutive low-affinity nitrate uptake) were present or nearly normal in the backcrossed mutant. Electrophysiological analysis of individual root cells showed that the nrt2 mutant showed little response to 0.25 mM of nitrate, whereas NRT2 wild-type cells showed an initial depolarization followed by recovery. At 10 mM of nitrate both the mutant and wild-type cells displayed similar, strong electrical responses. These results indicate that NRT2 is a critical and perhaps necessary gene for constitutive, high-affinity nitrate uptake in Arabidopsis, but not for inducible, high-affinity nor constitutive, low-affinity nitrate uptake. Thus, these systems are genetically distinct. PMID:8799195

Respiration of Nitrate and Nitrite.

PubMed

Cole, Jeffrey A; Richardson, David J

2008-09-01

Nitrate reduction to ammonia via nitrite occurs widely as an anabolic process through which bacteria, archaea, and plants can assimilate nitrate into cellular biomass. Escherichia coli and related enteric bacteria can couple the eight-electron reduction of nitrate to ammonium to growth by coupling the nitrate and nitrite reductases involved to energy-conserving respiratory electron transport systems. In global terms, the respiratory reduction of nitrate to ammonium dominates nitrate and nitrite reduction in many electron-rich environments such as anoxic marine sediments and sulfide-rich thermal vents, the human gastrointestinal tract, and the bodies of warm-blooded animals. This review reviews the regulation and enzymology of this process in E. coli and, where relevant detail is available, also in Salmonella and draws comparisons with and implications for the process in other bacteria where it is pertinent to do so. Fatty acids may be present in high levels in many of the natural environments of E. coli and Salmonella in which oxygen is limited but nitrate is available to support respiration. In E. coli, nitrate reduction in the periplasm involves the products of two seven-gene operons, napFDAGHBC, encoding the periplasmic nitrate reductase, and nrfABCDEFG, encoding the periplasmic nitrite reductase. No bacterium has yet been shown to couple a periplasmic nitrate reductase solely to the cytoplasmic nitrite reductase NirB. The cytoplasmic pathway for nitrate reduction to ammonia is restricted almost exclusively to a few groups of facultative anaerobic bacteria that encounter high concentrations of environmental nitrate.

Effect of high oral doses of nitrate on salivary recirculation of nitrates and nitrites and on bacterial diversity in the saliva of young pigs.

PubMed

Trevisi, P; Casini, L; Nisi, I; Messori, S; Bosi, P

2011-04-01

Ingested nitrate is absorbed in the small intestine, recirculated into the saliva and reduced to nitrite by oral bacteria. In pigs receiving a moderate dietary addition of nitrate, the recirculation into the saliva is modest, so we aimed to assess the effect of higher nitrate doses to find out how the animal reacts to this new situation and to evaluate if a higher nitrate level could enhance the nitrate reduction process, improving the nitrite production Trial 1. Six piglets received 100 g of a commercial diet with 2.45% KNO(3) . In relation to baseline values, nitrate in blood serum and saliva increased 15 times, and declined after 6 h vs. 2 h. Salivary nitrite increased seven times after the addition and declined after 6 h vs. 2 h. Trial 2. Six piglets were fed a diet with or without 1.22% KNO(3) for 2 weeks. Salivary nitrate and nitrite increased with the addition of KNO3: nitrate increased from d0 to the end of the trial, nitrite increased 15 times after 1 week, but decreased after 2 weeks to 4.5-fold the control. After 2 weeks, nitrate reduced Shan diversity index of salivary microbiota. The present results indicate that the long exposure to high quantities of nitrates impairs the oral reduction of nitrate to nitrite and engenders a reduction of the mouth's microbiota diversity. © 2010 Blackwell Verlag GmbH.

Nitrate decreases xanthine oxidoreductase-mediated nitrite reductase activity and attenuates vascular and blood pressure responses to nitrite.

PubMed

Damacena-Angelis, Célio; Oliveira-Paula, Gustavo H; Pinheiro, Lucas C; Crevelin, Eduardo J; Portella, Rafael L; Moraes, Luiz Alberto B; Tanus-Santos, Jose E

2017-08-01

Nitrite and nitrate restore deficient endogenous nitric oxide (NO) production as they are converted back to NO, and therefore complement the classic enzymatic NO synthesis. Circulating nitrate and nitrite must cross membrane barriers to produce their effects and increased nitrate concentrations may attenuate the nitrite influx into cells, decreasing NO generation from nitrite. Moreover, xanthine oxidoreductase (XOR) mediates NO formation from nitrite and nitrate. However, no study has examined whether nitrate attenuates XOR-mediated NO generation from nitrite. We hypothesized that nitrate attenuates the vascular and blood pressure responses to nitrite either by interfering with nitrite influx into vascular tissue, or by competing with nitrite for XOR, thus inhibiting XOR-mediated NO generation. We used two independent vascular function assays in rats (aortic ring preparations and isolated mesenteric arterial bed perfusion) to examine the effects of sodium nitrate on the concentration-dependent responses to sodium nitrite. Both assays showed that nitrate attenuated the vascular responses to nitrite. Conversely, the aortic responses to the NO donor DETANONOate were not affected by sodium nitrate. Further confirming these results, we found that nitrate attenuated the acute blood pressure lowering effects of increasing doses of nitrite infused intravenously in freely moving rats. The possibility that nitrate could compete with nitrite and decrease nitrite influx into cells was tested by measuring the accumulation of nitrogen-15-labeled nitrite ( 15 N-nitrite) by aortic rings using ultra-performance liquid chromatography tandem mass-spectrometry (UPLC-MS/MS). Nitrate exerted no effect on aortic accumulation of 15 N-nitrite. Next, we used chemiluminescence-based NO detection to examine whether nitrate attenuates XOR-mediated nitrite reductase activity. Nitrate significantly shifted the Michaelis Menten saturation curve to the right, with a 3-fold increase in the Michaelis constant. Together, our results show that nitrate inhibits XOR-mediated NO production from nitrite, and this mechanism may explain how nitrate attenuates the vascular and blood pressure responses to nitrite. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Nitrate vulnerability projections from Bayesian inference of multiple groundwater age tracers

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

Alikhani, Jamal; Deinhart, Amanda L.; Visser, Ate

Nitrate is a major source of contamination of groundwater in the United States and around the world. We tested the applicability of multiple groundwater age tracers ( 3H, 3He, 4He, 14C, 13C, and 85Kr) in projecting future trends of nitrate concentration in 9 long-screened, public drinking water wells in Turlock, California, where nitrate concentrations are increasing toward the regulatory limit. Very low 85Kr concentrations and apparent 3H/ 3He ages point to a relatively old modern fraction (40–50 years), diluted with pre-modern groundwater, corroborated by the onset and slope of increasing nitrate concentrations. An inverse Gaussian–Dirac model was chosen to representmore » the age distribution of the sampled groundwater at each well. Model parameters were estimated using a Bayesian inference, resulting in the posterior probability distribution – including the associated uncertainty – of the parameters and projected nitrate concentrations. Three scenarios were considered, including combined historic nitrate and age tracer data, the sole use of nitrate and the sole use of age tracer data. Each scenario was evaluated based on the ability of the model to reproduce the data and the level of reliability of the nitrate projections. The tracer-only scenario closely reproduced tracer concentrations, but not observed trends in the nitrate concentration. Both cases that included nitrate data resulted in good agreement with historical nitrate trends. Use of combined tracers and nitrate data resulted in a narrower range of projections of future nitrate levels. However, use of combined tracer and nitrate resulted in a larger discrepancy between modeled and measured tracers for some of the tracers. In conclusion, despite nitrate trend slopes between 0.56 and 1.73 mg/L/year in 7 of the 9 wells, the probability that concentrations will increase to levels above the MCL by 2040 are over 95% for only two of the wells, and below 15% in the other wells, due to a leveling off of reconstructed historical nitrate loadings to groundwater since about 1990.« less

Nitrate vulnerability projections from Bayesian inference of multiple groundwater age tracers

DOE PAGES

Alikhani, Jamal; Deinhart, Amanda L.; Visser, Ate; ...

2016-04-20

Nitrate is a major source of contamination of groundwater in the United States and around the world. We tested the applicability of multiple groundwater age tracers ( 3H, 3He, 4He, 14C, 13C, and 85Kr) in projecting future trends of nitrate concentration in 9 long-screened, public drinking water wells in Turlock, California, where nitrate concentrations are increasing toward the regulatory limit. Very low 85Kr concentrations and apparent 3H/ 3He ages point to a relatively old modern fraction (40–50 years), diluted with pre-modern groundwater, corroborated by the onset and slope of increasing nitrate concentrations. An inverse Gaussian–Dirac model was chosen to representmore » the age distribution of the sampled groundwater at each well. Model parameters were estimated using a Bayesian inference, resulting in the posterior probability distribution – including the associated uncertainty – of the parameters and projected nitrate concentrations. Three scenarios were considered, including combined historic nitrate and age tracer data, the sole use of nitrate and the sole use of age tracer data. Each scenario was evaluated based on the ability of the model to reproduce the data and the level of reliability of the nitrate projections. The tracer-only scenario closely reproduced tracer concentrations, but not observed trends in the nitrate concentration. Both cases that included nitrate data resulted in good agreement with historical nitrate trends. Use of combined tracers and nitrate data resulted in a narrower range of projections of future nitrate levels. However, use of combined tracer and nitrate resulted in a larger discrepancy between modeled and measured tracers for some of the tracers. In conclusion, despite nitrate trend slopes between 0.56 and 1.73 mg/L/year in 7 of the 9 wells, the probability that concentrations will increase to levels above the MCL by 2040 are over 95% for only two of the wells, and below 15% in the other wells, due to a leveling off of reconstructed historical nitrate loadings to groundwater since about 1990.« less

Induction of the Nitrate Assimilation nirA Operon and Protein-Protein Interactions in the Maturation of Nitrate and Nitrite Reductases in the Cyanobacterium Anabaena sp. Strain PCC 7120.

PubMed

Frías, José E; Flores, Enrique

2015-07-01

Nitrate is widely used as a nitrogen source by cyanobacteria, in which the nitrate assimilation structural genes frequently constitute the so-called nirA operon. This operon contains the genes encoding nitrite reductase (nirA), a nitrate/nitrite transporter (frequently an ABC-type transporter; nrtABCD), and nitrate reductase (narB). In the model filamentous cyanobacterium Anabaena sp. strain PCC 7120, which can fix N2 in specialized cells termed heterocysts, the nirA operon is expressed at high levels only in media containing nitrate or nitrite and lacking ammonium, a preferred nitrogen source. Here we examined the genes downstream of the nirA operon in Anabaena and found that a small open reading frame of unknown function, alr0613, can be cotranscribed with the operon. The next gene in the genome, alr0614 (narM), showed an expression pattern similar to that of the nirA operon, implying correlated expression of narM and the operon. A mutant of narM with an insertion mutation failed to produce nitrate reductase activity, consistent with the idea that NarM is required for the maturation of NarB. Both narM and narB mutants were impaired in the nitrate-dependent induction of the nirA operon, suggesting that nitrite is an inducer of the operon in Anabaena. It has previously been shown that the nitrite reductase protein NirA requires NirB, a protein likely involved in protein-protein interactions, to attain maximum activity. Bacterial two-hybrid analysis confirmed possible NirA-NirB and NarB-NarM interactions, suggesting that the development of both nitrite reductase and nitrate reductase activities in cyanobacteria involves physical interaction of the corresponding enzymes with their cognate partners, NirB and NarM, respectively. Nitrate is an important source of nitrogen for many microorganisms that is utilized through the nitrate assimilation system, which includes nitrate/nitrite membrane transporters and the nitrate and nitrite reductases. Many cyanobacteria assimilate nitrate, but regulation of the nitrate assimilation system varies in different cyanobacterial groups. In the N2-fixing, heterocyst-forming cyanobacteria, the nirA operon, which includes the structural genes for the nitrate assimilation system, is expressed in the presence of nitrate or nitrite if ammonium is not available to the cells. Here we studied the genes required for production of an active nitrate reductase, providing information on the nitrate-dependent induction of the operon, and found evidence for possible protein-protein interactions in the maturation of nitrate reductase and nitrite reductase. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

On the role of brain serotonin in expression of genetic predisposition to catalepsy in animal models

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

Popova, N.K.; Kulikov, A.V.

1995-06-19

The activity of the rate-limiting enzyme of serotonin biosynthesis, tryptophan hydroxylase, in the striatum but not in the hippocampus and midbrain of rats bred for predisposition to catalepsy was higher than in nonselected rats. Mice of the highly susceptible to catalepsy CBA strain also differed from other noncataleptic mouse strains by the highest tryptophan hydroxylase activity in the striatum. Inhibition of tryptophan hydroxylase with p-chlorophenylalanine and p-chloromethamphetamine drastically decreased immobility time in hereditary predisposed to catalepsy animals. A decrease in the {sup 3}H-ketanserin specific binding in the striatum of cataleptic rats and CBA mice was found. It was suggested thatmore » this decrease in 5-HT2A serotonin receptor density represented a down regulation of the receptors due to an activation of serotonergic transmission in striatum. It is suggested that hereditary catalepsy may be resulted from genetic changes in the regulation of serotonin metabolism in striatum. 32 refs., 6 figs.« less

Growth hormone, insulin and aging: The benefits of endocrine defects

PubMed Central

Bartke, Andrzej

2012-01-01

Longevity of mice can be increased by spontaneous or experimentally induced mutations that interfere with the biosynthesis or actions of growth hormone (GH), insulin-like growth factor 1 (IGF-1), or insulin in the adipose tissue. The effects of GH resistance and deficiency of GH (along with thyrotropin and prolactin) on aging and lifespan are the most pronounced and best established of these mutations. Potential mechanisms linking these endocrine deficits with delayed aging and extended longevity include increased stress resistance, alterations in insulin and mammalian target of rapamycin (mTOR) signaling and metabolic adjustments. Physiological relationships deduced from the extreme phenotypes of long-lived mouse mutants appear to apply broadly, encompassing genetically normal (“wild-type”) mice and other mammalian species. The role of GH in the control of human aging continues to be hotly debated, but recent data indicate that reduced somatotropic signaling provides protection from cancer and other age-related diseases and may promote old age survival. PMID:20851173

Clock Regulation of Metabolites Reveals Coupling between Transcription and Metabolism.

PubMed

Krishnaiah, Saikumari Y; Wu, Gang; Altman, Brian J; Growe, Jacqueline; Rhoades, Seth D; Coldren, Faith; Venkataraman, Anand; Olarerin-George, Anthony O; Francey, Lauren J; Mukherjee, Sarmistha; Girish, Saiveda; Selby, Christopher P; Cal, Sibel; Er, Ubeydullah; Sianati, Bahareh; Sengupta, Arjun; Anafi, Ron C; Kavakli, I Halil; Sancar, Aziz; Baur, Joseph A; Dang, Chi V; Hogenesch, John B; Weljie, Aalim M

2017-04-04

The intricate connection between the circadian clock and metabolism remains poorly understood. We used high temporal resolution metabolite profiling to explore clock regulation of mouse liver and cell-autonomous metabolism. In liver, ∼50% of metabolites were circadian, with enrichment of nucleotide, amino acid, and methylation pathways. In U2 OS cells, 28% were circadian, including amino acids and NAD biosynthesis metabolites. Eighteen metabolites oscillated in both systems and a subset of these in primary hepatocytes. These 18 metabolites were enriched in methylation and amino acid pathways. To assess clock dependence of these rhythms, we used genetic perturbation. BMAL1 knockdown diminished metabolite rhythms, while CRY1 or CRY2 perturbation generally shortened or lengthened rhythms, respectively. Surprisingly, CRY1 knockdown induced 8 hr rhythms in amino acid, methylation, and vitamin metabolites, decoupling metabolite from transcriptional rhythms, with potential impact on nutrient sensing in vivo. These results provide the first comprehensive views of circadian liver and cell-autonomous metabolism. Copyright © 2017 Elsevier Inc. All rights reserved.

Lack of Tryptophan Hydroxylase-1 in Mice Results in Gait Abnormalities

PubMed Central

Suidan, Georgette L.; Vanderhorst, Veronique; Hampton, Thomas G.; Wong, Siu Ling; Voorhees, Jaymie R.; Wagner, Denisa D.

2013-01-01

The role of peripheral serotonin in nervous system development is poorly understood. Tryptophan hydroxylase-1 (TPH1) is expressed by non-neuronal cells including enterochromaffin cells of the gut, mast cells and the pineal gland and is the rate-limiting enzyme involved in the biosynthesis of peripheral serotonin. Serotonin released into circulation is taken up by platelets via the serotonin transporter and stored in dense granules. It has been previously reported that mouse embryos removed from Tph1-deficient mothers present abnormal nervous system morphology. The goal of this study was to assess whether Tph1-deficiency results in behavioral abnormalities. We did not find any differences between Tph1-deficient and wild-type mice in general motor behavior as tested by rotarod, grip-strength test, open field and beam walk. However, here we report that Tph1 (−/−) mice display altered gait dynamics and deficits in rearing behavior compared to wild-type (WT) suggesting that tryptophan hydroxylase-1 expression has an impact on the nervous system. PMID:23516593

Lack of tryptophan hydroxylase-1 in mice results in gait abnormalities.

PubMed

Suidan, Georgette L; Duerschmied, Daniel; Dillon, Gregory M; Vanderhorst, Veronique; Hampton, Thomas G; Wong, Siu Ling; Voorhees, Jaymie R; Wagner, Denisa D

2013-01-01

The role of peripheral serotonin in nervous system development is poorly understood. Tryptophan hydroxylase-1 (TPH1) is expressed by non-neuronal cells including enterochromaffin cells of the gut, mast cells and the pineal gland and is the rate-limiting enzyme involved in the biosynthesis of peripheral serotonin. Serotonin released into circulation is taken up by platelets via the serotonin transporter and stored in dense granules. It has been previously reported that mouse embryos removed from Tph1-deficient mothers present abnormal nervous system morphology. The goal of this study was to assess whether Tph1-deficiency results in behavioral abnormalities. We did not find any differences between Tph1-deficient and wild-type mice in general motor behavior as tested by rotarod, grip-strength test, open field and beam walk. However, here we report that Tph1 (-/-) mice display altered gait dynamics and deficits in rearing behavior compared to wild-type (WT) suggesting that tryptophan hydroxylase-1 expression has an impact on the nervous system.

In vivo self-bio-imaging of tumors through in situ biosynthesized fluorescent gold nanoclusters

NASA Astrophysics Data System (ADS)

Wang, Jianling; Zhang, Gen; Li, Qiwei; Jiang, Hui; Liu, Chongyang; Amatore, Christian; Wang, Xuemei

2013-01-01

Fluorescence imaging in vivo allows non-invasive tumor diagnostic thus permitting a direct monitoring of cancer therapies progresses. It is established herein that fluorescent gold nanoclusters are spontaneously biosynthesized by cancerous cell (i.e., HepG2, human hepatocarcinoma cell line; K562, leukemia cell line) incubated with micromolar chloroauric acid solutions, a biocompatible molecular Au(III) species. Gold nanoparticles form by Au(III) reduction inside cells cytoplasms and ultimately concentrate around their nucleoli, thus affording precise cell imaging. Importantly, this does not occur in non-cancerous cells, as evidenced with human embryo liver cells (L02) used as controls. This dichotomy is exploited for a new strategy for in vivo self-bio-imaging of tumors. Subcutaneous injections of millimolar chloroauric acid solution near xenograft tumors of the nude mouse model of hepatocellular carcinoma or chronic myeloid leukemia led to efficient biosynthesis of fluorescent gold nanoclusters without significant dissemination to the surrounding normal tissues, hence allowing specific fluorescent self-bio-marking of the tumors.

Dual-seq transcriptomics reveals the battle for iron during Pseudomonas aeruginosa acute murine pneumonia

PubMed Central

Damron, F. Heath; Oglesby-Sherrouse, Amanda G.; Wilks, Angela; Barbier, Mariette

2016-01-01

Determining bacterial gene expression during infection is fundamental to understand pathogenesis. In this study, we used dual RNA-seq to simultaneously measure P. aeruginosa and the murine host’s gene expression and response to respiratory infection. Bacterial genes encoding products involved in metabolism and virulence were differentially expressed during infection and the type III and VI secretion systems were highly expressed in vivo. Strikingly, heme acquisition, ferric-enterobactin transport, and pyoverdine biosynthesis genes were found to be significantly up-regulated during infection. In the mouse, we profiled the acute immune response to P. aeruginosa and identified the pro-inflammatory cytokines involved in acute response to the bacterium in the lung. Additionally, we also identified numerous host iron sequestration systems upregulated during infection. Overall, this work sheds light on how P. aeruginosa triggers a pro-inflammatory response and competes for iron with the host during infection, as iron is one of the central elements for which both pathogen and host fight during acute pneumonia. PMID:27982111

Spaceflight Activates Autophagy Programs and the Proteasome in Mouse Liver

PubMed Central

Blaber, Elizabeth A.; Pecaut, Michael J.

2017-01-01

Increased oxidative stress is an unavoidable consequence of exposure to the space environment. Our previous studies showed that mice exposed to space for 13.5 days had decreased glutathione levels, suggesting impairments in oxidative defense. Here we performed unbiased, unsupervised and integrated multi-‘omic analyses of metabolomic and transcriptomic datasets from mice flown aboard the Space Shuttle Atlantis. Enrichment analyses of metabolite and gene sets showed significant changes in osmolyte concentrations and pathways related to glycerophospholipid and sphingolipid metabolism, likely consequences of relative dehydration of the spaceflight mice. However, we also found increased enrichment of aminoacyl-tRNA biosynthesis and purine metabolic pathways, concomitant with enrichment of genes associated with autophagy and the ubiquitin-proteasome. When taken together with a downregulation in nuclear factor (erythroid-derived 2)-like 2-mediated signaling, our analyses suggest that decreased hepatic oxidative defense may lead to aberrant tRNA post-translational processing, induction of degradation programs and senescence-associated mitochondrial dysfunction in response to the spaceflight environment. PMID:28953266

Selective and potent urea inhibitors of Cryptosporidium parvum inosine 5′ monophosphate dehydrogenase

PubMed Central

Gorla, Suresh Kumar; Kavitha, Mandapati; Zhang, Minjia; Liu, Xiaoping; Sharling, Lisa; Gollapalli, Deviprasad R.; Striepen, Boris; Hedstrom, Lizbeth; Cuny, Gregory D.

2012-01-01

Cryptosporidium parvum and related species are zoonotic intracellular parasites of the intestine. Cryptosporidium is a leading cause of diarrhea in small children around the world. Infection can cause severe pathology in children and immunocompromised patients. This waterborne parasite is resistant to common methods of water treatment and therefore a prominent threat to drinking and recreation water even in countries with strong water safety systems. The drugs currently used to combat these organisms are ineffective. Genomic analysis revealed that the parasite relies solely on inosine-5′-monophosphate dehydrogenase (IMPDH) for the biosynthesis of guanine nucleotides. Herein, we report a selective urea-based inhibitor of C. parvum IMPDH (CpIMPDH) identified by high throughput screening. We performed a SAR study of these inhibitors with some analogues exhibiting high potency (IC50 < 2 nM) against CpIMPDH, excellent selectivity > 1000-fold versus human IMPDH type 2 and good stability in mouse liver microsomes. A subset of inhibitors also displayed potent antiparasitic activity in a Toxoplasma gondii model. PMID:22950983

Nicotinamide riboside promotes Sir2 silencing and extends lifespan via Nrk and Urh1/Pnp1/Meu1 pathways to NAD+.

PubMed

Belenky, Peter; Racette, Frances G; Bogan, Katrina L; McClure, Julie M; Smith, Jeffrey S; Brenner, Charles

2007-05-04

Although NAD(+) biosynthesis is required for Sir2 functions and replicative lifespan in yeast, alterations in NAD(+) precursors have been reported to accelerate aging but not to extend lifespan. In eukaryotes, nicotinamide riboside is a newly discovered NAD(+) precursor that is converted to nicotinamide mononucleotide by specific nicotinamide riboside kinases, Nrk1 and Nrk2. In this study, we discovered that exogenous nicotinamide riboside promotes Sir2-dependent repression of recombination, improves gene silencing, and extends lifespan without calorie restriction. The mechanism of action of nicotinamide riboside is totally dependent on increased net NAD(+) synthesis through two pathways, the Nrk1 pathway and the Urh1/Pnp1/Meu1 pathway, which is Nrk1 independent. Additionally, the two nicotinamide riboside salvage pathways contribute to NAD(+) metabolism in the absence of nicotinamide-riboside supplementation. Thus, like calorie restriction in the mouse, nicotinamide riboside elevates NAD(+) and increases Sir2 function.

Characterization of mitochondrial ferritin in Drosophila.

PubMed

Missirlis, Fanis; Holmberg, Sara; Georgieva, Teodora; Dunkov, Boris C; Rouault, Tracey A; Law, John H

2006-04-11

Mitochondrial function depends on iron-containing enzymes and proteins, whose maturation requires available iron for biosynthesis of iron-sulfur clusters and heme. Little is known about how mitochondrial iron homeostasis is maintained, although the recent discovery of a mitochondrial ferritin in mammals and plants has uncovered a potential key player in the process. Here, we show that Drosophila melanogaster expresses mitochondrial ferritin from an intron-containing gene. It has high similarity to the mouse and human mitochondrial ferritin sequences and, as in mammals, is expressed mainly in testis. This ferritin contains a putative mitochondrial targeting sequence and an epitope-tagged version localizes to mitochondria in transfected cells. Overexpression of mitochondrial ferritin fails to alter both total-body iron levels and iron that is bound to secretory ferritins. However, the viability of iron-deficient flies is compromised by overexpression of mitochondrial ferritin, suggesting that it may sequester iron at the expense of other important cellular functions. The conservation of mitochondrial ferritin in an insect species underscores the importance of this iron-storage molecule.

Endocannabinoids in Liver Disease

PubMed Central

Tam, Joseph; Liu, Jie; Mukhopadhyay, Bani; Cinar, Resat; Godlewski, Grzegorz; Kunos, George

2010-01-01

Endocannabinoids are lipid mediators of the same cannabinoid (CB) receptors that mediate the effects of marijuana. The endocannabinoid system (ECS) consists of CB receptors, endocannabinoids, and the enzymes involved in their biosynthesis and degradation, and is present both in brain and peripheral tissues, including the liver. The hepatic ECS is activated in various liver diseases, which contributes to the underlying pathologies. In cirrhosis of various etiologies, activation of vascular and cardiac CB1 receptors by macrophage- and platelet-derived endocannabinoids contribute to the vasodilated state and cardiomyopathy, which can be reversed by CB1 blockade. In mouse models of liver fibrosis, activation of CB1 receptors on hepatic stellate cells is fibrogenic, and CB1 blockade slows the progression of fibrosis. Fatty liver induced by high-fat diets or chronic alcohol feeding depend on activation of peripheral, including hepatic CB1 receptors, which also contribute to insulin resistance and dyslipidemias. Although the documented therapeutic potential of CB1 blockade is limited by neuropsychiatric side effects, these may be mitigated by using novel, peripherally restricted CB1 antagonists. PMID:21254182

Spaceflight Activates Autophagy Programs and the Proteasome in Mouse Liver.

PubMed

Blaber, Elizabeth A; Pecaut, Michael J; Jonscher, Karen R

2017-09-27

Increased oxidative stress is an unavoidable consequence of exposure to the space environment. Our previous studies showed that mice exposed to space for 13.5 days had decreased glutathione levels, suggesting impairments in oxidative defense. Here we performed unbiased, unsupervised and integrated multi-'omic analyses of metabolomic and transcriptomic datasets from mice flown aboard the Space Shuttle Atlantis. Enrichment analyses of metabolite and gene sets showed significant changes in osmolyte concentrations and pathways related to glycerophospholipid and sphingolipid metabolism, likely consequences of relative dehydration of the spaceflight mice. However, we also found increased enrichment of aminoacyl-tRNA biosynthesis and purine metabolic pathways, concomitant with enrichment of genes associated with autophagy and the ubiquitin-proteasome. When taken together with a downregulation in nuclear factor (erythroid-derived 2)-like 2-mediated signaling, our analyses suggest that decreased hepatic oxidative defense may lead to aberrant tRNA post-translational processing, induction of degradation programs and senescence-associated mitochondrial dysfunction in response to the spaceflight environment.

High Ambient Temperature Represses Anthocyanin Biosynthesis through Degradation of HY5

PubMed Central

Kim, Sara; Hwang, Geonhee; Lee, Seulgi; Zhu, Jia-Ying; Paik, Inyup; Nguyen, Thom Thi; Kim, Jungmook; Oh, Eunkyoo

2017-01-01

Anthocyanins are flavonoid compounds that protect plant tissues from many environmental stresses including high light irradiance, freezing temperatures, and pathogen infection. Regulation of anthocyanin biosynthesis is intimately associated with environmental changes to enhance plant survival under stressful environmental conditions. Various factors, such as UV, visible light, cold, osmotic stress, and pathogen infection, can induce anthocyanin biosynthesis. In contrast, high temperatures are known to reduce anthocyanin accumulation in many plant species, even drastically in the skin of fruits such as grape berries and apples. However, the mechanisms by which high temperatures regulate anthocyanin biosynthesis in Arabidopsis thaliana remain largely unknown. Here, we show that high ambient temperatures repress anthocyanin biosynthesis through the E3 ubiquitin ligase CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) and the positive regulator of anthocyanin biosynthesis ELONGATED HYPOCOTYL5 (HY5). We show that an increase in ambient temperature decreases expression of genes required in both the early and late steps of the anthocyanin biosynthesis pathway in Arabidopsis seedlings. As a result, seedlings grown at a high temperature (28°C) accumulate less anthocyanin pigment than those grown at a low temperature (17°C). We further show that high temperature induces the degradation of the HY5 protein in a COP1 activity-dependent manner. In agreement with this finding, anthocyanin biosynthesis and accumulation do not respond to ambient temperature changes in cop1 and hy5 mutant plants. The degradation of HY5 derepresses the expression of MYBL2, which partially mediates the high temperature repression of anthocyanin biosynthesis. Overall, our study demonstrates that high ambient temperatures repress anthocyanin biosynthesis through a COP1-HY5 signaling module. PMID:29104579

Comparative Proteomic Analysis Reveals Proteins Putatively Involved in Toxin Biosynthesis in the Marine Dinoflagellate Alexandrium catenella

PubMed Central

Wang, Da-Zhi; Gao, Yue; Lin, Lin; Hong, Hua-Sheng

2013-01-01

Alexandrium is a neurotoxin-producing dinoflagellate genus resulting in paralytic shellfish poisonings around the world. However, little is known about the toxin biosynthesis mechanism in Alexandrium. This study compared protein profiles of A. catenella collected at different toxin biosynthesis stages (non-toxin synthesis, initial toxin synthesis and toxin synthesizing) coupled with the cell cycle, and identified differentially expressed proteins using 2-DE and MALDI-TOF-TOF mass spectrometry. The results showed that toxin biosynthesis of A. catenella occurred within a defined time frame in the G1 phase of the cell cycle. Proteomic analysis indicated that 102 protein spots altered significantly in abundance (P < 0.05), and 53 proteins were identified using database searching. These proteins were involved in a variety of biological processes, i.e., protein modification and biosynthesis, metabolism, cell division, oxidative stress, transport, signal transduction, and translation. Among them, nine proteins with known functions in paralytic shellfish toxin-producing cyanobacteria, i.e., methionine S-adenosyltransferase, chloroplast ferredoxin-NADP+ reductase, S-adenosylhomocysteinase, adenosylhomocysteinase, ornithine carbamoyltransferase, inorganic pyrophosphatase, sulfotransferase (similar to), alcohol dehydrogenase and arginine deiminase, varied significantly at different toxin biosynthesis stages and formed an interaction network, indicating that they might be involved in toxin biosynthesis in A. catenella. This study is the first step in the dissection of the behavior of the A. catenella proteome during different toxin biosynthesis stages and provides new insights into toxin biosynthesis in dinoflagellates. PMID:23340676

Active secretion and protective effect of salivary nitrate against stress in human volunteers and rats

PubMed Central

Jin, Luyuan; Qin, Lizheng; Xia, Dengsheng; Liu, Xibao; Fan, Zhipeng; Zhang, Chunmei; Gu, Liankun; He, Junqi; Ambudkar, Indu S.; Deng, Dajun; Wang, Songlin

2014-01-01

Up to 25% of the circulating nitrate in blood is actively taken up, concentrated, and secreted into saliva by the salivary glands. Salivary nitrate can be reduced to nitrite by the commensal bacteria in the oral cavity or stomach and then further converted to nitric oxide (NO) in vivo, which may play a role in gastric protection. However, whether salivary nitrate is actively secreted in human beings has not yet been determined. This study was designed to determine whether salivary nitrate is actively secreted in human beings as an acute stress response and what role salivary nitrate plays in stress-induced gastric injury. To observe salivary nitrate function under stress conditions, alteration of salivary nitrate and nitrite was analyzed among 22 healthy volunteers before and after a strong stress activity, jumping down from a platform at the height of 68m. A series of stress indexes was analyzed to monitor the stress situation. We found that both the concentration and the total amount of nitrate in mixed saliva were significantly increased in the human volunteers immediately after the jump, with an additional increase 1 h later (p < 0.01). Saliva nitrite reached a maximum immediately after the jump and was maintained 1 h later. To study the biological functions of salivary nitrate and nitrite in stress protection, we further carried out a water-immersion-restraint stress (WIRS) assay in male adult rats with bilateral parotid and submandibular duct ligature (BPSDL). Intragastric nitrate, nitrite, and NO; gastric mucosal blood flow; and gastric ulcer index (UI) were monitored and nitrate was administrated in drinking water to compensate for nitrate secretion in BPSDL animals. Significantly decreased levels of intragastric nitrate, nitrite, and NO and gastricmucosal blood flow were measured in BPSDL rats during the WIRS assay compared to sham control rats (p < 0.05). Recovery was observed in the BPSDL rats upon nitrate administration. The WIRS-induced UI was significantly higher in the BPSDL animals compared to controls, and nitrate administration rescued the WIRS-induced gastric injury in BPSDL rats. In conclusion, this study suggests that stress promotes salivary nitrate secretion and nitrite formation, which may play important roles in gastric protection against stress-induced injury via the nitrate-dependent NO pathway. PMID:23277147

Identifying nitrate sources and transformations in surface water by combining dual isotopes of nitrate and stable isotope mixing model in a watershed with different land uses and multi-tributaries

NASA Astrophysics Data System (ADS)

Wang, Meng; Lu, Baohong

2017-04-01

Nitrate is essential for the growth and survival of plants, animals and humans. However, excess nitrate in drinking water is regarded as a health hazard as it is linked to infant methemoglobinemia and esophageal cancer. Revealing nitrate characteristics and identifying its sources are fundamental for making effective water management strategies, but nitrate sources in multi-tributaries and mixed land covered watersheds remain unclear. It is difficult to determine the predominant NO3- sources using conventional water quality monitoring techniques. In our study, based on 20 surface water sampling sites for more than two years' monitoring from April 2012 to December 2014, water chemical and dual isotopic approaches (δ15N-NO3- and δ18O-NO3-) were integrated for the first time to evaluate nitrate characteristics and sources in the Huashan watershed, Jianghuai hilly region, East China. The results demonstrated that nitrate content in surface water was relatively low in the downstream (<10 mg/L), but spatial heterogeneities were remarkable among different sub-watersheds. Extremely high nitrate was observed at the source of the river in one of the sub-watersheds, which exhibited an exponential decline along the stream due to dilution, absorption by aquatic plants, and high forest cover. Although dramatically decline of nitrate occurred along the stream, denitrification was not found in surface water by analyzing δ15N-NO3- and δ18O-NO3- relationship. Proportional contributions of five potential nitrate sources (i.e., precipitation; manure and sewage; soil nitrogen; nitrate fertilizer; nitrate derived from ammonia fertilizer and rainfall) were estimated using a Bayesian isotope mixing model. Model results indicated nitrate sources varied significantly among different rainfall conditions, land use types, as well as anthropologic activities. In summary, coupling dual isotopes of nitrate (δ15N-NO3- and δ18O-NO3-, simultaneously) with a Bayesian isotope mixing model offers a useful and practical way to qualitatively analyze nitrate sources and transformations as well as quantitatively estimate the contributions of potential nitrate sources in surface water. With the assessment of nitrate sources and characteristics, effective management strategies can be implemented to reduce N export and improve water quality in this region.

Determination of the origin of groundwater nitrate at an air weapons range using the dual isotope approach.

PubMed

Bordeleau, Geneviève; Savard, Martine M; Martel, Richard; Ampleman, Guy; Thiboutot, Sonia

2008-06-06

Nitrate is one of the most common contaminants in shallow groundwater, and many sources may contribute to the nitrate load within an aquifer. Groundwater nitrate plumes have been detected at several ammunition production sites. However, the presence of multiple potential sources and the lack of existing isotopic data concerning explosive degradation-induced nitrate constitute a limitation when it comes to linking both types of contaminants. On military training ranges, high nitrate concentrations in groundwater were reported for the first time as part of the hydrogeological characterization of the Cold Lake Air Weapons Range (CLAWR), Alberta, Canada. Explosives degradation is thought to be the main source of nitrate contamination at CLAWR, as no other major source is present. Isotopic analyses of N and O in nitrate were performed on groundwater samples from the unconfined and confined aquifers; the dual isotopic analysis approach was used in order to increase the chances of identifying the source of nitrate. The isotopic ratios for the groundwater samples with low nitrate concentration suggested a natural origin with a strong contribution of anthropogenic atmospheric NOx. For the samples with nitrate concentration above the expected background level the isotopic ratios did not correspond to any source documented in the literature. Dissolved RDX samples were degraded in the laboratory and results showed that all reproduced degradation processes released nitrate with a strong fractionation. Laboratory isotopic values for RDX-derived NO(3)(-) produced a trend of high delta(18)O-low delta(15)N to low delta(18)O-high delta(15)N, and groundwater samples with nitrate concentrations above the expected background level appeared along this trend. Our results thus point toward a characteristic field of isotopic ratios for nitrate being derived from the degradation of RDX.

Does the evidence about health risks associated with nitrate ingestion warrant an increase of the nitrate standard for drinking water?

PubMed Central

van Grinsven, Hans JM; Ward, Mary H; Benjamin, Nigel; de Kok, Theo M

2006-01-01

Several authors have suggested that it is safe to raise the health standard for nitrate in drinking water, and save money on measures associated with nitrate pollution of drinking water resources. The major argument has been that the epidemiologic evidence for acute and chronic health effects related to drinking water nitrate at concentrations near the health standard is inconclusive. With respect to the chronic effects, the argument was motivated by the absence of evidence for adverse health effects related to ingestion of nitrate from dietary sources. An interdisciplinary discussion of these arguments led to three important observations. First, there have been only a few well-designed epidemiologic studies that evaluated ingestion of nitrate in drinking water and risk of specific cancers or adverse reproductive outcomes among potentially susceptible subgroups likely to have elevated endogenous nitrosation. Positive associations have been observed for some but not all health outcomes evaluated. Second, the epidemiologic studies of cancer do not support an association between ingestion of dietary nitrate (vegetables) and an increased risk of cancer, because intake of dietary nitrate is associated with intake of antioxidants and other beneficial phytochemicals. Third, 2–3 % of the population in Western Europe and the US could be exposed to nitrate levels in drinking water exceeding the WHO standard of 50 mg/l nitrate, particularly those living in rural areas. The health losses due to this exposure cannot be estimated. Therefore, we conclude that it is not possible to weigh the costs and benefits from changing the nitrate standard for drinking water and groundwater resources by considering the potential consequences for human health and by considering the potential savings due to reduced costs for nitrate removal and prevention of nitrate pollution. PMID:16989661

Dissimilatory nitrate reduction by Aspergillus terreus isolated from the seasonal oxygen minimum zone in the Arabian Sea

PubMed Central

2014-01-01

Background A wealth of microbial eukaryotes is adapted to life in oxygen-deficient marine environments. Evidence is accumulating that some of these eukaryotes survive anoxia by employing dissimilatory nitrate reduction, a strategy that otherwise is widespread in prokaryotes. Here, we report on the anaerobic nitrate metabolism of the fungus Aspergillus terreus (isolate An-4) that was obtained from sediment in the seasonal oxygen minimum zone in the Arabian Sea, a globally important site of oceanic nitrogen loss and nitrous oxide emission. Results Axenic incubations of An-4 in the presence and absence of oxygen and nitrate revealed that this fungal isolate is capable of dissimilatory nitrate reduction to ammonium under anoxic conditions. A 15N-labeling experiment proved that An-4 produced and excreted ammonium through nitrate reduction at a rate of up to 175 nmol 15NH4+ g-1 protein h-1. The products of dissimilatory nitrate reduction were ammonium (83%), nitrous oxide (15.5%), and nitrite (1.5%), while dinitrogen production was not observed. The process led to substantial cellular ATP production and biomass growth and also occurred when ammonium was added to suppress nitrate assimilation, stressing the dissimilatory nature of nitrate reduction. Interestingly, An-4 used intracellular nitrate stores (up to 6–8 μmol NO3- g-1 protein) for dissimilatory nitrate reduction. Conclusions Our findings expand the short list of microbial eukaryotes that store nitrate intracellularly and carry out dissimilatory nitrate reduction when oxygen is absent. In the currently spreading oxygen-deficient zones in the ocean, an as yet unexplored diversity of fungi may recycle nitrate to ammonium and nitrite, the substrates of the major nitrogen loss process anaerobic ammonium oxidation, and the potent greenhouse gas nitrous oxide. PMID:24517718

Vascular effects of dietary nitrate (as found in green leafy vegetables and beetroot) via the nitrate-nitrite-nitric oxide pathway.

PubMed

Lidder, Satnam; Webb, Andrew J

2013-03-01

The discovery that dietary (inorganic) nitrate has important vascular effects came from the relatively recent realization of the 'nitrate-nitrite-nitric oxide (NO) pathway'. Dietary nitrate has been demonstrated to have a range of beneficial vascular effects, including reducing blood pressure, inhibiting platelet aggregation, preserving or improving endothelial dysfunction, enhancing exercise performance in healthy individuals and patients with peripheral arterial disease. Pre-clinical studies with nitrate or nitrite also show the potential to protect against ischaemia-reperfusion injury and reduce arterial stiffness, inflammation and intimal thickness. However, there is a need for good evidence for hard endpoints beyond epidemiological studies. Whilst these suggest reduction in cardiovascular risk with diets high in nitrate-rich vegetables (such as a Mediterranean diet), others have suggested possible small positive and negative associations with dietary nitrate and cancer, but these remain unproven. Interactions with other nutrients, such as vitamin C, polyphenols and fatty acids may enhance or inhibit these effects. In order to provide simple guidance on nitrate intake from different vegetables, we have developed the Nitrate 'Veg-Table' with 'Nitrate Units' [each unit being 1 mmol of nitrate (62 mg)] to achieve a nitrate intake that is likely to be sufficient to derive benefit, but also to minimize the risk of potential side effects from excessive ingestion, given the current available evidence. The lack of data concerning the long term effects of dietary nitrate is a limitation, and this will need to be addressed in future trials. © 2012 The Authors. British Journal of Clinical Pharmacology © 2012 The British Pharmacological Society.

Nitrate ammonification in mangrove soils: a hidden source of nitrite?

PubMed Central

Balk, Melike; Laverman, Anniet M.; Keuskamp, Joost A.; Laanbroek, Hendrikus J.

2015-01-01

Nitrate reduction is considered to be a minor microbial pathway in the oxidation of mangrove-derived organic matter due to a limited supply of nitrate in mangrove soils. At a limited availability of this electron acceptor compared to the supply of degradable carbon, nitrate ammonification is thought to be the preferential pathway of nitrate reduction. Mangrove forest mutually differ in their productivity, which may lead to different available carbon to nitrate ratios in their soil. Hence, nitrate ammonification is expected to be of more importance in high- compared to low-productive forests. The hypothesis was tested in flow-through reactors that contain undisturbed mangrove soils from high-productive Avicennia germinans and Rhizophora mangle forests in Florida and low-productive Avicennia marina forests in Saudi Arabia. Nitrate was undetectable in the soils from both regions. It was assumed that a legacy of nitrate ammonification would be reflected by a higher ammonium production from these soils upon the addition of nitrate. Unexpectedly, the soils from the low-productive forests in Saudi Arabia produced considerably more ammonium than the soils from the high-productive forests in Florida. Hence, other environmental factors than productivity must govern the selection of nitrate ammonification or denitrification. A rather intriguing observation was the 1:1 production of nitrite and ammonium during the consumption of nitrate, more or less independent from sampling region, location, sampling depth, mangrove species and from the absence or presence of additional degradable carbon. This 1:1 ratio points to a coupled production of ammonium and nitrite by one group of nitrate-reducing microorganisms. Such a production of nitrite will be hidden by the presence of active nitrite-reducing microorganisms under the nitrate-limited conditions of most mangrove forest soils. PMID:25784903

Resolving the contributions of the membrane-bound and periplasmic nitrate reductase systems to nitric oxide and nitrous oxide production in Salmonella enterica serovar Typhimurium.

PubMed

Rowley, Gary; Hensen, Daniela; Felgate, Heather; Arkenberg, Anke; Appia-Ayme, Corinne; Prior, Karen; Harrington, Carl; Field, Sarah J; Butt, Julea N; Baggs, Elizabeth; Richardson, David J

2012-01-15

The production of cytotoxic nitric oxide (NO) and conversion into the neuropharmacological agent and potent greenhouse gas nitrous oxide (N₂O) is linked with anoxic nitrate catabolism by Salmonella enterica serovar Typhimurium. Salmonella can synthesize two types of nitrate reductase: a membrane-bound form (Nar) and a periplasmic form (Nap). Nitrate catabolism was studied under nitrate-rich and nitrate-limited conditions in chemostat cultures following transition from oxic to anoxic conditions. Intracellular NO production was reported qualitatively by assessing transcription of the NO-regulated genes encoding flavohaemoglobin (Hmp), flavorubredoxin (NorV) and hybrid cluster protein (Hcp). A more quantitative analysis of the extent of NO formation was gained by measuring production of N₂O, the end-product of anoxic NO-detoxification. Under nitrate-rich conditions, the nar, nap, hmp, norV and hcp genes were all induced following transition from the oxic to anoxic state, and 20% of nitrate consumed in steady-state was released as N₂O when nitrite had accumulated to millimolar levels. The kinetics of nitrate consumption, nitrite accumulation and N₂O production were similar to those of wild-type in nitrate-sufficient cultures of a nap mutant. In contrast, in a narG mutant, the steady-state rate of N₂O production was ~30-fold lower than that of the wild-type. Under nitrate-limited conditions, nap, but not nar, was up-regulated following transition from oxic to anoxic metabolism and very little N₂O production was observed. Thus a combination of nitrate-sufficiency, nitrite accumulation and an active Nar-type nitrate reductase leads to NO and thence N₂O production, and this can account for up to 20% of the nitrate catabolized.

Dissimilatory nitrate reduction by Aspergillus terreus isolated from the seasonal oxygen minimum zone in the Arabian Sea.

PubMed

Stief, Peter; Fuchs-Ocklenburg, Silvia; Kamp, Anja; Manohar, Cathrine-Sumathi; Houbraken, Jos; Boekhout, Teun; de Beer, Dirk; Stoeck, Thorsten

2014-02-11

A wealth of microbial eukaryotes is adapted to life in oxygen-deficient marine environments. Evidence is accumulating that some of these eukaryotes survive anoxia by employing dissimilatory nitrate reduction, a strategy that otherwise is widespread in prokaryotes. Here, we report on the anaerobic nitrate metabolism of the fungus Aspergillus terreus (isolate An-4) that was obtained from sediment in the seasonal oxygen minimum zone in the Arabian Sea, a globally important site of oceanic nitrogen loss and nitrous oxide emission. Axenic incubations of An-4 in the presence and absence of oxygen and nitrate revealed that this fungal isolate is capable of dissimilatory nitrate reduction to ammonium under anoxic conditions. A ¹⁵N-labeling experiment proved that An-4 produced and excreted ammonium through nitrate reduction at a rate of up to 175 nmol ¹⁵NH₄⁺ g⁻¹ protein h⁻¹. The products of dissimilatory nitrate reduction were ammonium (83%), nitrous oxide (15.5%), and nitrite (1.5%), while dinitrogen production was not observed. The process led to substantial cellular ATP production and biomass growth and also occurred when ammonium was added to suppress nitrate assimilation, stressing the dissimilatory nature of nitrate reduction. Interestingly, An-4 used intracellular nitrate stores (up to 6-8 μmol NO₃⁻ g⁻¹ protein) for dissimilatory nitrate reduction. Our findings expand the short list of microbial eukaryotes that store nitrate intracellularly and carry out dissimilatory nitrate reduction when oxygen is absent. In the currently spreading oxygen-deficient zones in the ocean, an as yet unexplored diversity of fungi may recycle nitrate to ammonium and nitrite, the substrates of the major nitrogen loss process anaerobic ammonium oxidation, and the potent greenhouse gas nitrous oxide.

Nitrate ammonification in mangrove soils: a hidden source of nitrite?

PubMed

Balk, Melike; Laverman, Anniet M; Keuskamp, Joost A; Laanbroek, Hendrikus J

2015-01-01

Nitrate reduction is considered to be a minor microbial pathway in the oxidation of mangrove-derived organic matter due to a limited supply of nitrate in mangrove soils. At a limited availability of this electron acceptor compared to the supply of degradable carbon, nitrate ammonification is thought to be the preferential pathway of nitrate reduction. Mangrove forest mutually differ in their productivity, which may lead to different available carbon to nitrate ratios in their soil. Hence, nitrate ammonification is expected to be of more importance in high- compared to low-productive forests. The hypothesis was tested in flow-through reactors that contain undisturbed mangrove soils from high-productive Avicennia germinans and Rhizophora mangle forests in Florida and low-productive Avicennia marina forests in Saudi Arabia. Nitrate was undetectable in the soils from both regions. It was assumed that a legacy of nitrate ammonification would be reflected by a higher ammonium production from these soils upon the addition of nitrate. Unexpectedly, the soils from the low-productive forests in Saudi Arabia produced considerably more ammonium than the soils from the high-productive forests in Florida. Hence, other environmental factors than productivity must govern the selection of nitrate ammonification or denitrification. A rather intriguing observation was the 1:1 production of nitrite and ammonium during the consumption of nitrate, more or less independent from sampling region, location, sampling depth, mangrove species and from the absence or presence of additional degradable carbon. This 1:1 ratio points to a coupled production of ammonium and nitrite by one group of nitrate-reducing microorganisms. Such a production of nitrite will be hidden by the presence of active nitrite-reducing microorganisms under the nitrate-limited conditions of most mangrove forest soils.

C{sub 1}-C{sub 15} alkyl nitrates, benzyl nitrate, and bifunctional nitrates: Measurements in California and South Atlantic air and global comparison using C{sub 2}Cl{sub 4} and CHBr{sub 3} as marker molecules

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

Schneider, M.; Luxenhofer, O.; Deissler, A.

1998-10-15

Measurements of C{sub 1}--C{sub 15} alkyl nitrates, perchloroethylene, and bromoform at two different sampling sites near Santa Cruz, CA, were conducted in 1995. The halocarbons were used as marker molecules to differentiate the air parcels collected into marine and continental groups. The average concentration of {Sigma}n/i-C{sub 3}--C{sub 12} alkyl nitrates at the California Coast was lower than the levels obtained in the coastal mountains. This difference was shown to be most significant for the long chain n/i-C{sub 6}--C{sub 12} alkyl nitrates. It is concluded that the {ge}C{sub 6} alkyl nitrates in continental air can contribute 1--2% to the total NO{submore » y}. The results are summarized together with earlier data sets to give a picture of contemporary levels and of the global occurrence of C{sub 3}--C{sub 12} alkyl nitrates. In comparison with South Atlantic air, pattern analysis of n-alkyl nitrates suggests a marine source of primary n-alkyl nitrates. It is also shown that liquid chromatographic preseparation of the air sample extracts leads to a fraction that contains more polar organic nitrates. Several alkyl dinitrates and benzyl nitrate are detected in air samples from California, the South Atlantic region, and Europe. The vicinal alkyl dinitrates show increased abundance in a nighttime sample. The relative abundance of benzyl nitrate compared to alkyl (mono) nitrates is used as a tool for global air mass characterization.« less

Contribution of atmospheric nitrate to stream-water nitrate in Japanese coniferous forests revealed by the oxygen isotope ratio of nitrate.

PubMed

Tobari, Y; Koba, K; Fukushima, K; Tokuchi, N; Ohte, N; Tateno, R; Toyoda, S; Yoshioka, T; Yoshida, N

2010-05-15

Evaluation of the openness of the nitrogen (N) cycle in forest ecosystems is important in efforts to improve forest management because the N supply often limits primary production. The use of the oxygen isotope ratio (delta(18)O) of nitrate is a promising approach to determine how effectively atmospheric nitrate can be retained in a forest ecosystem. We investigated the delta(18)O of nitrate in stream water in order to estimate the contribution of atmospheric NO(3) (-) in stream-water NO(3) (-) (f(atm)) from 26 watersheds with different stand ages (1-87 years) in Japan. The stream-water nitrate concentrations were high in young forests whereas, in contrast, old forests discharged low-nitrate stream water. These results implied a low f(atm) and a closed N cycle in older forests. However, the delta(18)O values of nitrate in stream water revealed that f(atm) values were higher in older forests than in younger forests. These results indicated that even in old forests, where the discharged N loss was small, atmospheric nitrate was not retained effectively. The steep slopes of the studied watersheds (>40 degrees ) which hinder the capturing of atmospheric nitrate by plants and microbes might be responsible for the inefficient utilization of atmospheric nitrate. Moreover, the unprocessed fraction of atmospheric nitrate in the stream-water nitrate in the forest (f(unprocessed)) was high in the young forest (78%), although f(unprocessed) was stable and low for other forests (5-13%). This high f(unprocessed) of the young forest indicated that the young forest retained neither atmospheric NO(3) (-) nor soil NO(3) (-) effectively, engendering high stream-water NO(3) (-) concentrations. Copyright (c) 2010 John Wiley & Sons, Ltd.

Contrasting nitrate adsorption in Andisols of two coffee plantations in Costa Rica.

PubMed

Ryan, M C; Graham, G R; Rudolph, D L

2001-01-01

Fertilizer use in coffee plantations is a suspected cause of rising ground water nitrate concentrations in the ground water-dependent Central Valley of Costa Rica. Nitrate adsorption was evaluated beneath two coffee (Coffea arabica L.) plantations in the Central Valley. Previous work at one site had identified unsaturated zone nitrate retardation relative to a tritium tracer. Differences in nitrate adsorption were assessed in cores to 4 m depth in Andisols at this and one other plantation using differences in KCl- and water-extractable nitrate as an index. Significant adsorption was confirmed at the site of the previous tracer test, but not at the second site. Anion exchange capacity, X-ray diffraction data, extractable Al and Si, and soil pH in NaF corroborated that differences in adsorption characteristics were related to subtle differences in clay mineralogy. Soils at the site with significant nitrate adsorption showed an Al-rich allophane clay content compared with a more weathered, Si-rich allophane and halloysite clay mineral content at the site with negligible adsorption. At the site with significant nitrate adsorption, nitrate occupied less than 10% of the total anion adsorption capacity, suggesting that adsorption may provide long-term potential for mitigation or delay of nitrate leaching. Evaluation of nitrate sorption potential of soil at local and landscape scales would be useful in development of nitrogen management practices to reduce nitrate leaching to ground water.

Factors influencing protein tyrosine nitration--structure-based predictive models.

PubMed

Bayden, Alexander S; Yakovlev, Vasily A; Graves, Paul R; Mikkelsen, Ross B; Kellogg, Glen E

2011-03-15

Models for exploring tyrosine nitration in proteins have been created based on 3D structural features of 20 proteins for which high-resolution X-ray crystallographic or NMR data are available and for which nitration of 35 total tyrosines has been experimentally proven under oxidative stress. Factors suggested in previous work to enhance nitration were examined with quantitative structural descriptors. The role of neighboring acidic and basic residues is complex: for the majority of tyrosines that are nitrated the distance to the heteroatom of the closest charged side chain corresponds to the distance needed for suspected nitrating species to form hydrogen bond bridges between the tyrosine and that charged amino acid. This suggests that such bridges play a very important role in tyrosine nitration. Nitration is generally hindered for tyrosines that are buried and for those tyrosines for which there is insufficient space for the nitro group. For in vitro nitration, closed environments with nearby heteroatoms or unsaturated centers that can stabilize radicals are somewhat favored. Four quantitative structure-based models, depending on the conditions of nitration, have been developed for predicting site-specific tyrosine nitration. The best model, relevant for both in vitro and in vivo cases, predicts 30 of 35 tyrosine nitrations (positive predictive value) and has a sensitivity of 60/71 (11 false positives). Copyright © 2010 Elsevier Inc. All rights reserved.

Effect of nitrate, acetate and hydrogen on native perchlorate-reducing microbial communities and their activity in vadose soil

PubMed Central

Nozawa-Inoue, Mamie; Jien, Mercy; Yang, Kun; Rolston, Dennis E.; Hristova, Krassimira R.; Scow, Kate M.

2011-01-01

Effect of nitrate, acetate and hydrogen on native perchlorate-reducing bacteria (PRB) was examined by conducting microcosm tests using vadose soil collected from a perchlorate-contaminated site. The rate of perchlorate reduction was enhanced by hydrogen amendment and inhibited by acetate amendment, compared to unamendment. Nitrate was reduced before perchlorate in all amendments. In hydrogen-amended and unamended soils, nitrate delayed perchlorate reduction, suggesting the PRB preferentially use nitrate as an electron acceptor. In contrast, nitrate eliminated the inhibitory effect of acetate amendment on perchlorate reduction and increased the rate and the extent, possibly because the preceding nitrate reduction/denitrification decreased the acetate concentration which was inhibitory to the native PRB. In hydrogen-amended and unamended soils, perchlorate reductase gene (pcrA) copies, representing PRB densities, increased with either perchlorate or nitrate reduction, suggesting either perchlorate or nitrate stimulates growth of the PRB. In contrast, in acetate-amended soil pcrA increased only when perchlorate was depleted: a large portion of the PRB may have not utilized nitrate in this amendment. Nitrate addition did not alter the distribution of the dominant pcrA clones in hydrogen-amended soil, likely because of the functional redundancy of PRB as nitrate-reducers/denitrifiers, whereas acetate selected different pcrA clones from those with hydrogen amendment. PMID:21284679

The Arabidopsis Vacuolar Sorting Receptor1 Is Required for Osmotic Stress-Induced Abscisic Acid Biosynthesis1[OPEN

PubMed Central

Wang, Zhen-Yu; Gehring, Chris; Zhu, Jianhua; Li, Feng-Min; Zhu, Jian-Kang; Xiong, Liming

2015-01-01

Osmotic stress activates the biosynthesis of the phytohormone abscisic acid (ABA) through a pathway that is rate limited by the carotenoid cleavage enzyme 9-cis-epoxycarotenoid dioxygenase (NCED). To understand the signal transduction mechanism underlying the activation of ABA biosynthesis, we performed a forward genetic screen to isolate mutants defective in osmotic stress regulation of the NCED3 gene. Here, we identified the Arabidopsis (Arabidopsis thaliana) Vacuolar Sorting Receptor1 (VSR1) as a unique regulator of ABA biosynthesis. The vsr1 mutant not only shows increased sensitivity to osmotic stress, but also is defective in the feedback regulation of ABA biosynthesis by ABA. Further analysis revealed that vacuolar trafficking mediated by VSR1 is required for osmotic stress-responsive ABA biosynthesis and osmotic stress tolerance. Moreover, under osmotic stress conditions, the membrane potential, calcium flux, and vacuolar pH changes in the vsr1 mutant differ from those in the wild type. Given that manipulation of the intracellular pH is sufficient to modulate the expression of ABA biosynthesis genes, including NCED3, and ABA accumulation, we propose that intracellular pH changes caused by osmotic stress may play a signaling role in regulating ABA biosynthesis and that this regulation is dependent on functional VSR1. PMID:25416474

Glycerol-3-phosphate O-acyltransferase is required for PBAN-induced sex pheromone biosynthesis in Bombyx mori

PubMed Central

Du, Mengfang; Liu, Xiaoguang; Liu, Xiaoming; Yin, Xinming; Han, Shuangyin; Song, Qisheng; An, Shiheng

2015-01-01

Female moths employ their own pheromone blends as a communicational medium in mating behavior. The biosynthesis and release of sex pheromone in female moths are regulated by pheromone biosynthesis activating neuropeptide (PBAN) and the corresponding action of PBAN has been well elucidated in Bombyx mori. However, very little is known about the molecular mechanism regarding the biosynthesis of sex pheromone precursor. In this study, quantitative proteomics was utilized to comprehensively elucidate the expression dynamics of pheromone glands (PGs) during development. Proteomic analysis revealed a serial of differentially expressed sex pheromone biosynthesis-associated proteins at the different time points of B. mori development. Most interestingly B. mori glycerol-3-phosphate O-acyltransferase (BmGPAT) was found to be expressed during the key periods of sex pheromone biosynthesis. RNAi knockdown of BmGPAT confirmed the important function of this protein in the biosynthesis of sex pheromone precursor, triacylglcerol (TAG), and subsequently PBAN-induced production of sex pheromone, bombykol. Behavioral analysis showed that RNAi knockdown of GPAT significantly impaired the ability of females to attract males. Our findings indicate that GPAT acts to regulate the biosynthesis of sex pheromone precursor, TAG, thus influencing PBAN-induced sex pheromone production and subsequent mating behavior. PMID:25630665

Characterization of Lipooligosaccharide-Biosynthetic Loci of Campylobacter jejuni Reveals New Lipooligosaccharide Classes: Evidence of Mosaic Organizations▿ †

PubMed Central

Parker, Craig T.; Gilbert, Michel; Yuki, Nobuhiro; Endtz, Hubert P.; Mandrell, Robert E.

2008-01-01

The lipooligosaccharide (LOS) biosynthesis region is one of the more variable genomic regions between strains of Campylobacter jejuni. Indeed, eight classes of LOS biosynthesis loci have been established previously based on gene content and organization. In this study, we characterize additional classes of LOS biosynthesis loci and analyze various mechanisms that result in changes to LOS structures. To gain further insights into the genomic diversity of C. jejuni LOS biosynthesis region, we sequenced the LOS biosynthesis loci of 15 strains that possessed gene content that was distinct from the eight classes. This analysis identified 11 new classes of LOS loci that exhibited examples of deletions and insertions of genes and cassettes of genes found in other LOS classes or capsular biosynthesis loci leading to mosaic LOS loci. The sequence analysis also revealed both missense mutations leading to “allelic” glycosyltransferases and phase-variable and non-phase-variable gene inactivation by the deletion or insertion of bases. Specifically, we demonstrated that gene inactivation is an important mechanism for altering the LOS structures of strains possessing the same class of LOS biosynthesis locus. Together, these observations suggest that LOS biosynthesis region is a hotspot for genetic exchange and variability, often leading to changes in the LOS produced. PMID:18556784

Functional analysis of aromatic biosynthetic pathways in Pseudomonas putida KT2440

PubMed Central

Molina‐Henares, M. Antonia; García‐Salamanca, Adela; Molina‐Henares, A. Jesús; De La Torre, Jesús; Herrera, M. Carmen; Ramos, Juan L.; Duque, Estrella

2009-01-01

Summary Pseudomonas putida KT2440 is a non‐pathogenic prototrophic bacterium with high potential for biotechnological applications. Despite all that is known about this strain, the biosynthesis of essential chemicals has not been fully analysed and auxotroph mutants are scarce. We carried out massive mini‐Tn5 random mutagenesis and screened for auxotrophs that require aromatic amino acids. The biosynthesis of aromatic amino acids was analysed in detail including physical and transcriptional organization of genes, complementation assays and feeding experiments to establish pathway intermediates. There is a single pathway from chorismate leading to the biosynthesis of tryptophan, whereas the biosynthesis of phenylalanine and tyrosine is achieved through multiple convergent pathways. Genes for tryptophan biosynthesis are grouped in unlinked regions with the trpBA and trpGDE genes organized as operons and the trpI, trpE and trpF genes organized as single transcriptional units. The pheA and tyrA gene‐encoding multifunctional enzymes for phenylalanine and tyrosine biosynthesis are linked in the chromosome and form an operon with the serC gene involved in serine biosynthesis. The last step in the biosynthesis of these two amino acids requires an amino transferase activity for which multiple tyrB‐like genes are present in the host chromosome. PMID:21261884

Glycoprotein biosynthesis in animal cells grown in suspension culture. Assembly of lipid-linked saccharides and formation of protein-bound 'high-mannose' oligosaccharides.

PubMed Central

Bailey, D S; Burke, J; Sinclair, R; Mukherjee, B B

1981-01-01

Glycoprotein biosynthesis was studied with mouse L-cells grown in suspension culture. Glucose-deprived cells incorporated [3H]mannose into 'high-mannose' protein-bound oligosaccharides and a few relatively high-molecular-weight lipid-linked oligosaccharides. The latter were retained by DEAE-cellulose and turned over quite slowly during pulse--chase experiments. Increased heterogeneity in size of lipid-linked oligosaccharides developed during prolonged glucose deprivation. Sequential elongation of lipid-linked oligosaccharides was also observed, and conditions that prevented the assembly of the higher lipid-linked oligosaccharides also prevented the formation of the larger protein-bound 'high-mannose' oligosaccharides. In parallel experiments, [3H]mannose was incorporated into a total polyribosome fraction, suggesting that mannose residues were transferred co-translationally to nascent protein. Membrane preparations from these cells catalysed the assembly from UDP-N-acetyl-D-[6-3H]glucosamine and GDP-D-[U-14C]mannose of polyisoprenyl diphosphate derivatives whose oligosaccharide moieties were heterogeneous in size. Elongation of the N-acetyl-D-[6-3H]glucosamine-initiated glycolipids with mannose residues produced several higher lipid-linked oligosaccharides similar to those seen during glucose deprivation in vivo. Glucosylation of these mannose-containing oligosaccharides from UDP-D-[6-3H]glucose was restricted to those of a relatively high molecular weight. Protein-bound saccharides formed in vitro were mainly smaller in size than those assembled on the lipid acceptors. These results support the involvement of lipid-linked saccharides in the synthesis of asparagine-linked glycoproteins, but show both in vivo and in vitro that protein-bound 'high-mannose' oligosaccharide formation can occur independently of higher lipid-linked oligosaccharide synthesis. PMID:7306042

Targeted Deletions of COX-2 and Atherogenesis in Mice

PubMed Central

Hui, Yiqun; Ricciotti, Emanuela; Crichton, Irene; Yu, Zhou; Wang, Dairong; Stubbe, Jane; Wang, Miao; Puré, Ellen; FitzGerald, Garret A.

2010-01-01

Background While the dominant product of vascular cyclooxygenase (COX)-2, prostacyclin (PGI2), restrains atherogenesis, inhibition and deletion of COX-2 have yielded conflicting results in mouse models of atherosclerosis. Floxed mice were used to parse distinct cellular contributions of COX-2 in macrophages (Mac) and T cells (TC) to atherogenesis. Methods and Results Deletion of Mac COX-2 (MacKO) was attained using LysMCre mice and suppressed completely lipopolysaccharide (LPS) stimulated Mac prostaglandin (PG) formation and LPS evoked systemic PG biosynthesis by ∼ 30%. LPS stimulated COX-2 expression was suppressed in polymorphonuclear leucocytes (PMN) isolated from MacKOs, but PG formation was not even detected in PMN supernatants from control mice. Atherogenesis was attenuated when MacKOs were crossed into hyperlipidemic LdlR KOs. Deletion of Mac COX-2 appeared to remove a restraint on COX-2 expression in lesional non-leukocyte (CD45 and CD11b negative) vascular cells that express vascular cell adhesion molecule and variably, α-smooth muscle actin and vimentin, portending a shift in PG profile and consequent atheroprotection. Basal expression of COX-2 was minimal in TCs, but use of CD4Cre to generate TC knockouts (TCKOs) depressed its modest upregulation by anti-CD3ε. However, biosynthesis of PGs, TC composition in lymphatic organs and atherogenesis in LDLR KOs were unaltered in TCKOs. Conclusions Mac COX-2, primarily a source of thromboxane A2 and PGE2, promotes atherogenesis and exerts a restraint on enzyme expression by lesional cells suggestive of vascular smooth muscle cells, a prominent source of atheroprotective PGI2. TC COX-2 does not influence detectably TC development or function nor atherogenesis in mice. PMID:20530000

Hyperforin, an Anti-Inflammatory Constituent from St. John's Wort, Inhibits Microsomal Prostaglandin E2 Synthase-1 and Suppresses Prostaglandin E2 Formation in vivo

PubMed Central

Koeberle, Andreas; Rossi, Antonietta; Bauer, Julia; Dehm, Friederike; Verotta, Luisella; Northoff, Hinnak; Sautebin, Lidia; Werz, Oliver

2010-01-01

The acylphloroglucinol hyperforin (Hyp) from St. John's wort possesses anti-inflammatory and anti-carcinogenic properties which were ascribed among others to the inhibition of 5-lipoxygenase. Here, we investigated whether Hyp also interferes with prostanoid generation in biological systems, particularly with key enzymes participating in prostaglandin (PG)E2 biosynthesis, i.e., cyclooxygenases (COX)-1/2 and microsomal PGE2 synthase (mPGES)-1 which play key roles in inflammation and tumorigenesis. Similar to the mPGES-1 inhibitors MK-886 and MD-52, Hyp significantly suppressed PGE2 formation in whole blood assays starting at 0.03–1 μM, whereas the concomitant generation of COX-derived 12(S)-hydroxy-5-cis-8,10-trans-heptadecatrienoic acid, thromboxane B2, and 6-keto PGF1α was not significantly suppressed up to 30 μM. In cell-free assays, Hyp efficiently blocked the conversion of PGH2 to PGE2 mediated by mPGES-1 (IC50 = 1 μM), and isolated COX enzymes were not (COX-2) or hardly (COX-1) suppressed. Intraperitoneal (i.p.) administration of Hyp (4 mg kg−1) to rats impaired exudate volume and leukocyte numbers in carrageenan-induced pleurisy associated with reduced PGE2 levels, and Hyp (given i.p.) inhibited carrageenan-induced mouse paw edema formation (ED50 = 1 mg kg−1) being superior over indomethacin (ED50 = 5 mg kg−1). We conclude that the suppression of PGE2 biosynthesis in vitro and in vivo by acting on mPGES-1 critically contributes to the anti-inflammatory efficiency of Hyp. PMID:21687502

TP53 modulating agent, CP-31398 enhances antitumor effects of ODC inhibitor in mouse model of urinary bladder transitional cell carcinoma.

PubMed

Madka, Venkateshwar; Mohammed, Altaf; Li, Qian; Zhang, Yuting; Kumar, Gaurav; Lightfoot, Stan; Wu, Xueru; Steele, Vernon; Kopelovich, Levy; Rao, Chinthalapally V

2015-01-01

Mutations of the tumor suppressor p53 and elevated levels of polyamines are known to play key roles in urothelial tumorigenesis. We investigated the inhibition of polyamines biosynthesis and the restoration of p53 signaling as a possible means of preventing muscle invasive urothelial tumors using DFMO, an ODC-inhibiting agent, and CP-31398 (CP), a p53 stabilizing agent. Transgenic UPII-SV40T male mice at 6weeks age (n=15/group) were fed control diet (AIN-76A) or experimental diets containing DFMO (1000 and 2000 ppm) or 150 ppm CP or both. At 40 weeks of age, all mice were euthanized and urinary bladders were evaluated to determine tumor weight and histopathology. Low-dose DFMO had a moderate significant inhibitory effect on tumor growth (38%, P<0.02) and tumor invasion (23%). High-dose DFMO had a 47% tumor inhibition (P<0.0001) and 40% inhibition tumor invasion. There was no significant difference between 1000 and 2000 ppm doses of DFMO (P>0.05). CP at 150 ppm alone had a strong inhibitory effect on tumor growth by 80% (P<0.0001); however, no effect on tumor invasion was observed. Interestingly, the combination of DFMO (1000 ppm) and CP (150 ppm) led to significant decrease in tumor weight (70%, P<0.0001) and tumor invasion (62.5%; P<0.005). Molecular analysis of the urothelial tumors suggested a modulation of polyamine biosynthesis, proliferation, cell cycle regulators resulting from the use of these agents. These results suggest that targeting two or more pathways could be an effective approach for chemoprevention. A combination of CP and DFMO appears to be a promising strategy for urothelial TCC prevention.

Identification of N-Acetyltaurine as a Novel Metabolite of Ethanol through Metabolomics-guided Biochemical Analysis*

PubMed Central

Shi, Xiaolei; Yao, Dan; Chen, Chi

2012-01-01

The influence of ethanol on the small molecule metabolome and the role of CYP2E1 in ethanol-induced hepatotoxicity were investigated using liquid chromatography-mass spectrometry (LC-MS)-based metabolomics platform and Cyp2e1-null mouse model. Histological and biochemical examinations of ethanol-exposed mice indicated that the Cyp2e1-null mice were more resistant to ethanol-induced hepatic steatosis and transaminase leakage than the wild-type mice, suggesting CYP2E1 contributes to ethanol-induced toxicity. Metabolomic analysis of urinary metabolites revealed time- and dose-dependent changes in the chemical composition of urine. Along with ethyl glucuronide and ethyl sulfate, N-acetyltaurine (NAT) was identified as a urinary metabolite that is highly responsive to ethanol exposure and is correlated with the presence of CYP2E1. Subsequent stable isotope labeling analysis using deuterated ethanol determined that NAT is a novel metabolite of ethanol. Among three possible substrates of NAT biosynthesis (taurine, acetyl-CoA, and acetate), the level of taurine was significantly reduced, whereas the levels of acetyl-CoA and acetate were dramatically increased after ethanol exposure. In vitro incubation assays suggested that acetate is the main precursor of NAT, which was further confirmed by the stable isotope labeling analysis using deuterated acetate. The incubations of tissues and cellular fractions with taurine and acetate indicated that the kidney has the highest NAT synthase activity among the tested organs, whereas the cytosol is the main site of NAT biosynthesis inside the cell. Overall, the combination of biochemical and metabolomic analysis revealed NAT is a novel metabolite of ethanol and a potential biomarker of hyperacetatemia. PMID:22228769

Bacterial Colonization of Host Cells in the Absence of Cholesterol

PubMed Central

Gilk, Stacey D.; Cockrell, Diane C.; Luterbach, Courtney; Hansen, Bryan; Knodler, Leigh A.; Ibarra, J. Antonio; Steele-Mortimer, Olivia; Heinzen, Robert A.

2013-01-01

Reports implicating important roles for cholesterol and cholesterol-rich lipid rafts in host-pathogen interactions have largely employed sterol sequestering agents and biosynthesis inhibitors. Because the pleiotropic effects of these compounds can complicate experimental interpretation, we developed a new model system to investigate cholesterol requirements in pathogen infection utilizing DHCR24−/− mouse embryonic fibroblasts (MEFs). DHCR24−/− MEFs lack the Δ24 sterol reductase required for the final enzymatic step in cholesterol biosynthesis, and consequently accumulate desmosterol into cellular membranes. Defective lipid raft function by DHCR24−/− MEFs adapted to growth in cholesterol-free medium was confirmed by showing deficient uptake of cholera-toxin B and impaired signaling by epidermal growth factor. Infection in the absence of cholesterol was then investigated for three intracellular bacterial pathogens: Coxiella burnetii, Salmonella enterica serovar Typhimurium, and Chlamydia trachomatis. Invasion by S. Typhimurium and C. trachomatis was unaltered in DHCR24−/− MEFs. In contrast, C. burnetii entry was significantly decreased in −cholesterol MEFs, and also in +cholesterol MEFs when lipid raft-associated αVβ3 integrin was blocked, suggesting a role for lipid rafts in C. burnetii uptake. Once internalized, all three pathogens established their respective vacuolar niches and replicated normally. However, the C. burnetii-occupied vacuole within DHCR24−/− MEFs lacked the CD63-postive material and multilamellar membranes typical of vacuoles formed in wild type cells, indicating cholesterol functions in trafficking of multivesicular bodies to the pathogen vacuole. These data demonstrate that cholesterol is not essential for invasion and intracellular replication by S. Typhimurium and C. trachomatis, but plays a role in C. burnetii-host cell interactions. PMID:23358892

21 CFR 170.60 - Nitrites and/or nitrates in curing premixes.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Nitrites and/or nitrates in curing premixes. 170... and Decisions § 170.60 Nitrites and/or nitrates in curing premixes. (a) Nitrites and/or nitrates are.... (b) Nitrites and/or nitrates, when packaged separately from flavoring and seasoning in curing...

21 CFR 176.320 - Sodium nitrate-urea complex.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Sodium nitrate-urea complex. 176.320 Section 176... Paperboard § 176.320 Sodium nitrate-urea complex. Sodium nitrate-urea complex may be safely used as a..., packaging, transporting, or holding food, subject to the provisions of this section. (a) Sodium nitrate-urea...

21 CFR 170.60 - Nitrites and/or nitrates in curing premixes.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Nitrites and/or nitrates in curing premixes. 170... and Decisions § 170.60 Nitrites and/or nitrates in curing premixes. (a) Nitrites and/or nitrates are.... (b) Nitrites and/or nitrates, when packaged separately from flavoring and seasoning in curing...

21 CFR 170.60 - Nitrites and/or nitrates in curing premixes.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Nitrites and/or nitrates in curing premixes. 170... and Decisions § 170.60 Nitrites and/or nitrates in curing premixes. (a) Nitrites and/or nitrates are.... (b) Nitrites and/or nitrates, when packaged separately from flavoring and seasoning in curing...