Liver glucose metabolism in humans

PubMed Central

Adeva-Andany, María M.; Pérez-Felpete, Noemi; Fernández-Fernández, Carlos; Donapetry-García, Cristóbal; Pazos-García, Cristina

2016-01-01

Information about normal hepatic glucose metabolism may help to understand pathogenic mechanisms underlying obesity and diabetes mellitus. In addition, liver glucose metabolism is involved in glycosylation reactions and connected with fatty acid metabolism. The liver receives dietary carbohydrates directly from the intestine via the portal vein. Glucokinase phosphorylates glucose to glucose 6-phosphate inside the hepatocyte, ensuring that an adequate flow of glucose enters the cell to be metabolized. Glucose 6-phosphate may proceed to several metabolic pathways. During the post-prandial period, most glucose 6-phosphate is used to synthesize glycogen via the formation of glucose 1-phosphate and UDP–glucose. Minor amounts of UDP–glucose are used to form UDP–glucuronate and UDP–galactose, which are donors of monosaccharide units used in glycosylation. A second pathway of glucose 6-phosphate metabolism is the formation of fructose 6-phosphate, which may either start the hexosamine pathway to produce UDP-N-acetylglucosamine or follow the glycolytic pathway to generate pyruvate and then acetyl-CoA. Acetyl-CoA may enter the tricarboxylic acid (TCA) cycle to be oxidized or may be exported to the cytosol to synthesize fatty acids, when excess glucose is present within the hepatocyte. Finally, glucose 6-phosphate may produce NADPH and ribose 5-phosphate through the pentose phosphate pathway. Glucose metabolism supplies intermediates for glycosylation, a post-translational modification of proteins and lipids that modulates their activity. Congenital deficiency of phosphoglucomutase (PGM)-1 and PGM-3 is associated with impaired glycosylation. In addition to metabolize carbohydrates, the liver produces glucose to be used by other tissues, from glycogen breakdown or from de novo synthesis using primarily lactate and alanine (gluconeogenesis). PMID:27707936

Influence of F- on stark splitting of Yb3+ and the thermal expansion of silica glass

NASA Astrophysics Data System (ADS)

Cao, Yabin; Chen, Si; Shao, Chongyun; Yu, Chunlei

2018-06-01

A local phosphate/fluoride environment of Yb3+ was created in silica glass using a multi-step method. The influence of F- on the Stark splitting of Yb3+ in Al3+/P5+/F- co-doped silica glass was studied at room-temperature, in addition to its effect on the thermal expansion performance of the glass matrix. The results indicate that Yb3+ ions in Al3+/P5+/F- co-doped silica glass have a larger Stark splitting energy of 2F7/2 compared to Al3+/P5+ co-doped silica glass. Moreover, a larger integrated absorption cross-section (34.58 pm2 × nm), stimulated emission cross-section (0.63 pm2), and better thermal expansion performance (1.3062 × 10-6 K- at 100 °C) are achieved in Al3+/P5+/F- co-doped silica glass. Finally, different function mechanisms of F- in silica and phosphate glasses were analyzed and the F-Si bond was used to explain the results in silica glass. The combination of low refractive index, large Stark splitting energy of 2F7/2, and small thermal expansion makes Al3+/P5+/F- co-doped silica glass a preferred material for large mode area fibers for high-power laser applications.

[Intensity of pentose phosphate metabolism of carbohydrates in various brain areas in normal and starved animals].

PubMed

Kerimov, B F

2002-01-01

The activities of key enzymes of pentose phosphate pathway, glucose-6-phosphate dehydrogenase (G-6 PD) and 6-phosphogluconate dehydrogenase (6-PGD), were studied in cytoplasmatic fractions of brain cortical (limbic, orbital, sensorimotor cortex) and subcortical (myelencefalon, mesencefalon, hypothalamus) structures of rats subjected to starvation for 1, 2, 3, 5 and 7 days. Short-term starvation (1-3 days) caused activation of 6-GPD and 6-PGD both in cortical and subcortical structures. Long-term starvation for 5-7 days caused a decrease of activities of the pentose phosphate pathway enzymes in all studied structures. It is suggested that enzymes of pentose phosphate pathway in nervous tissues are functionally and metabolically related to glutathione system and during starvation they indirectly participate in the regulation lipid peroxidation processes.

The role of NF-κB signaling pathway in polyhexamethylene guanidine phosphate induced inflammatory response in mouse macrophage RAW264.7 cells.

PubMed

Kim, Ha Ryong; Shin, Da Young; Chung, Kyu Hyuck

2015-03-04

Polyhexamethylene guanidine (PHMG) phosphate is a competitive disinfectant with strong antibacterial activity. However, epidemiologists revealed that inhaled PHMG-phosphate may increase the risk of pulmonary fibrosis associated with inflammation, resulting in the deaths of many people, including infants and pregnant women. In addition, in vitro and in vivo studies reported the inflammatory effects of PHMG-phosphate. Therefore, the aim of the present study was to clarify the inflammatory effects and its mechanism induced by PHMG-phosphate in murine RAW264.7 macrophages. Cell viability, inflammatory cytokine secretion, nuclear factor kappa B (NF-κB) activation, and reactive oxygen species (ROS) generation were investigated in macrophages exposed to PHMG-phosphate. PHMG-phosphate induced dose-dependent cytotoxicity, with LC50 values of 11.15-0.99mg/ml at 6 and 24h, respectively. PHMG-phosphate induced pro-inflammatory cytokines including IL-1β, IL-6, and IL-8. In particular, IL-8 expression was completely inhibited by the NF-κB inhibitor BAY11-7082. In addition, PHMG-phosphate decreased IκB-α protein expression and increased NF-κB-mediated luciferase activity, which was diminished by N-acetyl-l-cystein. However, abundant amounts of ROS were generated in the presence of PHMG-phosphate at high concentrations with a cytotoxic effect. Our results demonstrated that PHMG-phosphate triggered the activation of NF-κB signaling pathway by modulating the degradation of IκB-α. Furthermore, the NF-κB signaling pathway plays a critical role in the inflammatory responses induced by PHMG-phosphate. We assumed that ROS generated by PHMG-phosphate were associated with inflammatory responses as secondary mechanism. In conclusion, we suggest that PHMG-phosphate induces inflammatory responses via NF-κB signaling pathway. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Lactose metabolism by Staphylococcus aureus: characterization of lacABCD, the structural genes of the tagatose 6-phosphate pathway.

PubMed Central

Rosey, E L; Oskouian, B; Stewart, G C

1991-01-01

The nucleotide and deduced amino acid sequences of the lacA and lacB genes of the Staphylococcus aureus lactose operon (lacABCDFEG) are presented. The primary translation products are polypeptides of 142 (Mr = 15,425) and 171 (Mr = 18,953) amino acids, respectively. The lacABCD loci were shown to encode enzymes of the tagatose 6-phosphate pathway through both in vitro studies and complementation analysis in Escherichia coli. A serum aldolase assay, modified to allow detection of the tagatose 6-phosphate pathway enzymes utilizing galactose 6-phosphate or fructose phosphate analogs as substrate, is described. Expression of both lacA and lacB was required for galactose 6-phosphate isomerase activity. LacC (34 kDa) demonstrated tagatose 6-phosphate kinase activity and was found to share significant homology with LacC from Lactococcus lactis and with both the minor 6-phosphofructokinase (PfkB) and 1-phosphofructokinase (FruK) from E. coli. Detection of tagatose 1,6-bisphosphate aldolase activity was dependent on expression of the 36-kDa protein specified by lacD. The LacD protein is highly homologous with LacD of L. lactis. Thus, the lacABCD genes comprise the tagatose 6-phosphate pathway and are cotranscribed with genes lacFEG, which specify proteins for transport and cleavage of lactose in S. aureus. PMID:1655695

Phosphate starvation promoted the accumulation of phenolic acids by inducing the key enzyme genes in Salvia miltiorrhiza hairy roots.

PubMed

Liu, Lin; Yang, DongFeng; Liang, TongYao; Zhang, HaiHua; He, ZhiGui; Liang, ZongSuo

2016-09-01

Phosphate starvation increased the production of phenolic acids by inducing the key enzyme genes in a positive feedback pathway in Saliva miltiorrhiza hairy roots. SPX may be involved in this process. Salvia miltiorrhiza is a wildly popular traditional Chinese medicine used for the treatment of coronary heart diseases and inflammation. Phosphate is an essential plant macronutrient that is often deficient, thereby limiting crop yield. In this study, we investigated the effects of phosphate concentration on the biomass and accumulation of phenolic acid in S. miltiorrhiza. Results show that 0.124 mM phosphate was favorable for plant growth. Moreover, 0.0124 mM phosphate was beneficial for the accumulation of phenolic acids, wherein the contents of danshensu, caffeic acid, rosmarinic acid, and salvianolic acid B were, respectively, 2.33-, 1.02-, 1.68-, and 2.17-fold higher than that of the control. By contrast, 12.4 mM phosphate inhibited the accumulation of phenolic acids. The key enzyme genes in the phenolic acid biosynthesis pathway were investigated to elucidate the mechanism of phosphate starvation-induced increase of phenolic acids. The results suggest that phosphate starvation induced the gene expression from the downstream pathway to the upstream pathway, i.e., a feedback phenomenon. In addition, phosphate starvation response gene SPX (SYG1, Pho81, and XPR1) was promoted by phosphate deficiency (0.0124 mM). We inferred that SPX responded to phosphate starvation, which then affected the expression of later responsive key enzyme genes in phenolic acid biosynthesis, resulting in the accumulation of phenolic acids. Our findings provide a resource-saving and environmental protection strategy to increase the yield of active substance in herbal preparations. The relationship between SPX and key enzyme genes and the role they play in phenolic acid biosynthesis during phosphate deficiency need further studies.

Thermochemical cyclic system for splitting water and/or carbon dioxide by means of cerium compounds and reactions useful therein

DOEpatents

Bamberger, C.E.; Robinson, P.R.

A thermochemical cyclic process for producing hydrogen from water comprises reacting ceric oxide with monobasic or dibasic alkali metal phosphate to yield a solid reaction product, oxygen and water. The solid reaction product, alkali metal carbonate or bicarbonate, and water, are reacted to yield hydrogen, ceric oxide, carbon dioxide and trialkali metal phosphate. Ceric oxide is recycled. Trialkali metal phosphate, carbon dioxide and water are reacted to yield monobasic or dibasic alkali metal phosphate and alkali metal bicarbonate, which are recycled. The cyclic process can be modified for producing carbon monoxide from carbon dioxide by reacting the alkali metal cerous phosphate and alkali metal carbonate or bicarbonate in the absence of water to produce carbon monoxide, ceric oxide, carbon dioxide and trialkali metal phosphate. Carbon monoxide can be converted to hydrogen by the water gas shift reaction.

Thermochemical cyclic system for splitting water and/or carbon dioxide by means of cerium compounds and reactions useful therein

DOEpatents

Bamberger, Carlos E.; Robinson, Paul R.

1980-01-01

A thermochemical cyclic process for producing hydrogen from water comprises reacting ceric oxide with monobasic or dibasic alkali metal phosphate to yield a solid reaction product, oxygen and water. The solid reaction product, alkali metal carbonate or bicarbonate, and water, are reacted to yield hydrogen, ceric oxide, carbon dioxide and trialkali metal phosphate. Ceric oxide is recycled. Trialkali metal phosphate, carbon dioxide and water are reacted to yield monobasic or dibasic alkali metal phosphate and alkali metal bicarbonate, which are recycled. The cylic process can be modified for producing carbon monoxide from carbon dioxide by reacting the alkali metal cerous phosphate and alkali metal carbonate or bicarbonate in the absence of water to produce carbon monoxide, ceric oxide, carbon dioxide and trialkali metal phosphate. Carbon monoxide can be converted to hydrogen by the water gas shift reaction.

Novel bioassay for the discovery of inhibitors of the 2-C-Methyl-D-Erythritol 4-Phosphate (MEP) and terpenoid pathways leading to carotenoid biosynthesis

USDA-ARS?s Scientific Manuscript database

The 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway leads to the synthesis of isopentenyl-phosphate (IPP) in plastids. It is a major branch point providing precursors for the synthesis of carotenoids, tocopherols, plastoquinone and the phytyl chain of chlorophylls, as well as the hormones abscisi...

Evaluation of the pentose phosphate pathway from 14CO2 data. Fallibility of a classic equation when applied to non-homogeneous tissues.

PubMed

Larrabee, M G

1990-11-15

A classic equation that has frequently been used to estimate the fraction of glucose metabolized by the pentose phosphate pathway, using 14CO2 data, is more simply re-derived with careful consideration of the assumptions involved and the conditions under which it is applicable. The equation is shown to be unreliable for non-homogeneous tissues, depending on the fraction of triose phosphate converted to CO2. The formula in question is as follows: ([1]CO2/G-[6]CO2/G)/(1-[6]CO2/G) = 3Fmet./(1 + 2Fmet.) where [1]CO2 and [6]CO2 are output rates of carbons 1 and 6 of glucose respectively to CO2, G is the rate of glucose uptake and Fmet. is the fraction of the glucose that is metabolized to CO2 and triose phosphate by the pentose phosphate pathway, allowing for recycling of an appropriate fraction of the fructose-6-phosphate produced by the pathway. This analysis illustrates the importance of suitably testing any equation that assumes homogeneity before application to non-homogeneous tissues.

Prebiotic synthesis of phosphoenol pyruvate by α-phosphorylation-controlled triose glycolysis

NASA Astrophysics Data System (ADS)

Coggins, Adam J.; Powner, Matthew W.

2017-04-01

Phosphoenol pyruvate is the highest-energy phosphate found in living organisms and is one of the most versatile molecules in metabolism. Consequently, it is an essential intermediate in a wide variety of biochemical pathways, including carbon fixation, the shikimate pathway, substrate-level phosphorylation, gluconeogenesis and glycolysis. Triose glycolysis (generation of ATP from glyceraldehyde 3-phosphate via phosphoenol pyruvate) is among the most central and highly conserved pathways in metabolism. Here, we demonstrate the efficient and robust synthesis of phosphoenol pyruvate from prebiotic nucleotide precursors, glycolaldehyde and glyceraldehyde. Furthermore, phosphoenol pyruvate is derived within an α-phosphorylation controlled reaction network that gives access to glyceric acid 2-phosphate, glyceric acid 3-phosphate, phosphoserine and pyruvate. Our results demonstrate that the key components of a core metabolic pathway central to energy transduction and amino acid, sugar, nucleotide and lipid biosyntheses can be reconstituted in high yield under mild, prebiotically plausible conditions.

Mechano-chemical pathways to H2O and CO2 splitting

NASA Astrophysics Data System (ADS)

Vedadi, Mohammad H.; Haas, Stephan

2011-10-01

The shock-induced collapse of CO2-filled nanobubbles is investigated using molecular dynamics simulations based on a reactive force field. The energetic nanojet and high-pressure water hammer shock formed during and after collapse of the nanobubble trigger mechano-chemical H2O-CO2 reactions, some of which lead to splitting of water and formation of O2 molecules. The dominant pathways through which splitting of water molecules occur are identified.

Plasmid linkage of the D-tagatose 6-phosphate pathway in Streptococcus lactis: effect on lactose and galactose metabolism.

PubMed Central

Crow, V L; Davey, G P; Pearce, L E; Thomas, T D

1983-01-01

The three enzymes of the D-tagatose 6-phosphate pathway (galactose 6-phosphate isomerase, D-tagatose 6-phosphate kinase, and tagatose 1,6-diphosphate aldolase) were absent in lactose-negative (Lac-) derivatives of Streptococcus lactis C10, H1, and 133 grown on galactose. The lactose phosphoenolpyruvate-dependent phosphotransferase system and phospho-beta-galactosidase activities were also absent in Lac- derivatives of strains H1 and 133 and were low (possibly absent) in C10 Lac-. In all three Lac- derivatives, low galactose phosphotransferase system activity was found. On galactose, Lac- derivatives grew more slowly (presumably using the Leloir pathway) than the wild-type strains and accumulated high intracellular concentrations of galactose 6-phosphate (up to 49 mM); no intracellular tagatose 1,6-diphosphate was detected. The data suggest that the Lac phenotype is plasmid linked in the three strains studied, with the evidence being more substantial for strain H1. A Lac- derivative of H1 contained a single plasmid (33 megadaltons) which was absent from the Lac- mutant. We suggest that the genes linked to the lactose plasmid in S. lactis are more numerous than previously envisaged, coding for all of the enzymes involved in lactose metabolism from initial transport to the formation of triose phosphates via the D-tagatose 6-phosphate pathway. Images PMID:6294064

Lactose metabolism by Streptococcus mutans: evidence for induction of the tagatose 6-phosphate pathway.

PubMed Central

Hamilton, I R; Lebtag, H

1979-01-01

Growth on lactose by strains of Streptococcus mutans resulted in the induction of the lactose-phosphoenolpyruvate-phosphotransferase system, phospho-beta-galactosidase, and the enzymes of the tagatose 6-phosphate pathway. PMID:230175

On the role of GAPDH isoenzymes during pentose fermentation in engineered Saccharomyces cerevisiae.

PubMed

Linck, Annabell; Vu, Xuan-Khang; Essl, Christine; Hiesl, Charlotte; Boles, Eckhard; Oreb, Mislav

2014-05-01

In the metabolic network of the cell, many intermediary products are shared between different pathways. d-Glyceraldehyde-3-phosphate, a glycolytic intermediate, is a substrate of GAPDH but is also utilized by transaldolase and transketolase in the scrambling reactions of the nonoxidative pentose phosphate pathway. Recent efforts to engineer baker's yeast strains capable of utilizing pentose sugars present in plant biomass rely on increasing the carbon flux through this pathway. However, the competition between transaldolase and GAPDH for d-glyceraldehyde-3-phosphate produced in the first transketolase reaction compromises the carbon balance of the pathway, thereby limiting the product yield. Guided by the hypothesis that reduction in GAPDH activity would increase the availability of d-glyceraldehyde-3-phosphate for transaldolase and thereby improve ethanol production during fermentation of pentoses, we performed a comprehensive characterization of the three GAPDH isoenzymes in baker's yeast, Tdh1, Tdh2, and Tdh3 and analyzed the effect of their deletion on xylose utilization by engineered strains. Our data suggest that overexpression of transaldolase is a more promising strategy than reduction in GAPDH activity to increase the flux through the nonoxidative pentose phosphate pathway. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Polyphosphatase PPN1 of Saccharomyces cerevisiae: Switching of Exopolyphosphatase and Endopolyphosphatase Activities

PubMed Central

Andreeva, Nadezhda; Trilisenko, Ludmila; Eldarov, Mikhail; Kulakovskaya, Tatiana

2015-01-01

The polyphosphatase PPN1 of Saccharomyces cerevisiae shows an exopolyphosphatase activity splitting phosphate from chain end and an endopolyphosphatase activity fragmenting high molecular inorganic polyphosphates into shorter polymers. We revealed the compounds switching these activities of PPN1. Phosphate release and fragmentation of high molecular polyphosphate prevailed in the presence of Co2+ and Mg2+, respectively. Phosphate release and polyphosphate chain shortening in the presence of Co2+ were inhibited by ADP but not affected by ATP and argininе. The polyphosphate chain shortening in the presence of Mg2+ was activated by ADP and arginine but inhibited by ATP. PMID:25742176

SLC20A2 DEFICIENCY IN MICE LEADS TO ELEVATED PHOSPHATE LEVELS IN CEREBROSPINAL FLUID AND GLYMPHATIC PATHWAY-ASSOCIATED ARTERIOLAR CALCIFICATION, AND RECAPITULATES HUMAN IDIOPATHIC BASAL GANGLIA CALCIFICATION

PubMed Central

Wallingford, MC; Chia, J; Leaf, EM; Borgeia, S; Chavkin, NW; Sawangmake, C; Marro, K; Cox, TC; Speer, MY; Giachelli, CM

2016-01-01

Idiopathic basal ganglia calcification is a brain calcification disorder that has been genetically linked to autosomal dominant mutations in the sodium-dependent phosphate co-transporter, SLC20A2. The mechanisms whereby deficiency of Slc20a2 leads to basal ganglion calcification are unknown. In the mouse brain, we found that Slc20a2 was expressed in tissues that produce and/or regulate cerebrospinal fluid, including choroid plexus, ependyma and arteriolar smooth muscle cells. Haploinsufficient Slc20a2 +/− mice developed age-dependent basal ganglia calcification that formed in glymphatic pathway-associated arterioles. Slc20a2 deficiency uncovered phosphate homeostasis dysregulation characterized by abnormally high cerebrospinal fluid phosphate levels and hydrocephalus, in addition to basal ganglia calcification. Slc20a2 siRNA knockdown in smooth muscle cells revealed increased susceptibility to high phosphate-induced calcification. These data suggested that loss of Slc20a2 led to dysregulated phosphate homeostasis and enhanced susceptibility of arteriolar smooth muscle cells to elevated phosphate-induced calcification. Together, dysregulated cerebrospinal fluid phosphate and enhanced smooth muscle cell susceptibility may predispose to glymphatic pathway-associated arteriolar calcification. PMID:26822507

An Oral Load of [13C3]Glycerol and Blood NMR Analysis Detect Fatty Acid Esterification, Pentose Phosphate Pathway, and Glycerol Metabolism through the Tricarboxylic Acid Cycle in Human Liver.

PubMed

Jin, Eunsook S; Sherry, A Dean; Malloy, Craig R

2016-09-02

Drugs and other interventions for high impact hepatic diseases often target biochemical pathways such as gluconeogenesis, lipogenesis, or the metabolic response to oxidative stress. However, traditional liver function tests do not provide quantitative data about these pathways. In this study, we developed a simple method to evaluate these processes by NMR analysis of plasma metabolites. Healthy subjects ingested [U-(13)C3]glycerol, and blood was drawn at multiple times. Each subject completed three visits under differing nutritional states. High resolution (13)C NMR spectra of plasma triacylglycerols and glucose provided new insights into a number of hepatic processes including fatty acid esterification, the pentose phosphate pathway, and gluconeogenesis through the tricarboxylic acid cycle. Fasting stimulated pentose phosphate pathway activity and metabolism of [U-(13)C3]glycerol in the tricarboxylic acid cycle prior to gluconeogenesis or glyceroneogenesis. Fatty acid esterification was transient in the fasted state but continuous under fed conditions. We conclude that a simple NMR analysis of blood metabolites provides an important biomarker of pentose phosphate pathway activity, triacylglycerol synthesis, and flux through anaplerotic pathways in mitochondria of human liver. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Production of 2-deoxyribose 5-phosphate from fructose to demonstrate a potential of artificial bio-synthetic pathway using thermophilic enzymes.

PubMed

Honda, Kohsuke; Maya, Shohei; Omasa, Takeshi; Hirota, Ryuichi; Kuroda, Akio; Ohtake, Hisao

2010-08-02

Six thermophilic enzymes from Thermus thermophilus were used to construct an 'artificial bio-synthetic pathway' for the production of 2-deoxyribose 5-phosphate from fructose. By a simple operation using six recombinant Escherichia coli strains producing the thermophilic enzymes, respectively, fructose was converted to 2-deoxyribose 5-phosphate with a molar yield of 55%. Copyright 2010 Elsevier B.V. All rights reserved.

Genetic Evidence for the Physiological Significance of the d-Tagatose 6-Phosphate Pathway of Lactose and d-Galactose Degradation in Staphylococcus aureus1

PubMed Central

Bissett, Donald L.; Anderson, Richard L.

1974-01-01

Mutants of Staphylococcus aureus were isolated which were unable to utilize d-galactose or lactose, but which were able to utilize all other carbohydrates tested. Growth of the mutants on a peptone-containing medium was inhibited by d-galactose. Of those mutants selected for further study, one (tagI2) was missing d-galactose 6-phosphate isomerase, one (tagK3) was missing d-tagatose 6-phosphate kinase, and one (tagA4) was missing d-tagatose 1, 6-diphosphate aldolase. Each of these mutants accumulated the substrate of the missing enzyme intracellularly. Spontaneous revertants of each of the mutants simultaneously regained their ability to utilize d-galactose and lactose, lost their sensitivity to d-galactose, regained the missing enzymatic activities, and no longer accumulated intermediates of the d-tagatose 6-phosphate pathway. These data support our previous contention that the physiologically significant route for the metabolism of d-galactose and the d-galactosyl moiety of lactose in S. aureus is the d-tagatose 6-phosphate pathway. Furthermore, a mutant constitutive for all three enzymes of this pathway was isolated, indicating that the products of the tagI, tagK, and tagA genes are under common genetic control. This conclusion was supported by the demonstration that d-galactose 6-phosphate isomerase, d-tagatose 6-phosphate kinase, and d-tagatose 1, 6-diphosphate aldolase are coordinately induced in the parental strain. PMID:4277494

Starch Biosynthesis in Developing Wheat Grain 1

PubMed Central

Keeling, Peter L.; Wood, John R.; Tyson, R. Huw; Bridges, Ian G.

1988-01-01

We have used 13C-labeled sugars and nuclear magnetic resonance (NMR) spectrometry to study the metabolic pathway of starch biosynthesis in developing wheat grain (Triticum aestivum cv Mardler). Our aim was to examine the extent of redistribution of 13C between carbons atoms 1 and 6 of [1-13C] or [6-13C]glucose (or fructose) incorporated into starch, and hence provide evidence for or against the involvement of triose phosphates in the metabolic pathway. Starch synthesis in the endosperm tissue was studied in two experimental systems. First, the 13C sugars were supplied to isolated endosperm tissue incubated in vitro, and second the 13C sugars were supplied in vivo to the intact plant. The 13C starch produced by the endosperm tissue of the grain was isolated and enzymically degraded to glucose using amyloglucosidase, and the distribution of 13C in all glucosyl carbons was quantified by 13C-NMR spectrometry. In all of the experiments, irrespective of the incubation time or incubation conditions, there was a similar pattern of partial (between 15 and 20%) redistribution of label between carbons 1 and 6 of glucose recovered from starch. There was no detectable increase over background 13C incidence in carbons 2 to 5. Within each experiment, the same pattern of partial redistribution of label was found in the glucosyl and fructosyl moieties of sucrose extracted from the tissue. Since it is unlikely that sucrose is present in the amyloplast, we suggest that the observed redistribution of label occurred in the cytosolic compartment of the endosperm cells and that both sucrose and starch are synthesized from a common pool of intermediates, such as hexose phosphate. We suggest that redistribution of label occurs via a cytosolic pathway cycle involving conversion of hexose phosphate to triose phosphate, interconversion of triose phosphate by triose phosphate isomerase, and resynthesis of hexose phosphate in the cytosol. A further round of triose phosphate interconversion in the amyloplast could not be detected. These data seriously weaken the argument for the selective uptake of triose phosphates by the amyloplast as part of the pathway of starch biosynthesis from sucrose in plant storage tissues. Instead, we suggest that a hexose phosphate such as glucose 1-phosphate, glucose 6-phosphate, or fructose 6-phosphate is the most likely candidate for entry into the amyloplast. A pathway of starch biosynthesis is presented, which is consistent with our data and with the current information on the intracellular distribution of enzymes in plant storage tissues. PMID:16666140

Structure of L-Xylulose-5-Phosphate 3-Epimerase (UlaE) from the Anaerobic L-Ascorbate Utilization Pathway of Escherichia coli: Identification of a Novel Phosphate Binding Motif within a TIM Barrel Fold

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

Shi, Rong; Pineda, Marco; Ajamian, Eunice

2009-01-15

Three catabolic enzymes, UlaD, UlaE, and UlaF, are involved in a pathway leading to fermentation of L-ascorbate under anaerobic conditions. UlaD catalyzes a {beta}-keto acid decarboxylation reaction to produce L-xylulose-5-phosphate, which undergoes successive epimerization reactions with UlaE (L-xylulose-5-phosphate 3-epimerase) and UlaF (L-ribulose-5-phosphate 4-epimerase), yielding D-xylulose-5-phosphate, an intermediate in the pentose phosphate pathway. We describe here crystallographic studies of UlaE from Escherichia coli O157:H7 that complete the structural characterization of this pathway. UlaE has a triosephosphate isomerase (TIM) barrel fold and forms dimers. The active site is located at the C-terminal ends of the parallel {beta}-strands. The enzyme binds Zn{sup 2+},more » which is coordinated by Glu155, Asp185, His211, and Glu251. We identified a phosphate-binding site formed by residues from the {beta}1/{alpha}1 loop and {alpha}3' helix in the N-terminal region. This site differs from the well-characterized phosphate-binding motif found in several TIM barrel superfamilies that is located at strands {beta}7 and {beta}8. The intrinsic flexibility of the active site region is reflected by two different conformations of loops forming part of the substrate-binding site. Based on computational docking of the L-xylulose 5-phosphate substrate to UlaE and structural similarities of the active site of this enzyme to the active sites of other epimerases, a metal-dependent epimerization mechanism for UlaE is proposed, and Glu155 and Glu251 are implicated as catalytic residues. Mutation and activity measurements for structurally equivalent residues in related epimerases supported this mechanistic proposal.« less

Pho4 Is Essential for Dissemination of Cryptococcus neoformans to the Host Brain by Promoting Phosphate Uptake and Growth at Alkaline pH

PubMed Central

Kaufman-Francis, Keren; Desmarini, Desmarini; Juillard, Pierre G.; Li, Cecilia; Stifter, Sebastian A.; Feng, Carl G.; Sorrell, Tania C.; Grau, Georges E. R.; Bahn, Yong-Sun

2017-01-01

ABSTRACT Phosphate acquisition by fungi is regulated by the phosphate-sensing and acquisition (PHO) signaling pathway. Cryptococcus neoformans disseminates from the lung to the brain and is the commonest cause of fungal meningitis worldwide. To investigate the contribution of PHO signaling to cryptococcal dissemination, we characterized a transcription factor knockout strain (hlh3Δ/pho4Δ) defective in phosphate acquisition. Despite little similarity with other fungal Pho4 proteins, Hlh3/Pho4 functioned like a typical phosphate-responsive transcription factor in phosphate-deprived cryptococci, accumulating in nuclei and triggering expression of genes involved in phosphate acquisition. The pho4Δ mutant strain was susceptible to a number of stresses, the effect of which, except for alkaline pH, was alleviated by phosphate supplementation. Even in the presence of phosphate, the PHO pathway was activated in wild-type cryptococci at or above physiological pH, and under these conditions, the pho4Δ mutant had a growth defect and compromised phosphate uptake. The pho4Δ mutant was hypovirulent in a mouse inhalation model, where dissemination to the brain was reduced dramatically, and markedly hypovirulent in an intravenous dissemination model. The pho4Δ mutant was not detected in blood, nor did it proliferate significantly when cultured with peripheral blood monocytes. In conclusion, dissemination of infection and the pathogenesis of meningitis are dependent on cryptococcal phosphate uptake and stress tolerance at alkaline pH, both of which are Pho4 dependent. IMPORTANCE Cryptococcal meningitis is fatal without treatment and responsible for more than 500,000 deaths annually. To be a successful pathogen, C. neoformans must obtain an adequate supply of essential nutrients, including phosphate, from various host niches. Phosphate acquisition in fungi is regulated by the PHO signaling cascade, which is activated when intracellular phosphate decreases below a critical level. Induction of phosphate acquisition genes leads to the uptake of free phosphate via transporters. By blocking the PHO pathway using a Pho4 transcription factor mutant (pho4Δ mutant), we demonstrate the importance of the pathway for cryptococcal dissemination and the establishment of brain infection in murine models. Specifically, we show that reduced dissemination of the pho4Δ mutant to the brain is due to an alkaline pH tolerance defect, as alkaline pH mimics the conditions of phosphate deprivation. The end result is inhibited proliferation in host tissues, particularly in blood. Podcast: A podcast concerning this article is available. PMID:28144629

Erythrocytes of uranium miners: the activity of the pentose phosphate pathway

PubMed Central

Vích, Z.; Novosad, F.; Brychtová, V.

1970-01-01

Vích, Z., Novosad, F., and Brychtová, V. (1970).Brit. J. industr. Med.,27, 287-290. Erythrocytes of uranium miners: the activity of the pentose phosphate pathway. The functioning of erythrocytes was studied by determination of the activity of the pentose phosphate pathway in 431 individuals - 221 uranium miners, 42 employees of a uranium ore trimming station (30 of whom were exposed), 36 former uranium miners, 32 coal miners, and 100 persons not working in mines and with no previous exposure. In the groups exposed to long-term occupational radiation, the activity of the pentose phosphate cycle was found to be enhanced. This finding was interpreted as evidence for a change in the functional state of the erythrocytes in exposed persons due to the effects of radiation on the genesis of red cells in the bone marrow. PMID:5448126

Combination of Entner-Doudoroff Pathway with MEP Increases Isoprene Production in Engineered Escherichia coli

PubMed Central

Liu, Huaiwei; Sun, Yuanzhang; Ramos, Kristine Rose M.; Nisola, Grace M.; Valdehuesa, Kris Niño G.; Lee, Won–Keun; Park, Si Jae; Chung, Wook-Jin

2013-01-01

Embden-Meyerhof pathway (EMP) in tandem with 2-C-methyl-D-erythritol 4-phosphate pathway (MEP) is commonly used for isoprenoid biosynthesis in E. coli. However, this combination has limitations as EMP generates an imbalanced distribution of pyruvate and glyceraldehyde-3-phosphate (G3P). Herein, four glycolytic pathways—EMP, Entner-Doudoroff Pathway (EDP), Pentose Phosphate Pathway (PPP) and Dahms pathway were tested as MEP feeding modules for isoprene production. Results revealed the highest isoprene production from EDP containing modules, wherein pyruvate and G3P were generated simultaneously; isoprene titer and yield were more than three and six times higher than those of the EMP module, respectively. Additionally, the PPP module that generates G3P prior to pyruvate was significantly more effective than the Dahms pathway, in which pyruvate production precedes G3P. In terms of precursor generation and energy/reducing-equivalent supply, EDP+PPP was found to be the ideal feeding module for MEP. These findings may launch a new direction for the optimization of MEP-dependent isoprenoid biosynthesis pathways. PMID:24376679

Methods for the determination of intracellular levels of ribose phosphates.

PubMed

Camici, Marcella; Tozzi, Maria Grazia; Ipata, Piero Luigi

2006-10-31

Ribose phosphates are either synthesized through the oxidative branch of the pentose phosphate pathway or stem from the phosphorolytic cleavage of the N-glycosidic bond of ribonucleosides. The two major pentose phosphates, ribose-5-phosphate and ribose-1-phosphate, can be readily interconverted by phosphopentomutase. Ribose-5-phosphate is also the direct precursor of 5-phosphoribosyl-1-pyrophosphate, which is used for both de novo and salvage synthesis of nucleotides. On the other hand, the phosphorolysis of deoxyribonucleosides is the major source of deoxyribose phosphates. While the destiny of the nucleobase stemming from nucleoside phosphorolysis has been extensively investigated, the fate of the sugar moiety has been somehow neglected. However, extensive advances have been made in elucidating the pathways by which the pentose phosphates, arising from nucleoside phosphorolysis, are either recycled, without opening of their furanosidic ring, or catabolized as a carbon and energy source. Nevertheless, many aspects of pentose phosphate metabolism, and the possible involvement of these compounds in a number of cellular processes still remain obscure. The comprehension of the role played by pentose phosphates may be greatly facilitated by the knowledge of their steady-state intracellular levels and of their changes in response to variations of intra- and extracellular signals.

Silencing trehalose-6-phosphate synthase incapacitates adult mosquitoes by interfering with the biosynthetic pathway for flight fuel

USDA-ARS?s Scientific Manuscript database

Trehalose is a disaccharide comprised of two glucose molecules. It is the main blood sugar of insects and is essential for flight. Trehalose is synthesized by two enzymes: trehalose-6-phosphate synthase (T6PS) converts glucose-6-phosphate to trehalose-6-phosphate, and trehalose-6-phosphate phosphata...

What is and what is not electromagnetically induced transparency in whispering-gallery microcavities.

PubMed

Peng, Bo; Özdemir, Sahin Kaya; Chen, Weijian; Nori, Franco; Yang, Lan

2014-10-24

There has been an increasing interest in all-optical analogues of electromagnetically induced transparency and Autler-Townes splitting. Despite the differences in their underlying physics, both electromagnetically induced transparency and Autler-Townes splitting are quantified by a transparency window in the absorption or transmission spectrum, which often leads to a confusion about its origin. While the transparency window in electromagnetically induced transparency is a result of Fano interference among different transition pathways, in Autler-Townes splitting it is the result of strong field-driven interactions leading to the splitting of energy levels. Being able to tell objectively whether an observed transparency window is because of electromagnetically induced transparency or Autler-Townes splitting is crucial for applications and for clarifying the physics involved. Here we demonstrate the pathways leading to electromagnetically induced transparency, Fano resonances and Autler-Townes splitting in coupled whispering-gallery-mode resonators. Moreover, we report the application of the Akaike Information Criterion discerning between all-optical analogues of electromagnetically induced transparency and Autler-Townes splitting and clarifying the transition between them.

Effect of Reaction Pathway on the Extent and Mechanism of Uranium(VI) Immobilization with Calcium and Phosphate

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

Mehta, Vrajesh S.; Maillot, Fabien; Wang, Zheming

Phosphate addition to subsurface environments contaminated with uranium can be used as an in situ remediation approach. Batch experiments were conducted to evaluate the dependence of the extent and mechanism of uranium uptake on the pathway for reaction with calcium phosphates. At pH 4.0 and 6.0 uranium uptake occurred via autunite (Ca(UO2)(PO4)3) precipitation irrespective of the starting forms of calcium and phosphate. At pH 7.5, the uptake mechanism depended on the nature of the calcium and phosphate. When dissolved uranium, calcium, and phosphate were added simultaneously, uranium was structurally incorporated into a newly formed amorphous calcium phosphate solid. Adsorption wasmore » the dominant removal mechanism for uranium contacted with pre-formed amorphous calcium phosphate solids,. When U(VI) was added to a suspension containing amorphous calcium phosphate solids as well as dissolved calcium and phosphate, then removal occurred through precipitation (57±4 %) of autunite and adsorption (43±4 %) onto calcium phosphate. The solid phase speciation of the uranium was determined using X-ray absorption spectroscopy and laser induced fluorescence spectroscopy. Dissolved uranium, calcium, and phosphate concentrations with saturation index calculations helped identify removal mechanisms and determine thermodynamically favorable solid phases.« less

Engineering a functional 1-deoxy-D-xylulose 5-phosphate (DXP) pathway in Saccharomyces cerevisiae

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

Kirby, James; Dietzel, Kevin L.; Wichmann, Gale

2016-10-27

Isoprenoids are made by all free-living organisms and range from essential metabolites like sterols and quinones to more complex compounds like pinene and rubber. They are used in many commercial applications and much work has gone into engineering microbial hosts for their production. Isoprenoids are produced either from acetyl-CoA via the mevalonate pathway or from pyruvate and glyceraldehyde 3-phosphate via the 1-deoxy-D-xylulose 5-phosphate (DXP) pathway. Saccharomyces cerevisiae exclusively utilizes the mevalonate pathway to synthesize native isoprenoids and in fact the alternative DXP pathway has never been found or successfully reconstructed in the eukaryotic cytosol. There are, however, several advantages tomore » isoprenoid synthesis via the DXP pathway, such as a higher theoretical yield, and it has long been a goal to transplant the pathway into yeast. In this work, we investigate and address barriers to DXP pathway functionality in S. cerevisiae using a combination of synthetic biology, biochemistry and metabolomics. We report, for the first time, functional expression of the DXP pathway in S. cerevisiae. Under low aeration conditions, an engineered strain relying solely on the DXP pathway for isoprenoid biosynthesis achieved an endpoint biomass 80% of that of the same strain using the mevalonate pathway.« less

The pentose phosphate pathway and cancer

PubMed Central

Patra, Krushna C.; Hay, Nissim

2015-01-01

The pentose phosphate pathway (PPP), which branches from glycolysis at the first committed step of glucose metabolism, is required for the synthesis of ribonucleotides and is a major source of NADPH. NADPH is required for and consumed during fatty acid synthesis and the scavenging of reactive oxygen species. Therefore, the PPP plays a pivotal role in helping glycolytic cancer cells to meet their anabolic demands and combat oxidative stress. Recently, several neoplastic lesions were shown to have evolved to facilitate the flux of glucose into the pentose phosphate pathway. This review summarizes the fundamental functions of the PPP, its regulation in cancer cells, and its importance in cancer cell metabolism and survival. PMID:25037503

Estradiol promotes pentose phosphate pathway addiction and cell survival via reactivation of Akt in mTORC1 hyperactive cells.

PubMed

Sun, Y; Gu, X; Zhang, E; Park, M-A; Pereira, A M; Wang, S; Morrison, T; Li, C; Blenis, J; Gerbaudo, V H; Henske, E P; Yu, J J

2014-05-15

Lymphangioleiomyomatosis (LAM) is a female-predominant interstitial lung disease that can lead to respiratory failure. LAM cells typically have inactivating TSC2 mutations, leading to mTORC1 activation. The gender specificity of LAM suggests that estradiol contributes to disease development, yet the underlying pathogenic mechanisms are not completely understood. Using metabolomic profiling, we identified an estradiol-enhanced pentose phosphate pathway signature in Tsc2-deficient cells. Estradiol increased levels of cellular NADPH, decreased levels of reactive oxygen species, and enhanced cell survival under oxidative stress. Mechanistically, estradiol reactivated Akt in TSC2-deficient cells in vitro and in vivo, induced membrane translocation of glucose transporters (GLUT1 or GLUT4), and increased glucose uptake in an Akt-dependent manner. (18)F-FDG-PET imaging demonstrated enhanced glucose uptake in xenograft tumors of Tsc2-deficient cells from estradiol-treated mice. Expression array study identified estradiol-enhanced transcript levels of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the pentose phosphate pathway. Consistent with this, G6PD was abundant in xenograft tumors and lung metastatic lesions of Tsc2-deficient cells from estradiol-treated mice. Molecular depletion of G6PD attenuated estradiol-enhanced survival in vitro, and treatment with 6-aminonicotinamide, a competitive inhibitor of G6PD, reduced lung colonization of Tsc2-deficient cells. Collectively, these data indicate that estradiol promotes glucose metabolism in mTORC1 hyperactive cells through the pentose phosphate pathway via Akt reactivation and G6PD upregulation, thereby enhancing cell survival under oxidative stress. Interestingly, a strong correlation between estrogen exposure and G6PD was also found in breast cancer cells. Targeting the pentose phosphate pathway may have therapeutic benefit for LAM and possibly other hormonally dependent neoplasms.

Regulation of Mammary Tumor Formation and Lipid Biosynthesis by Spot14

DTIC Science & Technology

2014-06-01

consistent with these terms, showing enrichment in glycolysis/ gluconeogenesis , the pentose phosphate pathway, and the cell cycle in PyMT/S14-/- versus PyMT...Enrichment Glycolysis / Gluconeogenesis 0.0018 6.66 Pentose phosphate pathway 0.0045 11.62 Cell cycle 0.0254 3.54 Starch and sucrose metabolism 0.0799

Synthesis and crystal structure of the solid solution Co3(SeO3)3-x(PO3OH)x(H2O) involving crystallographic split positions of Se4+ and P5+.

PubMed

Zimmermann, Iwan; Johnsson, Mats

2013-10-21

Three new cobalt selenite hydroxo-phosphates laying in the solid solution Co3(SeO3)3-x(PO3OH)x(H2O), with x = 0.8, x = 1.0, and x = 1.2 are reported. Single crystals were obtained by hydrothermal synthesis and the crystal structure was determined by single crystal X-ray diffraction. The structure can be described as a 3D framework having selenite and hydroxo-phosphate groups protruding into channels in the crystal structure. Se(4+) and P(5+) share a split position in the structure so that either SeO3 groups having a stereochemically active lone pair or tetrahedrally coordinated PO3OH groups are present. The OH-group is thus only present when the split position is occupied by P(5+). The crystal water is coordinated to a cobalt atom and TG and IR measurements show that the water and hydroxyl groups leave the structure at unusually high temperatures (>450 °C). Magnetic susceptibility measurements show antiferromagnetic coupling below 16 K and a magnetic moment of 4.02(3) μB per Co atom was observed.

Mathematical modelling of metabolic pathways affected by an enzyme deficiency. Energy and redox metabolism of glucose-6-phosphate-dehydrogenase-deficient erythrocytes.

PubMed

Schuster, R; Jacobasch, G; Holzhütter, H G

1989-07-01

The effects of various forms of glucose-6-phosphate dehydrogenase deficiency on erythrocyte metabolism have been studied on the basis of a complex mathematical model which comprises the main pathways of this cell: glycolysis, pentose pathway, reactions of the glutathione and adenine nucleotide metabolism. The calculated flux rates through the oxidative pentose pathway with and without methylene blue are in good accord with experimental results. The degree of deficiency as predicted by the model on the basis of calculated upper oxidative load boundaries, as well as of maximal methylene blue stimulation, correlates with the individual clinical manifestation of the metabolic disease. Therefore, the model allows one to judge the degree of metabolic disorder in the presence of glucose-6-phosphate dehydrogenase enzymopathies if the kinetic properties of the defect enzyme are known. Experimentally accessible parameters for an assessment of the oxidative load capacity of cells in vivo are proposed. It is pointed out that the threshold of tolerance as to energetic load is drastically reduced in the case of severe glucose-6-phosphate dehydrogenase deficiency.

Regulatory Tasks of the Phosphoenolpyruvate-Phosphotransferase System of Pseudomonas putida in Central Carbon Metabolism

PubMed Central

Chavarría, Max; Kleijn, Roelco J.; Sauer, Uwe; Pflüger-Grau, Katharina; de Lorenzo, Víctor

2012-01-01

ABSTRACT Two branches of the phosphoenolpyruvate-phosphotransferase system (PTS) operate in the soil bacterium Pseudomonas putida KT2440. One branch encompasses a complete set of enzymes for fructose intake (PTSFru), while the other (N-related PTS, or PTSNtr) controls various cellular functions unrelated to the transport of carbohydrates. The potential of these two systems for regulating central carbon catabolism has been investigated by measuring the metabolic fluxes of isogenic strains bearing nonpolar mutations in PTSFru or PTSNtr genes and grown on either fructose (a PTS substrate) or glucose, the transport of which is not governed by the PTS in this bacterium. The flow of carbon from each sugar was distinctly split between the Entner-Doudoroff, pentose phosphate, and Embden-Meyerhof-Parnas pathways in a ratio that was maintained in each of the PTS mutants examined. However, strains lacking PtsN (EIIANtr) displayed significantly higher fluxes in the reactions of the pyruvate shunt, which bypasses malate dehydrogenase in the TCA cycle. This was consistent with the increased activity of the malic enzyme and the pyruvate carboxylase found in the corresponding PTS mutants. Genetic evidence suggested that such a metabolic effect of PtsN required the transfer of high-energy phosphate through the system. The EIIANtr protein of the PTSNtr thus helps adjust central metabolic fluxes to satisfy the anabolic and energetic demands of the overall cell physiology. PMID:22434849

A Janus cobalt-based catalytic material for electro-splitting of water

NASA Astrophysics Data System (ADS)

Cobo, Saioa; Heidkamp, Jonathan; Jacques, Pierre-André; Fize, Jennifer; Fourmond, Vincent; Guetaz, Laure; Jousselme, Bruno; Ivanova, Valentina; Dau, Holger; Palacin, Serge; Fontecave, Marc; Artero, Vincent

2012-09-01

The future of energy supply depends on innovative breakthroughs regarding the design of cheap, sustainable and efficient systems for the conversion and storage of renewable energy sources. The production of hydrogen through water splitting seems a promising and appealing solution. We found that a robust nanoparticulate electrocatalytic material, H2-CoCat, can be electrochemically prepared from cobalt salts in a phosphate buffer. This material consists of metallic cobalt coated with a cobalt-oxo/hydroxo-phosphate layer in contact with the electrolyte and mediates H2 evolution from neutral aqueous buffer at modest overpotentials. Remarkably, it can be converted on anodic equilibration into the previously described amorphous cobalt oxide film (O2-CoCat or CoPi) catalysing O2 evolution. The switch between the two catalytic forms is fully reversible and corresponds to a local interconversion between two morphologies and compositions at the surface of the electrode. After deposition, the noble-metal-free coating thus functions as a robust, bifunctional and switchable catalyst.

{open_quotes}The effects of diabetes on the activity of the enzyme glutamine: fructose-6-phosphate amindotransferase{close_quotes}

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

Nelson, S.P.

1994-12-31

Hexsoamine synthetic pathway (HexNSP) controls the supply of essential substrates for glycoprotein synthesis. In vitro studies suggest that increased flux of glucose via the hexsoamine synthetic pathway may play a role in glucose induced insulin resistance of glucose transport. Glutamine: fructose-6-phosphate amindotransferase (GFAT) controls flux into the hexsoamine synthetic pathway; the major products are UDPN-acetylhexosamines (UDP.HexNac=UDP.GlcNAc= UDP.GalNac). I examined whether diabetes ({approximately} 7 days post intravenous streptozotocin, and genetically linked) affects the activity of glutamine: fructose-6-phosphate in rat and mouse skeletal muscle in vivo. Nucleotide linked HexNAc were analyzed by high pressure liquid chromatography(HPLC) in deproteinized hind limb muscle extracts.

Crystal structure of 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase from the ESKAPE pathogen Acinetobacter baumannii

PubMed Central

Sutton, Kristin A.; Breen, Jennifer; Russo, Thomas A.; Schultz, L. Wayne; Umland, Timothy C.

2016-01-01

The enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase catalyzes the sixth step of the seven-step shikimate pathway. Chorismate, the product of the pathway, is a precursor for the biosynthesis of aromatic amino acids, siderophores and metabolites such as folate, ubiquinone and vitamin K. The shikimate pathway is present in bacteria, fungi, algae, plants and apicomplexan parasites, but is absent in humans. The EPSP synthase enzyme produces 5-enolpyruvylshikimate 3-phosphate and phosphate from phosphoenolpyruvate and shikimate 3-phosphate via a transferase reaction, and is the target of the herbicide glyphosate. The Acinetobacter baumannii gene encoding EPSP synthase, aroA, has previously been demonstrated to be essential during host infection for the growth and survival of this clinically important drug-resistant ESKAPE pathogen. Prephenate dehydrogenase is also encoded by the bifunctional A. baumannii aroA gene, but its activity is dependent upon EPSP synthase since it operates downstream of the shikimate pathway. As part of an effort to evaluate new antimicrobial targets, recombinant A. baumannii EPSP (AbEPSP) synthase, comprising residues Ala301–Gln756 of the aroA gene product, was overexpressed in Escherichia coli, purified and crystallized. The crystal structure, determined to 2.37 Å resolution, is described in the context of a potential antimicrobial target and in comparison to EPSP synthases that are resistant or sensitive to the herbicide glyphosate. PMID:26919521

Crystal structure of 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase from the ESKAPE pathogen Acinetobacter baumannii.

PubMed

Sutton, Kristin A; Breen, Jennifer; Russo, Thomas A; Schultz, L Wayne; Umland, Timothy C

2016-03-01

The enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase catalyzes the sixth step of the seven-step shikimate pathway. Chorismate, the product of the pathway, is a precursor for the biosynthesis of aromatic amino acids, siderophores and metabolites such as folate, ubiquinone and vitamin K. The shikimate pathway is present in bacteria, fungi, algae, plants and apicomplexan parasites, but is absent in humans. The EPSP synthase enzyme produces 5-enolpyruvylshikimate 3-phosphate and phosphate from phosphoenolpyruvate and shikimate 3-phosphate via a transferase reaction, and is the target of the herbicide glyphosate. The Acinetobacter baumannii gene encoding EPSP synthase, aroA, has previously been demonstrated to be essential during host infection for the growth and survival of this clinically important drug-resistant ESKAPE pathogen. Prephenate dehydrogenase is also encoded by the bifunctional A. baumannii aroA gene, but its activity is dependent upon EPSP synthase since it operates downstream of the shikimate pathway. As part of an effort to evaluate new antimicrobial targets, recombinant A. baumannii EPSP (AbEPSP) synthase, comprising residues Ala301-Gln756 of the aroA gene product, was overexpressed in Escherichia coli, purified and crystallized. The crystal structure, determined to 2.37 Å resolution, is described in the context of a potential antimicrobial target and in comparison to EPSP synthases that are resistant or sensitive to the herbicide glyphosate.

The pentose phosphate pathway leads to enhanced succinic acid flux in biofilms of wild-type Actinobacillus succinogenes.

PubMed

Bradfield, Michael F A; Nicol, Willie

2016-11-01

Increased pentose phosphate pathway flux, relative to total substrate uptake flux, is shown to enhance succinic acid (SA) yields under continuous, non-growth conditions of Actinobacillus succinogenes biofilms. Separate fermentations of glucose and xylose were conducted in a custom, continuous biofilm reactor at four different dilution rates. Glucose-6-phosphate dehydrogenase assays were performed on cell extracts derived from in situ removal of biofilm at each steady state. The results of the assays were coupled to a kinetic model that revealed an increase in oxidative pentose phosphate pathway (OPPP) flux relative to total substrate flux with increasing SA titre, for both substrates. Furthermore, applying metabolite concentration data to metabolic flux models that include the OPPP revealed similar flux relationships to those observed in the experimental kinetic analysis. A relative increase in OPPP flux produces additional reduction power that enables increased flux through the reductive branch of the TCA cycle, leading to increased SA yields, reduced by-product formation and complete closure of the overall redox balance.

Activity of Escherichia coli, Aspergillus niger, and Rye Phytase toward Partially Phosphorylated myo-Inositol Phosphates.

PubMed

Greiner, Ralf

2017-11-08

Kinetic parameters for the dephosphorylation of sodium phytate and a series of partially phosphorylated myo-inositol phosphates were determined at pH 3.0 and pH 5.0 for three phytase preparations (Aspergillus niger, Escherichia coli, rye). The enzymes showed lower affinity and turnover numbers at pH 3 compared to pH 5 toward all myo-inositol phosphates included in the study. The number and distribution of phosphate groups on the myo-inositol ring affected the kinetic parameters. Representatives of the individual phytate dephosphorylation pathways were identified as the best substrates of the phytases. Within the individual phytate dephosphorylation pathways, the pentakisphosphates were better substrates compared to the tetrakisphosphates or phytate itself. E. coli and rye phytase showed comparable activities at both pH values toward the tetrakis- and trisphosphate, whereas A. niger phytase exhibited a higher activity toward the tetrakisphosphate. A myo-inositol phosphate with alternate phosphate groups was shown to be not significantly dephosphorylated by the phytases.

(13)C metabolic flux analysis in neurons utilizing a model that accounts for hexose phosphate recycling within the pentose phosphate pathway.

PubMed

Gebril, Hoda M; Avula, Bharathi; Wang, Yan-Hong; Khan, Ikhlas A; Jekabsons, Mika B

2016-02-01

Glycolysis, mitochondrial substrate oxidation, and the pentose phosphate pathway (PPP) are critical for neuronal bioenergetics and oxidation-reduction homeostasis, but quantitating their fluxes remains challenging, especially when processes such as hexose phosphate (i.e., glucose/fructose-6-phosphate) recycling in the PPP are considered. A hexose phosphate recycling model was developed which exploited the rates of glucose consumption, lactate production, and mitochondrial respiration to infer fluxes through the major glucose consuming pathways of adherent cerebellar granule neurons by replicating [(13)C]lactate labeling from metabolism of [1,2-(13)C2]glucose. Flux calculations were predicated on a steady-state system with reactions having known stoichiometries and carbon atom transitions. Non-oxidative PPP activity and consequent hexose phosphate recycling, as well as pyruvate production by cytoplasmic malic enzyme, were optimized by the model and found to account for 28 ± 2% and 7.7 ± 0.2% of hexose phosphate and pyruvate labeling, respectively. From the resulting fluxes, 52 ± 6% of glucose was metabolized by glycolysis, compared to 19 ± 2% by the combined oxidative/non-oxidative pentose cycle that allows for hexose phosphate recycling, and 29 ± 8% by the combined oxidative PPP/de novo nucleotide synthesis reactions. By extension, 62 ± 6% of glucose was converted to pyruvate, the metabolism of which resulted in 16 ± 1% of glucose oxidized by mitochondria and 46 ± 6% exported as lactate. The results indicate a surprisingly high proportion of glucose utilized by the pentose cycle and the reactions synthesizing nucleotides, and exported as lactate. While the in vitro conditions to which the neurons were exposed (high glucose, no lactate or other exogenous substrates) limit extrapolating these results to the in vivo state, the approach provides a means of assessing a number of metabolic fluxes within the context of hexose phosphate recycling in the PPP from a minimal set of measurements. Copyright © 2015 Elsevier Ltd. All rights reserved.

Estradiol promotes pentose phosphate pathway addiction and cell survival via reactivation of Akt in mTORC1 hyperactive cells

PubMed Central

Sun, Y; Gu, X; Zhang, E; Park, M-A; Pereira, A M; Wang, S; Morrison, T; Li, C; Blenis, J; Gerbaudo, V H; Henske, E P; Yu, J J

2014-01-01

Lymphangioleiomyomatosis (LAM) is a female-predominant interstitial lung disease that can lead to respiratory failure. LAM cells typically have inactivating TSC2 mutations, leading to mTORC1 activation. The gender specificity of LAM suggests that estradiol contributes to disease development, yet the underlying pathogenic mechanisms are not completely understood. Using metabolomic profiling, we identified an estradiol-enhanced pentose phosphate pathway signature in Tsc2-deficient cells. Estradiol increased levels of cellular NADPH, decreased levels of reactive oxygen species, and enhanced cell survival under oxidative stress. Mechanistically, estradiol reactivated Akt in TSC2-deficient cells in vitro and in vivo, induced membrane translocation of glucose transporters (GLUT1 or GLUT4), and increased glucose uptake in an Akt-dependent manner. 18F-FDG-PET imaging demonstrated enhanced glucose uptake in xenograft tumors of Tsc2-deficient cells from estradiol-treated mice. Expression array study identified estradiol-enhanced transcript levels of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the pentose phosphate pathway. Consistent with this, G6PD was abundant in xenograft tumors and lung metastatic lesions of Tsc2-deficient cells from estradiol-treated mice. Molecular depletion of G6PD attenuated estradiol-enhanced survival in vitro, and treatment with 6-aminonicotinamide, a competitive inhibitor of G6PD, reduced lung colonization of Tsc2-deficient cells. Collectively, these data indicate that estradiol promotes glucose metabolism in mTORC1 hyperactive cells through the pentose phosphate pathway via Akt reactivation and G6PD upregulation, thereby enhancing cell survival under oxidative stress. Interestingly, a strong correlation between estrogen exposure and G6PD was also found in breast cancer cells. Targeting the pentose phosphate pathway may have therapeutic benefit for LAM and possibly other hormonally dependent neoplasms. PMID:24832603

SANS study of HC1 extraction by selected neutral organophosphorus compounds in n-octane.

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

Chiarizia, R.; Stepinski, D.; Antonio, M. R.

2010-01-01

The extraction of HCl by tri(2-ethylhexyl) phosphate (TEHP), tri-n-octyl phosphate (TOP), and tri-n-octylphosphine oxide (TOPO) in n-octane was investigated by liquid-liquid distribution of acid and water and small-angle neutron scattering (SANS) measurements. No formation of a heavy organic phase (third phase) was observed with TEHP and TOP under the experimental conditions used, whereas for 0.4 M TOPO the HCl limiting organic concentration (LOC) at 23 C was 0.32 M (with 5.1 M HCl in the equilibrium aqueous phase). For higher HCl concentrations in the aqueous phase, the organic phase splits into a light and a heavy layer. For TEHP andmore » TOP, the SANS results, interpreted using the Baxter model for hard spheres with surface adhesion, indicated the formation of only small reverse micelles with little intermicellar attraction. For TOPO, the scattering signals suggested the formation of much larger and strongly interacting micelles. The critical values of the stickiness parameter, {tau}{sup -1}, and the interaction potential energy, U(r), for the LOC sample in the TOPO system were consistent with the model for third-phase formation previously developed for tri-n-butyl phosphate (TBP). According to this model, organic phase splitting is due to van der Waals interactions between the polar cores of reverse micelles formed by the extractants in the organic phase.« less

Phage Display on the Anti‐infective Target 1‐Deoxy‐d‐xylulose‐5‐phosphate Synthase Leads to an Acceptor–Substrate Competitive Peptidic Inhibitor

PubMed Central

Marcozzi, Alessio; Masini, Tiziana; Zhu, Di; Pesce, Diego; Illarionov, Boris; Fischer, Markus

2017-01-01

Abstract Enzymes of the 2‐C‐methyl‐d‐erythritol‐4‐phosphate pathway for the biosynthesis of isoprenoid precursors are validated drug targets. By performing phage display on 1‐deoxy‐d‐xylulose‐5‐phosphate synthase (DXS), which catalyzes the first step of this pathway, we discovered several peptide hits and recognized false‐positive hits. The enriched peptide binder P12 emerged as a substrate (d‐glyceraldehyde‐3‐phosphate)‐competitive inhibitor of Deinococcus radiodurans DXS. The results indicate possible overlap of the cofactor‐ and acceptor‐substrate‐binding pockets and provide inspiration for the design of inhibitors of DXS with a unique and novel mechanism of inhibition. PMID:29119720

All solution-processed lead halide perovskite-BiVO4 tandem assembly for photolytic solar fuels production.

PubMed

Chen, Yong-Siou; Manser, Joseph S; Kamat, Prashant V

2015-01-21

The quest for economic, large-scale hydrogen production has motivated the search for new materials and device designs capable of splitting water using only energy from the sun. Here we introduce an all solution-processed tandem water splitting assembly composed of a BiVO4 photoanode and a single-junction CH3NH3PbI3 hybrid perovskite solar cell. This unique configuration allows efficient solar photon management, with the metal oxide photoanode selectively harvesting high energy visible photons, and the underlying perovskite solar cell capturing lower energy visible-near IR wavelengths in a single-pass excitation. Operating without external bias under standard AM 1.5G illumination, the photoanode-photovoltaic architecture, in conjunction with an earth-abundant cobalt phosphate catalyst, exhibits a solar-to-hydrogen conversion efficiency of 2.5% at neutral pH. The design of low-cost tandem water splitting assemblies employing single-junction hybrid perovskite materials establishes a potentially promising new frontier for solar water splitting research.

Nanocube-based hematite photoanode produced in the presence of Na2HPO4 for efficient solar water splitting

NASA Astrophysics Data System (ADS)

Liu, Kan; Wang, Hongyan; Wu, Quanping; Zhao, Jun; Sun, Zhe; Xue, Song

2015-06-01

A thin film of α-Fe2O3 on FTO substrate has been synthesized from hydrothermal process in an aqueous solution of FeCl3 and Na2HPO4. A nanocube structure of α-Fe2O3 is observed within the formed hematite films and coated with phosphate ions on the surface. For comparison, another phosphate modified hematite film has been prepared by soaking the bare hematite film in Na2HPO4 solution. A negative electrostatic field can be built up on the surface of both phosphate modified hematite which will promote charge separation and extraction of photoexcited holes to the electrode surface. It is found that different types of phosphate complex exist in the hematite films, which has been determined by the isoelectric point (IEP) of the hematite films, and consequently influences the formation and strength of the electrostatic field. The effects of phosphate ions on the morphology, surface characteristics and the photoelectrochemical properties of the hematite thin films are investigated and the mechanism is proposed.

GATA4-mediated cardiac hypertrophy induced by D-myo-inositol 1,4,5-tris-phosphate

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

Zhu Zhiming; Zhu Shanjun; Liu Daoyan

2005-12-16

We evaluated the effects of D-myo-inositol 1,4,5-tris-phosphate on cardiac hypertrophy. D-myo-inositol 1,4,5-tris-phosphate augmented cardiac hypertrophy as evidenced by its effects on DNA synthesis, protein synthesis, and expression of immediate-early genes c-myc and c-fos, {beta}-myosin heavy chain, and {alpha}-actin. The administration of D-myo-inositol 1,4,5-tris-phosphate increased the expression of nuclear factor of activated T-cells and cardiac-restricted zinc finger transcription factor (GATA4). Real-time quantitative RT-PCR showed that D-myo-inositol 1,4,5-tris-phosphate-induced GATA4 mRNA was significantly enhanced even in the presence of the calcineurin inhibitor, cyclosporine A. The effect of D-myo-inositol 1,4,5-tris-phosphate was blocked after inhibition of inositol-trisphosphate receptors but not after inhibition of c-Raf/mitogen-activated proteinmore » kinase kinase (MEK)/mitogen-activated protein kinase (ERK) or p38 mitogen-activated protein kinase pathways. The study shows that D-myo-inositol 1,4,5-tris-phosphate-induced cardiac hypertrophy is mediated by GATA4 but independent from the calcineurin pathway.« less

Pentose phosphates in nucleoside interconversion and catabolism.

PubMed

Tozzi, Maria G; Camici, Marcella; Mascia, Laura; Sgarrella, Francesco; Ipata, Piero L

2006-03-01

Ribose phosphates are either synthesized through the oxidative branch of the pentose phosphate pathway, or are supplied by nucleoside phosphorylases. The two main pentose phosphates, ribose-5-phosphate and ribose-1-phosphate, are readily interconverted by the action of phosphopentomutase. Ribose-5-phosphate is the direct precursor of 5-phosphoribosyl-1-pyrophosphate, for both de novo and 'salvage' synthesis of nucleotides. Phosphorolysis of deoxyribonucleosides is the main source of deoxyribose phosphates, which are interconvertible, through the action of phosphopentomutase. The pentose moiety of all nucleosides can serve as a carbon and energy source. During the past decade, extensive advances have been made in elucidating the pathways by which the pentose phosphates, arising from nucleoside phosphorolysis, are either recycled, without opening of their furanosidic ring, or catabolized as a carbon and energy source. We review herein the experimental knowledge on the molecular mechanisms by which (a) ribose-1-phosphate, produced by purine nucleoside phosphorylase acting catabolically, is either anabolized for pyrimidine salvage and 5-fluorouracil activation, with uridine phosphorylase acting anabolically, or recycled for nucleoside and base interconversion; (b) the nucleosides can be regarded, both in bacteria and in eukaryotic cells, as carriers of sugars, that are made available though the action of nucleoside phosphorylases. In bacteria, catabolism of nucleosides, when suitable carbon and energy sources are not available, is accomplished by a battery of nucleoside transporters and of inducible catabolic enzymes for purine and pyrimidine nucleosides and for pentose phosphates. In eukaryotic cells, the modulation of pentose phosphate production by nucleoside catabolism seems to be affected by developmental and physiological factors on enzyme levels.

TPhP exposure disturbs carbohydrate metabolism, lipid metabolism, and the DNA damage repair system in zebrafish liver

NASA Astrophysics Data System (ADS)

Du, Zhongkun; Zhang, Yan; Wang, Guowei; Peng, Jianbiao; Wang, Zunyao; Gao, Shixiang

2016-02-01

Triphenyl phosphate is a high production volume organophosphate flame retardant that has been detected in multiple environmental media at increasing concentrations. The environmental and health risks of triphenyl phosphate have drawn attention because of the multiplex toxicity of this chemical compound. However, few studies have paid close attention to the impacts of triphenyl phosphate on liver metabolism. We investigated hepatic histopathological, metabolomic and transcriptomic responses of zebrafish after exposure to 0.050 mg/L and 0.300 mg/L triphenyl phosphate for 7 days. Metabolomic analysis revealed significant changes in the contents of glucose, UDP-glucose, lactate, succinate, fumarate, choline, acetylcarnitine, and several fatty acids. Transcriptomic analysis revealed that related pathways, such as the glycosphingolipid biosynthesis, PPAR signaling pathway and fatty acid elongation, were significantly affected. These results suggest that triphenyl phosphate exposure markedly disturbs hepatic carbohydrate and lipid metabolism in zebrafish. Moreover, DNA replication, the cell cycle, and non-homologous end-joining and base excision repair were strongly affected, thus indicating that triphenyl phosphate hinders the DNA damage repair system in zebrafish liver cells. The present study provides a systematic analysis of the triphenyl phosphate-induced toxic effects in zebrafish liver and demonstrates that low concentrations of triphenyl phosphate affect normal metabolism and cell cycle.

TPhP exposure disturbs carbohydrate metabolism, lipid metabolism, and the DNA damage repair system in zebrafish liver

PubMed Central

Du, Zhongkun; Zhang, Yan; Wang, Guowei; Peng, Jianbiao; Wang, Zunyao; Gao, Shixiang

2016-01-01

Triphenyl phosphate is a high production volume organophosphate flame retardant that has been detected in multiple environmental media at increasing concentrations. The environmental and health risks of triphenyl phosphate have drawn attention because of the multiplex toxicity of this chemical compound. However, few studies have paid close attention to the impacts of triphenyl phosphate on liver metabolism. We investigated hepatic histopathological, metabolomic and transcriptomic responses of zebrafish after exposure to 0.050 mg/L and 0.300 mg/L triphenyl phosphate for 7 days. Metabolomic analysis revealed significant changes in the contents of glucose, UDP-glucose, lactate, succinate, fumarate, choline, acetylcarnitine, and several fatty acids. Transcriptomic analysis revealed that related pathways, such as the glycosphingolipid biosynthesis, PPAR signaling pathway and fatty acid elongation, were significantly affected. These results suggest that triphenyl phosphate exposure markedly disturbs hepatic carbohydrate and lipid metabolism in zebrafish. Moreover, DNA replication, the cell cycle, and non-homologous end-joining and base excision repair were strongly affected, thus indicating that triphenyl phosphate hinders the DNA damage repair system in zebrafish liver cells. The present study provides a systematic analysis of the triphenyl phosphate-induced toxic effects in zebrafish liver and demonstrates that low concentrations of triphenyl phosphate affect normal metabolism and cell cycle. PMID:26898711

Development of an in vivo glucosylation platform by coupling production to growth: Production of phenolic glucosides by a glycosyltransferase of Vitis vinifera.

PubMed

De Bruyn, Frederik; De Paepe, Brecht; Maertens, Jo; Beauprez, Joeri; De Cocker, Pieter; Mincke, Stein; Stevens, Christian; De Mey, Marjan

2015-08-01

Glycosylation of small molecules can significantly alter their properties such as solubility, stability, and/or bioactivity, making glycosides attractive and highly demanded compounds. Consequently, many biotechnological glycosylation approaches have been developed, with enzymatic synthesis and whole-cell biocatalysis as the most prominent techniques. However, most processes still suffer from low yields, production rates and inefficient UDP-sugar formation. To this end, a novel metabolic engineering strategy is presented for the in vivo glucosylation of small molecules in Escherichia coli W. This strategy focuses on the introduction of an alternative sucrose metabolism using sucrose phosphorylase for the direct and efficient generation of glucose 1-phosphate as precursor for UDP-glucose formation and fructose, which serves as a carbon source for growth. By targeted gene deletions, a split metabolism is created whereby glucose 1-phosphate is rerouted from the glycolysis to product formation (i.e., glucosylation). Further, the production pathway was enhanced by increasing and preserving the intracellular UDP-glucose pool. Expression of a versatile glucosyltransferase from Vitis vinifera (VvGT2) enabled the strain to efficiently produce 14 glucose esters of various hydroxycinnamates and hydroxybenzoates with conversion yields up to 100%. To our knowledge, this fast growing (and simultaneously producing) E. coli mutant is the first versatile host described for the glucosylation of phenolic acids in a fermentative way using only sucrose as a cheap and sustainable carbon source. © 2015 Wiley Periodicals, Inc.

Amorphous cobalt potassium phosphate microclusters as efficient photoelectrochemical water oxidation catalyst

NASA Astrophysics Data System (ADS)

Zhang, Ye; Zhao, Chunsong; Dai, Xuezeng; Lin, Hong; Cui, Bai; Li, Jianbao

2013-12-01

A novel amorphous cobalt potassium phosphate hydrate compound (KCoPO4·H2O) is identified to be active photocatalyst for oxygen evolution reaction (OER) to facilitate hydrogen generation from water photolysis. It has been synthesized through a facile and cost-effective solution-based precipitation method using earth-abundant materials. Its highly porous structure and large surface areas are found to be responsible for the excellent electrochemical performance featuring a low OER onset at ∼550 mVSCE and high current density in alkaline condition. Unlike traditional cobalt-based spinel oxides (Co3O4, NiCo2O4) and phosphate (Co-Pi, Co(PO3)2) electrocatalysts, with proper energy band alignment for light-assisted water oxidation, cobalt potassium phosphate hydrate also exhibits robust visible-light response, generating a photocurrent density of ∼200 μA cm-2 at 0.7 VSCE. This catalyst could thus be considered as a promising candidate to perform photoelectrochemical water splitting.

Evaluation of hypophosphatemia: lessons from patients with genetic disorders

PubMed Central

Bacchetta, Justine; Salusky, Isidro B

2014-01-01

Phosphate is a key element for several physiological pathways, such as skeletal development, bone mineralization, membrane composition, nucleotide structure, maintenance of plasma pH, and cellular signaling. The kidneys have a key role in phosphate homeostasis with three hormones playing important roles in renal phosphate handling (i.e., parathyroid hormone (PTH), fibroblast growth factor 23 (FGF23), and 1-25 dihydroxy-vitamin D). Independently of the genetic diseases affecting the FGF23 pathway (such as hypophosphatemic rickets), hypophosphatemia is a frequent condition in daily practice, and untreated severe hypophosphatemia can induce hemolysis, rhabdomyolysis, respiratory failure, cardiac dysfunction and neurological impairment, thus requiring a rapid correction to avoid severe complications. The aims of this case report are to summarize the etiologies and the biological evaluation of hypophosphatemia in adults, and to provide an overview of our current understanding of phosphate metabolism. PMID:22075221

Zinc disrupts central carbon metabolism and capsule biosynthesis in Streptococcus pyogenes.

PubMed

Ong, Cheryl-lynn Y; Walker, Mark J; McEwan, Alastair G

2015-06-01

Neutrophils release free zinc to eliminate the phagocytosed bacterial pathogen Streptococcus pyogenes (Group A Streptococcus; GAS). In this study, we investigated the mechanisms underpinning zinc toxicity towards this human pathogen, responsible for diseases ranging from pharyngitis and impetigo, to severe invasive infections. Using the globally-disseminated M1T1 GAS strain, we demonstrate that zinc stress impairs glucose metabolism through the inhibition of the glycolytic enzymes phosphofructokinase and glyceraldehyde-3-phosphate dehydrogenase. In the presence of zinc, a metabolic shift to the tagatose-6-phosphate pathway allows conversion of D-galactose to dihydroxyacetone phosphate and glyceraldehyde phosphate, partially bypassing impaired glycolytic enzymes to generate pyruvate. Additionally, zinc inhibition of phosphoglucomutase results in decreased capsule biosynthesis. These data indicate that zinc exerts it toxicity via mechanisms that inhibit both GAS central carbon metabolism and virulence pathways.

Zinc disrupts central carbon metabolism and capsule biosynthesis in Streptococcus pyogenes

PubMed Central

Ong, Cheryl-lynn Y.; Walker, Mark J.; McEwan, Alastair G.

2015-01-01

Neutrophils release free zinc to eliminate the phagocytosed bacterial pathogen Streptococcus pyogenes (Group A Streptococcus; GAS). In this study, we investigated the mechanisms underpinning zinc toxicity towards this human pathogen, responsible for diseases ranging from pharyngitis and impetigo, to severe invasive infections. Using the globally-disseminated M1T1 GAS strain, we demonstrate that zinc stress impairs glucose metabolism through the inhibition of the glycolytic enzymes phosphofructokinase and glyceraldehyde-3-phosphate dehydrogenase. In the presence of zinc, a metabolic shift to the tagatose-6-phosphate pathway allows conversion of D-galactose to dihydroxyacetone phosphate and glyceraldehyde phosphate, partially bypassing impaired glycolytic enzymes to generate pyruvate. Additionally, zinc inhibition of phosphoglucomutase results in decreased capsule biosynthesis. These data indicate that zinc exerts it toxicity via mechanisms that inhibit both GAS central carbon metabolism and virulence pathways. PMID:26028191

The plastidial 2-C-methyl-D-erythritol 4-phosphate pathway provides the isoprenyl moiety for protein geranylgeranylation in tobacco BY-2 cells.

PubMed

Gerber, Esther; Hemmerlin, Andréa; Hartmann, Michael; Heintz, Dimitri; Hartmann, Marie-Andrée; Mutterer, Jérôme; Rodríguez-Concepción, Manuel; Boronat, Albert; Van Dorsselaer, Alain; Rohmer, Michel; Crowell, Dring N; Bach, Thomas J

2009-01-01

Protein farnesylation and geranylgeranylation are important posttranslational modifications in eukaryotic cells. We visualized in transformed Nicotiana tabacum Bright Yellow-2 (BY-2) cells the geranylgeranylation and plasma membrane localization of GFP-BD-CVIL, which consists of green fluorescent protein (GFP) fused to the C-terminal polybasic domain (BD) and CVIL isoprenylation motif from the Oryza sativa calmodulin, CaM61. Treatment with fosmidomycin (Fos) or oxoclomazone (OC), inhibitors of the plastidial 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway, caused mislocalization of the protein to the nucleus, whereas treatment with mevinolin, an inhibitor of the cytosolic mevalonate pathway, did not. The nuclear localization of GFP-BD-CVIL in the presence of MEP pathway inhibitors was completely reversed by all-trans-geranylgeraniol (GGol). Furthermore, 1-deoxy-d-xylulose (DX) reversed the effects of OC, but not Fos, consistent with the hypothesis that OC blocks 1-deoxy-d-xylulose 5-phosphate synthesis, whereas Fos inhibits its conversion to 2-C-methyl-d-erythritol 4-phosphate. By contrast, GGol and DX did not rescue the nuclear mislocalization of GFP-BD-CVIL in the presence of a protein geranylgeranyltransferase type 1 inhibitor. Thus, the MEP pathway has an essential role in geranylgeranyl diphosphate (GGPP) biosynthesis and protein geranylgeranylation in BY-2 cells. GFP-BD-CVIL is a versatile tool for identifying pharmaceuticals and herbicides that interfere either with GGPP biosynthesis or with protein geranylgeranylation.

Evolutionary Importance of the Intramolecular Pathways of Hydrolysis of Phosphate Ester Mixed Anhydrides with Amino Acids and Peptides

NASA Astrophysics Data System (ADS)

Liu, Ziwei; Beaufils, Damien; Rossi, Jean-Christophe; Pascal, Robert

2014-12-01

Aminoacyl adenylates (aa-AMPs) constitute essential intermediates of protein biosynthesis. Their polymerization in aqueous solution has often been claimed as a potential route to abiotic peptides in spite of a highly efficient CO2-promoted pathway of hydrolysis. Here we investigate the efficiency and relevance of this frequently overlooked pathway from model amino acid phosphate mixed anhydrides including aa-AMPs. Its predominance was demonstrated at CO2 concentrations matching that of physiological fluids or that of the present-day ocean, making a direct polymerization pathway unlikely. By contrast, the occurrence of the CO2-promoted pathway was observed to increase the efficiency of peptide bond formation owing to the high reactivity of the N-carboxyanhydride (NCA) intermediate. Even considering CO2 concentrations in early Earth liquid environments equivalent to present levels, mixed anhydrides would have polymerized predominantly through NCAs. The issue of a potential involvement of NCAs as biochemical metabolites could even be raised. The formation of peptide-phosphate mixed anhydrides from 5(4H)-oxazolones (transiently formed through prebiotically relevant peptide activation pathways) was also observed as well as the occurrence of the reverse cyclization process in the reactions of these mixed anhydrides. These processes constitute the core of a reaction network that could potentially have evolved towards the emergence of translation.

Metabolic evolution of two reducing equivalent-conserving pathways for high-yield succinate production in Escherichia coli.

PubMed

Zhu, Xinna; Tan, Zaigao; Xu, Hongtao; Chen, Jing; Tang, Jinlei; Zhang, Xueli

2014-07-01

Reducing equivalents are an important cofactor for efficient synthesis of target products. During metabolic evolution to improve succinate production in Escherichia coli strains, two reducing equivalent-conserving pathways were activated to increase succinate yield. The sensitivity of pyruvate dehydrogenase to NADH inhibition was eliminated by three nucleotide mutations in the lpdA gene. Pyruvate dehydrogenase activity increased under anaerobic conditions, which provided additional NADH. The pentose phosphate pathway and transhydrogenase were activated by increased activities of transketolase and soluble transhydrogenase SthA. These data suggest that more carbon flux went through the pentose phosphate pathway, thus leading to production of more reducing equivalent in the form of NADPH, which was then converted to NADH through soluble transhydrogenase for succinate production. Reverse metabolic engineering was further performed in a parent strain, which was not metabolically evolved, to verify the effects of activating these two reducing equivalent-conserving pathways for improving succinate yield. Activating pyruvate dehydrogenase increased succinate yield from 1.12 to 1.31mol/mol, whereas activating the pentose phosphate pathway and transhydrogenase increased succinate yield from 1.12 to 1.33mol/mol. Activating these two pathways in combination led to a succinate yield of 1.5mol/mol (88% of theoretical maximum), suggesting that they exhibited a synergistic effect for improving succinate yield. Copyright © 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Tumor acidity-activatable manganese phosphate nanoplatform for amplification of photodynamic cancer therapy and magnetic resonance imaging.

PubMed

Hao, Yongwei; Zheng, Cuixia; Wang, Lei; Zhang, Jinjie; Niu, Xiuxiu; Song, Qingling; Feng, Qianhua; Zhao, Hongjuan; Li, Li; Zhang, Hongling; Zhang, Zhenzhong; Zhang, Yun

2017-10-15

Amorphous biodegradable metal phosphate nanomaterials are considered to possess great potential in cancer theranostic application due to their promise in providing ultra-sensitive pH-responsive therapeutic benefits and diagnostic functions simultaneously. Here we report the synthesis of photosensitising and acriflavine-carrying amorphous porous manganese phosphate (PMP) nanoparticles with ultra-sensitive pH-responsive degradability and their application for a photoactivable synergistic nanosystem that imparts reactive oxygen species (ROS) induced cytotoxicity in synchrony with hypoxia-inducible factor 1α/vascular endothelial growth factor (HIF1α/VEGF) inhibitor that suppresses tumor growth and treatment escape signalling pathway. Carboxymethyl dextran (CMD) is chemically anchored on the surface of porous manganese phosphate theranostic system through the pH-responsive boronate esters. Upon the stimulus of the tumor acid microenvironment, manganese phosphate disintegrates and releases Mn 2+ ions rapidly, which are responsible for the magnetic resonance imaging (MRI) effect. Meanwhile, the released photosensitizer chlorin e6 (Ce6) produces ROS under irradiation while acriflavine (ACF) inhibits the HIF-1α/VEGF pathway during the burst release of VEGF in tumour induced by photodynamic therapy (PDT), resulting in increased therapeutic efficacy. Considering the strong pH responsivity, MRI signal amplification and drug release profile, the PMP nanoparticles offer new prospects for tumor acidity-activatable theranostic application by amplifying the PDT through inhibiting the HIF-1α /VEGF pathway timely while enhancing the MRI effect. In this study, we report the synthesis of the tumor acidity-activatable amorphous porous manganese phosphate nanoparticles and their application for a photoactivable synergistic nanosystem that imparts reactive oxygen species (ROS) induced cytotoxicity in synchrony with hypoxia-inducible factor 1α/vascular endothelial growth factor (HIF-1α/VEGF) inhibitor that suppresses tumor growth and treatment escape signalling pathway. Besides, upon the stimulus of the tumor acid microenvironment, the manganese phosphate nanoparticles finally disintegrate and release Mn 2+ ions rapidly, which are responsible for the magnetic resonance imaging (MRI) effect. This nanoplatform is featured with distinctive advantages such as ultra pH-responsive drug release, MRI function and rational drug combination exploiting the blockage of the treatment escape signalling pathway. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Glutamine-dependent carbamoyl-phosphate synthetase and other enzyme activities related to the pyrimidine pathway in spleen of Squalus acanthias (spiny dogfish).

PubMed Central

Anderson, P M

1989-01-01

The first two steps of urea synthesis in liver of marine elasmobranchs involve formation of glutamine from ammonia and of carbamoyl phosphate from glutamine, catalysed by glutamine synthetase and carbamoyl-phosphate synthetase, respectively [Anderson & Casey (1984) J. Biol. Chem. 259, 456-462]; both of these enzymes are localized exclusively in the mitochondrial matrix. The objective of this study was to establish the enzymology of carbamoyl phosphate formation and utilization for pyrimidine nucleotide biosynthesis in Squalus acanthias (spiny dogfish), a representative elasmobranch. Aspartate carbamoyltransferase could not be detected in liver of dogfish. Spleen extracts, however, had glutamine-dependent carbamoyl-phosphate synthetase, aspartate carbamoyltransferase, dihydro-orotase, and glutamine synthetase activities, all localized in the cytosol; dihydro-orotate dehydrogenase, orotate phosphoribosyltransferase, and orotidine-5'-decarboxylase activities were also present. Except for glutamine synthetase, the levels of all activities were very low. The carbamoyl-phosphate synthetase activity is inhibited by UTP and is activated by 5-phosphoribosyl 1-pyrophosphate. The first three enzyme activities of the pyrimidine pathway were eluted in distinctly different positions during gel filtration chromatography under a number of different conditions; although complete proteolysis of inter-domain regions of a multifunctional complex during extraction cannot be excluded, the evidence suggests that in dogfish, in contrast to mammalian species, these three enzymes of the pyrimidine pathway exist as individual polypeptide chains. These results: (1) establish that dogfish express two different glutamine-dependent carbamoyl-phosphate synthetase activities, (2) confirm the report [Smith, Ritter & Campbell (1987) J. Biol. Chem. 262, 198-202] that dogfish express two different glutamine synthetases, and (3) provide indirect evidence that glutamine may not be available in liver for biosynthetic reactions other than urea formation. Images Fig. 1. PMID:2570570

Stabilized CdSe-CoPi composite photoanode for light-assisted water oxidation by transformation of a CdSe/cobalt metal thin film.

PubMed

Costi, Ronny; Young, Elizabeth R; Bulović, Vladimir; Nocera, Daniel G

2013-04-10

Integration of water splitting catalysts with visible-light-absorbing semiconductors would enable direct solar-energy-to-fuel conversion schemes such as those based on water splitting. A disadvantage of some common semiconductors that possess desirable optical bandgaps is their chemical instability under the conditions needed for oxygen evolution reaction (OER). In this study, we demonstrate the dual benefits gained from using a cobalt metal thin-film as the precursor for the preparation of cobalt-phosphate (CoPi) OER catalyst on cadmium chalcogenide photoanodes. The cobalt layer protects the underlying semiconductor from oxidation and degradation while forming the catalyst and simultaneously facilitates the advantageous incorporation of the cadmium chalcogenide layer into the CoPi layer during continued processing of the electrode. The resulting hybrid material forms a stable photoactive anode for light-assisted water splitting.

Synthesis and Physicochemical Characterization of D-Tagatose-1-Phosphate: The Substrate of the Tagatose-1-Phosphate Kinase in the Phosphotransferase System-Mediated D-Tagatose Catabolic Pathway of Bacillus licheniformis.

PubMed

Van der Heiden, Edwige; Delmarcelle, Michaël; Simon, Patricia; Counson, Melody; Galleni, Moreno; Freedberg, Darón I; Thompson, John; Joris, Bernard; Battistel, Marcos D

2015-01-01

We report the first enzymatic synthesis of D-tagatose-1-phosphate (Tag-1P) by the multicomponent phosphoenolpyruvate:sugar phosphotransferase system (PEP-PTS) present in tagatose-grown cells of Klebsiella pneumoniae. Physicochemical characterization by (31)P and (1)H nuclear magnetic resonance spectroscopy reveals that, in solution, this derivative is primarily in the pyranose form. Tag-1P was used to characterize the putative tagatose-1-phosphate kinase (TagK) of the Bacillus licheniformis PTS-mediated D-tagatose catabolic pathway (Bli-TagP). For this purpose, a soluble protein fusion was obtained with the 6 His-tagged trigger factor (TF(His6)) of Escherichia coli. The active fusion enzyme was named TagK-TF(His6). Tag-1P and D-fructose-1-phosphate are substrates for the TagK-TF(His6) enzyme, whereas the isomeric derivatives D-tagatose-6-phosphate and D-fructose-6-phosphate are inhibitors. Studies of catalytic efficiency (kcat/Km) reveal that the enzyme specificity is markedly in favor of Tag-1P as the substrate. Importantly, we show in vivo that the transfer of the phosphate moiety from PEP to the B. licheniformis tagatose-specific Enzyme II in E. coli is inefficient. The capability of the PTS general cytoplasmic components of B. subtilis, HPr and Enzyme I to restore the phosphate transfer is demonstrated. © 2015 S. Karger AG, Basel.

Synthesis and Physicochemical Characterization of D-Tagatose-1-phosphate: The Substrate of the Tagatose-1-Phosphate Kinase TagK in the PTS-mediated D-Tagatose Catabolic Pathway of Bacillus licheniformis

PubMed Central

Van der Heiden, Edwige; Delmarcelle, Michaël; Simon, Patricia; Counson, Melody; Galleni, Moreno; Freedberg, Darón I.; Thompson, John; Joris, Bernard; Battistel, Marcos D.

2015-01-01

We report the first enzymatic synthesis of D-tagatose-1-phosphate (Tag-1P) by the multi-component PEP-dependent:tag-PTS present in tagatose-grown cells of Klebsiella pneumoniae. Physicochemical characterization by 31P and 1H NMR spectroscopy reveals that, in solution, this derivative is primarily in the pyranose form. Tag-1P was used to characterize the putative tagatose-1-phosphate kinase (TagK) of the Bacillus licheniformis PTS-mediated D-Tagatose catabolic Pathway (Bli-TagP). For this purpose, a soluble protein fusion was obtained with the 6 His-tagged trigger factor (TFHis6) of Escherichia coli. The active fusion enzyme was named TagK-TFHis6. Tag-1P and D-fructose-1-phosphate (Fru-1P) are substrates for the TagK-TFHis6 enzyme, whereas the isomeric derivatives D-tagatose-6-phosphate (Tag-6P) and D-fructose-6-phosphate (Fru-6P) are inhibitors. Studies of catalytic efficiency (kcat/Km) reveal that the enzyme specificity is markedly in favor of Tag-1P as substrate. Importantly, we show in vivo that the transfer of the phosphate moiety from PEP to the B. licheniformis tagatose-specific enzyme II (EIITag) in E.coli is inefficient. The capability of the PTS general cytoplasmic components of B. subtilis, HPr and EI, to restore the phosphate transfer is demonstrated. PMID:26159072

Regulation of Cellular Diacylglycerol through Lipid Phosphate Phosphatases Is Required for Pathogenesis of the Rice Blast Fungus, Magnaporthe oryzae

PubMed Central

Mir, Albely Afifa; Choi, Jaeyoung; Choi, Jaehyuk; Lee, Yong-Hwan

2014-01-01

Considering implication of diacylglycerol in both metabolism and signaling pathways, maintaining proper levels of diacylglycerol (DAG) is critical to cellular homeostasis and development. Except the PIP2-PLC mediated pathway, metabolic pathways leading to generation of DAG converge on dephosphorylation of phosphatidic acid catalyzed by lipid phosphate phosphatases. Here we report the role of such enzymes in a model plant pathogenic fungus, Magnaporthe oryzae. We identified five genes encoding putative lipid phosphate phosphatases (MoLPP1 to MoLPP5). Targeted disruption of four genes (except MoLPP4) showed that MoLPP3 and MoLPP5 are required for normal progression of infection-specific development and proliferation within host plants, whereas MoLPP1 and MoLPP2 are indispensable for fungal pathogenicity. Reintroduction of MoLPP3 and MoLPP5 into individual deletion mutants restored all the defects. Furthermore, exogenous addition of saturated DAG not only restored defect in appressorium formation but also complemented reduced virulence in both mutants. Taken together, our data indicate differential roles of lipid phosphate phosphatase genes and requirement of proper regulation of cellular DAGs for fungal development and pathogenesis. PMID:24959955

Cross-talk between Phosphate Starvation and Other Environmental Stress Signaling Pathways in Plants

PubMed Central

Baek, Dongwon; Chun, Hyun Jin; Yun, Dae-Jin; Kim, Min Chul

2017-01-01

The maintenance of inorganic phosphate (Pi) homeostasis is essential for plant growth and yield. Plants have evolved strategies to cope with Pi starvation at the transcriptional, post-transcriptional, and post-translational levels, which maximizes its availability. Many transcription factors, miRNAs, and transporters participate in the Pi starvation signaling pathway where their activities are modulated by sugar and phytohormone signaling. Environmental stresses significantly affect the uptake and utilization of nutrients by plants, but their effects on the Pi starvation response remain unclear. Recently, we reported that Pi starvation signaling is affected by abiotic stresses such as salt, abscisic acid, and drought. In this review, we identified transcription factors, such as MYB, WRKY, and zinc finger transcription factors with functions in Pi starvation and other environmental stress signaling. In silico analysis of the promoter regions of Pi starvation-responsive genes, including phosphate transporters, microRNAs, and phosphate starvation–induced genes, suggest that their expression may be regulated by other environmental stresses, such as hormones, drought, cold, heat, and pathogens as well as by Pi starvation. Thus, we suggest the possibility of cross-talk between Pi starvation signaling and other environmental stress signaling pathways. PMID:29047263

Degradation of Phytate by the 6-Phytase from Hafnia alvei: A Combined Structural and Solution Study

PubMed Central

Blagova, Elena V.; Turkenburg, Johan P.; Waterman, Jitka; Roberts, Shirley M.; Vind, Jesper; Sjøholm, Carsten; Lassen, Søren F.; De Maria, Leonardo; Glitsoe, Vibe; Skov, Lars K.; Wilson, Keith S.

2013-01-01

Phytases hydrolyse phytate (myo-inositol hexakisphosphate), the principal form of phosphate stored in plant seeds to produce phosphate and lower phosphorylated myo-inositols. They are used extensively in the feed industry, and have been characterised biochemically and structurally with a number of structures in the PDB. They are divided into four distinct families: histidine acid phosphatases (HAP), β-propeller phytases, cysteine phosphatases and purple acid phosphatases and also split into three enzyme classes, the 3-, 5- and 6-phytases, depending on the position of the first phosphate in the inositol ring to be removed. We report identification, cloning, purification and 3D structures of 6-phytases from two bacteria, Hafnia alvei and Yersinia kristensenii, together with their pH optima, thermal stability, and degradation profiles for phytate. An important result is the structure of the H. alvei enzyme in complex with the substrate analogue myo-inositol hexakissulphate. In contrast to the only previous structure of a ligand-bound 6-phytase, where the 3-phosphate was unexpectedly in the catalytic site, in the H. alvei complex the expected scissile 6-phosphate (sulphate in the inhibitor) is placed in the catalytic site. PMID:23741456

Degradation of phytate by the 6-phytase from Hafnia alvei: a combined structural and solution study.

PubMed

Ariza, Antonio; Moroz, Olga V; Blagova, Elena V; Turkenburg, Johan P; Waterman, Jitka; Roberts, Shirley M; Vind, Jesper; Sjøholm, Carsten; Lassen, Søren F; De Maria, Leonardo; Glitsoe, Vibe; Skov, Lars K; Wilson, Keith S

2013-01-01

Phytases hydrolyse phytate (myo-inositol hexakisphosphate), the principal form of phosphate stored in plant seeds to produce phosphate and lower phosphorylated myo-inositols. They are used extensively in the feed industry, and have been characterised biochemically and structurally with a number of structures in the PDB. They are divided into four distinct families: histidine acid phosphatases (HAP), β-propeller phytases, cysteine phosphatases and purple acid phosphatases and also split into three enzyme classes, the 3-, 5- and 6-phytases, depending on the position of the first phosphate in the inositol ring to be removed. We report identification, cloning, purification and 3D structures of 6-phytases from two bacteria, Hafnia alvei and Yersinia kristensenii, together with their pH optima, thermal stability, and degradation profiles for phytate. An important result is the structure of the H. alvei enzyme in complex with the substrate analogue myo-inositol hexakissulphate. In contrast to the only previous structure of a ligand-bound 6-phytase, where the 3-phosphate was unexpectedly in the catalytic site, in the H. alvei complex the expected scissile 6-phosphate (sulphate in the inhibitor) is placed in the catalytic site.

Digital gene expression analysis of corky split vein caused by boron deficiency in 'Newhall' Navel Orange (Citrus sinensis Osbeck) for selecting differentially expressed genes related to vascular hypertrophy.

PubMed

Yang, Cheng-Quan; Liu, Yong-Zhong; An, Ji-Cui; Li, Shuang; Jin, Long-Fei; Zhou, Gao-Feng; Wei, Qing-Jiang; Yan, Hui-Qing; Wang, Nan-Nan; Fu, Li-Na; Liu, Xiao; Hu, Xiao-Mei; Yan, Ting-Shuai; Peng, Shu-Ang

2013-01-01

Corky split vein caused by boron (B) deficiency in 'Newhall' Navel Orange was studied in the present research. The boron-deficient citrus exhibited a symptom of corky split vein in mature leaves. Morphologic and anatomical surveys at four representative phases of corky split veins showed that the symptom was the result of vascular hypertrophy. Digital gene expression (DGE) analysis was performed based on the Illumina HiSeq™ 2000 platform, which was applied to analyze the gene expression profilings of corky split veins at four morphologic phases. Over 5.3 million clean reads per library were successfully mapped to the reference database and more than 22897 mapped genes per library were simultaneously obtained. Analysis of the differentially expressed genes (DEGs) revealed that the expressions of genes associated with cytokinin signal transduction, cell division, vascular development, lignin biosynthesis and photosynthesis in corky split veins were all affected. The expressions of WOL and ARR12 involved in the cytokinin signal transduction pathway were up-regulated at 1(st) phase of corky split vein development. Furthermore, the expressions of some cell cycle genes, CYCs and CDKB, and vascular development genes, WOX4 and VND7, were up-regulated at the following 2(nd) and 3(rd) phases. These findings indicated that the cytokinin signal transduction pathway may play a role in initiating symptom observed in our study.

Combined metabolic and transcriptional profiling identifies pentose phosphate pathway activation by HSP27 phosphorylation during cerebral ischemia.

PubMed

Imahori, Taichiro; Hosoda, Kohkichi; Nakai, Tomoaki; Yamamoto, Yusuke; Irino, Yasuhiro; Shinohara, Masakazu; Sato, Naoko; Sasayama, Takashi; Tanaka, Kazuhiro; Nagashima, Hiroaki; Kohta, Masaaki; Kohmura, Eiji

2017-05-04

The metabolic pathophysiology underlying ischemic stroke remains poorly understood. To gain insight into these mechanisms, we performed a comparative metabolic and transcriptional analysis of the effects of cerebral ischemia on the metabolism of the cerebral cortex using middle cerebral artery occlusion (MCAO) rat model. Metabolic profiling by gas-chromatography/mass-spectrometry analysis showed clear separation between the ischemia and control group. The decreases of fructose 6-phosphate and ribulose 5-phosphate suggested enhancement of the pentose phosphate pathway (PPP) during cerebral ischemia (120-min MCAO) without reperfusion. Transcriptional profiling by microarray hybridization indicated that the Toll-like receptor and mitogen-activated protein kinase (MAPK) signaling pathways were upregulated during cerebral ischemia without reperfusion. In relation to the PPP, upregulation of heat shock protein 27 (HSP27) was observed in the MAPK signaling pathway and was confirmed through real-time polymerase chain reaction. Immunoblotting showed a slight increase in HSP27 protein expression and a marked increase in HSP27 phosphorylation at serine 85 after 60-min and 120-min MCAO without reperfusion. Corresponding upregulation of glucose 6-phosphate dehydrogenase (G6PD) activity and an increase in the NADPH/NAD + ratio were also observed after 120-min MCAO. Furthermore, intracerebroventricular injection of ataxia telangiectasia mutated (ATM) kinase inhibitor (KU-55933) significantly reduced HSP27 phosphorylation and G6PD upregulation after MCAO, but that of protein kinase D inhibitor (CID755673) did not affect HSP27 phosphorylation. Consequently, G6PD activation via ischemia-induced HSP27 phosphorylation by ATM kinase may be part of an endogenous antioxidant defense neuroprotection mechanism during the earliest stages of ischemia. These findings have important therapeutic implications for the treatment of stroke. Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

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

PubMed

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

2013-08-01

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

Proteomic and biochemical basis for enhanced growth yield of Enterobacter sp. LCR1 on insoluble phosphate medium.

PubMed

Kumar, Arvind; Rai, Lal Chand

2015-01-01

Proteomics and biochemical analyses were used to unravel the basis for higher growth yield of Enterobacter sp. LCR1 on insoluble phosphate medium compared to soluble. Proteomic analysis using 2-DE, MALDI-TOF/MS and LC-MS revealed the involvement of nine proteins. Down-regulation of fructose bisphosphate aldolase with decreased concentrations of glucose-6-phosphate and fructose-6-phosphate indicated diminished glycolysis. However, up-regulation of phosphoglycerate mutase, increase in the activities of 6-phosphogluconate dehydratase, 2-keto-3-deoxy-6-phosphogluconate aldolase and 6-phosphogluconate dehydrogenase suggested induction of Entner-Doudoroff and pentose phosphate pathways. These pathways generate sufficient energy from gluconic acid, which is also used for biosynthesis as indicated by up-regulation of elongation factor Tu, elongation factor G and protein disulfide isomerase. Increased reactive oxygen species (ROS) formation resulting from organic acid oxidation leads to overexpressed manganese superoxide dismutase and increased activities of catalase and ascorbate peroxidase. Thus the organism uses gluconate instead of glucose for energy, while alleviating extra ROS formation by oxidative defense enzymes. Copyright © 2014 Elsevier GmbH. All rights reserved.

Nonenzymatic gluconeogenesis-like formation of fructose 1,6-bisphosphate in ice.

PubMed

Messner, Christoph B; Driscoll, Paul C; Piedrafita, Gabriel; De Volder, Michael F L; Ralser, Markus

2017-07-11

The evolutionary origins of metabolism, in particular the emergence of the sugar phosphates that constitute glycolysis, the pentose phosphate pathway, and the RNA and DNA backbone, are largely unknown. In cells, a major source of glucose and the large sugar phosphates is gluconeogenesis. This ancient anabolic pathway (re-)builds carbon bonds as cleaved in glycolysis in an aldol condensation of the unstable catabolites glyceraldehyde 3-phosphate and dihydroxyacetone phosphate, forming the much more stable fructose 1,6-bisphosphate. We here report the discovery of a nonenzymatic counterpart to this reaction. The in-ice nonenzymatic aldol addition leads to the continuous accumulation of fructose 1,6-bisphosphate in a permanently frozen solution as followed over months. Moreover, the in-ice reaction is accelerated by simple amino acids, in particular glycine and lysine. Revealing that gluconeogenesis may be of nonenzymatic origin, our results shed light on how glucose anabolism could have emerged in early life forms. Furthermore, the amino acid acceleration of a key cellular anabolic reaction may indicate a link between prebiotic chemistry and the nature of the first metabolic enzymes.

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

PubMed Central

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

2013-01-01

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

SKF83959 Produces Antidepressant Effects in a Chronic Social Defeat Stress Model of Depression through BDNF-TrkB Pathway

PubMed Central

Jiang, Bo; Wang, Fang; Yang, Si; Fang, Peng; Deng, Zhi-Fang; Xiao, Jun-Li; Hu, Zhuang-Li

2015-01-01

Background: SKF83959 stimulates the phospholipase Cβ/inositol phosphate 3 pathway, resulting in the activation of Ca2+/calmodulin-dependent kinase IIα, which affects the synthesis of brain-derived neurotrophic factor, a neurotrophic factor critical for the pathophysiology of depression. Previous reports showed that SKF83959 elicited antidepressant activity in the forced swim test and tail suspension test as a novel triple reuptake inhibitor. However, there are no studies showing the effects of SKF83959 in a chronic stress model of depression and the role of phospholipase C/inositol phosphate 3/calmodulin-dependent kinase IIα/brain-derived neurotrophic factor pathway in SKF83959-mediated antidepressant effects. Methods: In this study, SKF83959 was firstly investigated in the chronic social defeat stress model of depression. The changes in hippocampal neurogenesis, dendrite spine density, and brain-derived neurotrophic factor signaling pathway after chronic social defeat stress and SKF83959 treatment were then investigated. Pharmacological inhibitors and small interfering RNA/short hairpin RNA methods were further used to explore the antidepressive mechanisms of SKF83959. Results: We found that SKF83959 produced antidepressant effects in the chronic social defeat stress model and also restored the chronic social defeat stress-induced decrease in hippocampal brain-derived neurotrophic factor signaling pathway, dendritic spine density, and neurogenesis. By using various inhibitors and siRNA/shRNA methods, we further demonstrated that the hippocampal dopamine D5 receptor, phospholipase C/inositol phosphate 3/ calmodulin-dependent kinase IIα pathway, and brain-derived neurotrophic factor system are all necessary for the SKF83959 effects. Conclusion: These results suggest that SKF83959 can be developed as a novel antidepressant and produces antidepressant effects via the hippocampal D5/ phospholipase C/inositol phosphate 3/calmodulin-dependent kinase IIα/brain-derived neurotrophic factor pathway. PMID:25522427

Influenza Vaccine Manufacturing: Effect of Inactivation, Splitting and Site of Manufacturing. Comparison of Influenza Vaccine Production Processes

PubMed Central

Kon, Theone C.; Onu, Adrian; Berbecila, Laurentiu; Lupulescu, Emilia; Ghiorgisor, Alina; Kersten, Gideon F.; Cui, Yi-Qing; Amorij, Jean-Pierre; Van der Pol, Leo

2016-01-01

The aim of this study was to evaluate the impact of different inactivation and splitting procedures on influenza vaccine product composition, stability and recovery to support transfer of process technology. Four split and two whole inactivated virus (WIV) influenza vaccine bulks were produced and compared with respect to release criteria, stability of the bulk and haemagglutinin recovery. One clarified harvest of influenza H3N2 A/Uruguay virus prepared on 25.000 fertilized eggs was divided equally over six downstream processes. The main unit operation for purification was sucrose gradient zonal ultracentrifugation. The inactivation of the virus was performed with either formaldehyde in phosphate buffer or with beta-propiolactone in citrate buffer. For splitting of the viral products in presence of Tween®, either Triton™ X-100 or di-ethyl-ether was used. Removal of ether was established by centrifugation and evaporation, whereas removal of Triton-X100 was performed by hydrophobic interaction chromatography. All products were sterile filtered and subjected to a 5 months real time stability study. In all processes, major product losses were measured after sterile filtration; with larger losses for split virus than for WIV. The beta-propiolactone inactivation on average resulted in higher recoveries compared to processes using formaldehyde inactivation. Especially ether split formaldehyde product showed low recovery and least stability over a period of five months. PMID:26959983

Silencing of the pentose phosphate pathway genes influences DNA replication in human fibroblasts.

PubMed

Fornalewicz, Karolina; Wieczorek, Aneta; Węgrzyn, Grzegorz; Łyżeń, Robert

2017-11-30

Previous reports and our recently published data indicated that some enzymes of glycolysis and the tricarboxylic acid cycle can affect the genome replication process by changing either the efficiency or timing of DNA synthesis in human normal cells. Both these pathways are connected with the pentose phosphate pathway (PPP pathway). The PPP pathway supports cell growth by generating energy and precursors for nucleotides and amino acids. Therefore, we asked if silencing of genes coding for enzymes involved in the pentose phosphate pathway may also affect the control of DNA replication in human fibroblasts. Particular genes coding for PPP pathway enzymes were partially silenced with specific siRNAs. Such cells remained viable. We found that silencing of the H6PD, PRPS1, RPE genes caused less efficient enterance to the S phase and decrease in efficiency of DNA synthesis. On the other hand, in cells treated with siRNA against G6PD, RBKS and TALDO genes, the fraction of cells entering the S phase was increased. However, only in the case of G6PD and TALDO, the ratio of BrdU incorporation to DNA was significantly changed. The presented results together with our previously published studies illustrate the complexity of the influence of genes coding for central carbon metabolism on the control of DNA replication in human fibroblasts, and indicate which of them are especially important in this process. Copyright © 2017 Elsevier B.V. All rights reserved.

A systematic genetic screen for genes involved in sensing inorganic phosphate availability in Saccharomyces cerevisiae

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

Choi, Joonhyuk; Rajagopal, Abbhirami; Xu, Yi -Fan

Saccharomyces cerevisiae responds to changes in extracellular inorganic phosphate (Pi) availability by regulating the activity of the phosphate-responsive (PHO) signaling pathway, enabling cells to maintain intracellular levels of the essential nutrient P i. P i-limitation induces upregulation of inositol heptakisphosphate (IP 7) synthesized by the inositol hexakisphosphate kinase Vip1, triggering inhibition of the Pho80/Pho85 cyclin-cyclin dependent kinase (CDK) complex by the CDK inhibitor Pho81, which upregulates the PHO regulon through the CDK target and transcription factor Pho4. To identify genes that are involved in signaling upstream of the Pho80/Pho85/Pho81 complex and how they interact with each other to regulate themore » PHO pathway, we performed genome-wide screens with the synthetic genetic array method. We identified more than 300 mutants with defects in signaling upstream of the Pho80/Pho85/Pho81 complex, including AAH1, which encodes an adenine deaminase that negatively regulates the PHO pathway in a Vip1-dependent manner. Moreover, we showed that even in the absence of VIP1, the PHO pathway can be activated under prolonged periods of P i starvation, suggesting complexity in the mechanisms by which the PHO pathway is regulated.« less

A systematic genetic screen for genes involved in sensing inorganic phosphate availability in Saccharomyces cerevisiae

DOE PAGES

Choi, Joonhyuk; Rajagopal, Abbhirami; Xu, Yi -Fan; ...

2017-05-17

Saccharomyces cerevisiae responds to changes in extracellular inorganic phosphate (Pi) availability by regulating the activity of the phosphate-responsive (PHO) signaling pathway, enabling cells to maintain intracellular levels of the essential nutrient P i. P i-limitation induces upregulation of inositol heptakisphosphate (IP 7) synthesized by the inositol hexakisphosphate kinase Vip1, triggering inhibition of the Pho80/Pho85 cyclin-cyclin dependent kinase (CDK) complex by the CDK inhibitor Pho81, which upregulates the PHO regulon through the CDK target and transcription factor Pho4. To identify genes that are involved in signaling upstream of the Pho80/Pho85/Pho81 complex and how they interact with each other to regulate themore » PHO pathway, we performed genome-wide screens with the synthetic genetic array method. We identified more than 300 mutants with defects in signaling upstream of the Pho80/Pho85/Pho81 complex, including AAH1, which encodes an adenine deaminase that negatively regulates the PHO pathway in a Vip1-dependent manner. Moreover, we showed that even in the absence of VIP1, the PHO pathway can be activated under prolonged periods of P i starvation, suggesting complexity in the mechanisms by which the PHO pathway is regulated.« less

Dual Mechanism of Ion Permeation through VDAC Revealed with Inorganic Phosphate Ions and Phosphate Metabolites

PubMed Central

Krammer, Eva-Maria; Vu, Giang Thi; Homblé, Fabrice; Prévost, Martine

2015-01-01

In the exchange of metabolites and ions between the mitochondrion and the cytosol, the voltage-dependent anion channel (VDAC) is a key element, as it forms the major transport pathway for these compounds through the mitochondrial outer membrane. Numerous experimental studies have promoted the idea that VDAC acts as a regulator of essential mitochondrial functions. In this study, using a combination of molecular dynamics simulations, free-energy calculations, and electrophysiological measurements, we investigated the transport of ions through VDAC, with a focus on phosphate ions and metabolites. We showed that selectivity of VDAC towards small anions including monovalent phosphates arises from short-lived interactions with positively charged residues scattered throughout the pore. In dramatic contrast, permeation of divalent phosphate ions and phosphate metabolites (AMP and ATP) involves binding sites along a specific translocation pathway. This permeation mechanism offers an explanation for the decrease in VDAC conductance measured in the presence of ATP or AMP at physiological salt concentration. The binding sites occur at similar locations for the divalent phosphate ions, AMP and ATP, and contain identical basic residues. ATP features a marked affinity for a central region of the pore lined by two lysines and one arginine of the N-terminal helix. This cluster of residues together with a few other basic amino acids forms a “charged brush” which facilitates the passage of the anionic metabolites through the pore. All of this reveals that VDAC controls the transport of the inorganic phosphates and phosphate metabolites studied here through two different mechanisms. PMID:25860993

Metabolic changes associated with tumor metastasis, part 1: tumor pH, glycolysis and the pentose phosphate pathway.

PubMed

Payen, Valéry L; Porporato, Paolo E; Baselet, Bjorn; Sonveaux, Pierre

2016-04-01

Metabolic adaptations are intimately associated with changes in cell behavior. Cancers are characterized by a high metabolic plasticity resulting from mutations and the selection of metabolic phenotypes conferring growth and invasive advantages. While metabolic plasticity allows cancer cells to cope with various microenvironmental situations that can be encountered in a primary tumor, there is increasing evidence that metabolism is also a major driver of cancer metastasis. Rather than a general switch promoting metastasis as a whole, a succession of metabolic adaptations is more likely needed to promote different steps of the metastatic process. This review addresses the contribution of pH, glycolysis and the pentose phosphate pathway, and a companion paper summarizes current knowledge regarding the contribution of mitochondria, lipids and amino acid metabolism. Extracellular acidification, intracellular alkalinization, the glycolytic enzyme phosphoglucose isomerase acting as an autocrine cytokine, lactate and the pentose phosphate pathway are emerging as important factors controlling cancer metastasis.

Structural Basis for Substrate Specificity in Phosphate Binding (beta/alpha)8-Barrels: D-Allulose 6-Phosphate 3-Epimerase from Escherichia coli K-12

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

Chan,K.; Fedorov, A.; Almo, S.

2008-01-01

Enzymes that share the ({beta}/{alpha})8-barrel fold catalyze a diverse range of reactions. Many utilize phosphorylated substrates and share a conserved C-terminal ({beta}/a)2-quarter barrel subdomain that provides a binding motif for the dianionic phosphate group. We recently reported functional and structural studies of d-ribulose 5-phosphate 3-epimerase (RPE) from Streptococcus pyogenes that catalyzes the equilibration of the pentulose 5-phosphates d-ribulose 5-phosphate and d-xylulose 5-phosphate in the pentose phosphate pathway [J. Akana, A. A. Fedorov, E. Fedorov, W. R. P. Novack, P. C. Babbitt, S. C. Almo, and J. A. Gerlt (2006) Biochemistry 45, 2493-2503]. We now report functional and structural studies ofmore » d-allulose 6-phosphate 3-epimerase (ALSE) from Escherichia coli K-12 that catalyzes the equilibration of the hexulose 6-phosphates d-allulose 6-phosphate and d-fructose 6-phosphate in a catabolic pathway for d-allose. ALSE and RPE prefer their physiological substrates but are promiscuous for each other's substrate. The active sites (RPE complexed with d-xylitol 5-phosphate and ALSE complexed with d-glucitol 6-phosphate) are superimposable (as expected from their 39% sequence identity), with the exception of the phosphate binding motif. The loop following the eighth {beta}-strand in ALSE is one residue longer than the homologous loop in RPE, so the binding site for the hexulose 6-phosphate substrate/product in ALSE is elongated relative to that for the pentulose 5-phosphate substrate/product in RPE. We constructed three single-residue deletion mutants of the loop in ALSE, ?T196, ?S197 and ?G198, to investigate the structural bases for the differing substrate specificities; for each, the promiscuity is altered so that d-ribulose 5-phosphate is the preferred substrate. The changes in kcat/Km are dominated by changes in kcat, suggesting that substrate discrimination results from differential transition state stabilization. In both ALSE and RPE, the phosphate group hydrogen bonds not only with the conserved motif but also with an active site loop following the sixth {beta}-strand, providing a potential structural mechanism for coupling substrate binding with catalysis.« less

Synthesis, characterization, vibrational spectroscopy, and factor group analysis of partially metal-doped phosphate materials

NASA Astrophysics Data System (ADS)

Sronsri, Chuchai; Boonchom, Banjong

2018-04-01

A simple precipitating method was used to synthesize effectively a partially metal-doped phosphate hydrate (Mn0.9Mg0.1HPO4·3H2O), whereas the thermal decomposition process of the above hydrate precursor was used to obtain Mn1.8Mg0.2P2O7 and LiMn0.9Mg0.1PO4 compounds under different conditions. To separate the overlapping thermal decomposition peak, a deconvolution technique was used, and the separated peak was applied to calculate the water content. The factor group splitting analysis was used to exemplify their vibrational spectra obtained from normal vibrations of HPO42-, H2O, P2O74- and PO43- functional groups. Further, the deconvoluted bending mode of water was clearly observed. Mn0.9Mg0.1HPO4·3H2O was observed in the orthorhombic crystal system with the space group of Pbca (D2h15). The formula units per unit cell were found to be eight (Z = 8), and the site symmetric type of HPO42- was observed as Cs. For the HPO42- unit, the correlation filed splitting analysis of type C3v - Cs - D2h15 was calculated and had 96 internal modes, whereas H2O in the above hydrate was symbolized as C2v - Cs - D2h15 and had 24 modes. The symbol C2v - Cs - C2h3 was used for the correlation filed splitting analysis of P2O74- in Mn1.8Mg0.2P2O7 (monoclinic, C2/m (C2h3), Z = 2, and 42 modes). Finally, the symbol Td - Cs - D2h16 was used for the correlation filed splitting analysis of PO43- in LiMn0.9Mg0.1PO4 (orthorhombic, Pnma (D2h16), Z = 4, and 36 modes).

Lactobacillus casei 64H Contains a Phosphoenolpyruvate-Dependent Phosphotransferase System for Uptake of Galactose, as Confirmed by Analysis of ptsH and Different gal Mutants

PubMed Central

Bettenbrock, Katja; Siebers, Ulrike; Ehrenreich, Petra; Alpert, Carl-Alfred

1999-01-01

Galactose metabolism in Lactobacillus casei 64H was analyzed by genetic and biochemical methods. Mutants with defects in ptsH, galK, or the tagatose 6-phosphate pathway were isolated either by positive selection using 2-deoxyglucose or 2-deoxygalactose or by an enrichment procedure with streptozotocin. ptsH mutations abolish growth on lactose, cellobiose, N-acetylglucosamine, mannose, fructose, mannitol, glucitol, and ribitol, while growth on galactose continues at a reduced rate. Growth on galactose is also reduced, but not abolished, in galK mutants. A mutation in galK in combination with a mutation in the tagatose 6-phosphate pathway results in sensitivity to galactose and lactose, while a galK mutation in combination with a mutation in ptsH completely abolishes galactose metabolism. Transport assays, in vitro phosphorylation assays, and thin-layer chromatography of intermediates of galactose metabolism also indicate the functioning of a permease/Leloir pathway and a phosphoenolpyruvate-dependent phosphotransferase system (PTS)/tagatose 6-phosphate pathway. The galactose-PTS is induced by growth on either galactose or lactose, but the induction kinetics for the two substrates are different. PMID:9864334

Lysosomal enzymes and their receptors in invertebrates: an evolutionary perspective.

PubMed

Kumar, Nadimpalli Siva; Bhamidimarri, Poorna M

2015-01-01

Lysosomal biogenesis is an important process in eukaryotic cells to maintain cellular homeostasis. The key components that are involved in the biogenesis such as the lysosomal enzymes, their modifications and the mannose 6-phosphate receptors have been well studied and their evolutionary conservation across mammalian and non-mammalian vertebrates is clearly established. Invertebrate lysosomal biogenesis pathway on the other hand is not well studied. Although, details on mannose 6-phosphate receptors and enzymes involved in lysosomal enzyme modifications were reported earlier, a clear cut pathway has not been established. Recent research on the invertebrate species involving biogenesis of lysosomal enzymes suggests a possible conserved pathway in invertebrates. This review presents certain observations based on these processes that include biochemical, immunological and functional studies. Major conclusions include conservation of MPR-dependent pathway in higher invertebrates and recent evidence suggests that MPR-independent pathway might have been more prominent among lower invertebrates. The possible components of MPR-independent pathway that may play a role in lysosomal enzyme targeting are also discussed here.

Metabolic engineering of the pentose phosphate pathway for enhanced limonene production in the cyanobacterium Synechocysti s sp. PCC 6803.

PubMed

Lin, Po-Cheng; Saha, Rajib; Zhang, Fuzhong; Pakrasi, Himadri B

2017-12-13

Isoprenoids are diverse natural compounds, which have various applications as pharmaceuticals, fragrances, and solvents. The low yield of isoprenoids in plants makes them difficult for cost-effective production, and chemical synthesis of complex isoprenoids is impractical. Microbial production of isoprenoids has been considered as a promising approach to increase the yield. In this study, we engineered the model cyanobacterium Synechocystis sp. PCC 6803 for sustainable production of a commercially valuable isoprenoid, limonene. Limonene synthases from the plants Mentha spicata and Citrus limon were expressed in cyanobacteria for limonene production. Production of limonene was two-fold higher with limonene synthase from M. spicata than that from C. limon. To enhance isoprenoid production, computational strain design was conducted by applying the OptForce strain design algorithm on Synechocystis 6803. Based on the metabolic interventions suggested by this algorithm, genes (ribose 5-phosphate isomerase and ribulose 5-phosphate 3-epimerase) in the pentose phosphate pathway were overexpressed, and a geranyl diphosphate synthase from the plant Abies grandis was expressed to optimize the limonene biosynthetic pathway. The optimized strain produced 6.7 mg/L of limonene, a 2.3-fold improvement in productivity. Thus, this study presents a feasible strategy to engineer cyanobacteria for photosynthetic production of isoprenoids.

Enzymes involved in plastid-targeted phosphatidic acid synthesis are essential for Plasmodium yoelii liver stage development

PubMed Central

Lindner, Scott E.; Sartain, Mark J.; Hayes, Kiera; Harupa, Anke; Moritz, Robert L.; Kappe, Stefan H. I.; Vaughan, Ashley M.

2014-01-01

SUMMARY Malaria parasites scavenge nutrients from their host but also harbor enzymatic pathways for de novo macromolecule synthesis. One such pathway is apicoplast-targeted type II fatty acid synthesis, which is essential for late liver stage development in rodent malaria. It is likely that fatty acids synthesized in the apicoplast are ultimately incorporated into membrane phospholipids necessary for exoerythrocytic merozoite formation. We hypothesized that these synthesized fatty acids are being utilized for apicoplast-targeted phosphatidic acid synthesis, the phospholipid precursor. Phosphatidic acid is typically synthesized in a three-step reaction utilizing three enzymes: glycerol 3-phosphate dehydrogenase, glycerol 3-phosphate acyltransferase and lysophosphatidic acid acyltransferase. The Plasmodium genome is predicted to harbor genes for both apicoplast- and cytosol/endoplasmic reticulum-targeted phosphatidic synthesis. Our research shows that apicoplast-targeted P. yoelii glycerol 3-phosphate dehydrogenase and glycerol 3-phosphate acyltransferase are expressed only during liver stage development and deletion of the encoding genes resulted in late liver stage growth arrest and lack of merozoite differentiation. However, the predicted apicoplast-targeted lysophosphatidic acid acyltransferase gene was refractory to deletion and was expressed solely in the endoplasmic reticulum throughout the parasite lifecycle. Our results suggest that P. yoelii has an incomplete apicoplast-targeted phosphatidic acid synthesis pathway that is essential for liver stage maturation. PMID:24330260

TRAF6-Mediated SM22α K21 Ubiquitination Promotes G6PD Activation and NADPH Production, Contributing to GSH Homeostasis and VSMC Survival In Vitro and In Vivo.

PubMed

Dong, Li-Hua; Li, Liang; Song, Yu; Duan, Zhi-Li; Sun, Shao-Guang; Lin, Yan-Ling; Miao, Sui-Bing; Yin, Ya-Juan; Shu, Ya-Nan; Li, Huan; Chen, Peng; Zhao, Li-Li; Han, Mei

2015-09-25

Vascular smooth muscle cell (VSMC) survival under stressful conditions is integral to promoting vascular repair, but facilitates plaque stability during the development of atherosclerosis. The cytoskeleton-associated smooth muscle (SM) 22α protein is involved in the regulation of VSMC phenotypes, whereas the pentose phosphate pathway plays an essential role in cell proliferation through the production of dihydronicotinamide adenine dinucleotide phosphate. To identify the relationship between dihydronicotinamide adenine dinucleotide phosphate production and SM22α activity in the development and progression of vascular diseases. We showed that the expression and activity of glucose-6-phosphate dehydrogenase (G6PD) are promoted in platelet-derived growth factor (PDGF)-BB-induced proliferative VSMCs. PDGF-BB induced G6PD membrane translocation and activation in an SM22α K21 ubiquitination-dependent manner. Specifically, the ubiquitinated SM22α interacted with G6PD and mediated G6PD membrane translocation. Furthermore, we found that tumor necrosis factor receptor-associated factor (TRAF) 6 mediated SM22α K21 ubiquitination in a K63-linked manner on PDGF-BB stimulation. Knockdown of TRAF6 decreased the membrane translocation and activity of G6PD, in parallel with reduced SM22α K21 ubiquitination. Elevated levels of activated G6PD consequent to PDGF-BB induction led to increased dihydronicotinamide adenine dinucleotide phosphate generation through stimulation of the pentose phosphate pathway, which enhanced VSMC viability and reduced apoptosis in vivo and in vitro via glutathione homeostasis. We provide evidence that TRAF6-induced SM22α ubiquitination maintains VSMC survival through increased G6PD activity and dihydronicotinamide adenine dinucleotide phosphate production. The TRAF6-SM22α-G6PD pathway is a novel mechanism underlying the association between glucose metabolism and VSMC survival, which is beneficial for vascular repair after injury but facilitates atherosclerotic plaque stability. © 2015 American Heart Association, Inc.

OXIDATIVE STRESS, INFLAMMATION AND CARCINOGENESIS ARE CONTROLLED THROUGH THE PENTOSE PHOSPHATE PATHWAY BY TRANSALDOLASE

PubMed Central

Perl, Andras; Hanczko, Robert; Telarico, Tiffany; Oaks, Zachary; Landas, Steve

2011-01-01

Metabolism of glucose through the pentose phosphate pathway (PPP) influences the development of diverse pathologies. Hemolytic anemia due to deficiency of PPP enzyme glucose 6-phosphate dehydrogenase is the most common genetic disease in humans. Recently, inactivation of another PPP enzyme, transaldolase (TAL), has been implicated in male infertility and fatty liver progressing to steatohepatitis and cancer. Hepatocarcinogenesis was associated with activation of aldose reductase and redox-sensitive transcription factors and prevented by N-acetylcysteine. Here, we discuss how alternative formulations of the PPP with and without TAL reflect cell type-specific metabolic control of oxidative stress, a critical source of inflammation and carcinogenesis. Ongoing studies of TAL deficiency will identify new molecular targets for diagnosis and treatment in clinical practice. PMID:21376665

An aqueous, organic dye derivatized SnO 2 /TiO 2 core/shell photoanode

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

Wee, Kyung-Ryang; Sherman, Benjamin D.; Brennaman, M. Kyle

2016-01-01

Visible light driven water splitting in a dye-sensitized photoelectrochemical cell (DSPEC) based on a phosphonic acid-derivatized donor–π–acceptor (D–π–A) organic dye (P–A–π–D) is described with the dye anchored to an FTO|SnO 2/TiO 2core/shell photoanode in a pH 7 phosphate buffer solution.

Detection of membrane protein-protein interaction in planta based on dual-intein-coupled tripartite split-GFP association.

PubMed

Liu, Tzu-Yin; Chou, Wen-Chun; Chen, Wei-Yuan; Chu, Ching-Yi; Dai, Chen-Yi; Wu, Pei-Yu

2018-05-01

Despite the great interest in identifying protein-protein interactions (PPIs) in biological systems, only a few attempts have been made at large-scale PPI screening in planta. Unlike biochemical assays, bimolecular fluorescence complementation allows visualization of transient and weak PPIs in vivo at subcellular resolution. However, when the non-fluorescent fragments are highly expressed, spontaneous and irreversible self-assembly of the split halves can easily generate false positives. The recently developed tripartite split-GFP system was shown to be a reliable PPI reporter in mammalian and yeast cells. In this study, we adapted this methodology, in combination with the β-estradiol-inducible expression cassette, for the detection of membrane PPIs in planta. Using a transient expression assay by agroinfiltration of Nicotiana benthamiana leaves, we demonstrate the utility of the tripartite split-GFP association in plant cells and affirm that the tripartite split-GFP system yields no spurious background signal even with abundant fusion proteins readily accessible to the compartments of interaction. By validating a few of the Arabidopsis PPIs, including the membrane PPIs implicated in phosphate homeostasis, we proved the fidelity of this assay for detection of PPIs in various cellular compartments in planta. Moreover, the technique combining the tripartite split-GFP association and dual-intein-mediated cleavage of polyprotein precursor is feasible in stably transformed Arabidopsis plants. Our results provide a proof-of-concept implementation of the tripartite split-GFP system as a potential tool for membrane PPI screens in planta. © 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.

Metabolic Flux Analysis of Plastidic Isoprenoid Biosynthesis in Poplar Leaves Emitting and Nonemitting Isoprene1[W

PubMed Central

Ghirardo, Andrea; Wright, Louwrance Peter; Bi, Zhen; Rosenkranz, Maaria; Pulido, Pablo; Rodríguez-Concepción, Manuel; Niinemets, Ülo; Brüggemann, Nicolas; Gershenzon, Jonathan; Schnitzler, Jörg-Peter

2014-01-01

The plastidic 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway is one of the most important pathways in plants and produces a large variety of essential isoprenoids. Its regulation, however, is still not well understood. Using the stable isotope 13C-labeling technique, we analyzed the carbon fluxes through the MEP pathway and into the major plastidic isoprenoid products in isoprene-emitting and transgenic isoprene-nonemitting (NE) gray poplar (Populus × canescens). We assessed the dependence on temperature, light intensity, and atmospheric [CO2]. Isoprene biosynthesis was by far (99%) the main carbon sink of MEP pathway intermediates in mature gray poplar leaves, and its production required severalfold higher carbon fluxes compared with NE leaves with almost zero isoprene emission. To compensate for the much lower demand for carbon, NE leaves drastically reduced the overall carbon flux within the MEP pathway. Feedback inhibition of 1-deoxy-d-xylulose-5-phosphate synthase activity by accumulated plastidic dimethylallyl diphosphate almost completely explained this reduction in carbon flux. Our data demonstrate that short-term biochemical feedback regulation of 1-deoxy-d-xylulose-5-phosphate synthase activity by plastidic dimethylallyl diphosphate is an important regulatory mechanism of the MEP pathway. Despite being relieved from the large carbon demand of isoprene biosynthesis, NE plants redirected only approximately 0.5% of this saved carbon toward essential nonvolatile isoprenoids, i.e. β-carotene and lutein, most probably to compensate for the absence of isoprene and its antioxidant properties. PMID:24590857

Metabolic flux analysis of plastidic isoprenoid biosynthesis in poplar leaves emitting and nonemitting isoprene.

PubMed

Ghirardo, Andrea; Wright, Louwrance Peter; Bi, Zhen; Rosenkranz, Maaria; Pulido, Pablo; Rodríguez-Concepción, Manuel; Niinemets, Ülo; Brüggemann, Nicolas; Gershenzon, Jonathan; Schnitzler, Jörg-Peter

2014-05-01

The plastidic 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway is one of the most important pathways in plants and produces a large variety of essential isoprenoids. Its regulation, however, is still not well understood. Using the stable isotope 13C-labeling technique, we analyzed the carbon fluxes through the MEP pathway and into the major plastidic isoprenoid products in isoprene-emitting and transgenic isoprene-nonemitting (NE) gray poplar (Populus×canescens). We assessed the dependence on temperature, light intensity, and atmospheric [CO2]. Isoprene biosynthesis was by far (99%) the main carbon sink of MEP pathway intermediates in mature gray poplar leaves, and its production required severalfold higher carbon fluxes compared with NE leaves with almost zero isoprene emission. To compensate for the much lower demand for carbon, NE leaves drastically reduced the overall carbon flux within the MEP pathway. Feedback inhibition of 1-deoxy-D-xylulose-5-phosphate synthase activity by accumulated plastidic dimethylallyl diphosphate almost completely explained this reduction in carbon flux. Our data demonstrate that short-term biochemical feedback regulation of 1-deoxy-d-xylulose-5-phosphate synthase activity by plastidic dimethylallyl diphosphate is an important regulatory mechanism of the MEP pathway. Despite being relieved from the large carbon demand of isoprene biosynthesis, NE plants redirected only approximately 0.5% of this saved carbon toward essential nonvolatile isoprenoids, i.e. β-carotene and lutein, most probably to compensate for the absence of isoprene and its antioxidant properties.

Expression of the cytoplasmic mevalonate pathway in chloroplasts to reduce substrate limitations for cytoplasmically-produced terpenoid secondary products

USDA-ARS?s Scientific Manuscript database

All products of isoprenoid metabolism originate with the C5 non-allylic substrate, isopentenyl pyrophosphate (IPP). IPP is produced in plants by two distinct pathways, the mevalonate pathway (MEV) in the cytosol and the 2 C methyl-D-erythritol 4 phosphate (MEP) pathway in plastids. A multi-gene a...

Fructose metabolism in the cerebellum.

PubMed

Funari, Vincent A; Crandall, James E; Tolan, Dean R

2007-01-01

Under normal physiological conditions, the brain utilizes only a small number of carbon sources for energy. Recently, there is growing molecular and biochemical evidence that other carbon sources, including fructose, may play a role in neuro-energetics. Fructose is the number one commercial sweetener in Western civilization with large amounts of fructose being toxic, yet fructose metabolism remains relatively poorly characterized. Fructose is purportedly metabolized via either of two pathways, the fructose-1-phosphate pathway and/or the fructose-6-phosphate pathway. Many early metabolic studies could not clearly discriminate which of these two pathways predominates, nor could they distinguish which cell types in various tissues are capable of fructose metabolism. In addition, the lack of good physiological models, the diet-induced changes in gene expression in many tissues, the involvement of multiple genes in multiple pathways involved in fructose metabolism, and the lack of characterization of some genes involved in fructose metabolism have complicated our understanding of the physiological role of fructose in neuro-energetics. A recent neuro-metabolism study of the cerebellum demonstrated fructose metabolism and co-expression of the genes specific for the fructose 1-phosphate pathway, GLUT5 (glut5) and ketohexokinase (khk), in Purkinje cells suggesting this as an active pathway in specific neurons? Meanwhile, concern over the rapid increase in dietary fructose, particularly among children, has increased awareness about how fructose is metabolized in vivo and what effects a high fructose diet might have. In this regard, establishment of cellular and molecular studies and physiological characterization of the important and/or deleterious roles fructose plays in the brain is critical. This review will discuss the status of fructose metabolism in the brain with special reference to the cerebellum and the physiological roles of the different pathways.

A Pathway Closely Related to the d-Tagatose Pathway of Gram-Negative Enterobacteria Identified in the Gram-Positive Bacterium Bacillus licheniformis

PubMed Central

Van der Heiden, Edwige; Lebrun, Sarah; Freichels, Régine; Brans, Alain; Vastenavond, Christian M.; Galleni, Moreno; Joris, Bernard

2013-01-01

We report the first identification of a gene cluster involved in d-tagatose catabolism in Bacillus licheniformis. The pathway is closely related to the d-tagatose pathway of the Gram-negative bacterium Klebsiella oxytoca, in contrast to the d-tagatose 6-phosphate pathway described in the Gram-positive bacterium Staphylococcus aureus. PMID:23524682

A pathway closely related to the (D)-tagatose pathway of gram-negative enterobacteria identified in the gram-positive bacterium Bacillus licheniformis.

PubMed

Van der Heiden, Edwige; Delmarcelle, Michaël; Lebrun, Sarah; Freichels, Régine; Brans, Alain; Vastenavond, Christian M; Galleni, Moreno; Joris, Bernard

2013-06-01

We report the first identification of a gene cluster involved in d-tagatose catabolism in Bacillus licheniformis. The pathway is closely related to the d-tagatose pathway of the Gram-negative bacterium Klebsiella oxytoca, in contrast to the d-tagatose 6-phosphate pathway described in the Gram-positive bacterium Staphylococcus aureus.

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

Michalska, Karolina; Cuff, Marianne E.; Structural Biology Center, Biosciences Division, Argonne National Laboratory

The crystal structure of 2-oxo-3-deoxygalactonate kinase from the De Ley–Doudoroff pathway of galactose metabolism has been determined at 2.1 Å resolution. In most organisms, efficient d-galactose utilization requires the highly conserved Leloir pathway that converts d-galactose to d-glucose 1-phosphate. However, in some bacterial and fungal species alternative routes of d-galactose assimilation have been identified. In the so-called De Ley–Doudoroff pathway, d-galactose is metabolized into pyruvate and d-glyceraldehyde 3-phosphate in five consecutive reactions carried out by specific enzymes. The penultimate step in this pathway involves the phosphorylation of 2-oxo-3-deoxygalactonate to 2-oxo-3-deoxygalactonate 6-phosphate catalyzed by 2-oxo-3-deoxygalactonate kinase, with ATP serving as amore » phosphoryl-group donor. Here, a crystal structure of 2-oxo-3-deoxygalactonate kinase from Klebsiella pneumoniae determined at 2.1 Å resolution is reported, the first structure of an enzyme from the De Ley–Doudoroff pathway. Structural comparison indicates that the enzyme belongs to the ASKHA (acetate and sugar kinases/hsc70/actin) family of phosphotransferases. The protein is composed of two α/β domains, each of which contains a core common to all family members. Additional elements introduced between conserved structural motifs define the unique features of 2-oxo-3-deoxygalactonate kinase and possibly determine the biological function of the protein.« less

Facile formation of 2D Co2P@Co3O4 microsheets through in-situ toptactic conversion and surface corrosion: Bifunctional electrocatalysts towards overall water splitting

NASA Astrophysics Data System (ADS)

Yao, Lihua; Zhang, Nan; Wang, Yin; Ni, Yuanman; Yan, Dongpeng; Hu, Changwen

2018-01-01

Exploring efficient non-precious electrocatalysts for both the hydrogen evolution reaction (HER) and oxygen evolution reaction (OER) is crucial for many renewable energy conversion processes. In this work, we report that 2D Co2P@Co3O4 microsheets can be prepared through an in-situ toptactic conversion from single-crystal β-Co(OH)2 microplatelets, associated with a surface phosphatization and corrosion process. The resultant Co2P@Co3O4 2D hybrid materials can further serve as self-supported bifunctional catalytic electrodes to drive the overall water splitting for HER and OER simultaneously, with low overpotentials and high long-term stability. Furthermore, a water electrolyzer based on Co2P@Co3O4 hybrid as both anode and cathode is fabricated, which achieves 10 mA cm-2 current at only 1.57 V during water splitting process. Therefore, this work provides a facile strategy to obtain 2D Co2P-based micro/nanostructures, which act as low-cost and highly active electrocatalysts towards overall water splitting application.

Efficient photosensitized splitting of the thymine dimer/oxetane unit on its modifying beta-cyclodextrin by a binding electron donor.

PubMed

Tang, Wen-Jian; Song, Qin-Hua; Wang, Hong-Bo; Yu, Jing-Yu; Guo, Qing-Xiang

2006-07-07

Two modified beta-cyclodextrins (beta-CDs) with a thymine dimer and a thymine oxetane adduct respectively, TD-CD and Ox-CD, have been prepared, and utilized to bind an electron-rich chromophore, indole or N,N-dimethylaniline (DMA), to form a supramolecular complex. We have examined the photosensitized splitting of the dimer/oxetane unit in TD-CD/Ox-CD by indole or DMA via an electron-transfer pathway, and observed high splitting efficiencies of the dimer/oxetane unit. On the basis of measurements of fluorescence spectra and splitting quantum yields, it is suggested that the splitting reaction occurs in a supramolecular complex by an inclusion interaction between the modified beta-CDs and DMA or indole. The back electron transfer, which leads low splitting efficiencies for the covalently-linked chromophore-dimer/oxetane compounds, is suppressed in the non-covalently-bound complex, and the mechanism has been discussed.

The route of non-enzymic and enzymic breakdown of 5-phosphoribosyl 1-pyrophosphate to ribose 1-phosphate.

PubMed Central

Trembacz, H; Jezewska, M M

1990-01-01

Spontaneous decomposition of 5-phosphoribosyl 1-pyrophosphate at pH 5.5 was established to occur as follows: 5-Phosphoribosyl 1-pyrophosphate----5-phosphoribosyl 1,2-(cyclic)phosphate----ribose 1-phosphate----ribose Enzymic degradation of 5-phosphoribosyl 1-pyrophosphate by alkaline phosphatase from calf intestine and by acid phosphatases from potato and Aspergillus niger was found to proceed according to this pathway within the pH range 2.5-7.4 with accumulation of ribose 1-phosphate. In the case of alkaline phosphatase, Mg2+ ions inhibit the pyrophosphorolysis of 5-phosphoribosyl 1-pyrophosphate and stimulate the hydrolysis of ribose 1-phosphate. PMID:1700897

Pseudomonas putida KT2440 Strain Metabolizes Glucose through a Cycle Formed by Enzymes of the Entner-Doudoroff, Embden-Meyerhof-Parnas, and Pentose Phosphate Pathways.

PubMed

Nikel, Pablo I; Chavarría, Max; Fuhrer, Tobias; Sauer, Uwe; de Lorenzo, Víctor

2015-10-23

The soil bacterium Pseudomonas putida KT2440 lacks a functional Embden-Meyerhof-Parnas (EMP) pathway, and glycolysis is known to proceed almost exclusively through the Entner-Doudoroff (ED) route. To investigate the raison d'être of this metabolic arrangement, the distribution of periplasmic and cytoplasmic carbon fluxes was studied in glucose cultures of this bacterium by using (13)C-labeled substrates, combined with quantitative physiology experiments, metabolite quantification, and in vitro enzymatic assays under both saturating and non-saturating, quasi in vivo conditions. Metabolic flux analysis demonstrated that 90% of the consumed sugar was converted into gluconate, entering central carbon metabolism as 6-phosphogluconate and further channeled into the ED pathway. Remarkably, about 10% of the triose phosphates were found to be recycled back to form hexose phosphates. This set of reactions merges activities belonging to the ED, the EMP (operating in a gluconeogenic fashion), and the pentose phosphate pathways to form an unforeseen metabolic architecture (EDEMP cycle). Determination of the NADPH balance revealed that the default metabolic state of P. putida KT2440 is characterized by a slight catabolic overproduction of reducing power. Cells growing on glucose thus run a biochemical cycle that favors NADPH formation. Because NADPH is required not only for anabolic functions but also for counteracting different types of environmental stress, such a cyclic operation may contribute to the physiological heftiness of this bacterium in its natural habitats. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Dissecting the role of isoprene and stress-related hormones (ABA and ethylene) in Populus nigra exposed to unequal root zone water stress.

PubMed

Marino, Giovanni; Brunetti, Cecilia; Tattini, Massimiliano; Romano, Andrea; Biasioli, Franco; Tognetti, Roberto; Loreto, Francesco; Ferrini, Francesco; Centritto, Mauro

2017-12-01

Isoprene is synthesized through the 2-C-methylerythritol-5-phosphate (MEP) pathway that also produces abscisic acid (ABA). Increases in foliar free ABA concentration during drought induce stomatal closure and may also alter ethylene biosynthesis. We hypothesized a role of isoprene biosynthesis in protecting plants challenged by increasing water deficit, by influencing ABA production and ethylene evolution. We performed a split-root experiment on Populus nigra L. subjected to three water treatments: well-watered (WW) plants with both root sectors kept at pot capacity, plants with both root compartments allowed to dry for 5 days (DD) and plants with one-half of the roots irrigated to pot capacity, while the other half did not receive water (WD). WD and WW plants were similar in photosynthesis, water relations, foliar ABA concentration and isoprene emission, whereas these parameters were significantly affected in DD plants: leaf isoprene emission increased despite the fact that photosynthesis declined by 85% and the ABA-glucoside/free ABA ratio decreased significantly. Enhanced isoprene biosynthesis in water-stressed poplars may have contributed to sustaining leaf ABA biosynthesis by keeping the MEP pathway active. However, this enhancement in ABA was accompanied by no change in ethylene biosynthesis, likely confirming the antagonistic role between ABA and ethylene. These results may indicate a potential cross-talk among isoprene, ABA and ethylene under drought. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Teaching the Toolkit: A Laboratory Series to Demonstrate the Evolutionary Conservation of Metazoan Cell Signaling Pathways

ERIC Educational Resources Information Center

LeClair, Elizabeth E.

2008-01-01

A major finding of comparative genomics and developmental genetics is that metazoans share certain conserved, embryonically deployed signaling pathways that instruct cells as to their ultimate fate. Because the DNA encoding these pathways predates the evolutionary split of most animal groups, it should in principle be possible to clone…

Precise precursor rebalancing for isoprenoids production by fine control of gapA expression in Escherichia coli.

PubMed

Jung, Juyoung; Lim, Jae Hyung; Kim, Se Yeon; Im, Dae-Kyun; Seok, Joo Yeon; Lee, Seung-Jae V; Oh, Min-Kyu; Jung, Gyoo Yeol

2016-11-01

Biosynthesis of isoprenoids via the 1-deoxy-D-xylulose-5-phosphate (DXP) pathway requires equimolar glyceraldehyde 3-phosphate and pyruvate to divert carbon flux toward the products of interest. Here, we demonstrate that precursor balancing is one of the critical steps for the production of isoprenoids in Escherichia coli. First, the implementation of the synthetic lycopene production pathway as a model system and the amplification of the native DXP pathway were accomplished using synthetic constitutive promoters and redesigned 5'-untranslated regions (5'-UTRs). Next, fine-controlled precursor balancing was investigated by tuning phosphoenolpyruvate synthase (PpsA) or glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The results showed that tuning-down of gapA improved the specific lycopene content by 45% compared to the overexpression of ppsA. The specific lycopene content in the strains with down-regulated gapA increased by 97% compared to that in the parental strain. Our results indicate that gapA is the best target for precursor balancing to increase biosynthesis of isoprenoids. Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

The Pentose Phosphate Pathway as a Potential Target for Cancer Therapy

PubMed Central

Cho, Eunae Sandra; Cha, Yong Hoon; Kim, Hyun Sil; Kim, Nam Hee; Yook, Jong In

2018-01-01

During cancer progression, cancer cells are repeatedly exposed to metabolic stress conditions in a resource-limited environment which they must escape. Increasing evidence indicates the importance of nicotinamide adenine dinucleotide phosphate (NADPH) homeostasis in the survival of cancer cells under metabolic stress conditions, such as metabolic resource limitation and therapeutic intervention. NADPH is essential for scavenging of reactive oxygen species (ROS) mainly derived from oxidative phosphorylation required for ATP generation. Thus, metabolic reprogramming of NADPH homeostasis is an important step in cancer progression as well as in combinational therapeutic approaches. In mammalian, the pentose phosphate pathway (PPP) and one-carbon metabolism are major sources of NADPH production. In this review, we focus on the importance of glucose flux control towards PPP regulated by oncogenic pathways and the potential therein for metabolic targeting as a cancer therapy. We also summarize the role of Snail (Snai1), an important regulator of the epithelial mesenchymal transition (EMT), in controlling glucose flux towards PPP and thus potentiating cancer cell survival under oxidative and metabolic stress. PMID:29212304

AglH, a thermophilic UDP-N-acetylglucosamine-1-phosphate:dolichyl phosphate GlcNAc-1-phosphotransferase initiating protein N-glycosylation pathway in Sulfolobus acidocaldarius, is capable of complementing the eukaryal Alg7.

PubMed

Meyer, Benjamin H; Shams-Eldin, Hosam; Albers, Sonja-Verena

2017-01-01

AglH, a predicted UDP-GlcNAc-1-phosphate:dolichyl phosphate GlcNAc-1-phosphotransferase, is initiating the protein N-glycosylation pathway in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius. AglH successfully replaced the endogenous GlcNAc-1-phosphotransferase activity of Alg7 in a conditional lethal Saccharomyces cerevisiae strain, in which the first step of the eukaryal protein N-glycosylation process was repressed. This study is one of the few examples of cross-domain complementation demonstrating a conserved polyprenyl phosphate transferase reaction within the eukaryal and archaeal domain like it was demonstrated for Methanococcus voltae (Shams-Eldin et al. 2008). The topology prediction and the alignment of the AglH membrane protein with GlcNAc-1-phosphotransferases from the three domains of life show significant conservation of amino acids within the different proposed cytoplasmic loops. Alanine mutations of selected conserved amino acids in the putative cytoplasmic loops II (D 100 ), IV (F 220 ) and V (F 264 ) demonstrated the importance of these amino acids for cross-domain AlgH activity in in vitro complementation assays in S. cerevisiae. Furthermore, antibiotic treatment interfering directly with the activity of dolichyl phosphate GlcNAc-1-phosphotransferases confirmed the essentiality of N-glycosylation for cell survival.

Regulations and roles for alternative pathways of hexose metabolism in plants

Treesearch

Clanton C. Black; Laszlo Mustardy; S.S. Sung; P.P. Kormanik; D.-P. Xu; Nachman Paz

1987-01-01

Plants have two cytoplasmic pathways of glycolysis and gluconeogenesis for the reversible interconversion of fructose 6-phosphate (F-6-P) and fructose 1,6-bisphosphate (F-1,6,-P2). One pathway is described as a maintenance pathway that is catalyzed by a nucleotide triphosphate-dependent phosphofructokinase (EC 2.7.1.11; ATP-PFK) glycolytically and a F-1,6...

Development of inhibitors of the 2C-methyl-D-erythritol 4-phosphate (MEP) pathway enzymes as potential anti-infective agents.

PubMed

Masini, Tiziana; Hirsch, Anna K H

2014-12-11

Important pathogens such as Mycobacterium tuberculosis and Plasmodium falciparum, the causative agents of tuberculosis and malaria, respectively, and plants, utilize the 2C-methyl-D-erythritol 4-phosphate (MEP, 5) pathway for the biosynthesis of isopentenyl diphosphate (1) and dimethylallyl diphosphate (2), the universal precursors of isoprenoids, while humans exclusively utilize the alternative mevalonate pathway for the synthesis of 1 and 2. This distinct distribution, together with the fact that the MEP pathway is essential in numerous organisms, makes the enzymes of the MEP pathway attractive drug targets for the development of anti-infective agents and herbicides. Herein, we review the inhibitors reported over the past 2 years, in the context of the most important older developments and with a particular focus on the results obtained against enzymes of pathogenic organisms. We will also discuss new discoveries in terms of structural and mechanistic features, which can help to guide a rational development of inhibitors.

Inflammation, vitamin B6 and related pathways.

PubMed

Ueland, Per Magne; McCann, Adrian; Midttun, Øivind; Ulvik, Arve

2017-02-01

The active form of vitamin B6, pyridoxal 5'-phosphate (PLP), serves as a co-factor in more than 150 enzymatic reactions. Plasma PLP has consistently been shown to be low in inflammatory conditions; there is a parallel reduction in liver PLP, but minor changes in erythrocyte and muscle PLP and in functional vitamin B6 biomarkers. Plasma PLP also predicts the risk of chronic diseases like cardiovascular disease and some cancers, and is inversely associated with numerous inflammatory markers in clinical and population-based studies. Vitamin B6 intake and supplementation improve some immune functions in vitamin B6-deficient humans and experimental animals. A possible mechanism involved is mobilization of vitamin B6 to the sites of inflammation where it may serve as a co-factor in pathways producing metabolites with immunomodulating effects. Relevant vitamin B6-dependent inflammatory pathways include vitamin B6 catabolism, the kynurenine pathway, sphingosine 1-phosphate metabolism, the transsulfuration pathway, and serine and glycine metabolism. Copyright © 2016 Elsevier Ltd. All rights reserved.

Biosynthesis of 2-methyl-3-buten-2-ol emitted from needles of Pinus ponderosa via the non-mevalonate DOXP/MEP pathway of isoprenoid formation.

PubMed

Zeidler, J; Lichtenthaler, H K

2001-06-01

The volatile hemiterpene 2-methyl-3-buten-2-ol (MBO) is emitted from the needles of several pine species from the Western United States and contributes to ozone formation in the atmosphere. It is synthesised enzymatically from dimethylallyl diphosphate (DMAPP). We show here that needles of Pinus ponderosa Laws. incorporated [1-2H1]-1-deoxy-D-xylulose (d-DOX) into the emitted MBO, but not D,L-[2-13C]mevalonic acid lactone. Furthermore, MBO emission was inhibited by fosmidomycin, a specific inhibitor of the second enzyme of the mevalonate-independent pathway of isopentenyl diphosphate and DMAPP formation, i.e. the 1-deoxy-D-xylulose 5-phosphate/2-C-methyl-D-erythritol 4-phosphate (DOXP/MEP) pathway. We thus prove that MBO emitted from needles of P. ponderosa is primarily formed via the DOXP/MEP pathway.

Understanding D-Ribose and Mitochondrial Function.

PubMed

Mahoney, Diane E; Hiebert, John B; Thimmesch, Amanda; Pierce, John T; Vacek, James L; Clancy, Richard L; Sauer, Andrew J; Pierce, Janet D

2018-01-01

Mitochondria are important organelles referred to as cellular powerhouses for their unique properties of cellular energy production. With many pathologic conditions and aging, mitochondrial function declines, and there is a reduction in the production of adenosine triphosphate. The energy carrying molecule generated by cellular respiration and by pentose phosphate pathway, an alternative pathway of glucose metabolism. D-ribose is a naturally occurring monosaccharide found in the cells and particularly in the mitochondria is essential in energy production. Without sufficient energy, cells cannot maintain integrity and function. Supplemental D-ribose has been shown to improve cellular processes when there is mitochondrial dysfunction. When individuals take supplemental D-ribose, it can bypass part of the pentose pathway to produce D-ribose-5-phosphate for the production of energy. In this article, we review how energy is produced by cellular respiration, the pentose pathway, and the use of supplemental D-ribose.

An adaptation to life in acid through a novel mevalonate pathway

DOE PAGES

Vinokur, Jeffrey M.; Cummins, Matthew C.; Korman, Tyler P.; ...

2016-12-22

Here, extreme acidophiles are capable of growth at pH values near zero. Sustaining life in acidic environments requires extensive adaptations of membranes, proton pumps, and DNA repair mechanisms. Here we describe an adaptation of a core biochemical pathway, the mevalonate pathway, in extreme acidophiles. Two previously known mevalonate pathways involve ATP dependent decarboxylation of either mevalonate 5-phosphate or mevalonate 5-pyrophosphate, in which a single enzyme carries out two essential steps: (1) phosphorylation of the mevalonate moiety at the 3-OH position and (2) subsequent decarboxylation. We now demonstrate that in extreme acidophiles, decarboxylation is carried out by two separate steps: previouslymore » identified enzymes generate mevalonate 3,5-bisphosphate and a new decarboxylase we describe here, mevalonate 3,5-bisphosphate decarboxylase, produces isopentenyl phosphate. Why use two enzymes in acidophiles when one enzyme provides both functionalities in all other organisms examined to date? We find that at low pH, the dual function enzyme, mevalonate 5-phosphate decarboxylase is unable to carry out the first phosphorylation step, yet retains its ability to perform decarboxylation. We therefore propose that extreme acidophiles had to replace the dual-purpose enzyme with two specialized enzymes to efficiently produce isoprenoids in extremely acidic environments.« less

An adaptation to life in acid through a novel mevalonate pathway

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

Vinokur, Jeffrey M.; Cummins, Matthew C.; Korman, Tyler P.

Here, extreme acidophiles are capable of growth at pH values near zero. Sustaining life in acidic environments requires extensive adaptations of membranes, proton pumps, and DNA repair mechanisms. Here we describe an adaptation of a core biochemical pathway, the mevalonate pathway, in extreme acidophiles. Two previously known mevalonate pathways involve ATP dependent decarboxylation of either mevalonate 5-phosphate or mevalonate 5-pyrophosphate, in which a single enzyme carries out two essential steps: (1) phosphorylation of the mevalonate moiety at the 3-OH position and (2) subsequent decarboxylation. We now demonstrate that in extreme acidophiles, decarboxylation is carried out by two separate steps: previouslymore » identified enzymes generate mevalonate 3,5-bisphosphate and a new decarboxylase we describe here, mevalonate 3,5-bisphosphate decarboxylase, produces isopentenyl phosphate. Why use two enzymes in acidophiles when one enzyme provides both functionalities in all other organisms examined to date? We find that at low pH, the dual function enzyme, mevalonate 5-phosphate decarboxylase is unable to carry out the first phosphorylation step, yet retains its ability to perform decarboxylation. We therefore propose that extreme acidophiles had to replace the dual-purpose enzyme with two specialized enzymes to efficiently produce isoprenoids in extremely acidic environments.« less

Enzymes involved in plastid-targeted phosphatidic acid synthesis are essential for Plasmodium yoelii liver-stage development.

PubMed

Lindner, Scott E; Sartain, Mark J; Hayes, Kiera; Harupa, Anke; Moritz, Robert L; Kappe, Stefan H I; Vaughan, Ashley M

2014-02-01

Malaria parasites scavenge nutrients from their host but also harbour enzymatic pathways for de novo macromolecule synthesis. One such pathway is apicoplast-targeted type II fatty acid synthesis, which is essential for late liver-stage development in rodent malaria. It is likely that fatty acids synthesized in the apicoplast are ultimately incorporated into membrane phospholipids necessary for exoerythrocytic merozoite formation. We hypothesized that these synthesized fatty acids are being utilized for apicoplast-targeted phosphatidic acid synthesis, the phospholipid precursor. Phosphatidic acid is typically synthesized in a three-step reaction utilizing three enzymes: glycerol 3-phosphate dehydrogenase, glycerol 3-phosphate acyltransferase and lysophosphatidic acid acyltransferase. The Plasmodium genome is predicted to harbour genes for both apicoplast- and cytosol/endoplasmic reticulum-targeted phosphatidic acid synthesis. Our research shows that apicoplast-targeted Plasmodium yoelii glycerol 3-phosphate dehydrogenase and glycerol 3-phosphate acyltransferase are expressed only during liver-stage development and deletion of the encoding genes resulted in late liver-stage growth arrest and lack of merozoite differentiation. However, the predicted apicoplast-targeted lysophosphatidic acid acyltransferase gene was refractory to deletion and was expressed solely in the endoplasmic reticulum throughout the parasite life cycle. Our results suggest that P. yoelii has an incomplete apicoplast-targeted phosphatidic acid synthesis pathway that is essential for liver-stage maturation. © 2013 John Wiley & Sons Ltd.

Cannabidiol attenuates OGD/R-induced damage by enhancing mitochondrial bioenergetics and modulating glucose metabolism via pentose-phosphate pathway in hippocampal neurons.

PubMed

Sun, Shanshan; Hu, Fangyuan; Wu, Jihong; Zhang, Shenghai

2017-04-01

Deficient bioenergetics and diminished redox conservation have been implicated in the development of cerebral ischemia/reperfusion injury. In this study, the mechanisms underlying the neuroprotective effects of cannabidiol (CBD), a nonpsychotropic compound derived from Cannabis sativa with FDA-approved antiepilepsy properties, were studied in vitro using an oxygen-glucose-deprivation/reperfusion (OGD/R) model in a mouse hippocampal neuronal cell line. CBD supplementation during reperfusion rescued OGD/R-induced cell death, attenuated intracellular ROS generation and lipid peroxidation, and simultaneously reversed the abnormal changes in antioxidant biomarkers. Using the Seahorse XF e 24 Extracellular Flux Analyzer, we found that CBD significantly improved basal respiration, ATP-linked oxygen consumption rate, and the spare respiratory capacity, and augmented glucose consumption in OGD/R-injured neurons. The activation of glucose 6-phosphate dehydrogenase and the preservation of the NADPH/NADP + ratio implies that the pentose-phosphate pathway is stimulated by CBD, thus protecting hippocampal neurons from OGD/R injury. This study is the first to document the neuroprotective effects of CBD against OGD/R insult, which depend in part on attenuating oxidative stress, enhancing mitochondrial bioenergetics, and modulating glucose metabolism via the pentose-phosphate pathway, thus preserving both energy and the redox balance. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

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

Light-Induced Surface Reactions at the Bismuth Vanadate/Potassium Phosphate Interface.

PubMed

Favaro, Marco; Abdi, Fatwa F; Lamers, Marlene; Crumlin, Ethan J; Liu, Zhi; van de Krol, Roel; Starr, David E

2018-01-18

Bismuth vanadate has recently drawn significant research attention as a light-absorbing photoanode due to its performance for photoelectrochemical water splitting. In this study, we use in situ ambient pressure X-ray photoelectron spectroscopy with "tender" X-rays (4.0 keV) to investigate a polycrystalline bismuth vanadate (BiVO 4 ) electrode in contact with an aqueous potassium phosphate (KPi) solution at open circuit potential under both dark and light conditions. This is facilitated by the creation of a 25 to 30 nm thick electrolyte layer using the "dip-and-pull" method. We observe that under illumination bismuth phosphate forms on the BiVO 4 surface leading to an increase of the surface negative charge. The bismuth phosphate layer may act to passivate surface states observed in photoelectrochemical measurements. The repulsive interaction between the negatively charged surface under illumination and the phosphate ions in solution causes a shift in the distribution of ions in the thin aqueous electrolyte film, which is observed as an increase in their photoelectron signals. Interestingly, we find that such changes at the BiVO 4 /KPi electrolyte interface are reversible upon returning to dark conditions. By measuring the oxygen 1s photoelectron peak intensities from the phosphate ions and liquid water as a function of time under dark and light conditions, we determine the time scales for the forward and reverse reactions. Our results provide direct evidence for light-induced chemical modification of the BiVO 4 /KPi electrolyte interface.

The trehalose pathway in maize: conservation and gene regulation in response to the diurnal cycle and extended darkness

PubMed Central

Henry, Clémence; Bledsoe, Samuel W.; Siekman, Allison; Kollman, Alec; Waters, Brian M.; Feil, Regina; Stitt, Mark; Lagrimini, L. Mark

2014-01-01

Energy resources in plants are managed in continuously changing environments, such as changes occurring during the day/night cycle. Shading is an environmental disruption that decreases photosynthesis, compromises energy status, and impacts on crop productivity. The trehalose pathway plays a central but not well-defined role in maintaining energy balance. Here, we characterized the maize trehalose pathway genes and deciphered the impacts of the diurnal cycle and disruption of the day/night cycle on trehalose pathway gene expression and sugar metabolism. The maize genome encodes 14 trehalose-6-phosphate synthase (TPS) genes, 11 trehalose-6-phosphate phosphatase (TPP) genes, and one trehalase gene. Transcript abundance of most of these genes was impacted by the day/night cycle and extended dark stress, as were sucrose, hexose sugars, starch, and trehalose-6-phosphate (T6P) levels. After extended darkness, T6P levels inversely followed class II TPS and sucrose non-fermenting-related protein kinase 1 (SnRK1) target gene expression. Most significantly, T6P no longer tracked sucrose levels after extended darkness. These results showed: (i) conservation of the trehalose pathway in maize; (ii) that sucrose, hexose, starch, T6P, and TPS/TPP transcripts respond to the diurnal cycle; and(iii) that extended darkness disrupts the correlation between T6P and sucrose/hexose pools and affects SnRK1 target gene expression. A model for the role of the trehalose pathway in sensing of sucrose and energy status in maize seedlings is proposed. PMID:25271261

GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE-S, A SPERM-SPECIFIC GLYCOLYTIC ENZYME, IS REQUIRED FOR SPERM MOTILITY AND MALE FERTILITY

EPA Science Inventory

While glycolysis is highly conserved, it is remarkable that several novel isozymes in this central metabolic pathway are found in mammalian sperm. Glyceraldehyde 3-phosphate dehydrogenase-S (GAPDS) is the product of a mouse gene expressed only during spermatogenesis and, like it...

tortuga refines Notch pathway gene expression in the zebrafish presomitic mesoderm at the post-transcriptional level.

PubMed

Dill, Kariena K; Amacher, Sharon L

2005-11-15

We have identified the zebrafish tortuga (tor) gene by an ENU-induced mutation that disrupts the presomitic mesoderm (PSM) expression of Notch pathway genes. In tor mutants, Notch pathway gene expression persists in regions of the PSM where expression is normally off in wild type embryos. The expression of hairy/Enhancer of split-related 1 (her1) is affected first, followed by the delta genes deltaC and deltaD, and finally, by another hairy/Enhancer of split-related gene, her7. In situ hybridization with intron-specific probes for her1 and deltaC indicates that transcriptional bursts of expression are normal in tor mutants, suggesting that tor normally functions to refine her1 and deltaC message levels downstream of transcription. Despite the striking defects in Notch pathway gene expression, somite boundaries form normally in tor mutant embryos, although somitic mesoderm defects are apparent later, when cells mature to form muscle fibers. Thus, while the function of Notch pathway genes is required for proper somite formation, the tor mutant phenotype suggests that precise oscillations of Notch pathway transcripts are not essential for establishing segmental pattern in the presomitic mesoderm.

Interaction between Nitrogen and Phosphate Stress Responses in Sinorhizobium meliloti

DOE PAGES

Hagberg, Kelly L.; Yurgel, Svetlana N.; Mulder, Monika; ...

2016-11-30

Bacteria have developed various stress response pathways to improve their assimilation and allocation of limited nutrients, such as nitrogen and phosphate. While both the nitrogen stress response (NSR) and phosphate stress response (PSR) have been studied individually, there are few experiments reported that characterize effects of multiple stresses on one or more pathways in Sinorhizobium meliloti, a facultatively symbiotic, nitrogen-fixing bacteria. The PII proteins, GlnB and GlnK, regulate the NSR activity, but analysis of global transcription changes in a PII deficient mutant suggest that the S. meliloti PII proteins may also regulate the PSR. PII double deletion mutants grow verymore » slowly and pseudoreversion of the slow growth phenotype is common. In order to understand this phenomenon better, transposon mutants were isolated that had a faster growing phenotype. One mutation was in phoB, the response regulator for a two component regulatory system that is important in the PSR. phoB::Tn5 mutants had different phenotypes in the wild type compared to a PII deficient background. This led to the hypothesis that phosphate stress affects the NSR and conversely, that nitrogen stress affects the PSR. These results show that phosphate availability affects glutamine synthetase activity and expression, which are often used as indicators of NSR activity, but that nitrogen availability did not affect alkaline phosphatase activity and expression, which are indicators of PSR activity. Finally, we conclude that the NSR is co-regulated by nitrogen and phosphate, whereas the PSR does not appear to be co-regulated by nitrogen in addition to its known phosphate regulation.« less

Interaction between Nitrogen and Phosphate Stress Responses in Sinorhizobium meliloti

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

Hagberg, Kelly L.; Yurgel, Svetlana N.; Mulder, Monika

Bacteria have developed various stress response pathways to improve their assimilation and allocation of limited nutrients, such as nitrogen and phosphate. While both the nitrogen stress response (NSR) and phosphate stress response (PSR) have been studied individually, there are few experiments reported that characterize effects of multiple stresses on one or more pathways in Sinorhizobium meliloti, a facultatively symbiotic, nitrogen-fixing bacteria. The PII proteins, GlnB and GlnK, regulate the NSR activity, but analysis of global transcription changes in a PII deficient mutant suggest that the S. meliloti PII proteins may also regulate the PSR. PII double deletion mutants grow verymore » slowly and pseudoreversion of the slow growth phenotype is common. In order to understand this phenomenon better, transposon mutants were isolated that had a faster growing phenotype. One mutation was in phoB, the response regulator for a two component regulatory system that is important in the PSR. phoB::Tn5 mutants had different phenotypes in the wild type compared to a PII deficient background. This led to the hypothesis that phosphate stress affects the NSR and conversely, that nitrogen stress affects the PSR. These results show that phosphate availability affects glutamine synthetase activity and expression, which are often used as indicators of NSR activity, but that nitrogen availability did not affect alkaline phosphatase activity and expression, which are indicators of PSR activity. Finally, we conclude that the NSR is co-regulated by nitrogen and phosphate, whereas the PSR does not appear to be co-regulated by nitrogen in addition to its known phosphate regulation.« less

Glucose-6-phosphate dehydrogenase-6-phosphogluconolactonase: a unique bifunctional enzyme from Plasmodium falciparum.

PubMed

Jortzik, Esther; Mailu, Boniface M; Preuss, Janina; Fischer, Marina; Bode, Lars; Rahlfs, Stefan; Becker, Katja

2011-06-15

The survival of malaria parasites in human RBCs (red blood cells) depends on the pentose phosphate pathway, both in Plasmodium falciparum and its human host. G6PD (glucose-6-phosphate dehydrogenase) deficiency, the most common human enzyme deficiency, leads to a lack of NADPH in erythrocytes, and protects from malaria. In P. falciparum, G6PD is combined with the second enzyme of the pentose phosphate pathway to create a unique bifunctional enzyme named GluPho (glucose-6-phosphate dehydrogenase-6-phosphogluconolactonase). In the present paper, we report for the first time the cloning, heterologous overexpression, purification and kinetic characterization of both enzymatic activities of full-length PfGluPho (P. falciparum GluPho), and demonstrate striking structural and functional differences with the human enzymes. Detailed kinetic analyses indicate that PfGluPho functions on the basis of a rapid equilibrium random Bi Bi mechanism, where the binding of the second substrate depends on the first substrate. We furthermore show that PfGluPho is inhibited by S-glutathionylation. The availability of recombinant PfGluPho and the major differences to hG6PD (human G6PD) facilitate studies on PfGluPho as an excellent drug target candidate in the search for new antimalarial drugs.

The glycerol backbone of phospholipids derives from noncarbohydrate precursors in starved lung cancer cells.

PubMed

Leithner, Katharina; Triebl, Alexander; Trötzmüller, Martin; Hinteregger, Barbara; Leko, Petra; Wieser, Beatrix I; Grasmann, Gabriele; Bertsch, Alexandra L; Züllig, Thomas; Stacher, Elvira; Valli, Alessandro; Prassl, Ruth; Olschewski, Andrea; Harris, Adrian L; Köfeler, Harald C; Olschewski, Horst; Hrzenjak, Andelko

2018-06-12

Cancer cells are reprogrammed to consume large amounts of glucose to support anabolic biosynthetic pathways. However, blood perfusion and consequently the supply with glucose are frequently inadequate in solid cancers. PEPCK-M ( PCK2 ), the mitochondrial isoform of phosphoenolpyruvate carboxykinase (PEPCK), has been shown by us and others to be functionally expressed and to mediate gluconeogenesis, the reverse pathway of glycolysis, in different cancer cells. Serine and ribose synthesis have been identified as downstream pathways fed by PEPCK in cancer cells. Here, we report that PEPCK-M-dependent glycerol phosphate formation from noncarbohydrate precursors (glyceroneogenesis) occurs in starved lung cancer cells and supports de novo glycerophospholipid synthesis. Using stable isotope-labeled glutamine and lactate, we show that PEPCK-M generates phosphoenolpyruvate and 3-phosphoglycerate, which are at least partially converted to glycerol phosphate and incorporated into glycerophospholipids (GPL) under glucose and serum starvation. This pathway is required to maintain levels of GPL, especially phosphatidylethanolamine (PE), as shown by stable shRNA-mediated silencing of PEPCK-M in H23 lung cancer cells. PEPCK-M shRNA led to reduced colony formation after starvation, and the effect was partially reversed by the addition of dioleyl-PE. Furthermore, PEPCK-M silencing abrogated cancer growth in a lung cancer cell xenograft model. In conclusion, glycerol phosphate formation for de novo GPL synthesis via glyceroneogenesis is a newly characterized anabolic pathway in cancer cells mediated by PEPCK-M under conditions of severe nutrient deprivation. Copyright © 2018 the Author(s). Published by PNAS.

A de novo NADPH generation pathway for improving lysine production of Corynebacterium glutamicum by rational design of the coenzyme specificity of glyceraldehyde 3-phosphate dehydrogenase.

PubMed

Bommareddy, Rajesh Reddy; Chen, Zhen; Rappert, Sugima; Zeng, An-Ping

2014-09-01

Engineering the cofactor availability is a common strategy of metabolic engineering to improve the production of many industrially important compounds. In this work, a de novo NADPH generation pathway is proposed by altering the coenzyme specificity of a native NAD-dependent glyceraldehyde 3-phosphate dehydrogenase (GAPDH) to NADP, which consequently has the potential to produce additional NADPH in the glycolytic pathway. Specifically, the coenzyme specificity of GAPDH of Corynebacterium glutamicum is systematically manipulated by rational protein design and the effect of the manipulation for cellular metabolism and lysine production is evaluated. By a combinatorial modification of four key residues within the coenzyme binding sites, different GAPDH mutants with varied coenzyme specificity were constructed. While increasing the catalytic efficiency of GAPDH towards NADP enhanced lysine production in all of the tested mutants, the most significant improvement of lysine production (~60%) was achieved with the mutant showing similar preference towards both NAD and NADP. Metabolic flux analysis with (13)C isotope studies confirmed that there was no significant change of flux towards the pentose phosphate pathway and the increased lysine yield was mainly attributed to the NADPH generated by the mutated GAPDH. The present study highlights the importance of protein engineering as a key strategy in de novo pathway design and overproduction of desired products. Copyright © 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

The glycerol backbone of phospholipids derives from noncarbohydrate precursors in starved lung cancer cells

PubMed Central

Trötzmüller, Martin; Hinteregger, Barbara; Leko, Petra; Wieser, Beatrix I.; Grasmann, Gabriele; Bertsch, Alexandra L.; Züllig, Thomas; Stacher, Elvira; Valli, Alessandro; Prassl, Ruth; Olschewski, Andrea; Harris, Adrian L.; Köfeler, Harald C.; Olschewski, Horst; Hrzenjak, Andelko

2018-01-01

Cancer cells are reprogrammed to consume large amounts of glucose to support anabolic biosynthetic pathways. However, blood perfusion and consequently the supply with glucose are frequently inadequate in solid cancers. PEPCK-M (PCK2), the mitochondrial isoform of phosphoenolpyruvate carboxykinase (PEPCK), has been shown by us and others to be functionally expressed and to mediate gluconeogenesis, the reverse pathway of glycolysis, in different cancer cells. Serine and ribose synthesis have been identified as downstream pathways fed by PEPCK in cancer cells. Here, we report that PEPCK-M–dependent glycerol phosphate formation from noncarbohydrate precursors (glyceroneogenesis) occurs in starved lung cancer cells and supports de novo glycerophospholipid synthesis. Using stable isotope-labeled glutamine and lactate, we show that PEPCK-M generates phosphoenolpyruvate and 3-phosphoglycerate, which are at least partially converted to glycerol phosphate and incorporated into glycerophospholipids (GPL) under glucose and serum starvation. This pathway is required to maintain levels of GPL, especially phosphatidylethanolamine (PE), as shown by stable shRNA-mediated silencing of PEPCK-M in H23 lung cancer cells. PEPCK-M shRNA led to reduced colony formation after starvation, and the effect was partially reversed by the addition of dioleyl-PE. Furthermore, PEPCK-M silencing abrogated cancer growth in a lung cancer cell xenograft model. In conclusion, glycerol phosphate formation for de novo GPL synthesis via glyceroneogenesis is a newly characterized anabolic pathway in cancer cells mediated by PEPCK-M under conditions of severe nutrient deprivation. PMID:29844165

Gallic acid inhibits vascular calcification through the blockade of BMP2-Smad1/5/8 signaling pathway.

PubMed

Kee, Hae Jin; Cho, Soo-Na; Kim, Gwi Ran; Choi, Sin Young; Ryu, Yuhee; Kim, In Kyeom; Hong, Young Joon; Park, Hyung Wook; Ahn, Youngkeun; Cho, Jeong Gwan; Park, Jong Chun; Jeong, Myung Ho

2014-11-01

Vascular calcification is associated with increased risk of morbidity and mortality in patients with cardiovascular diseases, chronic kidney diseases, and diabetes. Gallic acid, a natural compound found in gallnut and green tea, is known to be antifungal, antioxidant, and anticancer. Here we investigated the effect of gallic acid on vascular smooth muscle cell (VSMC) calcification and the underlying mechanism. Gallic acid inhibited inorganic phosphate-induced osteoblast differentiation markers as well as calcification phenotypes (as determined by calcium deposition, Alizarin Red, and Von Kossa staining). Knockdown of BMP2 or Noggin blocked phosphate-induced calcification. Gallic acid suppressed phosphorylation of Smad1/5/8 protein induced by inorganic phosphate. Taken together, we suggest that gallic acid acts as a novel therapeutic agent of vascular calcification by mediating BMP2-Smad1/5/8 signaling pathway. Copyright © 2014 Elsevier Inc. All rights reserved.

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

PubMed

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

2008-11-01

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

Kinetic Characterization and Allosteric Inhibition of the Yersinia pestis 1-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase (MEP Synthase)

PubMed Central

Haymond, Amanda; Johny, Chinchu; Dowdy, Tyrone; Schweibenz, Brandon; Villarroel, Karen; Young, Richard; Mantooth, Clark J.; Patel, Trishal; Bases, Jessica; Jose, Geraldine San; Jackson, Emily R.; Dowd, Cynthia S.; Couch, Robin D.

2014-01-01

The methylerythritol phosphate (MEP) pathway found in many bacteria governs the synthesis of isoprenoids, which are crucial lipid precursors for vital cell components such as ubiquinone. Because mammals synthesize isoprenoids via an alternate pathway, the bacterial MEP pathway is an attractive target for novel antibiotic development, necessitated by emerging antibiotic resistance as well as biodefense concerns. The first committed step in the MEP pathway is the reduction and isomerization of 1-deoxy-D-xylulose-5-phosphate (DXP) to methylerythritol phosphate (MEP), catalyzed by MEP synthase. To facilitate drug development, we cloned, expressed, purified, and characterized MEP synthase from Yersinia pestis. Enzyme assays indicate apparent kinetic constants of KM DXP = 252 µM and KM NADPH = 13 µM, IC50 values for fosmidomycin and FR900098 of 710 nM and 231 nM respectively, and Ki values for fosmidomycin and FR900098 of 251 nM and 101 nM respectively. To ascertain if the Y. pestis MEP synthase was amenable to a high-throughput screening campaign, the Z-factor was determined (0.9) then the purified enzyme was screened against a pilot scale library containing rationally designed fosmidomycin analogs and natural product extracts. Several hit molecules were obtained, most notably a natural product allosteric affector of MEP synthase and a rationally designed bisubstrate derivative of FR900098 (able to associate with both the NADPH and DXP binding sites in MEP synthase). It is particularly noteworthy that allosteric regulation of MEP synthase has not been described previously. Thus, our discovery implicates an alternative site (and new chemical space) for rational drug development. PMID:25171339

Investigating the Contribution of the Phosphate Transport Pathway to Arsenic Accumulation in Rice1[W

PubMed Central

Wu, Zhongchang; Ren, Hongyan; McGrath, Steve P.; Wu, Ping; Zhao, Fang-Jie

2011-01-01

Arsenic (As) accumulation in rice (Oryza sativa) may pose a significant health risk to consumers. Plants take up different As species using various pathways. Here, we investigated the contribution of the phosphate (Pi) transport pathway to As accumulation in rice grown hydroponically or under flooded soil conditions. In hydroponic experiments, a rice mutant defective in OsPHF1 (for phosphate transporter traffic facilitator1) lost much of the ability to take up Pi and arsenate and to transport them from roots to shoots, whereas transgenic rice overexpressing either the Pi transporter OsPht1;8 (OsPT8) or the transcription factor OsPHR2 (for phosphate starvation response2) had enhanced abilities of Pi and arsenate uptake and translocation. OsPT8 was found to have a high affinity for both Pi and arsenate, and its overexpression increased the maximum influx by 3- to 5-fold. In arsenate-treated plants, both arsenate and arsenite were detected in the xylem sap, with the proportion of the latter increasing with the exposure time. Under the flooded soil conditions, the phf1 mutant took up less Pi whereas the overexpression lines took up more Pi. But there were no similar effects on As accumulation and distribution. Rice grain contained predominantly dimethylarsinic acid and arsenite, with arsenate being a minor species. These results suggest that the Pi transport pathway contributed little to As uptake and transport to grain in rice plants grown in flooded soil. Transgenic approaches to enhance Pi acquisition from paddy soil through the overexpression of Pi transporters may not increase As accumulation in rice grain. PMID:21715673

Glyphosate-induced oxidative stress in Arabidopsis thaliana affecting peroxisomal metabolism and triggers activity in the oxidative phase of the pentose phosphate pathway (OxPPP) involved in NADPH generation.

PubMed

de Freitas-Silva, Larisse; Rodríguez-Ruiz, Marta; Houmani, Hayet; da Silva, Luzimar Campos; Palma, José M; Corpas, Francisco J

2017-11-01

Glyphosate is a broad-spectrum systemic herbicide used worldwide. In susceptible plants, glyphosate affects the shikimate pathway and reduces aromatic amino acid synthesis. Using Arabidopsis seedlings grown in the presence of 20μM glyphosate, we analyzed H 2 O 2 , ascorbate, glutathione (GSH) and protein oxidation content as well as antioxidant catalase, superoxide dismutase (SOD) and ascorbate-glutathione cycle enzyme activity. We also examined the principal NADPH-generating system components, including glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), NADP-malic enzyme (NADP-ME) and NADP-isocitrate dehydrogenase (NADP-ICDH). Glyphosate caused a drastic reduction in growth parameters and an increase in protein oxidation. The herbicide also resulted in an overall increase in GSH content, antioxidant enzyme activity (catalase and all enzymatic components of the ascorbate-glutathione cycle) in addition to the two oxidative phase enzymes, G6PDH and 6PGDH, in the pentose phosphate pathway involved in NADPH generation. In this study, we provide new evidence on the participation of G6PDH and 6PGDH in the response to oxidative stress induced by glyphosate in Arabidopsis, in which peroxisomal enzymes, such as catalase and glycolate oxidase, are positively affected. We suggest that the NADPH provided by the oxidative phase of the pentose phosphate pathway (OxPPP) should serve to maintain glutathione reductase (GR) activity, thus preserving and regenerating the intracellular GSH pool under glyphosate-induced stress. It is particularly remarkable that the 6PGDH activity was unaffected by pro-oxidant and nitrating molecules such as H 2 0 2 , nitric oxide or peroxynitrite. Copyright © 2017 Elsevier GmbH. All rights reserved.

Fermentation of Xylose Causes Inefficient Metabolic State Due to Carbon/Energy Starvation and Reduced Glycolytic Flux in Recombinant Industrial Saccharomyces cerevisiae

PubMed Central

Matsushika, Akinori; Nagashima, Atsushi; Goshima, Tetsuya; Hoshino, Tamotsu

2013-01-01

In the present study, comprehensive, quantitative metabolome analysis was carried out on the recombinant glucose/xylose-cofermenting S. cerevisiae strain MA-R4 during fermentation with different carbon sources, including glucose, xylose, or glucose/xylose mixtures. Capillary electrophoresis time-of-flight mass spectrometry was used to determine the intracellular pools of metabolites from the central carbon pathways, energy metabolism pathways, and the levels of twenty amino acids. When xylose instead of glucose was metabolized by MA-R4, glycolytic metabolites including 3- phosphoglycerate, 2- phosphoglycerate, phosphoenolpyruvate, and pyruvate were dramatically reduced, while conversely, most pentose phosphate pathway metabolites such as sedoheptulose 7- phosphate and ribulose 5-phosphate were greatly increased. These results suggest that the low metabolic activity of glycolysis and the pool of pentose phosphate pathway intermediates are potential limiting factors in xylose utilization. It was further demonstrated that during xylose fermentation, about half of the twenty amino acids declined, and the adenylate/guanylate energy charge was impacted due to markedly decreased adenosine triphosphate/adenosine monophosphate and guanosine triphosphate/guanosine monophosphate ratios, implying that the fermentation of xylose leads to an inefficient metabolic state where the biosynthetic capabilities and energy balance are severely impaired. In addition, fermentation with xylose alone drastically increased the level of citrate in the tricarboxylic acid cycle and increased the aromatic amino acids tryptophan and tyrosine, strongly supporting the view that carbon starvation was induced. Interestingly, fermentation with xylose alone also increased the synthesis of the polyamine spermidine and its precursor S-adenosylmethionine. Thus, differences in carbon substrates, including glucose and xylose in the fermentation medium, strongly influenced the dynamic metabolism of MA-R4. These results provide a metabolic explanation for the low ethanol productivity on xylose compared to glucose. PMID:23874849

Arsenic Toxicity: The Effects on Plant Metabolism

PubMed Central

Finnegan, Patrick M.; Chen, Weihua

2012-01-01

The two forms of inorganic arsenic, arsenate (AsV) and arsenite (AsIII), are easily taken up by the cells of the plant root. Once in the cell, AsV can be readily converted to AsIII, the more toxic of the two forms. AsV and AsIII both disrupt plant metabolism, but through distinct mechanisms. AsV is a chemical analog of phosphate that can disrupt at least some phosphate-dependent aspects of metabolism. AsV can be translocated across cellular membranes by phosphate transport proteins, leading to imbalances in phosphate supply. It can compete with phosphate during phosphorylation reactions, leading to the formation of AsV adducts that are often unstable and short-lived. As an example, the formation and rapid autohydrolysis of AsV-ADP sets in place a futile cycle that uncouples photophosphorylation and oxidative phosphorylation, decreasing the ability of cells to produce ATP and carry out normal metabolism. AsIII is a dithiol reactive compound that binds to and potentially inactivates enzymes containing closely spaced cysteine residues or dithiol co-factors. Arsenic exposure generally induces the production of reactive oxygen species that can lead to the production of antioxidant metabolites and numerous enzymes involved in antioxidant defense. Oxidative carbon metabolism, amino acid and protein relationships, and nitrogen and sulfur assimilation pathways are also impacted by As exposure. Readjustment of several metabolic pathways, such as glutathione production, has been shown to lead to increased arsenic tolerance in plants. Species- and cultivar-dependent variation in arsenic sensitivity and the remodeling of metabolite pools that occurs in response to As exposure gives hope that additional metabolic pathways associated with As tolerance will be identified. PMID:22685440

Metabolic pathway engineering based on metabolomics confers acetic and formic acid tolerance to a recombinant xylose-fermenting strain of Saccharomyces cerevisiae

PubMed Central

2011-01-01

Background The development of novel yeast strains with increased tolerance toward inhibitors in lignocellulosic hydrolysates is highly desirable for the production of bio-ethanol. Weak organic acids such as acetic and formic acids are necessarily released during the pretreatment (i.e. solubilization and hydrolysis) of lignocelluloses, which negatively affect microbial growth and ethanol production. However, since the mode of toxicity is complicated, genetic engineering strategies addressing yeast tolerance to weak organic acids have been rare. Thus, enhanced basic research is expected to identify target genes for improved weak acid tolerance. Results In this study, the effect of acetic acid on xylose fermentation was analyzed by examining metabolite profiles in a recombinant xylose-fermenting strain of Saccharomyces cerevisiae. Metabolome analysis revealed that metabolites involved in the non-oxidative pentose phosphate pathway (PPP) [e.g. sedoheptulose-7-phosphate, ribulose-5-phosphate, ribose-5-phosphate and erythrose-4-phosphate] were significantly accumulated by the addition of acetate, indicating the possibility that acetic acid slows down the flux of the pathway. Accordingly, a gene encoding a PPP-related enzyme, transaldolase or transketolase, was overexpressed in the xylose-fermenting yeast, which successfully conferred increased ethanol productivity in the presence of acetic and formic acid. Conclusions Our metabolomic approach revealed one of the molecular events underlying the response to acetic acid and focuses attention on the non-oxidative PPP as a target for metabolic engineering. An important challenge for metabolic engineering is identification of gene targets that have material importance. This study has demonstrated that metabolomics is a powerful tool to develop rational strategies to confer tolerance to stress through genetic engineering. PMID:21219616

Robust Ordering of Anaphase Events by Adaptive Thresholds and Competing Degradation Pathways.

PubMed

Kamenz, Julia; Mihaljev, Tamara; Kubis, Armin; Legewie, Stefan; Hauf, Silke

2015-11-05

The splitting of chromosomes in anaphase and their delivery into the daughter cells needs to be accurately executed to maintain genome stability. Chromosome splitting requires the degradation of securin, whereas the distribution of the chromosomes into the daughter cells requires the degradation of cyclin B. We show that cells encounter and tolerate variations in the abundance of securin or cyclin B. This makes the concurrent onset of securin and cyclin B degradation insufficient to guarantee that early anaphase events occur in the correct order. We uncover that the timing of chromosome splitting is not determined by reaching a fixed securin level, but that this level adapts to the securin degradation kinetics. In conjunction with securin and cyclin B competing for degradation during anaphase, this provides robustness to the temporal order of anaphase events. Our work reveals how parallel cell-cycle pathways can be temporally coordinated despite variability in protein concentrations. Copyright © 2015 Elsevier Inc. All rights reserved.

Arabidopsis Chlorophyll Biosynthesis: An Essential Balance between the Methylerythritol Phosphate and Tetrapyrrole Pathways[C][W

PubMed Central

Kim, Se; Schlicke, Hagen; Van Ree, Kalie; Karvonen, Kristine; Subramaniam, Anant; Richter, Andreas; Grimm, Bernhard; Braam, Janet

2013-01-01

Chlorophyll, essential for photosynthesis, is composed of a chlorin ring and a geranylgeranyl diphosphate (GGPP)–derived isoprenoid, which are generated by the tetrapyrrole and methylerythritol phosphate (MEP) biosynthesis pathways, respectively. Although a functional MEP pathway is essential for plant viability, the underlying basis of the requirement has been unclear. We hypothesized that MEP pathway inhibition is lethal because a reduction in GGPP availability results in a stoichiometric imbalance in tetrapyrrolic chlorophyll precursors, which can cause deadly photooxidative stress. Consistent with this hypothesis, lethality of MEP pathway inhibition in Arabidopsis thaliana by fosmidomycin (FSM) is light dependent, and toxicity of MEP pathway inhibition is reduced by genetic and chemical impairment of the tetrapyrrole pathway. In addition, FSM treatment causes a transient accumulation of chlorophyllide and transcripts associated with singlet oxygen-induced stress. Furthermore, exogenous provision of the phytol molecule reduces FSM toxicity when the phytol can be modified for chlorophyll incorporation. These data provide an explanation for FSM toxicity and thereby provide enhanced understanding of the mechanisms of FSM resistance. This insight into MEP pathway inhibition consequences underlines the risk plants undertake to synthesize chlorophyll and suggests the existence of regulation, possibly involving chloroplast-to-nucleus retrograde signaling, that may monitor and maintain balance of chlorophyll precursor synthesis. PMID:24363312

Arabidopsis chlorophyll biosynthesis: an essential balance between the methylerythritol phosphate and tetrapyrrole pathways.

PubMed

Kim, Se; Schlicke, Hagen; Van Ree, Kalie; Karvonen, Kristine; Subramaniam, Anant; Richter, Andreas; Grimm, Bernhard; Braam, Janet

2013-12-01

Chlorophyll, essential for photosynthesis, is composed of a chlorin ring and a geranylgeranyl diphosphate (GGPP)-derived isoprenoid, which are generated by the tetrapyrrole and methylerythritol phosphate (MEP) biosynthesis pathways, respectively. Although a functional MEP pathway is essential for plant viability, the underlying basis of the requirement has been unclear. We hypothesized that MEP pathway inhibition is lethal because a reduction in GGPP availability results in a stoichiometric imbalance in tetrapyrrolic chlorophyll precursors, which can cause deadly photooxidative stress. Consistent with this hypothesis, lethality of MEP pathway inhibition in Arabidopsis thaliana by fosmidomycin (FSM) is light dependent, and toxicity of MEP pathway inhibition is reduced by genetic and chemical impairment of the tetrapyrrole pathway. In addition, FSM treatment causes a transient accumulation of chlorophyllide and transcripts associated with singlet oxygen-induced stress. Furthermore, exogenous provision of the phytol molecule reduces FSM toxicity when the phytol can be modified for chlorophyll incorporation. These data provide an explanation for FSM toxicity and thereby provide enhanced understanding of the mechanisms of FSM resistance. This insight into MEP pathway inhibition consequences underlines the risk plants undertake to synthesize chlorophyll and suggests the existence of regulation, possibly involving chloroplast-to-nucleus retrograde signaling, that may monitor and maintain balance of chlorophyll precursor synthesis.

The nutrient transceptor/PKA pathway functions independently of TOR and responds to leucine and Gcn2 in a TOR-independent manner.

PubMed

Conrad, Michaela; Kankipati, Harish Nag; Kimpe, Marlies; Van Zeebroeck, Griet; Zhang, Zhiqiang; Thevelein, Johan M

2017-08-01

Two nutrient-controlled signalling pathways, the PKA and TOR pathway, play a major role in nutrient regulation of growth as well as growth-correlated properties in yeast. The relationship between the two pathways is not well understood. We have used Gap1 and Pho84 transceptor-mediated activation of trehalase and phosphorylation of fragmented Sch9 as a read-out for rapid nutrient activation of PKA or TORC1, respectively. We have identified conditions in which L-citrulline-induced activation of Sch9 phosphorylation is compromised, but not activation of trehalase: addition of the TORC1 inhibitor, rapamycin and low levels of L-citrulline. The same disconnection was observed for phosphate activation in phosphate-starved cells. The leu2 auxotrophic mutation reduces amino acid activation of trehalase, which is counteracted by deletion of GCN2. Both effects were also independent of TORC1. Our results show that rapid activation of the TOR pathway by amino acids is not involved in rapid activation of the PKA pathway and that effects of Gcn2 inactivation as well as leu2 auxotrophy all act independently of the TOR pathway. Hence, rapid nutrient signalling to PKA and TOR in cells arrested by nutrient starvation acts through parallel pathways. © FEMS 2017.

In vitro metabolic engineering of bioelectricity generation by the complete oxidation of glucose.

PubMed

Zhu, Zhiguang; Zhang, Y-H Percival

2017-01-01

The direct generation of electricity from the most abundant renewable sugar, glucose, is an appealing alternative to the production of liquid biofuels and biohydrogen. However, enzyme-catalyzed bioelectricity generation from glucose suffers from low yields due to the incomplete oxidation of the six-carbon compound glucose via one or few enzymes. Here, we demonstrate a synthetic ATP- and CoA-free 12-enzyme pathway to implement the complete oxidation of glucose in vitro. This pathway is comprised of glucose phosphorylation via polyphosphate glucokinase, NADH generation catalyzed by glucose 6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH), electron transfer from NADH to the anode, and glucose 6-phosphate regeneration via the non-oxidative pentose phosphate pathway and gluconeogenesis. The faraday efficiency from glucose to electrons via this pathway was as high as 98.8%, suggesting the generation of nearly 24 electrons per molecule of glucose. The generated current density was greatly increased from 2.8 to 6.9mAcm -2 by replacing a low-activity G6PDH with a high-activity G6PDH and introducing a new enzyme, 6-phosphogluconolactonase, between G6PDH and 6PGDH. These results suggest the great potential of high-yield bioelectricity generation through in vitro metabolic engineering. Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Allosteric Inhibitors at the Heterodimer Interface of Imidazole Glycerol Phosphate Synthase

NASA Astrophysics Data System (ADS)

Snoeberger, Ning-Shiuan Nicole

Imidazole glycerol phosphate synthase (IGPS) from Thermotoga maritima is a heterodimeric enzyme composed of the HisH and HisF proteins. It is attractive as a pathological target since it is absent in mammals but found in plant and opportunistic human pathogens. IGPS was experimentally determined to be a V-type allosteric enzyme that is involved in an essential biosynthetic pathway of microorganisms. The enzyme catalyzes the hydrolysis of glutamine to form NH3 in the HisH protein, followed by cyclization of NH3 with N'-[(5'-phosphoribulosyl)imino]-5-aminoimidazole-4-carboxamide-ribonucleotide (PRFAR) in the HisF subunit, forming imidazole glycerol phosphate (IGP) and 5-aminoimidazole-4-carboxamide ribotide (AICAR) that enter the histidine and purine biosynthetic pathways. Allosteric motions induced upon the binding of the effector PRFAR to HisF propagate through the non-covalent HisH/HisF interface and synchronize catalytic activity at the two distant active sites. However, the nature of the allosteric pathway and the feasibility of manipulating signal transduction by using allosteric drug-like molecules remain to be established. Molecular docking studies of commercial drugs at the HisH/HisF interface were used to identify stable candidates with a potential allosteric effect on the reaction mechanism. Molecular dynamic simulations and calculations of NMR chemical shifts were combined to elucidate the allosteric pathway of IGPS.

A Set of Activators and Repressors Control Peripheral Glucose Pathways in Pseudomonas putida To Yield a Common Central Intermediate▿

PubMed Central

del Castillo, Teresa; Duque, Estrella; Ramos, Juan L.

2008-01-01

Pseudomonas putida KT2440 channels glucose to the central Entner-Doudoroff intermediate 6-phosphogluconate through three convergent pathways. The genes for these convergent pathways are clustered in three independent regions on the host chromosome. A number of monocistronic units and operons coexist within each of these clusters, favoring coexpression of catabolic enzymes and transport systems. Expression of the three pathways is mediated by three transcriptional repressors, HexR, GnuR, and PtxS, and by a positive transcriptional regulator, GltR-2. In this study, we generated mutants in each of the regulators and carried out transcriptional assays using microarrays and transcriptional fusions. These studies revealed that HexR controls the genes that encode glucokinase/glucose 6-phosphate dehydrogenase that yield 6-phosphogluconate; the genes for the Entner-Doudoroff enzymes that yield glyceraldehyde-3-phosphate and pyruvate; and gap-1, which encodes glyceraldehyde-3-phosphate dehydrogenase. GltR-2 is the transcriptional regulator that controls specific porins for the entry of glucose into the periplasmic space, as well as the gtsABCD operon for glucose transport through the inner membrane. GnuR is the repressor of gluconate transport and gluconokinase responsible for the conversion of gluconate into 6-phosphogluconate. PtxS, however, controls the enzymes for oxidation of gluconate to 2-ketogluconate, its transport and metabolism, and a set of genes unrelated to glucose metabolism. PMID:18245293

Solanesol Biosynthesis in Plants.

PubMed

Yan, Ning; Liu, Yanhua; Zhang, Hongbo; Du, Yongmei; Liu, Xinmin; Zhang, Zhongfeng

2017-03-23

Solanesol is a non-cyclic terpene alcohol composed of nine isoprene units that mainly accumulates in solanaceous plants. Solanesol plays an important role in the interactions between plants and environmental factors such as pathogen infections and moderate-to-high temperatures. Additionally, it is a key intermediate for the pharmaceutical synthesis of ubiquinone-based drugs such as coenzyme Q10 and vitamin K2, and anti-cancer agent synergizers such as N-solanesyl-N,N'-bis(3,4-dimethoxybenzyl) ethylenediamine (SDB). In plants, solanesol is formed by the 2- C -methyl-d-erythritol 4-phosphate (MEP) pathway within plastids. Solanesol's biosynthetic pathway involves the generation of C5 precursors, followed by the generation of direct precursors, and then the biosynthesis and modification of terpenoids; the first two stages of this pathway are well understood. Based on the current understanding of solanesol biosynthesis, we here review the key enzymes involved, including 1-deoxy-d-xylulose 5-phosphate synthase (DXS), 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR), isopentenyl diphosphate isomerase (IPI), geranyl geranyl diphosphate synthase (GGPPS), and solanesyl diphosphate synthase (SPS), as well as their biological functions. Notably, studies on microbial heterologous expression and overexpression of key enzymatic genes in tobacco solanesol biosynthesis are of significant importance for medical uses of tobacco.

Structure of the Bacillus anthracis dTDP- L -rhamnose-biosynthetic enzyme glucose-1-phosphate thymidylyltransferase (RfbA)

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

Baumgartner, Jackson; Lee, Jesi; Halavaty, Andrei S.

L-Rhamnose is a ubiquitous bacterial cell-wall component. The biosynthetic pathway for its precursor dTDP-L-rhamnose is not present in humans, which makes the enzymes of the pathway potential drug targets. In this study, the three-dimensional structure of the first protein of this pathway, glucose-1-phosphate thymidylyltransferase (RfbA), fromBacillus anthraciswas determined. In other organisms this enzyme is referred to as RmlA. RfbA was co-crystallized with the products of the enzymatic reaction, dTDP-α-D-glucose and pyrophosphate, and its structure was determined at 2.3 Å resolution. This is the first reported thymidylyltransferase structure from a Gram-positive bacterium. RfbA shares overall structural characteristics with known RmlA homologs.more » However, RfbA exhibits a shorter sequence at its C-terminus, which results in the absence of three α-helices involved in allosteric site formation. Consequently, RfbA was observed to exhibit a quaternary structure that is unique among currently reported glucose-1-phosphate thymidylyltransferase bacterial homologs. These structural analyses suggest that RfbA may not be allosterically regulated in some organisms and is structurally distinct from other RmlA homologs.« less

Data mining and pathway analysis of glucose-6-phosphate dehydrogenase with natural language processing.

PubMed

Chen, Long; Zhang, Chunhua; Wang, Yanling; Li, Yuqian; Han, Qiaoqiao; Yang, Huixin; Zhu, Yuechun

2017-08-01

Human glucose-6-phosphate dehydrogenase (G6PD) is a crucial enzyme in the pentose phosphate pathway, and serves an important role in biosynthesis and the redox balance. G6PD deficiency is a major cause of neonatal jaundice and acute hemolyticanemia, and recently, G6PD has been associated with diseases including inflammation and cancer. The aim of the present study was to conduct a search of the National Center for Biotechnology Information PubMed library for articles discussing G6PD. Genes that were identified to be associated with G6PD were recorded, and the frequency at which each gene appeared was calculated. Gene ontology (GO), pathway and network analyses were then performed. A total of 98 G6PD‑associated genes and 33 microRNAs (miRNAs) that potentially regulate G6PD were identified. The 98 G6PD‑associated genes were then sub‑classified into three functional groups by GO analysis, followed by analysis of function, pathway, network, and disease association. Out of the 47 signaling pathways identified, seven were significantly correlated with G6PD‑associated genes. At least two out of four independent programs identified the 33 miRNAs that were predicted to target G6PD. miR‑1207‑5P, miR‑1 and miR‑125a‑5p were predicted by all four software programs to target G6PD. The results of the present study revealed that dysregulation of G6PD was associated with cancer, autoimmune diseases, and oxidative stress‑induced disorders. These results revealed the potential roles of G6PD‑regulated signaling and metabolic pathways in the etiology of these diseases.

The trehalose pathway in maize: conservation and gene regulation in response to the diurnal cycle and extended darkness.

PubMed

Henry, Clémence; Bledsoe, Samuel W; Siekman, Allison; Kollman, Alec; Waters, Brian M; Feil, Regina; Stitt, Mark; Lagrimini, L Mark

2014-11-01

Energy resources in plants are managed in continuously changing environments, such as changes occurring during the day/night cycle. Shading is an environmental disruption that decreases photosynthesis, compromises energy status, and impacts on crop productivity. The trehalose pathway plays a central but not well-defined role in maintaining energy balance. Here, we characterized the maize trehalose pathway genes and deciphered the impacts of the diurnal cycle and disruption of the day/night cycle on trehalose pathway gene expression and sugar metabolism. The maize genome encodes 14 trehalose-6-phosphate synthase (TPS) genes, 11 trehalose-6-phosphate phosphatase (TPP) genes, and one trehalase gene. Transcript abundance of most of these genes was impacted by the day/night cycle and extended dark stress, as were sucrose, hexose sugars, starch, and trehalose-6-phosphate (T6P) levels. After extended darkness, T6P levels inversely followed class II TPS and sucrose non-fermenting-related protein kinase 1 (SnRK1) target gene expression. Most significantly, T6P no longer tracked sucrose levels after extended darkness. These results showed: (i) conservation of the trehalose pathway in maize; (ii) that sucrose, hexose, starch, T6P, and TPS/TPP transcripts respond to the diurnal cycle; and(iii) that extended darkness disrupts the correlation between T6P and sucrose/hexose pools and affects SnRK1 target gene expression. A model for the role of the trehalose pathway in sensing of sucrose and energy status in maize seedlings is proposed. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Data mining and pathway analysis of glucose-6-phosphate dehydrogenase with natural language processing

PubMed Central

Chen, Long; Zhang, Chunhua; Wang, Yanling; Li, Yuqian; Han, Qiaoqiao; Yang, Huixin; Zhu, Yuechun

2017-01-01

Human glucose-6-phosphate dehydrogenase (G6PD) is a crucial enzyme in the pentose phosphate pathway, and serves an important role in biosynthesis and the redox balance. G6PD deficiency is a major cause of neonatal jaundice and acute hemolyticanemia, and recently, G6PD has been associated with diseases including inflammation and cancer. The aim of the present study was to conduct a search of the National Center for Biotechnology Information PubMed library for articles discussing G6PD. Genes that were identified to be associated with G6PD were recorded, and the frequency at which each gene appeared was calculated. Gene ontology (GO), pathway and network analyses were then performed. A total of 98 G6PD-associated genes and 33 microRNAs (miRNAs) that potentially regulate G6PD were identified. The 98 G6PD-associated genes were then sub-classified into three functional groups by GO analysis, followed by analysis of function, pathway, network, and disease association. Out of the 47 signaling pathways identified, seven were significantly correlated with G6PD-associated genes. At least two out of four independent programs identified the 33 miRNAs that were predicted to target G6PD. miR-1207-5P, miR-1 and miR-125a-5p were predicted by all four software programs to target G6PD. The results of the present study revealed that dysregulation of G6PD was associated with cancer, autoimmune diseases, and oxidative stress-induced disorders. These results revealed the potential roles of G6PD-regulated signaling and metabolic pathways in the etiology of these diseases. PMID:28627690

Enhancement of anthraquinone production in Morinda citrifolia cell suspension cultures after stimulation of the proline cycle with two proline analogs.

PubMed

Quevedo, Carla V; Perassolo, María; Giulietti, Ana M; Rodríguez Talou, Julián

2012-03-01

Synthesis of anthraquinones (AQs) involves the shikimate and 2-C-methyl-D-erythritol 4-phosphate pathways. The proline cycle is linked to the pentose phosphate pathway (PPP) to generate NADPH needed in the first steps of this pathway. The effect of two proline analogs, azetidine-2-carboxylic acid (A2C) and thiazolidine-4-carboxylic acid (T4C), were evaluated in Morinda citrifolia suspension cultures. Both analogs gave higher proline accumulation after 6 and 10 days (68 and 179% after 6 days with A2C at 25 and 50 μM, respectively, and 111% with T4C added at 100 μM). Induction of the proline cycle increased the AQ content after 6 days (~40% for 50 μM A2C and 100 μM T4C). Whereas A2C (50 μM) increased only AQ production, T4C also enhanced total phenolics. However, no induction of the PPP was observed with any of the treatments. This pathway therefore does not limit the supply of carbon skeletons to secondary metabolic pathways.

Handling Uncertainty in Dynamic Models: The Pentose Phosphate Pathway in Trypanosoma brucei

PubMed Central

Alibu, Vincent P.; Burchmore, Richard J.; Gilbert, Ian H.; Trybiło, Maciej; Driessen, Nicole N.; Gilbert, David; Breitling, Rainer; Bakker, Barbara M.; Barrett, Michael P.

2013-01-01

Dynamic models of metabolism can be useful in identifying potential drug targets, especially in unicellular organisms. A model of glycolysis in the causative agent of human African trypanosomiasis, Trypanosoma brucei, has already shown the utility of this approach. Here we add the pentose phosphate pathway (PPP) of T. brucei to the glycolytic model. The PPP is localized to both the cytosol and the glycosome and adding it to the glycolytic model without further adjustments leads to a draining of the essential bound-phosphate moiety within the glycosome. This phosphate “leak” must be resolved for the model to be a reasonable representation of parasite physiology. Two main types of theoretical solution to the problem could be identified: (i) including additional enzymatic reactions in the glycosome, or (ii) adding a mechanism to transfer bound phosphates between cytosol and glycosome. One example of the first type of solution would be the presence of a glycosomal ribokinase to regenerate ATP from ribose 5-phosphate and ADP. Experimental characterization of ribokinase in T. brucei showed that very low enzyme levels are sufficient for parasite survival, indicating that other mechanisms are required in controlling the phosphate leak. Examples of the second type would involve the presence of an ATP:ADP exchanger or recently described permeability pores in the glycosomal membrane, although the current absence of identified genes encoding such molecules impedes experimental testing by genetic manipulation. Confronted with this uncertainty, we present a modeling strategy that identifies robust predictions in the context of incomplete system characterization. We illustrate this strategy by exploring the mechanism underlying the essential function of one of the PPP enzymes, and validate it by confirming the model predictions experimentally. PMID:24339766

Can short-term oral fine motor training affect precision of task performance and induce cortical plasticity of the jaw muscles?

PubMed

Zhang, Hong; Kumar, Abhishek; Kothari, Mohit; Luo, Xiaoping; Trulsson, Mats; Svensson, Krister G; Svensson, Peter

2016-07-01

The aim was to test the hypothesis that short-term oral sensorimotor training of the jaw muscles would increase the precision of task performance and induce neuroplastic changes in the corticomotor pathways, related to the masseter muscle. Fifteen healthy volunteers performed six series with ten trials of an oral sensorimotor task. The task was to manipulate and position a spherical chocolate candy in between the anterior teeth and split it into two equal halves. The precision of the task performance was evaluated by comparing the ratio between the two split halves. A series of "hold-and-split" tasks was also performed before and after the training. The hold force and split force along with the electromyographic (EMG) activity of jaw muscles were recorded. Motor-evoked potentials and cortical motor maps of the right masseter muscle were evoked by transcranial magnetic stimulation. There was a significant effect of series on the precision of the task performance during the short-term oral sensorimotor training (P < 0.002). The hold force during the "hold-and-split" task was significantly lower after training than before the short-term training (P = 0.011). However, there was no change in the split force and the EMG activity of the jaw muscles before and after the training. Further, there was a significant increase in the amplitude of the motor-evoked potentials (P < 0.016) and in the motor cortex map areas (P = 0.033), after the short-term oral sensorimotor training. Therefore, short-term oral sensorimotor task training increased the precision of task performance and induced signs of neuroplastic changes in the corticomotor pathways, related to the masseter muscle.

Enhancing solubility of deoxyxylulose phosphate pathway enzymes for microbial isoprenoid production

PubMed Central

2012-01-01

Background Recombinant proteins are routinely overexpressed in metabolic engineering. It is well known that some over-expressed heterologous recombinant enzymes are insoluble with little or no enzymatic activity. This study examined the solubility of over-expressed homologous enzymes of the deoxyxylulose phosphate pathway (DXP) and the impact of inclusion body formation on metabolic engineering of microbes. Results Four enzymes of this pathway (DXS, ISPG, ISPH and ISPA), but not all, were highly insoluble, regardless of the expression systems used. Insoluble dxs (the committed enzyme of DXP pathway) was found to be inactive. Expressions of fusion tags did not significantly improve the solubility of dxs. However, hypertonic media containing sorbitol, an osmolyte, successfully doubled the solubility of dxs, with the concomitant improvement in microbial production of the metabolite, DXP. Similarly, sorbitol significantly improved the production of soluble and functional ERG12, the committed enzyme in the mevalonate pathway. Conclusion This study demonstrated the unanticipated findings that some over-expressed homologous enzymes of the DXP pathway were highly insoluble, forming inclusion bodies, which affected metabolite formation. Sorbitol was found to increase both the solubility and function of some of these over-expressed enzymes, a strategy to increase the production of secondary metabolites. PMID:23148661

Isoprenoid biosynthesis in higher plants and in Escherichia coli: on the branching in the methylerythritol phosphate pathway and the independent biosynthesis of isopentenyl diphosphate and dimethylallyl diphosphate.

PubMed Central

Hoeffler, Jean-François; Hemmerlin, Andréa; Grosdemange-Billiard, Catherine; Bach, Thomas J; Rohmer, Michel

2002-01-01

In the bacterium Escherichia coli, the mevalonic-acid (MVA)-independent 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway is characterized by two branches leading separately to isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). The signature of this branching is the retention of deuterium in DMAPP and the deuterium loss in IPP after incorporation of 1-[4-(2)H]deoxy-d-xylulose ([4-(2)H]DX). Feeding tobacco BY-2 cell-suspension cultures with [4-(2)H]DX resulted in deuterium retention in the isoprene units derived from DMAPP, as well as from IPP in the plastidial isoprenoids, phytoene and plastoquinone, synthesized via the MEP pathway. This labelling pattern represents direct evidence for the presence of the DMAPP branch of the MEP pathway in a higher plant, and shows that IPP can be synthesized from DMAPP in plant plastids, most probably via a plastidial IPP isomerase. PMID:12010124

Phosphatidylinositol 3-phosphates-at the interface between cell signalling and membrane traffic.

PubMed

Marat, Andrea L; Haucke, Volker

2016-03-15

Phosphoinositides (PIs) form a minor class of phospholipids with crucial functions in cell physiology, ranging from cell signalling and motility to a role as signposts of compartmental membrane identity. Phosphatidylinositol 3-phosphates are present at the plasma membrane and within the endolysosomal system, where they serve as key regulators of both cell signalling and of intracellular membrane traffic. Here, we provide an overview of the metabolic pathways that regulate cellular synthesis of PI 3-phosphates at distinct intracellular sites and discuss the mechanisms by which these lipids regulate cell signalling and membrane traffic. Finally, we provide a framework for how PI 3-phosphate metabolism is integrated into the cellular network. © 2016 The Authors.

Modular pathway engineering of Corynebacterium glutamicum to improve xylose utilization and succinate production.

PubMed

Jo, Suah; Yoon, Jinkyung; Lee, Sun-Mi; Um, Youngsoon; Han, Sung Ok; Woo, Han Min

2017-09-20

Xylose-negative Corynebacterium glutamicum has been engineered to utilize xylose as the sole carbon source via either the xylose isomerase (XI) pathway or the Weimberg pathway. Heterologous expression of xylose isomerase and overexpression of a gene encoding for xylulose kinase enabled efficient xylose utilization. In this study, we show that two functionally-redundant transcriptional regulators (GntR1 and GntR2) present on xylose repress the pentose phosphate pathway genes. For efficient xylose utilization, pentose phosphate pathway genes and a phosphoketolase gene were overexpressed with the XI pathway in C. glutamicum. Overexpression of the genes encoding for transaldolase (Tal), 6-phosphogluconate dehydrogenase (Gnd), or phosphoketolase (XpkA) enhanced the growth and xylose consumption rates compared to the wild-type with the XI pathway alone. However, co-expression of these genes did not have a synergetic effect on xylose utilization. For the succinate production from xylose, overexpression of the tal gene with the XI pathway in a succinate-producing strain improved xylose utilization and increased the specific succinate production rate by 2.5-fold compared to wild-type with the XI pathway alone. Thus, overexpression of the tal, gnd, or xpkA gene could be helpful for engineering C. glutamicum toward production of value-added chemicals with efficient xylose utilization. Copyright © 2017 Elsevier B.V. All rights reserved.

Effects of long-term exposure to Cu2+ and Cd2+ on the pentose phosphate pathway dehydrogenase activities in the ovary of adult Bufo arenarum: possible role as biomarker for Cu2+ toxicity.

PubMed

Carattino, Marcelo D; Peralta, Susana; Pérez-Coll, Cristina; Naab, Fabián; Burlón, Alejandro; Kreiner, Andrés J; Preller, Ana F; de Schroeder, Teresa M Fonovich

2004-03-01

The effects of copper and cadmium on metabolism through the pentose phosphate pathway were evaluated in Bufo arenarum toad ovary. The effects of the two metals on dehydrogenases from this pathway were evaluated by three experiments: (1) in samples obtained from control females with addition of the metals to the reaction mixture (in vitro), (2) in samples obtained from control females and after long-term exposure of females to 4 and 100 microg/L of Cu or Cd in the incubation media (in vitro after exposure to the metals in vivo), and (3) 14CO2 production through the pentose phosphate pathway was evaluated after [U-14C]glucose microinjection on ovulated oocytes (in vivo after microinjection of the metals). Results from (1) evidenced inhibition of both enzyme activities but only above 1.5 mM Cu and Cd added to the reaction mixture. In (2) both glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities decreased in samples from the ovaries of females exposed in vivo to Cu, in a concentration-dependent manner (up to 90% in females exposed to 100 microg/L Cu: 2.12 +/- 1.57 NADPH micromol/min microg protein x 10(-5) vs 19.97 +/- 8.54 in control females). Cd treatment of the toads only rendered an inhibitory effect on 6-phosphogluconate dehydrogenase activity after exposure to 4 microg/L of the bivalent cation. (3) In vivo 14CO2 evolution significantly decreased in oocytes coinjected with 6.3 x 10(-3) mM Cu (calculated intracellular final concentration of the metal injected) and radioactive glucose. Cu and Cd concentration in samples from exposed females were always under detection limit by particle-induced X-ray emission. The results presented here are in agreement with a role for both glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities determination as biomarkers of effect and exposure for Cu but not for Cd toxicity.

A unique combination of genetic systems for the synthesis of trehalose in Rubrobacter xylanophilus: properties of a rare actinobacterial TreT.

PubMed

Nobre, Ana; Alarico, Susana; Fernandes, Chantal; Empadinhas, Nuno; da Costa, Milton S

2008-12-01

Trehalose is the primary organic solute in Rubrobacter xylanophilus under all conditions tested, including those for optimal growth. We detected genes of four different pathways for trehalose synthesis in the genome of this organism, namely, the trehalose-6-phosphate synthase (Tps)/trehalose-6-phosphate phosphatase (Tpp), TreS, TreY/TreZ, and TreT pathways. Moreover, R. xylanophilus is the only known member of the phylum Actinobacteria to harbor TreT. The Tps sequence is typically bacterial, but the Tpp sequence is closely related to eukaryotic counterparts. Both the Tps/Tpp and the TreT pathways were active in vivo, while the TreS and the TreY/TreZ pathways were not active under the growth conditions tested and appear not to contribute to the levels of trehalose observed. The genes from the active pathways were functionally expressed in Escherichia coli, and Tps was found to be highly specific for GDP-glucose, a rare feature among these enzymes. The trehalose-6-phosphate formed was specifically dephosphorylated to trehalose by Tpp. The recombinant TreT synthesized trehalose from different nucleoside diphosphate-glucose donors and glucose, but the activity in R. xylanophilus cell extracts was specific for ADP-glucose. The TreT could also catalyze trehalose hydrolysis in the presence of ADP, but with a very high K(m). Here, we functionally characterize two systems for the synthesis of trehalose in R. xylanophilus, a representative of an ancient lineage of the actinobacteria, and discuss a possible scenario for the exceptional occurrence of treT in this extremophilic bacterium.

Identification of acyl-CoA synthetases involved in the mammalian sphingosine 1-phosphate metabolic pathway.

PubMed

Ohkuni, Aya; Ohno, Yusuke; Kihara, Akio

2013-12-13

Sphingosine 1-phosphate (S1P) plays important roles both as a bioactive lipid molecule and an intermediate of the sphingolipid-to-glycerophospholipid metabolic pathway. To identify human acyl-CoA synthetases (ACSs) involved in S1P metabolism, we cloned all 26 human ACS genes and examined their abilities to restore deficient sphingolipid-to-glycerophospholipid metabolism in a yeast mutant lacking two ACS genes, FAA1 and FAA4. Here, in addition to the previously identified ACSL family members (ACSL1, 3, 4, 5, and 6), we found that ACSVL1, ACSVL4, and ACSBG1 also restored metabolism. All 8 ACSs were localized either exclusively or partly to the endoplasmic reticulum (ER), where S1P metabolism takes place. We previously proposed the entire S1P metabolic pathway from results obtained using yeast cells, i.e., S1P is metabolized to glycerophospholipids via trans-2-hexadecenal, trans-2-hexadecenoic acid, trans-2-hexadecenoyl-CoA, and palmitoyl-CoA. However, as S1P is not a naturally occurring long-chain base 1-phosphate in yeast, the validity of this pathway required further verification using mammalian cells. In the present study, we treated HeLa cells with the ACS inhibitor triacsin C and found that inhibition of ACSs resulted in accumulation of trans-2-hexadecenoic acid as in ACS mutant yeast. From these results, we conclude that S1P is metabolized by a common pathway in eukaryotes. Copyright © 2013 Elsevier Inc. All rights reserved.

Phosphate Solubilization and Gene Expression of Phosphate-Solubilizing Bacterium Burkholderia multivorans WS-FJ9 under Different Levels of Soluble Phosphate.

PubMed

Zeng, Qingwei; Wu, Xiaoqin; Wang, Jiangchuan; Ding, Xiaolei

2017-04-28

Phosphate-solubilizing bacteria (PSB) have the ability to dissolve insoluble phosphate and enhance soil fertility. However, the growth and mineral phosphate solubilization of PSB could be affected by exogenous soluble phosphate and the mechanism has not been fully understood. In the present study, the growth and mineral phosphate-solubilizing characteristics of PSB strain Burkholderia multivorans WS-FJ9 were investigated at six levels of exogenous soluble phosphate (0, 0.5, 1, 5, 10, and 20 mM). The WS-FJ9 strain showed better growth at high levels of soluble phosphate. The phosphate-solubilizing activity of WS-FJ9 was reduced as the soluble phosphate concentration increased, as well as the production of pyruvic acid. Transcriptome profiling of WS-FJ9 at three levels of exogenous soluble phosphate (0, 5, and 20 mM) identified 446 differentially expressed genes, among which 44 genes were continuously up-regulated when soluble phosphate concentration was increased and 81 genes were continuously down-regulated. Some genes related to cell growth were continuously up-regulated, which would account for the better growth of WS-FJ9 at high levels of soluble phosphate. Genes involved in glucose metabolism, including glycerate kinase, 2-oxoglutarate dehydrogenase, and sugar ABC-type transporter, were continuously down-regulated, which indicates that metabolic channeling of glucose towards the phosphorylative pathway was negatively regulated by soluble phosphate. These findings represent an important first step in understanding the molecular mechanisms of soluble phosphate effects on the growth and mineral phosphate solubilization of PSB.

[Modification of retinal photoreceptor membranes and Ca ion binding].

PubMed

Korchagin, V P; Berman, A L; Shukoliukov, S A; Rychkova, M P; Etingof, R N

1978-10-01

Calcium binding by modified photoreceptor membranes of cattle retina has been studied. Ca2+-binding the membranes significantly changes after C-phospholipase treatment, displaying the initial growth (less than 65% of lipid phosphorus removed) with subsequent decrease (more than 65% of phosphorus removed). Liposomes of the photoreceptor membranes lipids were found to bind more calcium than do the native photoreceptor membranes. Proteolytic enzymes (papaine, pronase) splitting some rhodopsin fragments do not affect the ability of the membrane to bind Ca2+. The increase of light-induced Ca-binding is observed only after the outer segments preincubation under conditions providing for rhodopsin phosphorylation. This effect was observed also after the splitting of the rhodopsin fragment by papaine. It is concluded that calcium binding in the photoreceptor membranes is mainly due to the phosphate groups of phospholipids.

Assessing the utility of bipolar membranes for use in photoelectrochemical water-splitting cells.

PubMed

Vargas-Barbosa, Nella M; Geise, Geoffrey M; Hickner, Michael A; Mallouk, Thomas E

2014-11-01

Membranes are important in water-splitting solar cells because they prevent crossover of hydrogen and oxygen. Here, bipolar membranes (BPMs) were tested as separators in water electrolysis cells. Steady-state membrane and solution resistances, electrode overpotentials, and pH gradients were measured at current densities relevant to solar photoelectrolysis. Under forward bias conditions, electrodialysis of phosphate buffer ions creates a pH gradient across a BPM. Under reverse bias, the BPM can maintain a constant buffer pH on both sides of the cell, but a large membrane potential develops. Thus, the BPM does not present a viable solution for electrolysis in buffered electrolytes. However, the membrane potential is minimized when the anode and cathode compartments of the cell contain strongly basic and acidic electrolytes, respectively. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Distinct domains within the NITROGEN LIMITATION ADAPTATION protein mediate its subcellular localization and function in the nitrate-dependent phosphate homeostasis pathway

USDA-ARS?s Scientific Manuscript database

The NITROGEN LIMITATION ADAPTATION (NLA) protein is a RING-type E3 ubiquitin ligase that plays an essential role in the regulation of nitrogen and phosphate homeostasis. NLA is localized to two distinct subcellular sites, the plasma membrane and nucleus, and contains four distinct domains: i) a RING...

Flux of Nutrients Between the Middle and Southern Adriatic Sea (Gargano-Split section)

DTIC Science & Technology

2013-04-28

silico molybdate in acid solution to mo- lybdenum blue by ascorbic acid . Oxalic acid is introduced to the sam- ple to minimize interferences from...sensitivity of this method. Nitrite is measured by reacting the sample under acidic conditions with sulfanilamide to form a diazo compound that then...colorimetric method in which a blue compound is formed by the reaction of phosphate, molybdate and antimony followed by reduction with ascorbic acid . The reduced

The missing step of the L-galactose pathway of ascorbate biosynthesis in plants, an L-galactose guanyltransferase, increases leaf ascorbate content.

PubMed

Laing, William A; Wright, Michele A; Cooney, Janine; Bulley, Sean M

2007-05-29

The gene for one postulated enzyme that converts GDP-L-galactose to L-galactose-1-phosphate is unknown in the L-galactose pathway of ascorbic acid biosynthesis and a possible candidate identified through map-based cloning is the uncharacterized gene At4g26850. We identified a putative function for At4g26850 using PSI-Blast and motif searching to show it was a member of the histidine triad superfamily, which includes D-galactose uridyltransferase. We cloned and expressed this Arabidopsis gene and the homologous gene from Actinidia chinensis in Escherichia coli and assayed the expressed protein for activities related to converting GDP-L-galactose to L-galactose-1-P. The expressed protein is best described as a GDP-L-galactose-hexose-1-phosphate guanyltransferase (EC 2.7.7.), catalyzing the transfer of GMP from GDP-l-galactose to a hexose-1-P, most likely D-mannose-1-phosphate in vivo. Transient expression of this A. chinensis gene in tobacco leaves resulted in a >3-fold increase in leaf ascorbate as well as a 50-fold increase in GDP-L-galactose-D-mannose-1-phosphate guanyltransferase activity.

The missing step of the l-galactose pathway of ascorbate biosynthesis in plants, an l-galactose guanyltransferase, increases leaf ascorbate content

PubMed Central

Laing, William A.; Wright, Michele A.; Cooney, Janine; Bulley, Sean M.

2007-01-01

The gene for one postulated enzyme that converts GDP-l-galactose to l-galactose-1-phosphate is unknown in the l-galactose pathway of ascorbic acid biosynthesis and a possible candidate identified through map-based cloning is the uncharacterized gene At4g26850. We identified a putative function for At4g26850 using PSI-Blast and motif searching to show it was a member of the histidine triad superfamily, which includes d-galactose uridyltransferase. We cloned and expressed this Arabidopsis gene and the homologous gene from Actinidia chinensis in Escherichia coli and assayed the expressed protein for activities related to converting GDP-l-galactose to l-galactose-1-P. The expressed protein is best described as a GDP-l-galactose-hexose-1-phosphate guanyltransferase (EC 2.7.7.), catalyzing the transfer of GMP from GDP-l-galactose to a hexose-1-P, most likely d-mannose-1-phosphate in vivo. Transient expression of this A. chinensis gene in tobacco leaves resulted in a >3-fold increase in leaf ascorbate as well as a 50-fold increase in GDP-l-galactose-d-mannose-1-phosphate guanyltransferase activity. PMID:17485667

Exogenous fatty acids affect CDP-choline pathway to increase phosphatidylcholine synthesis in granular pneumocytes

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

Chander, A.; Gullo, J.; Reicherter, J.

1987-05-01

Regulation of phosphatidylcholine (PC) synthesis in rat granular pneumocytes isolated by tryptic digestion of lungs and maintained in primary culture for 24 h was investigated by following effects of exogenous fatty acids on (/sup 3/H-methyl)choline incorporation into PC and disaturated PC (DSPC). At 0.1 mM choline, the rate of choline incorporation into PC and DSPC was 440 +/- and 380 +/- 50 pmol/h/ug Pi (mean +/- SE, n=3-5), respectively, and was linear for up to 3 h. PC synthesis was significantly increased by 0.1 mM each of palmitic, oleic, linoleic, or linolenic acid. However, synthesis of DSPC was increased onlymore » by palmitic acid and this increase was prevented by addition of oleic acid suggesting lack of effect on the remodeling pathway. Pulse-chase experiments with choline in absence or presence of palmitic or oleic acid showed that the label declined in choline phosphate and increased in PC more rapidly in presence of either of the fatty acids, suggesting rapid conversion of choline phosphate to PC. Microsomal choline phosphate cytidyltransferase activity in cells preincubated without or with palmitic acid for 3 h was 0.81 +/- 0.07 and 1.81 +/- 0.09 nmol choline phosphate converted/min/mg protein (n=4). These results suggest that in granular pneumocytes, exogenous fatty acids modulate PC synthesis by increasing choline phosphate cytidyltransferase activity.« less

Skeletal secretion of FGF-23 regulates phosphate and vitamin D metabolism

PubMed Central

Quarles, L. Darryl

2014-01-01

The discovery of fibroblast growth factor 23 (FGF-23) has expanded our understanding of phosphate and vitamin D homeostasis and provided new insights into the pathogenesis of hereditary hypophosphatemic and hyperphosphatemic disorders, as well as acquired disorders of phosphate metabolism, such as chronic kidney disease. FGF-23 is secreted by osteoblasts and osteocytes in bone and principally targets the kidney to regulate the reabsorption of phosphate, the production and catabolism of 1,25-dihydroxyvitamin D and the expression of α-Klotho, an anti-ageing hormone. Secreted FGF-23 plays a central role in complex endocrine networks involving local bone-derived factors that regulate mineralization of extracellular matrix and systemic hormones involved in mineral metabolism. Inactivating mutations of PHEX, DMP1 and ENPP1, which cause hereditary hypophosphatemic disorders and primary defects in bone mineralization, stimulate FGF23 gene transcription in osteoblasts and osteocytes, at least in part, through canonical and intracrine FGF receptor pathways. These FGF-23 regulatory pathways may enable systemic phosphate and vitamin D homeostasis to be coordinated with bone mineralization. FGF-23 also functions as a counter-regulatory hormone for 1,25-dihydroxyvitamin D in a bone–kidney endocrine loop. FGF-23, through regulation of additional genes in the kidney and extrarenal tissues, probably has broader physiological functions beyond regulation of mineral metabolism that account for the association between FGF-23 and increased mortality and morbidity in chronic kidney disease. PMID:22249518

Preferable adsorption of phosphate using lanthanum-incorporated porous zeolite: Characteristics and mechanism

NASA Astrophysics Data System (ADS)

He, Yinhai; Lin, Hai; Dong, Yingbo; Wang, Liang

2017-12-01

The adsorbent, where lanthanum oxide was incorporated onto porous zeolite (La-Z), of preferable adsorption towards phosphate was prepared by hydrothermal synthesis. Based on pH effect results, La-Z would effectively sequestrate phosphate over wider pH range of 3.0-7.0, alkaline conditions were unfavorable for phosphate. The adsorption of phosphate was not significantly influenced by ionic strength and by coexisting anions of chloride, nitrate and sulfate but bicarbonate showed slightly greater negative effects, indicating La-Z possessed highly selectivity to phosphate. Adsorption of phosphate could be well fitted by pseudo-second-order model and the process was mainly controlled by intra-particle diffusion. Equilibrium adsorption demonstrated that Langmuir model was more suitable than Freundlich model for description phosphate adsorption and the adsorption capacity was 17.2 mg P g-1, which exhibited 95% utilization of incorporated La. Over 95% phosphate was eliminated in real effluent treatment when the dose was 2 g L-1. The underlying mechanism for phosphate capture was probed with Zeta potential and X-ray photoelectron spectroscope analysis, and the formation of La-P inner-sphere complexation was testified to be the dominant pathway. All the results suggested that the porous zeolite-supported lanthanum oxide can serve as a promising adsorbent for phosphate removal in realistic application.

Computer-Aided Drug Design Applied to Marine Drug Discovery: Meridianins as Alzheimer's Disease Therapeutic Agents.

PubMed

Llorach-Pares, Laura; Nonell-Canals, Alfons; Sanchez-Martinez, Melchor; Avila, Conxita

2017-11-27

Computer-aided drug discovery/design (CADD) techniques allow the identification of natural products that are capable of modulating protein functions in pathogenesis-related pathways, constituting one of the most promising lines followed in drug discovery. In this paper, we computationally evaluated and reported the inhibitory activity found in meridianins A-G, a group of marine indole alkaloids isolated from the marine tunicate Aplidium , against various protein kinases involved in Alzheimer's disease (AD), a neurodegenerative pathology characterized by the presence of neurofibrillary tangles (NFT). Balance splitting between tau kinase and phosphate activities caused tau hyperphosphorylation and, thereby, its aggregation and NTF formation. Inhibition of specific kinases involved in its phosphorylation pathway could be one of the key strategies to reverse tau hyperphosphorylation and would represent an approach to develop drugs to palliate AD symptoms. Meridianins bind to the adenosine triphosphate (ATP) binding site of certain protein kinases, acting as ATP competitive inhibitors. These compounds show very promising scaffolds to design new drugs against AD, which could act over tau protein kinases Glycogen synthetase kinase-3 Beta (GSK3β) and Casein kinase 1 delta (CK1δ, CK1D or KC1D), and dual specificity kinases as dual specificity tyrosine phosphorylation regulated kinase 1 (DYRK1A) and cdc2-like kinases (CLK1). This work is aimed to highlight the role of CADD techniques in marine drug discovery and to provide precise information regarding the binding mode and strength of meridianins against several protein kinases that could help in the future development of anti-AD drugs.

Wnt7a interaction with Fzd5 and detection of signaling activation using a split eGFP

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

Carmon, Kendra S.; Loose, David S.

2008-04-04

Wnts are secreted glycoproteins that regulate important cellular processes including proliferation, differentiation, and cell fate. In the {beta}-catenin/canonical pathway, Wnt interacts with Fzd receptors to inhibit degradation of {beta}-catenin and promote its translocation into the nucleus where it regulates transcription of a number of genes. Dysregulation of this pathway has been attributed to a host of diseases including cancer. As a result, components of the {beta}-catenin/canonical pathway have been gaining recognition as promising targets for the discovery of novel therapeutic agents. Here, we show, using an ELISA-based protein-protein binding assay that purified Wnt7a binds to the extracellular cysteine-rich domain ofmore » Fzd5 in the nanomolar range. We have developed a novel split eGFP complementation assay to visually detect Wnt7a-Fzd5 interactions and subsequent pathway activation in cells. These biological tools could help lead to a better understanding of Wnt-Fzd interactions and the identification of new modulators of Wnt signaling.« less

Development of petri net-based dynamic model for improved production of farnesyl pyrophosphate by integrating mevalonate and methylerythritol phosphate pathways in yeast.

PubMed

Baadhe, Rama Raju; Mekala, Naveen Kumar; Palagiri, Satwik Reddy; Parcha, Sreenivasa Rao

2012-07-01

In this case study, we designed a farnesyl pyrophosphate (FPP) biosynthetic network using hybrid functional Petri net with extension (HFPNe) which is derived from traditional Petri net theory and allows easy modeling with graphical approach of various types of entities in the networks together. Our main objective is to improve the production of FPP in yeast, which is further converted to amorphadiene (AD), a precursor of artemisinin (antimalarial drug). Natively, mevalonate (MEV) pathway is present in yeast. Methyl erythritol phosphate pathways (MEP) are present only in higher plant plastids and eubacteria, but not present in yeast. IPP and DAMP are common isomeric intermediate in these two pathways, which immediately yields FPP. By integrating these two pathways in yeast, we augmented the FPP synthesis approximately two folds higher (431.16 U/pt) than in MEV pathway alone (259.91 U/pt) by using HFPNe technique. Further enhanced FPP levels converted to AD by amorphadiene synthase gene yielding 436.5 U/pt of AD which approximately two folds higher compared to the AD (258.5 U/pt) synthesized by MEV pathway exclusively. Simulation and validation processes performed using these models are reliable with identified biological information and data.

Textbook Errors & Misconceptions in Biology: Cell Metabolism.

ERIC Educational Resources Information Center

Storey, Richard D.

1991-01-01

The idea that errors and misconceptions in biology textbooks are often slow to be discovered and corrected is discussed. Selected errors, misconceptions, and topics of confusion about cell metabolism are described. Fermentation, respiration, Krebs cycle, pentose phosphate pathway, uniformity of catabolism, and metabolic pathways as models are…

The pentose phosphate pathway of glucose metabolism. Hormonal and dietary control of the oxidative and non-oxidative reactions of the cycle in liver

PubMed Central

Novello, F.; Gumaa, J. A.; McLean, Patricia

1969-01-01

1. Measurements were made of the non-oxidative reactions of the pentose phosphate cycle in liver (transketolase, transaldolase, ribulose 5-phosphate epimerase and ribose 5-phosphate isomerase activities) in a variety of hormonal and nutritional conditions. In addition, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities were measured for comparison with the oxidative reactions of the cycle; hexokinase, glucokinase and phosphoglucose isomerase activities were also included. Starvation for 2 days caused significant lowering of activity of all the enzymes of the pentose phosphate cycle based on activity in the whole liver. Re-feeding with a high-carbohydrate diet restored all the enzyme activities to the range of the control values with the exception of that of glucose 6-phosphate dehydrogenase, which showed the well-known `overshoot' effect. Re-feeding with a high-fat diet also restored the activities of all the enzymes of the pentose phosphate cycle and of hexokinase; glucokinase activity alone remained unchanged. Expressed as units/g. of liver or units/mg. of protein hexokinase, glucose 6-phosphate dehydrogenase, transketolase and pentose phosphate isomerase activities were unchanged by starvation; both 6-phosphogluconate dehydrogenase and ribulose 5-phosphate epimerase activities decreased faster than the liver weight or protein content. 2. Alloxan-diabetes resulted in a decrease of approx. 30–40% in the activities of 6-phosphogluconate dehydrogenase, ribose 5-phosphate isomerase, ribulose 5-phosphate epimerase and transketolase; in contrast with this glucose 6-phosphate dehydrogenase, transaldolase and phosphoglucose isomerase activities were unchanged. Treatment of alloxan-diabetic rats with protamine–zinc–insulin for 3 days caused a very marked increase to above normal levels of activity in all the enzymes of the pentose phosphate pathway except ribulose 5-phosphate epimerase, which was restored to the control value. Hexokinase activity was also raised by this treatment. After 7 days treatment of alloxan-diabetic rats with protamine–zinc–insulin the enzyme activities returned towards the control values. 3. In adrenalectomized rats the two most important changes were the rise in hexokinase activity and the fall in transketolase activity; in addition, ribulose 5-phosphate epimerase activity was also decreased. These effects were reversed by cortisone treatment. In addition, in cortisone-treated adrenalectomized rats glucokinase activity was significantly lower than the control value. 4. In thyroidectomized rats both ribose 5-phosphate isomerase and transketolase activities were decreased; in contrast with this transaldolase activity did not change significantly. Hypophysectomy caused a 50% fall in transketolase activity that was partially reversed by treatment with thyroxine and almost fully reversed by treatment with growth hormone for 8 days. 5. The results are discussed in relation to the hormonal control of the non-oxidative reactions of the pentose phosphate cycle, the marked changes in transketolase activity being particularly outstanding. PMID:5791534

Batch culture characterization and metabolic flux analysis of succinate-producing Escherichia coli strains.

PubMed

Sánchez, Ailen M; Bennett, George N; San, Ka-Yiu

2006-05-01

This study presents an in-depth analysis of the anaerobic metabolic fluxes of various mutant strains of Escherichia coli overexpressing the Lactococcus lactis pyruvate carboxylase (PYC) for the production of succinate. Previously, a metabolic network design that includes an active glyoxylate pathway implemented in vivo increased succinate yield from glucose in an E. coli mutant to 1.6 mol/mol under fully anaerobic conditions. The design consists of a dual succinate synthesis route, which diverts required quantities of NADH through the traditional fermentative pathway and maximizes the carbon converted to succinate by balancing the carbon flux through the fermentative pathway and the glyoxylate pathway (which has a lower NADH requirement). Mutant strains previously constructed during the development of high-yield succinate-producing strains were selected for further characterization to understand their metabolic response as a result of several genetic manipulations and to determine the significance of the fermentative and the glyoxylate pathways in the production of succinate. Measured fluxes obtained under batch cultivation conditions were used to estimate intracellular fluxes and identify critical branch point flux split ratios. The comparison of changes in branch point flux split ratios to the glyoxylate pathway and the fermentative pathway at the oxaloacetate (OAA) node as a result of different mutations revealed the sensitivity of succinate yield to these manipulations. The most favorable split ratio to obtain the highest succinate yield was the fractional partition of OAA to glyoxylate of 0.32 and 0.68 to the fermentative pathway obtained in strains SBS550MG (pHL413) and SBS990MG (pHL413). The succinate yields achieved in these two strains were 1.6 and 1.7 mol/mol, respectively. In addition, an active glyoxylate pathway in an ldhA, adhE, ack-pta mutant strain is shown to be responsible for the high succinate yields achieved anaerobically. Furthermore, in vitro activity measurements of seven crucial enzymes involved in the pathways studied and intracellular measurements of key intermediate metabolite pools provided additional insights on the physiological perturbations caused by these mutations. The characterization of these recombinant mutant strains in terms of flux distribution pattern, in vitro enzyme activity and intracellular metabolite pools provides useful information for the rational modification of metabolic fluxes to improve succinate production.

Alterations in Energy/Redox Metabolism Induced by Mitochondrial and Environmental Toxins: A Specific Role for Glucose-6-Phosphate-Dehydrogenase and the Pentose Phosphate Pathway in Paraquat Toxicity

PubMed Central

2015-01-01

Parkinson’s disease (PD) is a multifactorial disorder with a complex etiology including genetic risk factors, environmental exposures, and aging. While energy failure and oxidative stress have largely been associated with the loss of dopaminergic cells in PD and the toxicity induced by mitochondrial/environmental toxins, very little is known regarding the alterations in energy metabolism associated with mitochondrial dysfunction and their causative role in cell death progression. In this study, we investigated the alterations in the energy/redox-metabolome in dopaminergic cells exposed to environmental/mitochondrial toxins (paraquat, rotenone, 1-methyl-4-phenylpyridinium [MPP+], and 6-hydroxydopamine [6-OHDA]) in order to identify common and/or different mechanisms of toxicity. A combined metabolomics approach using nuclear magnetic resonance (NMR) and direct-infusion electrospray ionization mass spectrometry (DI-ESI-MS) was used to identify unique metabolic profile changes in response to these neurotoxins. Paraquat exposure induced the most profound alterations in the pentose phosphate pathway (PPP) metabolome. 13C-glucose flux analysis corroborated that PPP metabolites such as glucose-6-phosphate, fructose-6-phosphate, glucono-1,5-lactone, and erythrose-4-phosphate were increased by paraquat treatment, which was paralleled by inhibition of glycolysis and the TCA cycle. Proteomic analysis also found an increase in the expression of glucose-6-phosphate dehydrogenase (G6PD), which supplies reducing equivalents by regenerating nicotinamide adenine dinucleotide phosphate (NADPH) levels. Overexpression of G6PD selectively increased paraquat toxicity, while its inhibition with 6-aminonicotinamide inhibited paraquat-induced oxidative stress and cell death. These results suggest that paraquat “hijacks” the PPP to increase NADPH reducing equivalents and stimulate paraquat redox cycling, oxidative stress, and cell death. Our study clearly demonstrates that alterations in energy metabolism, which are specific for distinct mitochondiral/environmental toxins, are not bystanders to energy failure but also contribute significant to cell death progression. PMID:24937102

Regulation of Phospholipid Synthesis in Escherichia coli by Guanosine Tetraphosphate

PubMed Central

Merlie, John P.; Pizer, Lewis I.

1973-01-01

Phospholipid synthesis has been reported to be subject to stringent control in Escherichia coli. We present evidence that demonstrates a strict correlation between guanosine tetraphosphate accumulation and inhibition of phospholipid synthesis. In vivo experiments designed to examine the pattern of phospholipid labeling with 32P-inorganic phosphate and 32P-sn-glycerol-3-phosphate suggest that regulation must occur at the glycerol-3-phosphate acyltransferase step. Assay of phospholipid synthesis by cell-free extracts and semipurified preparations revealed that guanosine tetraphosphate inhibits at least two enzymes specific for the biosynthetic pathway, sn-glycerol-3-phosphate acyltransferase as well as sn-glycerol-3-phosphate phosphatidyl transferase. These findings provide a biochemical basis for the stringent control of lipid synthesis as well as regulation of steady-state levels of phospholipid in growing cells. Images PMID:4583220

A Whole-Cell Phenotypic Screening Platform for Identifying Methylerythritol Phosphate Pathway-Selective Inhibitors as Novel Antibacterial Agents

PubMed Central

Johnson, L. Jeffrey

2012-01-01

Isoprenoid biosynthesis is essential for survival of all living organisms. More than 50,000 unique isoprenoids occur naturally, with each constructed from two simple five-carbon precursors: isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Two pathways for the biosynthesis of IPP and DMAPP are found in nature. Humans exclusively use the mevalonate (MVA) pathway, while most bacteria, including all Gram-negative and many Gram-positive species, use the unrelated methylerythritol phosphate (MEP) pathway. Here we report the development of a novel, whole-cell phenotypic screening platform to identify compounds that selectively inhibit the MEP pathway. Strains of Salmonella enterica serovar Typhimurium were engineered to have separately inducible MEP (native) and MVA (nonnative) pathways. These strains, RMC26 and CT31-7d, were then used to differentiate MVA pathway- and MEP pathway-specific perturbation. Compounds that inhibit MEP pathway-dependent bacterial growth but leave MVA-dependent growth unaffected represent MEP pathway-selective antibacterials. This screening platform offers three significant results. First, the compound is antibacterial and is therefore cell permeant, enabling access to the intracellular target. Second, the compound inhibits one or more MEP pathway enzymes. Third, the MVA pathway is unaffected, suggesting selectivity for targeting the bacterial versus host pathway. The cell lines also display increased sensitivity to two reported MEP pathway-specific inhibitors, further biasing the platform toward inhibitors selective for the MEP pathway. We demonstrate development of a robust, high-throughput screening platform that combines phenotypic and target-based screening that can identify MEP pathway-selective antibacterials simply by monitoring optical density as the readout for cell growth/inhibition. PMID:22777049

Ultraviolet and infrared absorption spectra of Cr2O3 doped-sodium metaphosphate, lead metaphosphate and zinc metaphosphate glasses and effects of gamma irradiation: a comparative study.

PubMed

Marzouk, M A; ElBatal, F H; Abdelghany, A M

2013-10-01

The effects of gamma irradiation on spectral properties of Cr2O3-doped phosphate glasses of three varieties, namely sodium metaphosphate, lead metaphosphate and zinc metaphosphate have been investigated. Optical spectra of the undoped samples reveal strong UV absorption bands which are attributed to the presence of trace iron impurities in both the sodium and zinc phosphate glasses while the lead phosphate glass exhibits broad UV near visible bands due to combined absorption of both trace iron impurities and divalent lead ions. The effect of chromium oxide content has been investigated. The three different Cr2O3-doped phosphate glasses reveal spectral visible bands varying in their position and intensity and splitting due to the different field strengths of the Na(+), Pb(2+), Zn(2+) cations, together with the way they are housed in the network and their effects on the polarisability of neighboring oxygens ligands. The effects of gamma irradiation on the optical spectral properties of the various glasses have been compared. The different effects for lead and zinc phosphate are related to the ability of Pb(2+), and Zn(2+) to form additional structural units causing stability of the network towards gamma irradiation. Also, the introduction of the transition metal chromium ions reveals some shielding behavior towards irradiation. Infrared absorption spectra of the three different base phosphate glasses show characteristic vibrations due to various phosphate groups depending on the type of glass and Cr2O3 is observed to slightly affect the IR spectra. Gamma irradiation causes minor variations in some of the intensities of the IR spectra but the main characteristic bands due to phosphate groups remain in their number and position. Copyright © 2013 Elsevier B.V. All rights reserved.

Overexpression of hypoxia-inducible factor and metabolic pathways: possible targets of cancer.

PubMed

Singh, Davinder; Arora, Rohit; Kaur, Pardeep; Singh, Balbir; Mannan, Rahul; Arora, Saroj

2017-01-01

Cancer, the main cause of human deaths in the modern world is a group of diseases. Anticancer drug discovery is a challenge for scientists because of involvement of multiple survival pathways of cancer cells. An extensive study on the regulation of each step of these pathways may help find a potential cancer target. Up-regulated HIF-1 expression and altered metabolic pathways are two classical characteristics of cancer. Oxygen-dependent (through pVHL, PHDs, calcium-mediated) and independent (through growth factor signaling pathway, mdm2 pathway, HSP90) regulation of HIF-1α leads to angiogenesis, metastasis, and cell survival. The two subunits of HIF-1 regulates in the same fashion through different mechanisms. HIF-1α translation upregulates via mammalian target of rapamycin and mitogen-activated protein kinase signaling pathways, whereas HIF-1β through calmodulin kinase. Further, the stabilized interactions of these two subunits are important for proper functioning. Also, metabolic pathways crucial for the formation of building blocks (pentose phosphate pathway) and energy generation (glycolysis, TCA cycle and catabolism of glutamine) are altered in cancer cells to protect them from oxidative stress and to meet the reduced oxygen and nutrient supply. Up-regulated anaerobic metabolism occurs through enhanced expression of hexokinase, phosphofructokinase, triosephosphate isomerase, glucose 6-phosphate dehydrogenase and down-regulation of aerobic metabolism via pyruvate dehydrogenase kinase and lactate dehydrogenase which compensate energy requirements along with high glucose intake. Controlled expression of these two pathways through their common intermediate may serve as potent cancer target in future.

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

PubMed

Choi, Yun-Nam; Park, Jong Moon

2016-08-01

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

Co-production of hydrogen and ethanol from glucose by modification of glycolytic pathways in Escherichia coli - from Embden-Meyerhof-Parnas pathway to pentose phosphate pathway.

PubMed

Seol, Eunhee; Sekar, Balaji Sundara; Raj, Subramanian Mohan; Park, Sunghoon

2016-02-01

Hydrogen (H2) production from glucose by dark fermentation suffers from the low yield. As a solution to this problem, co-production of H2 and ethanol, both of which are good biofuels, has been suggested. To this end, using Escherichia coli, activation of pentose phosphate (PP) pathway, which can generate more NADPH than the Embden-Meyhof-Parnas (EMP) pathway, was attempted. Overexpression of two key enzymes in the branch nodes of the glycolytic pathway, Zwf and Gnd, significantly improved the co-production of H2 and ethanol with concomitant reduction of pyruvate secretion. Gene expression analysis and metabolic flux analysis (MFA) showed that, upon overexpression of Zwf and Gnd, glucose assimilation through the PP pathway, compared with that of the EMP or Entner-Doudoroff (ED) pathway, was greatly enhanced. The maximum co-production yields were 1.32 mol H2 mol(-1) glucose and 1.38 mol ethanol mol(-1) glucose, respectively. It is noteworthy that the glycolysis and the amount of NAD(P)H formed under anaerobic conditions could be altered by modifying (the activity of) several key enzymes. Our strategy could be applied for the development of industrial strains for biological production of reduced chemicals and biofuels which suffers from lack of reduced co-factors. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Terpenoids and Their Biosynthesis in Cyanobacteria

PubMed Central

Pattanaik, Bagmi; Lindberg, Pia

2015-01-01

Terpenoids, or isoprenoids, are a family of compounds with great structural diversity which are essential for all living organisms. In cyanobacteria, they are synthesized from the methylerythritol-phosphate (MEP) pathway, using glyceraldehyde 3-phosphate and pyruvate produced by photosynthesis as substrates. The products of the MEP pathway are the isomeric five-carbon compounds isopentenyl diphosphate and dimethylallyl diphosphate, which in turn form the basic building blocks for formation of all terpenoids. Many terpenoid compounds have useful properties and are of interest in the fields of pharmaceuticals and nutrition, and even potentially as future biofuels. The MEP pathway, its function and regulation, and the subsequent formation of terpenoids have not been fully elucidated in cyanobacteria, despite its relevance for biotechnological applications. In this review, we summarize the present knowledge about cyanobacterial terpenoid biosynthesis, both regarding the native metabolism and regarding metabolic engineering of cyanobacteria for heterologous production of non-native terpenoids. PMID:25615610

Regulation of Mammary Tumor Formation and Lipid Biosynthesis by Spot 14

DTIC Science & Technology

2013-10-01

1     Introduction: THRSP/Spot14/S14 is a small cytoplasmic protein that is highly expressed in tissues that synthesize fatty acids de novo...cycle regulation and also with various metabolic pathways, including glycolysis /gluconeogenesis and the pentose phosphate pathway (Table 1). The

Relationship between Glycolysis and Exopolysaccharide Biosynthesis in Lactococcus lactis

PubMed Central

Ramos, Ana; Boels, Ingeborg C.; de Vos, Willem M.; Santos, Helena

2001-01-01

The relationships between glucose metabolism and exopolysaccharide (EPS) production in a Lactococcus lactis strain containing the EPS gene cluster (Eps+) and in nonproducer strain MG5267 (Eps−) were characterized. The concentrations of relevant phosphorylated intermediates in EPS and cell wall biosynthetic pathways or glycolysis were determined by 31P nuclear magnetic resonance. The concentrations of two EPS precursors, UDP-glucose and UDP-galactose, were significantly lower in the Eps+ strain than in the Eps− strain. The precursors of the peptidoglycan pathway, UDP-N-acetylglucosamine and UDP-N-acetylmuramoyl-pentapeptide, were the major UDP-sugar derivatives detected in the two strains examined, but the concentration of the latter was greater in the Eps+ strain, indicating that there is competition between EPS synthesis and cell growth. An intermediate in biosynthesis of histidine and nucleotides, 5-phosphorylribose 1-pyrophosphate, accumulated at concentrations in the millimolar range, showing that the pentose phosphate pathway was operating. Fructose 1,6-bisphosphate and glucose 6-phosphate were the prominent glycolytic intermediates during exponential growth of both strains, whereas in the stationary phase the main metabolites were 3-phosphoglyceric acid, 2-phosphoglyceric acid, and phosphoenolpyruvate. The activities of relevant enzymes, such as phosphoglucose isomerase, α-phosphoglucomutase, and UDP-glucose pyrophosphorylase, were identical in the two strains. 13C enrichment on the sugar moieties of pure EPS showed that glucose 6-phosphate is the key metabolite at the branch point between glycolysis and EPS biosynthesis and ruled out involvement of the triose phosphate pool. This study provided clues for ways to enhance EPS production by genetic manipulation. PMID:11133425

Effects of glucose metabolism pathways on sperm motility and oxidative status during long-term liquid storage of goat semen.

PubMed

Qiu, Jian-Hua; Li, You-Wei; Xie, Hong-Li; Li, Qing; Dong, Hai-Bo; Sun, Ming-Ju; Gao, Wei-Qiang; Tan, Jing-He

2016-08-01

Although great efforts were made to prolong the fertility of liquid-stored semen, limited improvements have been achieved in different species. Although it is expected that energy supply and the redox potential will play an essential role in sperm function, there are few reports on the impact of specific energy substrates on spermatozoa during liquid semen storage. Furthermore, although it is accepted that glucose metabolism through glycolysis provides energy, roles of pentose phosphate pathway (PPP) and tricarboxylic acid cycle remain to be unequivocally found in spermatozoa. We have studied the pathways by which spermatozoa metabolize glucose during long-term liquid storage of goat semen. The results indicated that among the substrates tested, glucose and pyruvate were better than lactate in maintaining goat sperm motility. Although both glycolysis and PPP were essential, PPP was more important than glycolysis to maintain sperm motility. Pentose phosphate pathway reduced oxidative stress and provided glycolysis with more intermediate products such as fructose-6-phosphate. Pyruvate entered goat spermatozoa through monocarboxylate transporters and was oxidized by the tricarboxylic acid cycle and electron transfer to sustain sperm motility. Long-term liquid semen storage can be used as a good model to study sperm glucose metabolism. The data are important for an optimal control of sperm survival during semen handling and preservation not only in the goat but also in other species. Copyright © 2016 Elsevier Inc. All rights reserved.

Glycolytic intermediates and adenosine phosphates in rat liver at high altitude /3,800 m/.

NASA Technical Reports Server (NTRS)

Cipriano, L. F.; Pace, N.

1973-01-01

Liver tissue obtained from adult rats exposed to 3800 m altitude for intervals ranging from 1.5 hr to 63 days was examined by enzymatic analysis. During the first 3 hr of exposure, an immediate decrease in rephosphorylation of high-energy phosphates led to reduced glycogenesis and eventual pileup of AMP, pyruvate, fructose 1,6-diphosphate, glucose 6-phosphate, and glucose. This was accompanied by a reduction of pentose phosphate pathway activity. After 3 to 6 hr, a secondary adjustment of substrate concentrations occurred along with the apparent facilitation of phosphofructokinase. This secondary adjustment appears to increase anaerobic production of ATP and represents a significant intracellular contribution to the acclimatization process at high altitude.

The role of glucose-6-phosphate dehydrogenase in adipose tissue inflammation in obesity.

PubMed

Park, Yoon Jeong; Choe, Sung Sik; Sohn, Jee Hyung; Kim, Jae Bum

2017-04-03

Obesity is closely associated with metabolic diseases including type 2 diabetes. One hallmark characteristics of obesity is chronic inflammation that is coordinately controlled by complex signaling networks in adipose tissues. Compelling evidence indicates that reactive oxygen species (ROS) and its related signaling pathways play crucial roles in the progression of chronic inflammation in obesity. The pentose phosphate pathway (PPP) is an anabolic pathway that utilizes the glucoses to generate molecular building blocks and reducing equivalents in the form of NADPH. In particular, NADPH acts as one of the key modulators in the control of ROS through providing an electron for both ROS generation and scavenging. Recently, we have reported that glucose-6-phosphate dehydrogenase (G6PD), a rate-limiting enzyme of the PPP, is implicated in adipose tissue inflammation and systemic insulin resistance in obesity. Mechanistically, G6PD potentiates generation of ROS that augments pro-inflammatory responses in adipose tissue macrophages, leading to systemic insulin resistance. Here, we provide an overview of cell type- specific roles of G6PD in the regulation of ROS balance as well as additional details on the significance of G6PD that contributes to pro-oxidant NADPH generation in obesity-related chronic inflammation and insulin resistance.

Metabolic switches and adaptations deduced from the proteomes of Streptomyces coelicolor wild type and phoP mutant grown in batch culture.

PubMed

Thomas, Louise; Hodgson, David A; Wentzel, Alexander; Nieselt, Kay; Ellingsen, Trond E; Moore, Jonathan; Morrissey, Edward R; Legaie, Roxane; Wohlleben, Wolfgang; Rodríguez-García, Antonio; Martín, Juan F; Burroughs, Nigel J; Wellington, Elizabeth M H; Smith, Margaret C M

2012-02-01

Bacteria in the genus Streptomyces are soil-dwelling oligotrophs and important producers of secondary metabolites. Previously, we showed that global messenger RNA expression was subject to a series of metabolic and regulatory switches during the lifetime of a fermentor batch culture of Streptomyces coelicolor M145. Here we analyze the proteome from eight time points from the same fermentor culture and, because phosphate availability is an important regulator of secondary metabolite production, compare this to the proteome of a similar time course from an S. coelicolor mutant, INB201 (ΔphoP), defective in the control of phosphate utilization. The proteomes provide a detailed view of enzymes involved in central carbon and nitrogen metabolism. Trends in protein expression over the time courses were deduced from a protein abundance index, which also revealed the importance of stress pathway proteins in both cultures. As expected, the ΔphoP mutant was deficient in expression of PhoP-dependent genes, and several putatively compensatory metabolic and regulatory pathways for phosphate scavenging were detected. Notably there is a succession of switches that coordinately induce the production of enzymes for five different secondary metabolite biosynthesis pathways over the course of the batch cultures.

The specific role of plastidial glycolysis in photosynthetic and heterotrophic cells under scrutiny through the study of glyceraldehyde-3-phosphate dehydrogenase.

PubMed

Anoman, Armand Djoro; Flores-Tornero, María; Rosa-Telléz, Sara; Muñoz-Bertomeu, Jesús; Segura, Juan; Ros, Roc

2016-01-01

The cellular compartmentalization of metabolic processes is an important feature in plants where the same pathways could be simultaneously active in different compartments. Plant glycolysis occurs in the cytosol and plastids of green and non-green cells in which the requirements of energy and precursors may be completely different. Because of this, the relevance of plastidial glycolysis could be very different depending on the cell type. In the associated study, we investigated the function of plastidial glycolysis in photosynthetic and heterotrophic cells by specifically driving the expression of plastidial glyceraldehyde-3-phosphate dehydrogenase (GAPCp) in a glyceraldehyde-3-phosphate dehydrogenase double mutant background (gapcp1gapcp2). We showed that GAPCp is not functionally significant in photosynthetic cells, while it plays a crucial function in heterotrophic cells. We also showed that (i) GAPCp activity expression in root tips is necessary for primary root growth, (ii) its expression in heterotrophic cells of aerial parts and roots is necessary for plant growth and development, and (iii) GAPCp is an important metabolic connector of carbon and nitrogen metabolism through the phosphorylated pathway of serine biosynthesis (PPSB). We discuss here the role that this pathway could play in the control of plant growth and development.

Balancing of B6 Vitamers Is Essential for Plant Development and Metabolism in Arabidopsis

PubMed Central

Tohge, Takayuki; Fernie, Alisdair R.

2016-01-01

Vitamin B6 comprises a family of compounds that is essential for all organisms, most notable among which is the cofactor pyridoxal 5′-phosphate (PLP). Other forms of vitamin B6 include pyridoxamine 5′-phosphate (PMP), pyridoxine 5′-phosphate (PNP), and the corresponding nonphosphorylated derivatives. While plants can biosynthesize PLP de novo, they also have salvage pathways that serve to interconvert the different vitamers. The selective contribution of these various pathways to cellular vitamin B6 homeostasis in plants is not fully understood. Although biosynthesis de novo has been extensively characterized, the salvage pathways have received comparatively little attention in plants. Here, we show that the PMP/PNP oxidase PDX3 is essential for balancing B6 vitamer levels in Arabidopsis thaliana. In the absence of PDX3, growth and development are impaired and the metabolite profile is altered. Surprisingly, RNA sequencing reveals strong induction of stress-related genes in pdx3, particularly those associated with biotic stress that coincides with an increase in salicylic acid levels. Intriguingly, exogenous ammonium rescues the growth and developmental phenotype in line with a severe reduction in nitrate reductase activity that may be due to the overaccumulation of PMP in pdx3. Our analyses demonstrate an important link between vitamin B6 homeostasis and nitrogen metabolism. PMID:26858304

Direct determination of phosphate sugars in biological material by (1)H high-resolution magic-angle-spinning NMR spectroscopy.

PubMed

Diserens, Gaëlle; Vermathen, Martina; Gjuroski, Ilche; Eggimann, Sandra; Precht, Christina; Boesch, Chris; Vermathen, Peter

2016-08-01

The study aim was to unambiguously assign nucleotide sugars, mainly UDP-X that are known to be important in glycosylation processes as sugar donors, and glucose-phosphates that are important intermediate metabolites for storage and transfer of energy directly in spectra of intact cells, as well as in skeletal muscle biopsies by (1)H high-resolution magic-angle-spinning (HR-MAS) NMR. The results demonstrate that sugar phosphates can be determined quickly and non-destructively in cells and biopsies by HR-MAS, which may prove valuable considering the importance of phosphate sugars in cell metabolism for nucleic acid synthesis. As proof of principle, an example of phosphate-sugar reaction and degradation kinetics after unfreezing the sample is shown for a cardiac muscle, suggesting the possibility to follow by HR-MAS NMR some metabolic pathways. Graphical abstract Glucose-phosphate sugars (Glc-1P and Glc-6P) detected in muscle by 1H HR-MAS NMR.

Characterization of two transketolases encoded on the chromosome and the plasmid pBM19 of the facultative ribulose monophosphate cycle methylotroph Bacillus methanolicus

PubMed Central

2014-01-01

Background Transketolase (TKT) is a key enzyme of the pentose phosphate pathway (PPP), the Calvin cycle and the ribulose monophosphate (RuMP) cycle. Bacillus methanolicus is a facultative RuMP pathway methylotroph. B. methanolicus MGA3 harbors two genes putatively coding for TKTs; one located on the chromosome (tkt C ) and one located on the natural occurring plasmid pBM19 (tkt P ). Results Both enzymes were produced in recombinant Escherichia coli, purified and shown to share similar biochemical parameters in vitro. They were found to be active as homotetramers and require thiamine pyrophosphate for catalytic activity. The inactive apoform of the TKTs, yielded by dialysis against buffer containing 10 mM EDTA, could be reconstituted most efficiently with Mn2+ and Mg2+. Both TKTs were thermo stable at physiological temperature (up to 65°C) with the highest activity at neutral pH. Ni2+, ATP and ADP significantly inhibited activity of both TKTs. Unlike the recently characterized RuMP pathway enzymes fructose 1,6-bisphosphate aldolase (FBA) and fructose 1,6-bisphosphatase/sedoheptulose 1,7-bisphosphatase (FBPase/SBPase) from B. methanolicus MGA3, both TKTs exhibited similar kinetic parameters although they only share 76% identical amino acids. The kinetic parameters were determined for the reaction with the substrates xylulose 5-phosphate (TKTC: kcat/KM: 264 s-1 mM-1; TKTP: kcat/KM: 231 s-1 mM) and ribulose 5-phosphate (TKTC: kcat/KM: 109 s-1 mM; TKTP: kcat/KM: 84 s-1 mM) as well as for the reaction with the substrates glyceraldehyde 3-phosphate (TKTC: kcat/KM: 108 s-1 mM; TKTP: kcat/KM: 71 s-1 mM) and fructose 6-phosphate (TKTC kcat/KM: 115 s-1 mM; TKTP: kcat/KM: 448 s-1 mM). Conclusions Based on the kinetic parameters no major TKT of B. methanolicus could be determined. Increased expression of tkt P , but not of tkt C during growth with methanol [J Bacteriol 188:3063–3072, 2006] argues for TKTP being the major TKT relevant in the RuMP pathway. Neither TKT exhibited activity as dihydroxyacetone synthase, as found in methylotrophic yeast, or as the evolutionary related 1-deoxyxylulose-5-phosphate synthase. The biological significance of the two TKTs for B. methanolicus methylotrophy is discussed. PMID:24405865

Characterization of two transketolases encoded on the chromosome and the plasmid pBM19 of the facultative ribulose monophosphate cycle methylotroph Bacillus methanolicus.

PubMed

Markert, Benno; Stolzenberger, Jessica; Brautaset, Trygve; Wendisch, Volker F

2014-01-09

Transketolase (TKT) is a key enzyme of the pentose phosphate pathway (PPP), the Calvin cycle and the ribulose monophosphate (RuMP) cycle. Bacillus methanolicus is a facultative RuMP pathway methylotroph. B. methanolicus MGA3 harbors two genes putatively coding for TKTs; one located on the chromosome (tkt(C)) and one located on the natural occurring plasmid pBM19 (tkt(P)). Both enzymes were produced in recombinant Escherichia coli, purified and shown to share similar biochemical parameters in vitro. They were found to be active as homotetramers and require thiamine pyrophosphate for catalytic activity. The inactive apoform of the TKTs, yielded by dialysis against buffer containing 10 mM EDTA, could be reconstituted most efficiently with Mn(2+) and Mg(2+). Both TKTs were thermo stable at physiological temperature (up to 65°C) with the highest activity at neutral pH. Ni(2+), ATP and ADP significantly inhibited activity of both TKTs. Unlike the recently characterized RuMP pathway enzymes fructose 1,6-bisphosphate aldolase (FBA) and fructose 1,6-bisphosphatase/sedoheptulose 1,7-bisphosphatase (FBPase/SBPase) from B. methanolicus MGA3, both TKTs exhibited similar kinetic parameters although they only share 76% identical amino acids. The kinetic parameters were determined for the reaction with the substrates xylulose 5-phosphate (TKT(C): kcat/KM: 264 s(-1) mM(-1); TKT(P): kcat/KM: 231 s(-1) mM) and ribulose 5-phosphate (TKT(C): kcat/KM: 109 s(-1) mM; TKT(P): kcat/KM: 84 s(-1) mM) as well as for the reaction with the substrates glyceraldehyde 3-phosphate (TKT(C): kcat/KM: 108 s(-1) mM; TKT(P): kcat/KM: 71 s(-1) mM) and fructose 6-phosphate (TKT(C) kcat/KM: 115 s(-1) mM; TKT(P): kcat/KM: 448 s(-1) mM). Based on the kinetic parameters no major TKT of B. methanolicus could be determined. Increased expression of tkt(P), but not of tkt(C) during growth with methanol [J Bacteriol 188:3063-3072, 2006] argues for TKT(P) being the major TKT relevant in the RuMP pathway. Neither TKT exhibited activity as dihydroxyacetone synthase, as found in methylotrophic yeast, or as the evolutionary related 1-deoxyxylulose-5-phosphate synthase. The biological significance of the two TKTs for B. methanolicus methylotrophy is discussed.

Wnt5a Increases the Glycolytic Rate and the Activity of the Pentose Phosphate Pathway in Cortical Neurons

PubMed Central

Cisternas, Pedro; Salazar, Paulina; Silva-Álvarez, Carmen; Barros, L. Felipe

2016-01-01

In the last few years, several reports have proposed that Wnt signaling is a general metabolic regulator, suggesting a role for this pathway in the control of metabolic flux. Wnt signaling is critical for several neuronal functions, but little is known about the correlation between this pathway and energy metabolism. The brain has a high demand for glucose, which is mainly used for energy production. Neurons use energy for highly specific processes that require a high energy level, such as maintaining the electrical potential and synthesizing neurotransmitters. Moreover, an important metabolic impairment has been described in all neurodegenerative disorders. Despite the key role of glucose metabolism in the brain, little is known about the cellular pathways involved in regulating this process. We report here that Wnt5a induces an increase in glucose uptake and glycolytic rate and an increase in the activity of the pentose phosphate pathway; the effects of Wnt5a require the intracellular generation of nitric oxide. Our data suggest that Wnt signaling stimulates neuronal glucose metabolism, an effect that could be important for the reported neuroprotective role of Wnt signaling in neurodegenerative disorders. PMID:27688915

Wnt5a Increases the Glycolytic Rate and the Activity of the Pentose Phosphate Pathway in Cortical Neurons.

PubMed

Cisternas, Pedro; Salazar, Paulina; Silva-Álvarez, Carmen; Barros, L Felipe; Inestrosa, Nibaldo C

In the last few years, several reports have proposed that Wnt signaling is a general metabolic regulator, suggesting a role for this pathway in the control of metabolic flux. Wnt signaling is critical for several neuronal functions, but little is known about the correlation between this pathway and energy metabolism. The brain has a high demand for glucose, which is mainly used for energy production. Neurons use energy for highly specific processes that require a high energy level, such as maintaining the electrical potential and synthesizing neurotransmitters. Moreover, an important metabolic impairment has been described in all neurodegenerative disorders. Despite the key role of glucose metabolism in the brain, little is known about the cellular pathways involved in regulating this process. We report here that Wnt5a induces an increase in glucose uptake and glycolytic rate and an increase in the activity of the pentose phosphate pathway; the effects of Wnt5a require the intracellular generation of nitric oxide. Our data suggest that Wnt signaling stimulates neuronal glucose metabolism, an effect that could be important for the reported neuroprotective role of Wnt signaling in neurodegenerative disorders.

Mannitol metabolism during pathogenic fungal–host interactions under stressed conditions

PubMed Central

Meena, Mukesh; Prasad, Vishal; Zehra, Andleeb; Gupta, Vijai K.; Upadhyay, Ram S.

2015-01-01

Numerous plants and fungi produce mannitol, which may serve as an osmolyte or metabolic store; furthermore, mannitol also acts as a powerful quencher of reactive oxygen species (ROS). Some phytopathogenic fungi use mannitol to stifle ROS-mediated plant resistance. Mannitol is essential in pathogenesis to balance cell reinforcements produced by both plants and animals. Mannitol likewise serves as a source of reducing power, managing coenzymes, and controlling cytoplasmic pH by going about as a sink or hotspot for protons. The metabolic pathways for mannitol biosynthesis and catabolism have been characterized in filamentous fungi by direct diminishment of fructose-6-phosphate into mannitol-1-phosphate including a mannitol-1-phosphate phosphatase catalyst. In plants mannitol is integrated from mannose-6-phosphate to mannitol-1-phosphate, which then dephosphorylates to mannitol. The enzyme mannitol dehydrogenase plays a key role in host–pathogen interactions and must be co-localized with pathogen-secreted mannitol to resist the infection. PMID:26441941

Light-regulation of enzyme activity in anacystis nidulans (Richt.).

PubMed

Duggan, J X; Anderson, L E

1975-01-01

The effect of light on the levels of activity of six enzymes which are light-modulated in higher plants was examined in the photosynthetic procaryot Anacystis nidulans. Ribulose-5-phosphate kinase (EC 2.7.1.19) was found to be light-activated in vivo and dithiothreitol-activated in vitro while glucose-6-phosphate dehydrogenase (EC 1.1.1.49) was light-inactivated and dithiothreitol-inactivated. The enzymes fructose-1,6-diphosphate phosphatase (EC 3.1.3.11), sedoheptulose-1,7-diphosphate phosphatase, NAD- and NADP-linked glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12; EC 1.2.1.13) were not affected by light treatment of the intact algae, but sedoheptulose-diphosphate phosphatase and the glyceraldehyde-3-phosphate dehydrogenases were dithiothreitol-activated in crude extracts. Light apparently controls the activity of the reductive and oxidative pentose phosphate pathway in this photosynthetic procaryot as in higher plants, through a process which probably involves reductive modulation of enzyme activity.

Alkaline hydrolysis of ethylene phosphate: an ab initio study by supermolecule model and polarizable continuum approach.

PubMed

Xia, Futing; Zhu, Hua

2011-09-01

The alkaline hydrolysis reaction of ethylene phosphate (EP) has been investigated using a supermolecule model, in which several explicit water molecules are included. The structures and single-point energies for all of the stationary points are calculated in the gas phase and in solution at the B3LYP/6-31++G(df,p) and MP2/6-311++G(df,2p) levels. The effect of water bulk solvent is introduced by the polarizable continuum model (PCM). Water attack and hydroxide attack pathways are taken into account for the alkaline hydrolysis of EP. An associative mechanism is observed for both of the two pathways with a kinetically insignificant intermediate. The water attack pathway involves a water molecule attacking and a proton transfer from the attacking water to the hydroxide in the first step, followed by an endocyclic bond cleavage to the leaving group. While in the first step of the hydroxide attack pathway the nucleophile is the hydroxide anion. The calculated barriers in aqueous solution for the water attack and hydroxide attack pathways are all about 22 kcal/mol. The excellent agreement between the calculated and observed values demonstrates that both of the two pathways are possible for the alkaline hydrolysis of EP. Copyright © 2011 Wiley Periodicals, Inc.

The role of grain boundaries and transient porosity increase as fluid pathways for reaction front propagation

NASA Astrophysics Data System (ADS)

Jonas, Laura; John, Timm; Geisler, Thorsten; Putnis, Andrew

2013-04-01

The pseudomorphic replacement of Carrara marble by calcium phosphates was studied as a model system to examine the influence of different fluid pathways for reaction front propagation induced by fluid-rock interaction. In this model system, the grain boundaries present in the rock and the transient porosity structures developing throughout the replacement reaction enable the reaction front to progress further into the rock as well as to the center of each single grain until complete transformation. Hydrothermal treatment of the marble using phosphate bearing solutions at temperature levels of 150° C and 200° C for different durations lead to the formation of two product phases which were identified as hydroxyapatite [Ca5(PO4)3OH] as well as β-tricalcium phosphate [β-Ca3(PO4)2] (β-TCP). The formation of β-TCP was probably favored by the presence of ~0.6wt.% of Mg in the parent phase. Completely transformed single grains show a distinctive zoning, both in composition and texture. Whereas areas next to the grain boundary consist of nearly pure hydroxyapatite and show a coarse porosity, areas close to the center of the single grains show a high amount of β-TCP and a very fine porous microstructure. If F was added as an additional solution component, the formation of β-TCP was avoided and up to 3wt.% of F were incorporated into the product apatite. The use of the isotope 18O as a chronometer for the replacement reaction makes it possible to reconstruct the chronological development of the calcium phosphate reaction front. Raman analysis revealed that the incorporation of 18O in the PO4 tetrahedron of hydroxyapatite results in the development of distinct profiles in the calcium phosphate reaction front perpendicular to the grain boundaries of the marble. Through the use of the 18O chronometer, it is possible to estimate and compare the time effectiveness of the different fluid pathways in this model system. The results show that the grain boundaries serve as a very effective pathway that enable the fluid to penetrate the rock more than one order of magnitude faster compared to the newly developing channel-like porosity structures which act as pathways towards the center of single mineral grains. Thus, it may be possible for the fluid to progress relatively large distances along the grain boundaries after only short reaction durations without producing broad reaction fronts along the path.

Phosphate limitation induces sporulation in the chytridiomycete Blastocladiella emersonii.

PubMed

Bongiorno, Vagner Alexandre; Ferreira da Cruz, Angela; Nunis da Silva, Antonio; Corrêa, Luiz Carlos

2012-09-01

The cell cycle is controlled by numerous mechanisms that ensure correct cell division. If growth is not possible, cells may eventually promote autophagy, differentiation, or apoptosis. Microorganisms interrupt their growth and differentiate under general nutrient limitation. We analyzed the effects of phosphate limitation on growth and sporulation in the chytridiomycete Blastocladiella emersonii using kinetic data, phase-contrast, and laser confocal microscopy. Under phosphate limitation, zoospores germinated and subsequently formed 2-4 spores, regardless of the nutritional content of the medium. The removal of phosphate at any time during growth induced sporulation of vegetative cells. If phosphate was later added to the same cultures, growth was restored if the cells were not yet committed to sporulation. The cycles of addition and withdrawal of phosphate from growth medium resulted in cycles of germination-growth, germination-sporulation, or germination-growth-sporulation. These results show that phosphate limitation is sufficient to interrupt cell growth and to induce complete sporulation in B. emersonii. We concluded that the determination of growth or sporulation in this microorganism is linked to phosphate availability when other nutrients are not limiting. This result provides a new tool for the dissection of nutrient-energy and signal pathways in cell growth and differentiation.

Microbiome-Metabolome Responses in the Cecum and Colon of Pig to a High Resistant Starch Diet.

PubMed

Sun, Yue; Su, Yong; Zhu, Weiyun

2016-01-01

Currently, knowledge about the impact of long-term intake of high resistant starch diet on pig hindgut microbiota and metabolite profile is limited. In this study, a combination of the pyrosequencing and the mass spectrometry (MS)-based metabolomics techniques were used to investigate the effects of a raw potato starch (RPS, high in resistant starch) diet on microbial composition and microbial metabolites in the hindgut of pig. The results showed that Coprococcus, Ruminococcus, and Turicibacter increased significantly, while Sarcina and Clostridium decreased in relative abundances in the hindgut of pigs fed RPS. The metabolimic analysis revealed that RPS significantly affected starch and sucrose metabolites, amino acid turnover or protein biosynthesis, lipid metabolites, glycolysis, the pentose phosphate pathway, inositol phosphate metabolism, and nucleotide metabolism. Furthermore, a Pearson's correlation analysis showed that Ruminococcus and Coprococcus were positively correlated with glucose-6-phosphate, maltose, arachidonic acid, 9, 12-octadecadienoic acid, oleic acid, phosphate, but negatively correlated with α-aminobutyric acid. However, the correlation of Clostridium and Sarcina with these compounds was in the opposite direction. The results suggest that RPS not only alters the composition of the gut microbial community but also modulates the metabolic pathway of microbial metabolism, which may further affect the hindgut health of the host.

Microbiome-Metabolome Responses in the Cecum and Colon of Pig to a High Resistant Starch Diet

PubMed Central

Sun, Yue; Su, Yong; Zhu, Weiyun

2016-01-01

Currently, knowledge about the impact of long-term intake of high resistant starch diet on pig hindgut microbiota and metabolite profile is limited. In this study, a combination of the pyrosequencing and the mass spectrometry (MS)-based metabolomics techniques were used to investigate the effects of a raw potato starch (RPS, high in resistant starch) diet on microbial composition and microbial metabolites in the hindgut of pig. The results showed that Coprococcus, Ruminococcus, and Turicibacter increased significantly, while Sarcina and Clostridium decreased in relative abundances in the hindgut of pigs fed RPS. The metabolimic analysis revealed that RPS significantly affected starch and sucrose metabolites, amino acid turnover or protein biosynthesis, lipid metabolites, glycolysis, the pentose phosphate pathway, inositol phosphate metabolism, and nucleotide metabolism. Furthermore, a Pearson's correlation analysis showed that Ruminococcus and Coprococcus were positively correlated with glucose-6-phosphate, maltose, arachidonic acid, 9, 12-octadecadienoic acid, oleic acid, phosphate, but negatively correlated with α-aminobutyric acid. However, the correlation of Clostridium and Sarcina with these compounds was in the opposite direction. The results suggest that RPS not only alters the composition of the gut microbial community but also modulates the metabolic pathway of microbial metabolism, which may further affect the hindgut health of the host. PMID:27303373

Phosphate flame retardants and novel brominated flame retardants in home-produced eggs from an e-waste recycling region in China.

PubMed

Zheng, Xiaobo; Xu, Fuchao; Luo, Xiaojun; Mai, Bixian; Covaci, Adrian

2016-05-01

Phosphate flame retardants (PFRs) and novel brominated flame retardants (NBFRs) (2-ethylhexyl-2,3,4,5-tetrabromo-benzoate (EH-TBB) and bis-(2-ethylhexyl)-3,4,5,6-tetrabromo-phthalate (BEH-TEBP)) were measured in free-range chicken eggs from three e-waste recycling sites and a negative control site located in Guangdong province, Southern China. BEH-TEBP, tris-(chloroethyl)-phosphate (TCEP), tris-(chloropropyl)-phosphate (∑TCPP, two isomers) and tris-(1,3-dichloroisopropyl)-phosphate (TDCIPP) were detected in more than 50% of eggs samples with low concentrations. The median values of BEH-TEBP and total PFRs were 0.17-0.46 ng/g ww (wet weight) and 1.62-2.59 ng/g ww in eggs from the e-waste sites, respectively. The results indicate that EH-TBB, BEH-TEBP and PFRs are less persistent and bioaccumulative than polybrominated diphenyl ethers (PBDEs) in chicken eggs, and possibly also in other bio-matrices. Triphenyl phosphate (TPHP) were identified in albumen with higher frequencies, but at similar concentrations compared to yolk, while BEH-TEBP was mainly detected in yolk. The estimated daily intake (EDI) of BEH-TEBP and total PFRs from consumption of chicken eggs ranged from 0.03 to 0.09 and 0.32-0.52 ng/kg bw/day for adults, and 0.20-0.54 and 1.89-3.02 ng/kg bw/day for children in e-waste sites, respectively. Indoor dust ingestion seems to be a more important pathway for the intake of these FRs, while egg consumption is probably a more important exposure pathway for PBDEs. Copyright © 2015 Elsevier Ltd. All rights reserved.

Trehalose biosynthesis in Thermus thermophilus RQ-1: biochemical properties of the trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase.

PubMed

Silva, Zélia; Alarico, Susana; da Costa, Milton S

2005-02-01

The genes for trehalose synthesis in Thermus thermophilus RQ-1, namely otsA [trehalose-phosphate synthase (TPS)], otsB [trehalose-phosphate phosphatase (TPP)], and treS [trehalose synthase (maltose converting) (TreS)] genes are structurally linked. The TPS/TPP pathway plays a role in osmoadaptation, since mutants unable to synthesize trehalose via this pathway were less osmotolerant, in trehalose-deprived medium, than the wild-type strain. The otsA and otsB genes have now been individually cloned and overexpressed in Escherichia coli and the corresponding recombinant enzymes purified. The apparent molecular masses of TPS and TPP were 52 and 26 kDa, respectively. The recombinant TPS utilized UDP-glucose, TDP-glucose, ADP-glucose, or GDP-glucose, in this order as glucosyl donors, and glucose-6-phosphate as the glucosyl acceptor to produce trehalose-6-phosphate (T6P). The recombinant TPP catalyzed the dephosphorylation of T6P to trehalose. This enzyme also dephosphorylated G6P, and this activity was enhanced by NDP-glucose. TPS had an optimal activity at about 98 degrees C and pH near 6.0; TPP had a maximal activity near 70 degrees C and at pH 7.0. The enzymes were extremely thermostable: at 100 degrees C, TPS had a half-life of 31 min, and TPP had a half-life of 40 min. The enzymes did not require the presence of divalent cations for activity; however, the presence of Co2+ and Mg2+ stimulates both TPS and TPP. This is the first report of the characterization of TPS and TPP from a thermophilic organism.

Disruption of the GDP-mannose synthesis pathway in Streptomyces coelicolor results in antibiotic hyper-susceptible phenotypes.

PubMed

Howlett, Robert; Anttonen, Katri; Read, Nicholas; Smith, Margaret C M

2018-04-01

Actinomycete bacteria use polyprenol phosphate mannose as a lipid linked sugar donor for extra-cytoplasmic glycosyl transferases that transfer mannose to cell envelope polymers, including glycoproteins and glycolipids. We showed recently that strains of Streptomyces coelicolor with mutations in the gene ppm1 encoding polyprenol phosphate mannose synthase were both resistant to phage φC31 and have greatly increased susceptibility to antibiotics that mostly act on cell wall biogenesis. Here we show that mutations in the genes encoding enzymes that act upstream of Ppm1 in the polyprenol phosphate mannose synthesis pathway can also confer phage resistance and antibiotic hyper-susceptibility. GDP-mannose is a substrate for Ppm1 and is synthesised by GDP-mannose pyrophosphorylase (GMP; ManC) which uses GTP and mannose-1-phosphate as substrates. Phosphomannomutase (PMM; ManB) converts mannose-6-phosphate to mannose-1-phosphate. S. coelicolor strains with knocked down GMP activity or with a mutation in sco3028 encoding PMM acquire phenotypes that resemble those of the ppm1 - mutants i.e. φC31 resistant and susceptible to antibiotics. Differences in the phenotypes of the strains were observed, however. While the ppm1 - strains have a small colony phenotype, the sco3028 :: Tn5062 mutants had an extremely small colony phenotype indicative of an even greater growth defect. Moreover we were unable to generate a strain in which GMP activity encoded by sco3039 and sco4238 is completely knocked out, indicating that GMP is also an important enzyme for growth. Possibly GDP-mannose is at a metabolic branch point that supplies alternative nucleotide sugar donors.

The coupling of glycolysis and the Rubisco-based pathway through the non-oxidative pentose phosphate pathway to achieve low carbon dioxide emission fermentation.

PubMed

Li, Ya-Han; Ou-Yang, Fan-Yu; Yang, Cheng-Han; Li, Si-Yu

2015-01-01

In this study, Rubisco-based engineered Escherichia coli, containing two heterologous enzymes of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and phosphoribulokinase (PrkA), has been shown to be capable of the in situ recycling of carbon dioxide (CO2) during glycolysis. Two alternative approaches have been proposed to further enhance the carbon flow from glycolysis to a Rubisco-based pathway through the non-oxidative pentose phosphate pathway (NOPPP). The first is achieved by elevating the expression of transketolase I (TktA) and the second by blocking the native oxidation-decarboxylation reaction of E. coli by deleting the zwf gene from the chromosome (designated as JB/pTA and MZB, respectively). Decreases in the CO2 yield and the CO2 evolution per unit mole of ethanol production by at least 81% and 40% are observed. It is demonstrated in this study that the production of one mole of ethanol using E. coli strain MZB, the upper limit of CO2 emission is 0.052mol. Copyright © 2015 Elsevier Ltd. All rights reserved.

Chemogenetic Characterization of Inositol Phosphate Metabolic Pathway Reveals Druggable Enzymes for Targeting Kinetoplastid Parasites.

PubMed

Cestari, Igor; Haas, Paige; Moretti, Nilmar Silvio; Schenkman, Sergio; Stuart, Ken

2016-05-19

Kinetoplastids cause Chagas disease, human African trypanosomiasis, and leishmaniases. Current treatments for these diseases are toxic and inefficient, and our limited knowledge of drug targets and inhibitors has dramatically hindered the development of new drugs. Here we used a chemogenetic approach to identify new kinetoplastid drug targets and inhibitors. We conditionally knocked down Trypanosoma brucei inositol phosphate (IP) pathway genes and showed that almost every pathway step is essential for parasite growth and infection. Using a genetic and chemical screen, we identified inhibitors that target IP pathway enzymes and are selective against T. brucei. Two series of these inhibitors acted on T. brucei inositol polyphosphate multikinase (IPMK) preventing Ins(1,4,5)P3 and Ins(1,3,4,5)P4 phosphorylation. We show that IPMK is functionally conserved among kinetoplastids and that its inhibition is also lethal for Trypanosoma cruzi. Hence, IP enzymes are viable drug targets in kinetoplastids, and IPMK inhibitors may aid the development of new drugs. Copyright © 2016 Elsevier Ltd. All rights reserved.

Biomethylation and Volatilization of Arsenic by Model Protozoan Tetrahymena pyriformis under Different Phosphate Regimes

PubMed Central

Yin, Xixiang; Wang, Lihong; Zhang, Zhanchao; Fan, Guolan; Liu, Jianjun; Sun, Kaizhen; Sun, Guo-Xin

2017-01-01

Tetrahymena pyriformis, a freshwater protozoan, is common in aquatic systems. Arsenic detoxification through biotransformation by T. pyriformis is important but poorly understood. Arsenic metabolic pathways (including cellular accumulation, effluxion, biomethylation, and volatilization) of T. pyriformis were investigated at various phosphate concentrations. The total intracellular As concentration increased markedly as the external phosphate concentration decreased. The highest concentration was 168.8 mg·kg−1 dry weight, after exposure to As(V) for 20 h. Inorganic As was dominant at low phosphate concentrations (3, 6, and 15 mg·L−1), but the concentration was much lower at 30 mg·L−1 phosphate, and As(V) contributed only ~7% of total cellular As. Methylated As contributed 84% of total As at 30 mg·L−1 phosphate, and dimethylarsenate (DMAs(V)) was dominant, contributing up to 48% of total As. Cellular As effluxion was detected, including inorganic As(III), methylarsenate (MAs(V)) and DMAs(V). Volatile As was determined at various phosphate concentrations in the medium. All methylated As concentrations (intracellular, extracellular, and volatilized) had significant linear positive relationships with the initial phosphate concentration. To the best of our knowledge, this is the first study of As biotransformation by protozoa at different phosphate concentrations. PMID:28216593

Arbuscular Mycorrhizal Symbiosis Requires a Phosphate Transceptor in the Gigaspora margarita Fungal Symbiont.

PubMed

Xie, Xianan; Lin, Hui; Peng, Xiaowei; Xu, Congrui; Sun, Zhongfeng; Jiang, Kexin; Huang, Antian; Wu, Xiaohui; Tang, Nianwu; Salvioli, Alessandra; Bonfante, Paola; Zhao, Bin

2016-12-05

The majority of terrestrial vascular plants are capable of forming mutualistic associations with obligate biotrophic arbuscular mycorrhizal (AM) fungi from the phylum Glomeromycota. This mutualistic symbiosis provides carbohydrates to the fungus, and reciprocally improves plant phosphate uptake. AM fungal transporters can acquire phosphate from the soil through the hyphal networks. Nevertheless, the precise functions of AM fungal phosphate transporters, and whether they act as sensors or as nutrient transporters, in fungal signal transduction remain unclear. Here, we report a high-affinity phosphate transporter GigmPT from Gigaspora margarita that is required for AM symbiosis. Host-induced gene silencing of GigmPT hampers the development of G. margarita during AM symbiosis. Most importantly, GigmPT functions as a phosphate transceptor in G. margarita regarding the activation of the phosphate signaling pathway as well as the protein kinase A signaling cascade. Using the substituted-cysteine accessibility method, we identified residues A 146 (in transmembrane domain [TMD] IV) and Val 357 (in TMD VIII) of GigmPT, both of which are critical for phosphate signaling and transport in yeast during growth induction. Collectively, our results provide significant insights into the molecular functions of a phosphate transceptor from the AM fungus G. margarita. Copyright © 2016 The Author. Published by Elsevier Inc. All rights reserved.

Harmonics added to a flickering light can upset the balance between ON and OFF pathways to produce illusory colors.

PubMed

Rider, Andrew T; Henning, G Bruce; Eskew, Rhea T; Stockman, Andrew

2018-04-24

The neural signals generated by the light-sensitive photoreceptors in the human eye are substantially processed and recoded in the retina before being transmitted to the brain via the optic nerve. A key aspect of this recoding is the splitting of the signals within the two major cone-driven visual pathways into distinct ON and OFF branches that transmit information about increases and decreases in the neural signal around its mean level. While this separation is clearly important physiologically, its effect on perception is unclear. We have developed a model of the ON and OFF pathways in early color processing. Using this model as a guide, we can produce imbalances in the ON and OFF pathways by changing the shapes of time-varying stimulus waveforms and thus make reliable and predictable alterations to the perceived average color of the stimulus-although the physical mean of the waveforms does not change. The key components in the model are the early half-wave rectifying synapses that split retinal photoreceptor outputs into the ON and OFF pathways and later sigmoidal nonlinearities in each pathway. The ability to systematically vary the waveforms to change a perceptual quality by changing the balance of signals between the ON and OFF visual pathways provides a powerful psychophysical tool for disentangling and investigating the neural workings of human vision. Copyright © 2018 the Author(s). Published by PNAS.

Glycerophosphocholine metabolism in higher plant cells. Evidence of a new glyceryl-phosphodiester phosphodiesterase.

PubMed

van der Rest, Benoît; Boisson, Anne-Marie; Gout, Elisabeth; Bligny, Richard; Douce, Roland

2002-09-01

Glycerophosphocholine (GroPCho) is a diester that accumulates in different physiological processes leading to phospholipid remodeling. However, very little is known about its metabolism in higher plant cells. (31)P-Nuclear magnetic resonance spectroscopy and biochemical analyses performed on carrot (Daucus carota) cells fed with GroPCho revealed the existence of an extracellular GroPCho phosphodiesterase. This enzymatic activity splits GroPCho into sn-glycerol-3-phosphate and free choline. In vivo, sn-glycerol-3-phosphate is further hydrolyzed into glycerol and inorganic phosphate by acid phosphatase. We visualized the incorporation and the compartmentation of choline and observed that the major choline pool was phosphorylated and accumulated in the cytosol, whereas a minor fraction was incorporated in the vacuole as free choline. Isolation of plasma membranes, culture medium, and cell wall proteins enabled us to localize this phosphodiesterase activity on the cell wall. We also report the existence of an intracellular glycerophosphodiesterase. This second activity is localized in the vacuole and hydrolyzes GroPCho in a similar fashion to the cell wall phosphodiesterase. Both extra- and intracellular phosphodiesterases are widespread among different plant species and are often enhanced during phosphate deprivation. Finally, competition experiments on the extracellular phosphodiesterase suggested a specificity for glycerophosphodiesters (apparent K(m) of 50 microM), which distinguishes it from other phosphodiesterases previously described in the literature.

Systemic and local regulation of phosphate and nitrogen transporter genes by arbuscular mycorrhizal fungi in roots of winter wheat (Triticum aestivum L.).

PubMed

Duan, Jianfeng; Tian, Hui; Drijber, Rhae A; Gao, Yajun

2015-11-01

Previous studies have reported that the expression of phosphate (Pi) or nitrogen (N) transporter genes in roots of plants could be regulated by arbuscular mycorrhizal (AM) fungi, but little is known whether the regulation is systemic or not. The present study investigated the systemic and local regulation of multiple phosphate and nitrogen transporter genes by four AM fungal species belonging to four genera in the roots of winter wheat. A split-root culture system with AM inoculated (MR) and non-inoculated root compartments (NR) was used to investigate the systemic or local responses of phosphate and nitrogen transporter genes to colonization by four AM fungi in the roots of wheat. The expression of four Pi transporter, five nitrate transporter, and three ammonium transporter genes was quantified using real-time PCR. Of the four AM fungi tested, all locally increased expression of the AM-inducible Pi transporter genes, and most locally decreased expression of a Pi-starvation inducible Pi transporter gene. The addition of N in soil increased the expression of either Pi starvation inducible Pi transporters or AM inducible Pi transporters. Inoculation with AM fungi either had no effect, or could locally or systemically down-regulate expression of nitrogen transporter genes depending on gene type and AM fungal species. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Serum phosphate is associated with aortic valve calcification in the Multi-ethnic Study of Atherosclerosis (MESA).

PubMed

Linefsky, Jason P; O'Brien, Kevin D; Sachs, Michael; Katz, Ronit; Eng, John; Michos, Erin D; Budoff, Matthew J; de Boer, Ian; Kestenbaum, Bryan

2014-04-01

This study sought to investigate associations of phosphate metabolism biomarkers with aortic valve calcification (AVC). Calcific aortic valve disease (CAVD) is a common progressive condition that involves inflammatory and calcification mediators. Currently there are no effective medical treatments, but mineral metabolism pathways may be important in the development and progression of disease. We examined associations of phosphate metabolism biomarkers, including serum phosphate, urine phosphate, parathyroid hormone (PTH) and serum fibroblast growth factor (FGF)-23, with CT-assessed AVC at study baseline and in short-term follow-up in 6814 participants of the Multi-Ethnic Study of Atherosclerosis (MESA). At baseline, AVC prevalence was 13.2%. Higher serum phosphate levels were associated with significantly greater AVC prevalence (relative risk 1.3 per 1 mg/dL increment, 95% confidence incidence: 1.1 to 1.5, p<0.001). Serum FGF-23, serum PTH, and urine phosphate were not associated with prevalent AVC. Average follow-up CT evaluation was 2.4 years (range 0.9-4.9 years) with an AVC incidence of 4.1%. Overall, phosphate metabolism biomarkers were not associated with incident AVC except in the top FGF-23 quartile. Serum phosphate levels are significantly associated with AVC prevalence. Further study of phosphate metabolism as a modifiable risk factor for AVC is warranted. Published by Elsevier Ireland Ltd.

Serum Phosphate is Associated with Aortic Valve Calcification in the Multi-Ethnic Study of Atherosclerosis (MESA)

PubMed Central

Linefsky, Jason P.; O’Brien, Kevin D.; Sachs, Michael; Katz, Ronit; Eng, John; Michos, Erin D.; Budoff, Matthew J.; de Boer, Ian; Kestenbaum, Bryan

2014-01-01

Objectives This study sought to investigate associations of phosphate metabolism biomarkers with aortic valve calcification (AVC). Background Calcific aortic valve disease (CAVD) is a common progressive condition that involves inflammatory and calcification mediators. Currently there are no effective medical treatments, but mineral metabolism pathways may be important in the development and progression of disease. Methods We examined associations of phosphate metabolism biomarkers, including serum phosphate, urine phosphate, parathyroid hormone (PTH) and serum fibroblast growth factor (FGF)-23, with CT-assessed AVC at study baseline and in short-term follow-up in 6,814 participants of the Multi-Ethnic Study of Atherosclerosis (MESA). Results At baseline, AVC prevalence was 13.2%. Higher serum phosphate levels were associated with significantly greater AVC prevalence (relative risk 1.3 per 1mg/dL increment, 95% confidence incidence: 1.1 to 1.5, p < 0.001). Serum FGF-23, serum PTH, and urine phosphate were not associated with prevalent AVC. Average follow-up CT evaluation was 2.4 years (range 0.9–4.9 years) with an AVC incidence of 4.1%. Overall, phosphate metabolism biomarkers were not associated with incident AVC except in the top FGF-23 quartile. Conclusions Serum phosphate levels are significantly associated with AVC prevalence. Further study of phosphate metabolism as a modifiable risk factor for AVC is warranted. PMID:24530958

Effect of light on respiration and development of photosynthetic cells. Progress report, September 1, 1977--August 31, 1978

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

Gibbs, M

1977-08-31

The biophotolysis of water by photosynthetic cells resulting in the formation of hydrogen gas is of prime concern. That algal cells require both photosystems to complete this process is established. That a reduced carbon source can be photoxidized to release hydrogen and carbon dioxide has been proven. On the other hand, whether water is split to hydrogen and oxygen by the intact cell adapted to a hydrogen metabolism is an open question. A reconstituted preparation of higher plants can split water into its two components. A reconstituted algal preparation will be evaluated with respect to a similar reaction. If hydrogenmore » and oxygen are produced in vitro, what then regulates the cell into controlling this reaction during the onset of a hydrogen metabolism. The substrate for photorespiration is glycolic acid. The synthesis of this simple acid remain controversial. A new preparation of the spinach chloroplast has been developed which allows many compounds hitherto uncapable of crossing the organelle envelope to affect directly the carbon metabolism. We plan to use this preparation to evaluate the many proposed mechanisms of glycolate formation. Thus ribulose-1,5-diphosphate, hydroxypyruvate, hydroxypyruvate phosphate, oxaloacetate, and fructose-6-phosphate will be incubated under varying conditions and glycolate yields will be monitored. Conditions such as pH, substrate concentration, and oxygen partial pressure will be varied to determine accordance with in vivo conditions.« less

Synthesis, characterization, vibrational spectroscopy, and factor group analysis of partially metal-doped phosphate materials.

PubMed

Sronsri, Chuchai; Boonchom, Banjong

2018-04-05

A simple precipitating method was used to synthesize effectively a partially metal-doped phosphate hydrate (Mn 0.9 Mg 0.1 HPO 4 ·3H 2 O), whereas the thermal decomposition process of the above hydrate precursor was used to obtain Mn 1.8 Mg 0.2 P 2 O 7 and LiMn 0.9 Mg 0.1 PO 4 compounds under different conditions. To separate the overlapping thermal decomposition peak, a deconvolution technique was used, and the separated peak was applied to calculate the water content. The factor group splitting analysis was used to exemplify their vibrational spectra obtained from normal vibrations of HPO 4 2- , H 2 O, P 2 O 7 4- and PO 4 3- functional groups. Further, the deconvoluted bending mode of water was clearly observed. Mn 0.9 Mg 0.1 HPO 4 ·3H 2 O was observed in the orthorhombic crystal system with the space group of Pbca (D 2h 15 ). The formula units per unit cell were found to be eight (Z = 8), and the site symmetric type of HPO 4 2- was observed as C s . For the HPO 4 2- unit, the correlation filed splitting analysis of type C 3v  - C s  - D 2h 15 was calculated and had 96 internal modes, whereas H 2 O in the above hydrate was symbolized as C 2v  - C s  - D 2h 15 and had 24 modes. The symbol C 2v  - C s  - C 2h 3 was used for the correlation filed splitting analysis of P 2 O 7 4- in Mn 1.8 Mg 0.2 P 2 O 7 (monoclinic, C2/m (C 2h 3 ), Z = 2, and 42 modes). Finally, the symbol T d  - C s  - D 2h 16 was used for the correlation filed splitting analysis of PO 4 3- in LiMn 0.9 Mg 0.1 PO 4 (orthorhombic, Pnma (D 2h 16 ), Z = 4, and 36 modes). Copyright © 2018 Elsevier B.V. All rights reserved.

Engineered monoculture and co-culture of methylotrophic yeast for de novo production of monacolin J and lovastatin from methanol.

PubMed

Liu, Yiqi; Tu, Xiaohu; Xu, Qin; Bai, Chenxiao; Kong, Chuixing; Liu, Qi; Yu, Jiahui; Peng, Qiangqiang; Zhou, Xiangshan; Zhang, Yuanxing; Cai, Menghao

2018-01-01

As a promising one-carbon renewable substrate for industrial biotechnology, methanol has attracted much attention. However, engineering of microorganisms for industrial production of pharmaceuticals using a methanol substrate is still in infancy. In this study, the methylotrophic yeast Pichia pastoris was used to produce anti-hypercholesterolemia pharmaceuticals, lovastatin and its precursor monacolin J, from methanol. The biosynthetic pathways for monacolin J and lovastatin were first assembled and optimized in single strains using single copies of the relevant biosynthetic genes, and yields of 60.0mg/L monacolin J and 14.4mg/L lovastatin were obtained using methanol following pH controlled monoculture. To overcome limitations imposed by accumulation of intermediates and metabolic stress in monoculture, approaches using pathway splitting and co-culture were developed. Two pathway splitting strategies for monacolin J, and four for lovastatin were tested at different metabolic nodes. Biosynthesis of monacolin J and lovastatin was improved by 55% and 71%, respectively, when the upstream and downstream modules were separately accommodated in two different fluorescent strains, split at the metabolic node of dihydromonacolin L. However, pathway distribution at monacolin J blocked lovastatin biosynthesis in all designs, mainly due to its limited ability of crossing cellular membranes. Bioreactor fermentations were tested for the optimal co-culture strategies, and yields of 593.9mg/L monacolin J and 250.8mg/L lovastatin were achieved. This study provides an alternative method for production of monacolin J and lovastatin and reveals the potential of a methylotrophic yeast to produce complicated pharmaceuticals from methanol. Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Characterization of Central Carbon Metabolism of Streptococcus pneumoniae by Isotopologue Profiling*

PubMed Central

Härtel, Tobias; Eylert, Eva; Schulz, Christian; Petruschka, Lothar; Gierok, Philipp; Grubmüller, Stephanie; Lalk, Michael; Eisenreich, Wolfgang; Hammerschmidt, Sven

2012-01-01

The metabolism of Streptococcus pneumoniae was studied by isotopologue profiling after bacterial cultivation in chemically defined medium supplemented with [U-13C6]- or [1,2-13C2]glucose. GC/MS analysis of protein-derived amino acids showed lack of 13C label in amino acids that were also essential for pneumococcal growth. Ala, Ser, Asp, and Thr displayed high 13C enrichments, whereas Phe, Tyr, and Gly were only slightly labeled. The analysis of the labeling patterns showed formation of triose phosphate and pyruvate via the Embden-Meyerhof-Parnas pathway. The labeling patterns of Asp and Thr suggested formation of oxaloacetate exclusively via the phosphoenolpyruvate carboxylase reaction. Apparently, α-ketoglutarate was generated from unlabeled glutamate via the aspartate transaminase reaction. A fraction of Phe and Tyr obtained label via the chorismate route from erythrose 4-phosphate, generated via the pentose phosphate pathway, and phosphoenolpyruvate. Strikingly, the data revealed no significant flux from phosphoglycerate to Ser and Gly but showed formation of Ser via the reverse reaction, namely by hydroxymethylation of Gly. The essential Gly was acquired from the medium, and the biosynthesis pathway was confirmed in experiments using [U-13C2]glycine as a tracer. The hydroxymethyl group in Ser originated from formate, which was generated by the pyruvate formate-lyase. Highly similar isotopologue profiles were observed in corresponding experiments with pneumococcal mutants deficient in PavA, CodY, and glucose-6-phosphate dehydrogenase pointing to the robustness of the core metabolic network used by these facultative pathogenic bacteria. In conclusion, this study demonstrates the dual utilization of carbohydrates and amino acids under in vitro conditions and identifies the unconventional de novo biosynthesis of serine by pneumococci. PMID:22167202

Sphingosine-1-Phosphate Lyase Deficient Cells as a Tool to Study Protein Lipid Interactions

PubMed Central

Gerl, Mathias J.; Bittl, Verena; Kirchner, Susanne; Sachsenheimer, Timo; Brunner, Hanna L.; Lüchtenborg, Christian; Özbalci, Cagakan; Wiedemann, Hannah; Wegehingel, Sabine; Nickel, Walter; Haberkant, Per; Schultz, Carsten; Krüger, Marcus; Brügger, Britta

2016-01-01

Cell membranes contain hundreds to thousands of individual lipid species that are of structural importance but also specifically interact with proteins. Due to their highly controlled synthesis and role in signaling events sphingolipids are an intensely studied class of lipids. In order to investigate their metabolism and to study proteins interacting with sphingolipids, metabolic labeling based on photoactivatable sphingoid bases is the most straightforward approach. In order to monitor protein-lipid-crosslink products, sphingosine derivatives containing a reporter moiety, such as a radiolabel or a clickable group, are used. In normal cells, degradation of sphingoid bases via action of the checkpoint enzyme sphingosine-1-phosphate lyase occurs at position C2-C3 of the sphingoid base and channels the resulting hexadecenal into the glycerolipid biosynthesis pathway. In case the functionalized sphingosine looses the reporter moiety during its degradation, specificity towards sphingolipid labeling is maintained. In case degradation of a sphingosine derivative does not remove either the photoactivatable or reporter group from the resulting hexadecenal, specificity towards sphingolipid labeling can be achieved by blocking sphingosine-1-phosphate lyase activity and thus preventing sphingosine derivatives to be channeled into the sphingolipid-to-glycerolipid metabolic pathway. Here we report an approach using clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated nuclease Cas9 to create a sphingosine-1-phosphate lyase (SGPL1) HeLa knockout cell line to disrupt the sphingolipid-to-glycerolipid metabolic pathway. We found that the lipid and protein compositions as well as sphingolipid metabolism of SGPL1 knock-out HeLa cells only show little adaptations, which validates these cells as model systems to study transient protein-sphingolipid interactions. PMID:27100999

Prediction of Metabolite Concentrations, Rate Constants and Post-Translational Regulation Using Maximum Entropy-Based Simulations with Application to Central Metabolism of Neurospora crassa

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

Cannon, William; Zucker, Jeremy; Baxter, Douglas

We report the application of a recently proposed approach for modeling biological systems using a maximum entropy production rate principle in lieu of having in vivo rate constants. The method is applied in four steps: (1) a new ODE-based optimization approach based on Marcelin’s 1910 mass action equation is used to obtain the maximum entropy distribution, (2) the predicted metabolite concentrations are compared to those generally expected from experiment using a loss function from which post-translational regulation of enzymes is inferred, (3) the system is re-optimized with the inferred regulation from which rate constants are determined from the metabolite concentrationsmore » and reaction fluxes, and finally (4) a full ODE-based, mass action simulation with rate parameters and allosteric regulation is obtained. From the last step, the power characteristics and resistance of each reaction can be determined. The method is applied to the central metabolism of Neurospora crassa and the flow of material through the three competing pathways of upper glycolysis, the non-oxidative pentose phosphate pathway, and the oxidative pentose phosphate pathway are evaluated as a function of the NADP/NADPH ratio. It is predicted that regulation of phosphofructokinase (PFK) and flow through the pentose phosphate pathway are essential for preventing an extreme level of fructose 1, 6-bisphophate accumulation. Such an extreme level of fructose 1,6-bisphophate would otherwise result in a glassy cytoplasm with limited diffusion, dramatically decreasing the entropy and energy production rate and, consequently, biological competitiveness.« less

Novel method for early investigation of bioactivity in different borate bio-glasses

NASA Astrophysics Data System (ADS)

Abdelghany, A. M.

Some ternary borate glasses were prepared and corrosion behavior of such ternary borate glasses after immersion in aqueous dilute phosphate solution was studied using different immersion times. Fourier transform infrared (FTIR) absorption spectral measurements were done before and after immersion in the mentioned solution for extended times up to 2 days to justify the appearance of the characteristic FTIR bands due to calcium phosphate (hydroxyapatite (HA)) which is considered as the potential indication of bioactivity. Experimental IR data confirm the beginning of the appearance of FTIR bands at about 580 and 620 cm-1 after 3 days and the complete resolution with its characteristic split form after 1 week and more. Deconvolution analysis technique (DAT) of the FTIR spectrum was employed to investigate the bioactivity of such ternary borate system after a short period of immersion. The corrosion behavior of such glasses is explained in relation to a suggested hydrolysis followed by direct dissolution mechanism. The ease of dissolution of all the borate glasses constituents explains the formation of calcium phosphate and conversion to crystalline hydroxyapatite within the borate glass matrix. X-ray diffraction may be used to retrace the structural changes and degree of crystallinity of the prepared glasses.

A Metabolic Shift toward Pentose Phosphate Pathway Is Necessary for Amyloid Fibril- and Phorbol 12-Myristate 13-Acetate-induced Neutrophil Extracellular Trap (NET) Formation*

PubMed Central

Azevedo, Estefania P.; Rochael, Natalia C.; Guimarães-Costa, Anderson B.; de Souza-Vieira, Thiago S.; Ganilho, Juliana; Saraiva, Elvira M.; Palhano, Fernando L.; Foguel, Debora

2015-01-01

Neutrophils are the main defense cells of the innate immune system. Upon stimulation, neutrophils release their chromosomal DNA to trap and kill microorganisms and inhibit their dissemination. These chromatin traps are termed neutrophil extracellular traps (NETs) and are decorated with granular and cytoplasm proteins. NET release can be induced by several microorganism membrane components, phorbol 12-myristate 13-acetate as well as by amyloid fibrils, insoluble proteinaceous molecules associated with more than 40 different pathologies among other stimuli. The intracellular signaling involved in NET formation is complex and remains unclear for most tested stimuli. Herein we demonstrate that a metabolic shift toward the pentose phosphate pathway (PPP) is necessary for NET release because glucose-6-phosphate dehydrogenase (G6PD), an important enzyme from PPP, fuels NADPH oxidase with NADPH to produce superoxide and thus induce NETs. In addition, we observed that mitochondrial reactive oxygen species, which are NADPH-independent, are not effective in producing NETs. These data shed new light on how the PPP and glucose metabolism contributes to NET formation. PMID:26198639

Improved Xylitol Production from D-Arabitol by Enhancing the Coenzyme Regeneration Efficiency of the Pentose Phosphate Pathway in Gluconobacter oxydans.

PubMed

Li, Sha; Zhang, Jinliang; Xu, Hong; Feng, Xiaohai

2016-02-10

Gluconobacter oxydans is used to produce xylitol from D-arabitol. This study aims to improve xylitol production by increasing the coenzyme regeneration efficiency of the pentose phosphate pathway in G. oxydans. Glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) were overexpressed in G. oxydans. Real-time PCR and enzyme activity assays revealed that G6PDH/6PGDH activity and coenzyme regeneration efficiency increased in the recombinant G. oxydans strains. Approximately 29.3 g/L xylitol was obtained, with a yield of 73.2%, from 40 g/L d-arabitol in the batch biotransformation with the G. oxydans PZ strain. Moreover, the xylitol productivity (0.62 g/L/h) was 3.26-fold of the wild type strain (0.19 g/L/h). In repetitive batch biotransformation, the G. oxydans PZ cells were used for five cycles without incurring a significant loss in productivity. These results indicate that the recombinant G. oxydans PZ strain is economically feasible for xylitol production in industrial bioconversion.

Utilization of Glyphosate as Phosphate Source: Biochemistry and Genetics of Bacterial Carbon-Phosphorus Lyase

PubMed Central

Zechel, David L.; Jochimsen, Bjarne

2014-01-01

SUMMARY After several decades of use of glyphosate, the active ingredient in weed killers such as Roundup, in fields, forests, and gardens, the biochemical pathway of transformation of glyphosate phosphorus to a useful phosphorus source for microorganisms has been disclosed. Glyphosate is a member of a large group of chemicals, phosphonic acids or phosphonates, which are characterized by a carbon-phosphorus bond. This is in contrast to the general phosphorus compounds utilized and metabolized by microorganisms. Here phosphorus is found as phosphoric acid or phosphate ion, phosphoric acid esters, or phosphoric acid anhydrides. The latter compounds contain phosphorus that is bound only to oxygen. Hydrolytic, oxidative, and radical-based mechanisms for carbon-phosphorus bond cleavage have been described. This review deals with the radical-based mechanism employed by the carbon-phosphorus lyase of the carbon-phosphorus lyase pathway, which involves reactions for activation of phosphonate, carbon-phosphorus bond cleavage, and further chemical transformation before a useful phosphate ion is generated in a series of seven or eight enzyme-catalyzed reactions. The phn genes, encoding the enzymes for this pathway, are widespread among bacterial species. The processes are described with emphasis on glyphosate as a substrate. Additionally, the catabolism of glyphosate is intimately connected with that of aminomethylphosphonate, which is also treated in this review. Results of physiological and genetic analyses are combined with those of bioinformatics analyses. PMID:24600043

Acceleration of bone regeneration by activating Wnt/β-catenin signalling pathway via lithium released from lithium chloride/calcium phosphate cement in osteoporosis

NASA Astrophysics Data System (ADS)

Li, Li; Peng, Xiaozhong; Qin, Yongbao; Wang, Renchong; Tang, Jingli; Cui, Xu; Wang, Ting; Liu, Wenlong; Pan, Haobo; Li, Bing

2017-03-01

By virtue of its excellent bioactivity and osteoconductivity, calcium phosphate cement (CPC) has been applied extensively in bone engineering. Doping a trace element into CPC can change physical characteristics and enhance osteogenesis. The trace element lithium has been demonstrated to stimulate the proliferation and differentiation of osteoblasts. We investigated the fracture-healing effect of osteoporotic defects with lithium-doped calcium phosphate cement (Li/CPC) and the underlying mechanism. Li/CPC bodies immersed in simulated body fluid converted gradually to hydroxyapatite. Li/CPC extracts stimulated the proliferation and differentiation of osteoblasts upon release of lithium ions (Li+) at 25.35 ± 0.12 to 50.74 ± 0.13 mg/l through activation of the Wnt/β-catenin pathway in vitro. We also examined the effect of locally administered Li+ on defects in rat tibia between CPC and Li/CPC in vivo. Micro-computed tomography and histological staining showed that Li/CPC had better osteogenesis by increasing bone mass and promoting repair in defects compared with CPC (P < 0.05). Li/CPC also showed better osteoconductivity and osseointegration. These findings suggest that local release of Li+ from Li/CPC may accelerate bone regeneration from injury through activation of the Wnt/β-catenin pathway in osteoporosis.

Glucose metabolism and astrocyte-neuron interactions in the neonatal brain.

PubMed

Brekke, Eva; Morken, Tora Sund; Sonnewald, Ursula

2015-03-01

Glucose is essentially the sole fuel for the adult brain and the mapping of its metabolism has been extensive in the adult but not in the neonatal brain, which is believed to rely mainly on ketone bodies for energy supply. However, glucose is absolutely indispensable for normal development and recent studies have shed light on glycolysis, the pentose phosphate pathway and metabolic interactions between astrocytes and neurons in the 7-day-old rat brain. Appropriately (13)C labeled glucose was used to distinguish between glycolysis and the pentose phosphate pathway during development. Experiments using (13)C labeled acetate provided insight into the GABA-glutamate-glutamine cycle between astrocytes and neurons. It could be shown that in the neonatal brain the part of this cycle that transfers glutamine from astrocytes to neurons is operating efficiently while, in contrast, little glutamate is shuttled from neurons to astrocytes. This lack of glutamate for glutamine synthesis is compensated for by anaplerosis via increased pyruvate carboxylation relative to that in the adult brain. Furthermore, compared to adults, relatively more glucose is prioritized to the pentose phosphate pathway than glycolysis and pyruvate dehydrogenase activity. The reported developmental differences in glucose metabolism and neurotransmitter synthesis may determine the ability of the brain at various ages to resist excitotoxic insults such as hypoxia-ischemia. Copyright © 2015 Elsevier Ltd. All rights reserved.

Lipid Metabolism, Apoptosis and Cancer Therapy

PubMed Central

Huang, Chunfa; Freter, Carl

2015-01-01

Lipid metabolism is regulated by multiple signaling pathways, and generates a variety of bioactive lipid molecules. These bioactive lipid molecules known as signaling molecules, such as fatty acid, eicosanoids, diacylglycerol, phosphatidic acid, lysophophatidic acid, ceramide, sphingosine, sphingosine-1-phosphate, phosphatidylinositol-3 phosphate, and cholesterol, are involved in the activation or regulation of different signaling pathways. Lipid metabolism participates in the regulation of many cellular processes such as cell growth, proliferation, differentiation, survival, apoptosis, inflammation, motility, membrane homeostasis, chemotherapy response, and drug resistance. Bioactive lipid molecules promote apoptosis via the intrinsic pathway by modulating mitochondrial membrane permeability and activating different enzymes including caspases. In this review, we discuss recent data in the fields of lipid metabolism, lipid-mediated apoptosis, and cancer therapy. In conclusion, understanding the underlying molecular mechanism of lipid metabolism and the function of different lipid molecules could provide the basis for cancer cell death rationale, discover novel and potential targets, and develop new anticancer drugs for cancer therapy. PMID:25561239

Bioinformatics approaches for structural and functional analysis of proteins in secondary metabolism in Withania somnifera.

PubMed

Sanchita; Singh, Swati; Sharma, Ashok

2014-11-01

Withania somnifera (Ashwagandha) is an affluent storehouse of large number of pharmacologically active secondary metabolites known as withanolides. These secondary metabolites are produced by withanolide biosynthetic pathway. Very less information is available on structural and functional aspects of enzymes involved in withanolides biosynthetic pathways of Withiana somnifera. We therefore performed a bioinformatics analysis to look at functional and structural properties of these important enzymes. The pathway enzymes taken for this study were 3-Hydroxy-3-methylglutaryl coenzyme A reductase, 1-Deoxy-D-xylulose-5-phosphate synthase, 1-Deoxy-D-xylulose-5-phosphate reductase, farnesyl pyrophosphate synthase, squalene synthase, squalene epoxidase, and cycloartenol synthase. The prediction of secondary structure was performed for basic structural information. Three-dimensional structures for these enzymes were predicted. The physico-chemical properties such as pI, AI, GRAVY and instability index were also studied. The current information will provide a platform to know the structural attributes responsible for the function of these protein until experimental structures become available.

Expression of heterologous non-oxidative pentose phosphate pathway from Bacillus methanolicus and phosphoglucose isomerase deletion improves methanol assimilation and metabolite production by a synthetic Escherichia coli methylotroph

DOE PAGES

Bennett, R. Kyle; Gonzalez, Jacqueline E.; Whitaker, W. Brian; ...

2017-12-05

Synthetic methylotrophy aims to develop non-native methylotrophic microorganisms to utilize methane or methanol to produce chemicals and biofuels. We report two complimentary strategies to further engineer a previously engineered methylotrophic E. coli strain for improved methanol utilization. First, we demonstrate improved methanol assimilation in the presence of small amounts of yeast extract by expressing the non-oxidative pentose phosphate pathway (PPP) from Bacillus methanolicus. Second, we demonstrate improved co-utilization of methanol and glucose by deleting the phosphoglucose isomerase gene ( pgi), which rerouted glucose carbon flux through the oxidative PPP. Both strategies led to significant improvements in methanol assimilation as determinedmore » by 13C-labeling in intracellular metabolites. As a result, introduction of an acetone-formation pathway in the pgi-deficient methylotrophic E. coli strain led to improved methanol utilization and acetone titers during glucose fed-batch fermentation.« less

Expression of heterologous non-oxidative pentose phosphate pathway from Bacillus methanolicus and phosphoglucose isomerase deletion improves methanol assimilation and metabolite production by a synthetic Escherichia coli methylotroph

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

Bennett, R. Kyle; Gonzalez, Jacqueline E.; Whitaker, W. Brian

Synthetic methylotrophy aims to develop non-native methylotrophic microorganisms to utilize methane or methanol to produce chemicals and biofuels. We report two complimentary strategies to further engineer a previously engineered methylotrophic E. coli strain for improved methanol utilization. First, we demonstrate improved methanol assimilation in the presence of small amounts of yeast extract by expressing the non-oxidative pentose phosphate pathway (PPP) from Bacillus methanolicus. Second, we demonstrate improved co-utilization of methanol and glucose by deleting the phosphoglucose isomerase gene ( pgi), which rerouted glucose carbon flux through the oxidative PPP. Both strategies led to significant improvements in methanol assimilation as determinedmore » by 13C-labeling in intracellular metabolites. As a result, introduction of an acetone-formation pathway in the pgi-deficient methylotrophic E. coli strain led to improved methanol utilization and acetone titers during glucose fed-batch fermentation.« less

Oxidative Pentose Phosphate Pathway Inhibition Is A Key Determinant of Antimalarial Induced Cancer Cell Death

PubMed Central

Salas, Eduardo; Roy, Srirupa; Marsh, Timothy; Rubin, Brian; Debnath, Jayanta

2015-01-01

Despite immense interest in employing antimalarials as autophagy inhibitors to treat cancer, it remains unclear if these agents act predominantly via autophagy inhibition or whether other pathways direct their anti-cancer properties. By comparing the treatment effects of the antimalarials chloroquine (CQ) and quinacrine (Q) on KRAS mutant lung cancer cells, we demonstrate that inhibition of the oxidative arm of the pentose phosphate pathway (oxPPP) is required for antimalarial induced apoptosis. Despite inhibiting autophagy, neither CQ treatment nor RNAi against autophagy regulators (ATGs) promote cell death. In contrast, Q triggers high levels of apoptosis, both in vitro and in vivo, and this phenotype requires both autophagy inhibition and p53-dependent inhibition of the oxPPP. Simultaneous genetic targeting of the oxPPP and autophagy is sufficient to trigger apoptosis in lung cancer cells, including cells lacking p53. Thus, in addition to reduced autophagy, oxPPP inhibition serves as an important determinant of antimalarial cytotoxicity in cancer cells. PMID:26434592

Cytidine derivatives as IspF inhibitors of Burkolderia pseudomallei

PubMed Central

Zhang, Zheng; Jakkaraju, Sriram; Blain, Joy; Gogol, Kenneth; Zhao, Lei; Hartley, Robert C.; Karlsson, Courtney A.; Staker, Bart L.; Stewart, Lance J.; Myler, Peter J.; Clare, Michael; Begley, Darren W.; Horn, James R.; Hagen, Timothy J

2013-01-01

Published biological data suggest that the methyl erythritol phosphate (MEP) pathway, a non-mevalonate isoprenoid biosynthetic pathway, is essential for certain bacteria and other infectious disease organisms. One highly conserved enzyme in the MEP pathway is 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF). Fragment-bound complexes of IspF from Burkholderia pseudomallei were used to design and synthesize a series of molecules linking the cytidine moiety to different zinc pocket fragment binders. Testing by surface plasmon resonance (SPR) found one molecule in the series to possess binding affinity equal to that of cytidine diphosphate, despite lacking any metal-coordinating phosphate groups. Close inspection of the SPR data suggest different binding stoichiometries between IspF and test compounds. Crystallographic analysis shows important variations between the binding mode of one synthesized compound and the pose of the bound fragment from which it was designed. The binding modes of these molecules add to our structural knowledge base for IspF and suggest future refinements in this compound series. PMID:24157367

Fibroblast growth factor-23 increases mouse PGE2 production in vivo and in vitro.

PubMed

Syal, Ashu; Schiavi, Susan; Chakravarty, Sumana; Dwarakanath, Vangipuram; Quigley, Raymond; Baum, Michel

2006-02-01

Fibroblast growth factor-23 (FGF-23) has been implicated in the renal phosphate wasting in X-linked hypophosphatemia, tumor-induced osteomalacia, and autosomal dominant hypophosphatemic rickets. Recently, we demonstrated that Hyp mice have greater urinary PGE2 levels compared with C57/B6 mice and that indomethacin administration in vivo and in vitro ameliorates the phosphate transport defect in Hyp mice. To determine further whether altered prostaglandin metabolism plays a role in the renal phosphate transport defect in Hyp mice, we incubated renal proximal tubules with arachidonic acid. We find that PGE2 production was higher in Hyp mice than in C57/B6 mice. Incubation of C57/B6 mouse renal proximal tubules with FGF-23R176Q, an active mutant form of FGR23, increased tubular PGE2 production, an effect that was inhibited by 50 microM PD-98059 and 10 microM SB-203580, inhibitors of the MAP kinase pathway. C57/B6 mice injected with FGF-23R176Q had a approximately 10-fold increase in PGE2 excretion 24 h after intraperitoneal injection of FGF-23R176Q compared with vehicle-treated controls. Finally, we show that PGE2 inhibited both phosphate and volume absorption in mouse proximal convoluted tubules perfused in vitro and reduced brush-border membrane vesicle NaPi-2a protein abundance from renal cortex incubated in vitro with PGE2. In conclusion, FGF-23 increases urinary and renal tubular PGE2 production via the MAP kinase pathway and PGE2 inhibits proximal tubule phosphate transport.

Fibroblast growth factor-23 increases mouse PGE2 production in vivo and in vitro

PubMed Central

Syal, Ashu; Schiavi, Susan; Chakravarty, Sumana; Dwarakanath, Vangipuram; Quigley, Raymond; Baum, Michel

2014-01-01

Fibroblast growth factor-23 (FGF-23) has been implicated in the renal phosphate wasting in X-linked hypophosphatemia, tumor-induced osteomalacia, and autosomal dominant hypophosphatemic rickets. Recently, we demonstrated that Hyp mice have greater urinary PGE2 levels compared with C57/B6 mice and that indomethacin administration in vivo and in vitro ameliorates the phosphate transport defect in Hyp mice. To determine further whether altered prostaglandin metabolism plays a role in the renal phosphate transport defect in Hyp mice, we incubated renal proximal tubules with arachidonic acid. We find that PGE2 production was higher in Hyp mice than in C57/B6 mice. Incubation of C57/B6 mouse renal proximal tubules with FGF-23R176Q, an active mutant form of FGR23, increased tubular PGE2 production, an effect that was inhibited by 50 μM PD-98059 and 10 μM SB-203580, inhibitors of the MAP kinase pathway. C57/B6 mice injected with FGF-23R176Q had a ~10-fold increase in PGE2 excretion 24 h after intraperitoneal injection of FGF-23R176Q compared with vehicle-treated controls. Finally, we show that PGE2 inhibited both phosphate and volume absorption in mouse proximal convoluted tubules perfused in vitro and reduced brush-border membrane vesicle NaPi-2a protein abundance from renal cortex incubated in vitro with PGE2. In conclusion, FGF-23 increases urinary and renal tubular PGE2 production via the MAP kinase pathway and PGE2 inhibits proximal tubule phosphate transport. PMID:16144964

Stability and migration of large oxygen clusters in UO(2+x): density functional theory calculations.

PubMed

Andersson, D A; Espinosa-Faller, F J; Uberuaga, B P; Conradson, S D

2012-06-21

Using ab initio molecular dynamics simulations and nudged elastic band calculations we examine the finite temperature stability, transition pathways, and migration mechanisms of large oxygen clusters in UO(2+x). Here we specifically consider the recently proposed split quad-interstitial and cuboctahedral oxygen clusters. It is shown that isolated cuboctahedral clusters may transform into more stable configurations that are closely linked to the split quad-interstitial. The split quad-interstitial is stable with respect to single interstitials occupying the empty octahedral holes of the UO(2) lattice. In order to better understand discrepancies between theory and experiments, the simulated atomic pair distribution functions for the split quad-interstitial structures are analyzed with respect to the distribution function for U(4)O(9) previously obtained from neutron diffraction data. Our nudged elastic band calculations suggest that the split quad-interstitial may migrate by translating one of its constituent di-interstitial clusters via a barrier that is lower than the corresponding barrier for individual interstitials, but higher than the barrier for the most stable di-interstitial cluster.

Synthetic Routes to Methylerythritol Phosphate Pathway Intermediates and Downstream Isoprenoids

PubMed Central

Jarchow-Choy, Sarah K; Koppisch, Andrew T; Fox, David T

2014-01-01

Isoprenoids constitute the largest class of natural products with greater than 55,000 identified members. They play essential roles in maintaining proper cellular function leading to maintenance of human health, plant defense mechanisms against predators, and are often exploited for their beneficial properties in the pharmaceutical and nutraceutical industries. Most impressively, all known isoprenoids are derived from one of two C5-precursors, isopentenyl diphosphate (IPP) or dimethylallyl diphosphate (DMAPP). In order to study the enzyme transformations leading to the extensive structural diversity found within this class of compounds there must be access to the substrates. Sometimes, intermediates within a biological pathway can be isolated and used directly to study enzyme/pathway function. However, the primary route to most of the isoprenoid intermediates is through chemical catalysis. As such, this review provides the first exhaustive examination of synthetic routes to isoprenoid and isoprenoid precursors with particular emphasis on the syntheses of intermediates found as part of the 2C-methylerythritol 4-phosphate (MEP) pathway. In addition, representative syntheses are presented for the monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20), triterpenes (C30) and tetraterpenes (C40). Finally, in some instances, the synthetic routes to substrate analogs found both within the MEP pathway and downstream isoprenoids are examined. PMID:25009443

The Sjögren-Larsson syndrome gene encodes a hexadecenal dehydrogenase of the sphingosine 1-phosphate degradation pathway.

PubMed

Nakahara, Kanae; Ohkuni, Aya; Kitamura, Takuya; Abe, Kensuke; Naganuma, Tatsuro; Ohno, Yusuke; Zoeller, Raphael A; Kihara, Akio

2012-05-25

Sphingosine 1-phosphate (S1P) functions not only as a bioactive lipid molecule, but also as an important intermediate of the sole sphingolipid-to-glycerolipid metabolic pathway. However, the precise reactions and the enzymes involved in this pathway remain unresolved. We report here that yeast HFD1 and the Sjögren-Larsson syndrome (SLS)-causative mammalian gene ALDH3A2 are responsible for conversion of the S1P degradation product hexadecenal to hexadecenoic acid. The absence of ALDH3A2 in CHO-K1 mutant cells caused abnormal metabolism of S1P/hexadecenal to ether-linked glycerolipids. Moreover, we demonstrate that yeast Faa1 and Faa4 and mammalian ACSL family members are acyl-CoA synthetases involved in the sphingolipid-to-glycerolipid metabolic pathway and that hexadecenoic acid accumulates in Δfaa1 Δfaa4 mutant cells. These results unveil the entire S1P metabolic pathway: S1P is metabolized to glycerolipids via hexadecenal, hexadecenoic acid, hexadecenoyl-CoA, and palmitoyl-CoA. From our results we propose a possibility that accumulation of the S1P metabolite hexadecenal contributes to the pathogenesis of SLS. Copyright © 2012 Elsevier Inc. All rights reserved.

Dual Functions of the Trans-2-Enoyl-CoA Reductase TER in the Sphingosine 1-Phosphate Metabolic Pathway and in Fatty Acid Elongation*

PubMed Central

Wakashima, Takeshi; Abe, Kensuke; Kihara, Akio

2014-01-01

The sphingolipid metabolite sphingosine 1-phosphate (S1P) functions as a lipid mediator and as a key intermediate of the sole sphingolipid to glycerophospholipid metabolic pathway (S1P metabolic pathway). In this pathway, S1P is converted to palmitoyl-CoA through 4 reactions, then incorporated mainly into glycerophospholipids. Although most of the genes responsible for the S1P metabolic pathway have been identified, the gene encoding the trans-2-enoyl-CoA reductase, responsible for the saturation step (conversion of trans-2-hexadecenoyl-CoA to palmitoyl-CoA) remains unidentified. In the present study, we show that TER is the missing gene in mammals using analyses involving yeast cells, deleting the TER homolog TSC13, and TER-knockdown HeLa cells. TER is known to be involved in the production of very long-chain fatty acids (VLCFAs). A significant proportion of the saturated and monounsaturated VLCFAs are used for sphingolipid synthesis. Therefore, TER is involved in both the production of VLCFAs used in the fatty acid moiety of sphingolipids as well as in the degradation of the sphingosine moiety of sphingolipids via S1P. PMID:25049234

Gluconeogenesis in Leishmania mexicana: contribution of glycerol kinase, phosphoenolpyruvate carboxykinase, and pyruvate phosphate dikinase.

PubMed

Rodriguez-Contreras, Dayana; Hamilton, Nicklas

2014-11-21

Gluconeogenesis is an active pathway in Leishmania amastigotes and is essential for their survival within the mammalian cells. However, our knowledge about this pathway in trypanosomatids is very limited. We investigated the role of glycerol kinase (GK), phosphoenolpyruvate carboxykinase (PEPCK), and pyruvate phosphate dikinase (PPDK) in gluconeogenesis by generating the respective Leishmania mexicana Δgk, Δpepck, and Δppdk null mutants. Our results demonstrated that indeed GK, PEPCK, and PPDK are key players in the gluconeogenesis pathway in Leishmania, although stage-specific differences in their contribution to this pathway were found. GK participates in the entry of glycerol in promastigotes and amastigotes; PEPCK participates in the entry of aspartate in promastigotes, and PPDK is involved in the entry of alanine in amastigotes. Furthermore, the majority of alanine enters into the pathway via decarboxylation of pyruvate in promastigotes, whereas pathway redundancy is suggested for the entry of aspartate in amastigotes. Interestingly, we also found that l-lactate, an abundant glucogenic precursor in mammals, was used by Leishmania amastigotes to synthesize mannogen, entering the pathway through PPDK. On the basis of these new results, we propose a revision in the current model of gluconeogenesis in Leishmania, emphasizing the differences between amastigotes and promastigotes. This work underlines the importance of studying the trypanosomatid intracellular life cycle stages to gain a better understanding of the pathologies caused in humans. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

The pattern of shikimate pathway and phenylpropanoids after inhibition by glyphosate or quinate feeding in pea roots.

PubMed

Zabalza, Ana; Orcaray, Luis; Fernández-Escalada, Manuel; Zulet-González, Ainhoa; Royuela, Mercedes

2017-09-01

The shikimate pathway is a metabolic route for the biosynthesis of aromatic amino acids (AAAs) (i.e. phenylalanine, tyrosine, and tryptophan). A key enzyme of shikimate pathway (5-enolpyruvylshikimate-3-phosphate synthase, EPSPS) is the target of the widely used herbicide glyphosate. Quinate is a compound synthesized in plants through a side branch of the shikimate pathway. Glyphosate provokes quinate accumulation and exogenous quinate application to plants shows a potential role of quinate in the toxicity of the herbicide glyphosate. Based on this, we hypothesized that the role of quinate accumulation in the toxicity of the glyphosate would be mediated by a deregulation of the shikimate pathway. In this study the effect of the glyphosate and of the exogenous quinate was evaluated in roots of pea plants by analyzing the time course of a full metabolic map of several metabolites of shikimate and phenylpropanoid pathways. Glyphosate application induced an increase of the 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (DAHPS, first enzyme of the shikimate pathway) protein and accumulation of metabolites upstream of the enzyme EPSPS. No common effects on the metabolites and regulation of shikimate pathway were detected between quinate and glyphosate treatments, supporting that the importance of quinate in the mode of action of glyphosate is not mediated by a common alteration of the regulation of the shikimate pathway. Contrary to glyphosate, the exogenous quinate supplied was probably incorporated into the main trunk from the branch pathway and accumulated in the final products, such as lignin, concomitant with a decrease in the amount of DAHPS protein. Copyright © 2016 Elsevier B.V. All rights reserved.

Enhanced performance of the methylerythritol phosphate pathway by manipulation of redox reactions relevant to IspC, IspG, and IspH.

PubMed

Zhou, Jia; Yang, Liyang; Wang, Chonglong; Choi, Eui-Sung; Kim, Seon-Won

2017-04-20

The 2C-methyl-D-erythritol 4-phosphate (MEP) pathway is a carbon-efficient route for synthesis of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), the building blocks of isoprenoids. However, practical application of a native or recombinant MEP pathway for the mass production of isoprenoids in Escherichia coli has been unsatisfactory. In this study, the entire recombinant MEP pathway was established with plasmids and used for the production of an isoprenoid, protoilludene. E. coli harboring the recombinant MEP pathway plasmid (ME) and a protoilludene synthesis pathway plasmid (AO) produced 10.4mg/L of protoilludene after 48h of culture. To determine the rate-limiting gene on plasmid ME, each constituent gene of the MEP pathway was additionally overexpressed on the plasmid AO. The additional overexpression of IPP isomerase (IDI) enhanced protoilludene production to 67.4mg/L. Overexpression of the Fpr and FldA protein complex, which could mediate electron transfer from NADPH to Fe-S cluster proteins such as IspG and IspH of the MEP pathway, increased protoilludene production to 318.8mg/L. Given that it is required for IspC as well as IspG/H, the MEP pathway has high demand for NADPH. To increase the supply of NADPH, a NADH kinase from Saccharomyces cerevisiae (tPos5p) that converts NADH to NADPH was introduced along with the deletion of a promiscuous NADPH-dependent aldehyde reductase (YjgB) that consumes NADPH. This resulted in a protoilludene production of 512.7mg/L. The results indicate that IDI, Fpr-FldA redox proteins, and NADPH regenerators are key engineering points for boosting the metabolic flux toward a recombinant MEP pathway. Copyright © 2017 Elsevier B.V. All rights reserved.

Utilization of d-Ribitol by Lactobacillus casei BL23 Requires a Mannose-Type Phosphotransferase System and Three Catabolic Enzymes

PubMed Central

Bourand, A.; Yebra, M. J.; Boël, G.; Mazé, A.

2013-01-01

Lactobacillus casei strains 64H and BL23, but not ATCC 334, are able to ferment d-ribitol (also called d-adonitol). However, a BL23-derived ptsI mutant lacking enzyme I of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) was not able to utilize this pentitol, suggesting that strain BL23 transports and phosphorylates d-ribitol via a PTS. We identified an 11-kb region in the genome sequence of L. casei strain BL23 (LCABL_29160 to LCABL_29270) which is absent from strain ATCC 334 and which contains the genes for a GlpR/IolR-like repressor, the four components of a mannose-type PTS, and six metabolic enzymes potentially involved in d-ribitol metabolism. Deletion of the gene encoding the EIIB component of the presumed ribitol PTS indeed prevented d-ribitol fermentation. In addition, we overexpressed the six catabolic genes, purified the encoded enzymes, and determined the activities of four of them. They encode a d-ribitol-5-phosphate (d-ribitol-5-P) 2-dehydrogenase, a d-ribulose-5-P 3-epimerase, a d-ribose-5-P isomerase, and a d-xylulose-5-P phosphoketolase. In the first catabolic step, the protein d-ribitol-5-P 2-dehydrogenase uses NAD+ to oxidize d-ribitol-5-P formed during PTS-catalyzed transport to d-ribulose-5-P, which, in turn, is converted to d-xylulose-5-P by the enzyme d-ribulose-5-P 3-epimerase. Finally, the resulting d-xylulose-5-P is split by d-xylulose-5-P phosphoketolase in an inorganic phosphate-requiring reaction into acetylphosphate and the glycolytic intermediate d-glyceraldehyde-3-P. The three remaining enzymes, one of which was identified as d-ribose-5-P-isomerase, probably catalyze an alternative ribitol degradation pathway, which might be functional in L. casei strain 64H but not in BL23, because one of the BL23 genes carries a frameshift mutation. PMID:23564164

The Mechanism of Starch Over-Accumulation in Chlamydomonas reinhardtii High-Starch Mutants Identified by Comparative Transcriptome Analysis

PubMed Central

Koo, Kwang M.; Jung, Sera; Lee, Beom S.; Kim, Jin-Baek; Jo, Yeong D.; Choi, Hong-Il; Kang, Si-Yong; Chung, Gook-H.; Jeong, Won-Joong; Ahn, Joon-Woo

2017-01-01

The focus of this study was the mechanism of starch accumulation in Chlamydomonas reinhardtii high-starch mutants. Three C. reinhardtii mutants showing high-starch content were generated using gamma irradiation. When grown under nitrogen-deficient conditions, these mutants had more than twice as much starch than a wild-type control. The mechanism of starch over-accumulation in these mutants was studied with comparative transcriptome analysis. In all mutants, induction of phosphoglucomutase 1 (PGM1) expression was detected; PGM1 catalyzes the inter-conversion of glucose 1-phosphate and glucose 6-phosphate in both starch biosynthetic and glycolytic pathway. Interestingly, transcript levels of phosphoglucose isomerase 1 (PGI1), fructose 1,6-bisphosphate aldolase 1 and 2 (FBA1 and FBA2) were down-regulated in all mutants; PGI1, FBA1, and FBA2 act on downstream of glucose 6-phosphate conversion in glycolytic pathway. Therefore, down-regulations of PGI1, FBA1, and FBA2 may lead to accumulation of upstream metabolites, notably glucose 6-phosphate, resulting in induction of PGM1 expression through feed-forward regulation and that PGM1 overexpression caused starch over-accumulation in mutants. These results suggest that PGI1, FBA1, FBA2, and PGM1 correlate with each other in terms of coordinated transcriptional regulation and play central roles for starch over-accumulation in C. reinhardtii. PMID:28588557

Proteomics analysis of high lipid-producing strain Mucor circinelloides WJ11: an explanation for the mechanism of lipid accumulation at the proteomic level.

PubMed

Tang, Xin; Zan, Xinyi; Zhao, Lina; Chen, Haiqin; Chen, Yong Q; Chen, Wei; Song, Yuanda; Ratledge, Colin

2016-02-11

The oleaginous fungus, Mucor circinelloides, is attracting considerable interest as it produces oil rich in γ-linolenic acid. Nitrogen (N) deficiency is a common strategy to trigger the lipid accumulation in oleaginous microorganisms. Although a simple pathway from N depletion in the medium to lipid accumulation has been elucidated at the enzymatic level, global changes at protein levels upon N depletion have not been investigated. In this study, we have systematically analyzed the changes at the levels of protein expression in M. circinelloides WJ11, a high lipid-producing strain (36 %, lipid/cell dry weight), during lipid accumulation. Proteomic analysis demonstrated that N depletion increased the expression of glutamine synthetase, involved in ammonia assimilation, for the supply of cellular nitrogen but decreased the metabolism of amino acids. Upon N deficiency, many proteins (e.g., fructose-bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, enolase, pyruvate kinase) involved in glycolytic pathway were up-regulated while proteins involved in the tricarboxylic acid cycle (e.g., isocitrate dehydrogenase, succinyl-CoA ligase, succinate dehydrogenase, fumarate hydratase) were down-regulated, indicating this activity was retarded thereby leading to a greater flux of carbon into fatty acid biosynthesis. Moreover, glucose-6-phosphate dehydrogenase, transaldolase and transketolase, which participate in the pentose phosphate pathway, were up-regulated, leading to the increased production of NADPH, the reducing power for fatty acid biosynthesis. Furthermore, protein and nucleic acid metabolism were down-regulated and some proteins involved in energy metabolism, signal transduction, molecular chaperone and redox homeostasis were up-regulated upon N depletion, which may be the cellular response to the stress produced by the onset of N deficiency. N limitation increased those expressions of the proteins involved in ammonia assimilation but decreased that involved in the biosynthesis of amino acids. Upon N deprivation, the glycolytic pathway was up-regulated, while the activity of the tricarboxylic acid cycle was retarded, thus, leading more carbon flux to fatty acid biosynthesis. Moreover, the pentose phosphate pathway was up-regulated, then this would increase the production of NADPH. Together, coordinated regulation of central carbon metabolism upon N limitation, provides more carbon flux to acetyl-CoA and NADPH for fatty acid biosynthesis.

Central Role of the Trehalose Biosynthesis Pathway in the Pathogenesis of Human Fungal Infections: Opportunities and Challenges for Therapeutic Development

PubMed Central

Thammahong, Arsa; Puttikamonkul, Srisombat; Perfect, John R.; Brennan, Richard G.

2017-01-01

SUMMARY Invasive fungal infections cause significant morbidity and mortality in part due to a limited antifungal drug arsenal. One therapeutic challenge faced by clinicians is the significant host toxicity associated with antifungal drugs. Another challenge is the fungistatic mechanism of action of some drugs. Consequently, the identification of fungus-specific drug targets essential for fitness in vivo remains a significant goal of medical mycology research. The trehalose biosynthetic pathway is found in a wide variety of organisms, including human-pathogenic fungi, but not in humans. Genes encoding proteins involved in trehalose biosynthesis are mechanistically linked to the metabolism, cell wall homeostasis, stress responses, and virulence of Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus. While there are a number of pathways for trehalose production across the tree of life, the TPS/TPP (trehalose-6-phosphate synthase/trehalose-6-phosphate phosphatase) pathway is the canonical pathway found in human-pathogenic fungi. Importantly, data suggest that proteins involved in trehalose biosynthesis play other critical roles in fungal metabolism and in vivo fitness that remain to be fully elucidated. By further defining the biology and functions of trehalose and its biosynthetic pathway components in pathogenic fungi, an opportunity exists to leverage this pathway as a potent antifungal drug target. The goal of this review is to cover the known roles of this important molecule and its associated biosynthesis-encoding genes in the human-pathogenic fungi studied to date and to employ these data to critically assess the opportunities and challenges facing development of this pathway as a therapeutic target. PMID:28298477

BIOCHEMICAL AND GENETIC CHARACTERIZATION OF AN EARLY STEP IN A NOVEL PATHWAY FOR THE BIOSYNTHESIS OF AROMATIC AMINO ACIDS AND P-AMINOBENZOIC ACID IN THE ARCHAEON METHANOCOCCUS MARIPALUDIS

EPA Science Inventory

Methanococcus maripaludis is a strictly anaerobic, methane-producing archaeon and facultative autotroph capable of biosynthesizing all the amino acids and vitamins required for growth. In this work, the novel 6-deoxy-5-ketofructose-1-phosphate (DKFP) pathway for the biosynthesis ...

Traffic to the malaria parasite food vacuole: a novel pathway involving a phosphatidylinositol 3-phosphate-binding protein.

PubMed

McIntosh, Michael T; Vaid, Ankush; Hosgood, H Dean; Vijay, Justin; Bhattacharya, Anindita; Sahani, Mayurbhai H; Baevova, Pavlina; Joiner, Keith A; Sharma, Pushkar

2007-04-13

Phosphatidylinositol 3-phosphate (PI3P) is a key ligand for recruitment of endosomal regulatory proteins in higher eukaryotes. Subsets of these endosomal proteins possess a highly selective PI3P binding zinc finger motif belonging to the FYVE domain family. We have identified a single FYVE domain-containing protein in Plasmodium falciparum which we term FCP. Expression and mutagenesis studies demonstrate that key residues are involved in specific binding to PI3P. In contrast to FYVE proteins in other organisms, endogenous FCP localizes to a lysosomal compartment, the malaria parasite food vacuole (FV), rather than to cytoplasmic endocytic organelles. Transfections of deletion mutants further indicate that FCP is essential for trophozoite and FV maturation and that it traffics to the FV via a novel constitutive cytoplasmic to vacuole targeting pathway. This newly discovered pathway excludes the secretory pathway and is directed by a C-terminal 44-amino acid peptide domain. We conclude that an FYVE protein that might be expected to participate in vesicle targeting in the parasite cytosol instead has a vital and functional role in the malaria parasite FV.

The Split Virus Influenza Vaccine rapidly activates immune cells through Fcγ receptors.

PubMed

O'Gorman, William E; Huang, Huang; Wei, Yu-Ling; Davis, Kara L; Leipold, Michael D; Bendall, Sean C; Kidd, Brian A; Dekker, Cornelia L; Maecker, Holden T; Chien, Yueh-Hsiu; Davis, Mark M

2014-10-14

Seasonal influenza vaccination is one of the most common medical procedures and yet the extent to which it activates the immune system beyond inducing antibody production is not well understood. In the United States, the most prevalent formulations of the vaccine consist of degraded or "split" viral particles distributed without any adjuvants. Based on previous reports we sought to determine whether the split influenza vaccine activates innate immune receptors-specifically Toll-like receptors. High-dimensional proteomic profiling of human whole-blood using Cytometry by Time-of-Flight (CyTOF) was used to compare signaling pathway activation and cytokine production between the split influenza vaccine and a prototypical TLR response ex vivo. This analysis revealed that the split vaccine rapidly and potently activates multiple immune cell types but yields a proteomic signature quite distinct from TLR activation. Importantly, vaccine induced activity was dependent upon the presence of human sera indicating that a serum factor was necessary for vaccine-dependent immune activation. We found this serum factor to be human antibodies specific for influenza proteins and therefore immediate immune activation by the split vaccine is immune-complex dependent. These studies demonstrate that influenza virus "splitting" inactivates any potential adjuvants endogenous to influenza, such as RNA, but in previously exposed individuals can elicit a potent immune response by facilitating the rapid formation of immune complexes. Copyright © 2014 Elsevier Ltd. All rights reserved.

Biosynthesis of rare ketoses through constructing a recombination pathway in an engineered Corynebacterium glutamicum.

PubMed

Yang, Jiangang; Zhu, Yueming; Li, Jitao; Men, Yan; Sun, Yuanxia; Ma, Yanhe

2015-01-01

Rare sugars have various known biological functions and potential for applications in pharmaceutical, cosmetics, and food industries. Here we designed and constructed a recombination pathway in Corynebacterium glutamicum, in which dihydroxyacetone phosphate (DHAP), an intermediate of the glycolytic pathway, and a variety of aldehydes were condensed to synthesize rare ketoses sequentially by rhamnulose-1-phosphate aldolase (RhaD) and fructose-1-phosphatase (YqaB) obtained from Escherichia coli. A wild-type strain harboring this artificial pathway had the ability to produce D-sorbose and D-psicose using D-glyceraldehyde and glucose as the substrates. The tpi gene, encoding triose phosphate isomerase was further deleted, and the concentration of DHAP increased to nearly 20-fold relative to that of the wild-type. After additional optimization of expression levels from rhaD and yqaB genes and of the fermentation conditions, the engineered strain SY6(pVRTY) exhibited preferable performance for rare ketoses production. Its yield increased to 0.59 mol/mol D-glyceraldehyde from 0.33 mol/mol D-glyceraldehyde and productivity to 2.35 g/L h from 0.58 g/L h. Moreover, this strain accumulated 19.5 g/L of D-sorbose and 13.4 g/L of D-psicose using a fed-batch culture mode under the optimal conditions. In addition, it was verified that the strain SY6(pVRTY) meanwhile had the ability to synthesize C4, C5, C6, and C7 rare ketoses when a range of representative achiral and homochiral aldehydes were applied as the substrates. Therefore, the platform strain exhibited the potential for microbial production of rare ketoses and deoxysugars. © 2014 Wiley Periodicals, Inc.

Fe{sub 2.5}[BP{sub 2}O{sub 7}(OH){sub 2}][PO{sub 3}(OH)][PO{sub 3}(O{sub 0.5}OH{sub 0.5})] · H{sub 2}O, a new phosphate-borophosphate with a microporous structure

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

Belokoneva, E. L., E-mail: elbel@geol.msu.ru; Dimitrova, O. V.

2015-05-15

A new phosphate-borophosphate Fe{sub 2.5}{sup 3+}[BP{sub 2}O{sub 7}(OH){sub 2}][PO{sub 3}(OH)][PO{sub 3}(O{sub 0.5}OH{sub 0.5})] · H{sub 2}O, space group P12{sub 1}/n, is obtained under hydrothermal conditions. Blocks (Fe{sup 3+}(PO{sub 4}){sub 6}){sup 15−} consisting of vertex-sharing (Fe1,Fe2)O{sub 6} octahedra and six PO{sub 4} tetrahedra are distinguished in the structure of the new phase, which was determined without preliminary knowledge of the chemical formula. Such blocks are known for many phosphates (borophosphates), germanates, gallates, and silicates. Blocks form layers connected by BO{sub 4} tetrahedra into a framework with large pores reaching ∼9.5 Å, which are occupied by water molecules. The out-of-layer octahedral positionmore » of the Fe3 atom is split and occupied statistically. The anion radical is characterized as a phosphate-borophosphate: it consists of two isolated PO4 tetrahedra and a borophosphate soro group [BP{sub 2}O{sub 7}(OH){sub 2}] first found in NaIn[BP{sub 2}O{sub 8}(OH)]. A layer of octahedra is characterized by higher local symmetry corresponding to the orthorhombic group Pm2{sub 1}n.« less

Mixed chimerism and split tolerance

PubMed Central

Al-Adra, David P.

2011-01-01

Establishing hematopoietic mixed chimerism can lead to donor-specific tolerance to transplanted organs and may eliminate the need for long-term immunosuppressive therapy, while also preventing chronic rejection. In this review, we discuss central and peripheral mechanisms of chimerism induced tolerance. However, even in the long-lasting presence of a donor organ or donor hematopoietic cells, some allogeneic tissues from the same donor can be rejected; a phenomenon known as split tolerance. With the current goal of creating mixed chimeras using clinically feasible amounts of donor bone marrow and with minimal conditioning, split tolerance may become more prevalent and its mechanisms need to be explored. Some predisposing factors that may increase the likelihood of split tolerance are immunogenicity of the graft, certain donor-recipient combinations, prior sensitization, location and type of graft and minimal conditioning chimerism induction protocols. Additionally, split tolerance may occur due to a differential susceptibility of various types of tissues to rejection. The mechanisms involved in a tissue’s differential susceptibility to rejection include the presence of polymorphic tissue-specific antigens and variable sensitivity to indirect pathway effector mechanisms. Finally, we review the clinical attempts at allograft tolerance through the induction of chimerism; studies that are revealing the complex relationship between chimerism and tolerance. This relationship often displays split tolerance, and further research into its mechanisms is warranted. PMID:22509425

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

Dernotte, Jeremie; Dec, John E.; Ji, Chunsheng

A detailed understanding of the various factors affecting the trends in gross-indicated thermal efficiency with changes in key operating parameters has been carried out, applied to a one-liter displacement single-cylinder boosted Low-Temperature Gasoline Combustion (LTGC) engine. This work systematically investigates how the supplied fuel energy splits into the following four energy pathways: gross-indicated thermal efficiency, combustion inefficiency, heat transfer and exhaust losses, and how this split changes with operating conditions. Additional analysis is performed to determine the influence of variations in the ratio of specific heat capacities (γ) and the effective expansion ratio, related to the combustion-phasing retard (CA50), onmore » the energy split. Heat transfer and exhaust losses are computed using multiple standard cycle analysis techniques. Furthermore, the various methods are evaluated in order to validate the trends.« less

Lipopolysaccharide and Aldoheptose Biosynthesis in Transketolase Mutants of Salmonella typhimurium

PubMed Central

Eidels, L.; Osborn, M. J.

1971-01-01

Genetic and biochemical evidence that sedoheptulose-7-phosphate is an obligatory precursor of the L-glycero-D-mannoheptose residues of the lipopolysaccharide of Salmonella was obtained by isolation and characterization of transketolase-negative mutants of Salmonella typhimurium. These mutants, which are defective in synthesis of sedoheptulose-7-phosphate, were found to produce an incomplete heptose-deficient lipopolysaccharide, and were also sensitive to bile salts, a characteristic property of heptose-deficient mutants. Phenotypic repair of the defect in lipopolysaccharide synthesis was obtained by addition of exogenous sedoheptulose-7-phosphate to growing cultures of the mutant strains. Characterization of revertants isolated either as transketolase-positive or heptose-positive provided further evidence that the heptose deficiency resulted from mutation at the transketolase locus. On the basis of these findings a possible pathway for conversion of sedoheptulose-7-phosphate to L-glycero-D-mannoheptose is proposed. PMID:4942911

Effect of Enzyme Inhibitors on Terpene Trilactones Biosynthesis and Gene Expression Profiling in Ginkgo biloba Cultured Cells.

PubMed

Chen, Lijia; Tong, Hui; Wang, Mingxuan; Zhu, Jianhua; Zi, Jiachen; Song, Liyan; Yu, Rongmin

2015-12-01

The biosynthetic pathway of terpene trilactones of Ginkgo biloba is unclear. In this present study, suspension cultured cells of G. biloba were used to explore the regulation of the mevalonic acid (MVA) and methylerythritol 4-phosphate (MEP) pathways in response to specific enzyme inhibitors (lovastatin and clomazone). The results showed that the biosynthesis of bilobalide was more highly correlated with the MVA pathway, and the biosynthesis of ginkgolides was more highly correlated with the MEP pathway. Meanwhile, according to the results, it could be speculated that bilobalide might be a product of ginkgolide metabolism.

System-Wide Adaptations of Desulfovibrio alaskensis G20 to Phosphate-Limited Conditions

DOE PAGES

Bosak, Tanja; Schubotz, Florence; de Santiago-Torio, Ana; ...

2016-12-28

The prevalence of lipids devoid of phosphorus suggests that the availability of phosphorus limits microbial growth and activity in many anoxic, stratified environments. To better understand the response of anaerobic bacteria to phosphate limitation and starvation, this study combines microscopic and lipid analyses with the measurements of fitness of pooled barcoded transposon mutants of the model sulfate reducing bacterium Desulfovibrio alaskensis G20. Phosphate-limited G20 has lower growth rates and replaces more than 90% of its membrane phospholipids by a mixture of monoglycosyl diacylglycerol (MGDG), glycuronic acid diacylglycerol (GADG) and ornithine lipids, lacks polyphosphate granules, and synthesizes other cellular inclusions. Analysesmore » of pooled and individual mutants reveal the importance of the high-affinity phosphate transport system (the Pst system), PhoR, and glycolipid and ornithine lipid synthases during phosphate limitation. The phosphate-dependent synthesis of MGDG in G20 and the widespread occurrence of the MGDG/GADG synthase among sulfate reducing @-Proteobacteria implicate these microbes in the production of abundant MGDG in anaerobic environments where the concentrations of phosphate are lower than 10 μM. Numerous predicted changes in the composition of the cell envelope and systems involved in transport, maintenance of cytoplasmic redox potential, central metabolism and regulatory pathways also suggest an impact of phosphate limitation on the susceptibility of sulfate reducing bacteria to other anthropogenic or environmental stresses.« less

System-Wide Adaptations of Desulfovibrio alaskensis G20 to Phosphate-Limited Conditions

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

Bosak, Tanja; Schubotz, Florence; de Santiago-Torio, Ana

The prevalence of lipids devoid of phosphorus suggests that the availability of phosphorus limits microbial growth and activity in many anoxic, stratified environments. To better understand the response of anaerobic bacteria to phosphate limitation and starvation, this study combines microscopic and lipid analyses with the measurements of fitness of pooled barcoded transposon mutants of the model sulfate reducing bacterium Desulfovibrio alaskensis G20. Phosphate-limited G20 has lower growth rates and replaces more than 90% of its membrane phospholipids by a mixture of monoglycosyl diacylglycerol (MGDG), glycuronic acid diacylglycerol (GADG) and ornithine lipids, lacks polyphosphate granules, and synthesizes other cellular inclusions. Analysesmore » of pooled and individual mutants reveal the importance of the high-affinity phosphate transport system (the Pst system), PhoR, and glycolipid and ornithine lipid synthases during phosphate limitation. The phosphate-dependent synthesis of MGDG in G20 and the widespread occurrence of the MGDG/GADG synthase among sulfate reducing @-Proteobacteria implicate these microbes in the production of abundant MGDG in anaerobic environments where the concentrations of phosphate are lower than 10 μM. Numerous predicted changes in the composition of the cell envelope and systems involved in transport, maintenance of cytoplasmic redox potential, central metabolism and regulatory pathways also suggest an impact of phosphate limitation on the susceptibility of sulfate reducing bacteria to other anthropogenic or environmental stresses.« less

Arginine mediated purification of trehalose-6-phosphate synthase (TPS) from Candida utilis: Its characterization and regulation.

PubMed

Sengupta, Shinjinee; Lahiri, Sagar; Banerjee, Shakri; Bashistha, Bipasha; Ghosh, Anil K

2011-12-01

Trehalose is the most important multifunctional, non-reducing disaccharide found in nature. It is synthesized in yeast by an enzyme complex: trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP). In the present study TPS is purified using a new methodology from Candida utilis cells by inclusion of 100mM l-arginine during cell lysis and in the mobile phase of high performance gel filtration liquid chromatography (HPGFLC). An electrophoretically homogenous TPS that was purified was a 60 kDa protein with 22.1 fold purification having a specific activity of 2.03 U/mg. Alignment of the N-terminal sequence with TPS from Saccharomyces cerevisiae confirmed the 60 kDa protein to be TPS. Optimum activity of TPS was observed at a protein concentration of 1 μg, at a temperature of 37°C and pH 8.5. Aggregation mediated enzyme regulation was indicated. Metal cofactors, especially MnCl₂, MgCl₂ and ZnSO₄, acted as stimulators. Metal chelators like CDTA and EGTA stimulated enzyme activity. Among the four glucosyl donors, the highest V(max) and lowest K(m) values were calculated as 2.96 U/mg and 1.36 mM when adenosine di phosphate synthase (ADPG) was used as substrate. Among the glucosyl acceptors, glucose-6-phosphate (G-6-P) showed maximum activity followed by fructose-6-phosphate (F-6-P). Polyanions heparin and chondroitin sulfate were seen to stimulate TPS activity with different glucosyl donors. Substrate specificity, V(max) and K(m) values provided an insight into an altered trehalose metabolic pathway in the C. utilis strain where ADPG is the preferred substrate rather than the usual substrate uridine diphosphaphate glucose (UDPG). The present work employs a new purification strategy as well as highlights an altered pathway in C. utilis. 2011 Elsevier B.V. All rights reserved.

Metabolomics of Clostridial Biofuel Production

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

Rabinowitz, Joshua D; Aristilde, Ludmilla; Amador-Noguez, Daniel

2015-09-08

Members of the genus Clostridium collectively have the ideal set of the metabolic capabilities for fermentative biofuel production: cellulose degradation, hydrogen production, and solvent excretion. No single organism, however, can effectively convert cellulose into biofuels. Here we developed, using metabolomics and isotope tracers, basic science knowledge of Clostridial metabolism of utility for future efforts to engineer such an organism. In glucose fermentation carried out by the biofuel producer Clostridium acetobutylicum, we observed a remarkably ordered series of metabolite concentration changes as the fermentation progressed from acidogenesis to solventogenesis. In general, high-energy compounds decreased while low-energy species increased during solventogenesis. Thesemore » changes in metabolite concentrations were accompanied by large changes in intracellular metabolic fluxes, with pyruvate directed towards acetyl-CoA and solvents instead of oxaloacetate and amino acids. Thus, the solventogenic transition involves global remodeling of metabolism to redirect resources from biomass production into solvent production. In contrast to C. acetobutylicum, which is an avid fermenter, C. cellulolyticum metabolizes glucose only slowly. We find that glycolytic intermediate concentrations are radically different from fast fermenting organisms. Associated thermodynamic and isotope tracer analysis revealed that the full glycolytic pathway in C. cellulolyticum is reversible. This arises from changes in cofactor utilization for phosphofructokinase and an alternative pathway from phosphoenolpyruvate to pyruvate. The net effect is to increase the high-energy phosphate bond yield of glycolysis by 150% (from 2 to 5) at the expense of lower net flux. Thus, C. cellulolyticum prioritizes glycolytic energy efficiency over speed. Degradation of cellulose results in other sugars in addition to glucose. Simultaneous feeding of stable isotope-labeled glucose and unlabeled pentose sugars (xylose or arabinose) to C. acetobutylicum revealed that, as expected, glucose was preferred, with the pentose sugar selectively assimilated into the pentose phosphate pathway (PPP). Simultaneous feeding of xylose and arabinose revealed an unexpected hierarchy among these pentose sugars, with arabinose utilized preferentially over xylose. Pentose catabolism occurred via the phosphoketolase pathway (PKP), an alternative route of pentose catabolism that directly converts xylulose-5-phosphate into acetyl-phosphate and glyceraldehyde-3-phosphate. Taken collectively, these findings reveal two hierarchies in Clostridial pentose metabolism: xylose is subordinate to arabinose, and the PPP is used less than the PKP. Thus, in addition to massively expanding the available data on Clostridial metabolism, we identified three key regulatory points suitable for targeting in future bioengineering efforts: phosphofructokinase for enhancing fermentation, the pyruvate-oxaloacetate node for controlling solventogenesis, and the phosphoketolase reaction for driving pentose catabolism.« less

Potential-sensing electrochemical atomic force microscopy for in operando analysis of water-splitting catalysts and interfaces

NASA Astrophysics Data System (ADS)

Nellist, Michael R.; Laskowski, Forrest A. L.; Qiu, Jingjing; Hajibabaei, Hamed; Sivula, Kevin; Hamann, Thomas W.; Boettcher, Shannon W.

2018-01-01

Heterogeneous electrochemical phenomena, such as (photo)electrochemical water splitting to generate hydrogen using semiconductors and/or electrocatalysts, are driven by the accumulated charge carriers and thus the interfacial electrochemical potential gradients that promote charge transfer. However, measurements of the "surface" electrochemical potential during operation are not generally possible using conventional electrochemical techniques, which measure/control the potential of a conducting electrode substrate. Here we show that the nanoscale conducting tip of an atomic force microscope cantilever can sense the surface electrochemical potential of electrocatalysts in operando. To demonstrate utility, we measure the potential-dependent and thickness-dependent electronic properties of cobalt (oxy)hydroxide phosphate (CoPi). We then show that CoPi, when deposited on illuminated haematite (α-Fe2O3) photoelectrodes, acts as both a hole collector and an oxygen evolution catalyst. We demonstrate the versatility of the technique by comparing surface potentials of CoPi-decorated planar and mesoporous haematite and discuss viability for broader application in the study of electrochemical phenomena.

Adverse Effects of Simulated Hyper- and Hypo-Phosphatemia on Endothelial Cell Function and Viability

PubMed Central

Zeng, Caihong; Rakheja, Dinesh; Zhu, Jiankun; Ye, Ting; Hutcheson, Jack; Vaziri, Nosratola D.; Liu, Zhihong; Mohan, Chandra; Zhou, Xin J.

2011-01-01

Background Dysregulaiton of phosphate homeostasis as occurs in chronic kidney disease is associated with cardiovascular complications. It has been suggested that both hyperphosphatemia and hypophosphatemia can cause cardiovascular disease. The molecular mechanisms by which high or low serum phosphate levels adversely affect cardiovascular function are poorly understood. The purpose of this study was to explore the mechanisms of endothelial dysfunction in the presence of non-physiologic phosphate levels. Methodology/Principal Findings We studied the effects of simulated hyper- and hypophosphatemia in human umbilical vein endothelial cells in vitro. We found both simulated hyperphosphatemia and hypophosphatemia decrease eNOS expression and NO production. This was associated with reduced intracellular calcium, increased protein kinase C β2 (PKCβ2), reduced cell viability, and increased apoptosis. While simulated hyperphosphatemia was associated with decreased Akt/p-Akt, Bcl-xl/Bax ratios, NFkB-p65 and p-Erk abundance, simulated hypophosphatemia was associated with increased Akt/p-Akt and Bcl-xl/Bax ratios and p-Mek, p38, and p-p38 abundance. Conclusions/Significance This is the first demonstration of endothelial dysfunction with hypophosphatemia. Our data suggests that both hyperphosphatemia and hypophosphatemia decrease eNOS activity via reduced intracellular calcium and increased PKCβ2. Hyperphosphatemia also appears to reduce eNOS transcription via reduced signaling through PI3K/Akt/NF-kB and MAPK/NF-kB pathways. On the other hand, hypophosphatemia appears to activate these pathways. Our data provides the basis for further studies to elucidate the relationship between altered phosphate homeostasis and cardiovascular disease. As a corollary, our data suggests that the level of phosphate in the culture media, if not in the physiologic range, may inadvertently affect experimental results. PMID:21858050

Domain Organization in Candida glabrata THI6, a Bifunctional Enzyme Required for Thiamin Biosynthesis in Eukaryotes

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

Paul, Debamita; Chatterjee, Abhishek; Begley, Tadhg P.

2010-11-15

THI6 is a bifunctional enzyme found in the thiamin biosynthetic pathway in eukaryotes. The N-terminal domain of THI6 catalyzes the ligation of the thiamin thiazole and pyrimidine moieties to form thiamin phosphate, and the C-terminal domain catalyzes the phosphorylation of 4-methyl-5-hydroxyethylthiazole in a salvage pathway. In prokaryotes, thiamin phosphate synthase and 4-methyl-5-hydroxyethylthiazole kinase are separate gene products. Here we report the first crystal structure of a eukaryotic THI6 along with several complexes that characterize the active sites responsible for the two chemical reactions. THI6 from Candida glabrata is a homohexamer in which the six protomers form a cage-like structure. Eachmore » protomer is composed of two domains, which are structurally homologous to their monofunctional bacterial counterparts. Two loop regions not found in the bacterial enzymes provide interactions between the two domains. The structures of different protein-ligand complexes define the thiazole and ATP binding sites of the 4-methyl-5-hydroxyethylthiazole kinase domain and the thiazole phosphate and 4-amino-5-hydroxymethyl-2-methylpyrimidine pyrophosphate binding sites of the thiamin phosphate synthase domain. Our structural studies reveal that the active sites of the two domains are 40 {angstrom} apart and are not connected by an obvious channel. Biochemical studies show 4-methyl-5-hydroxyethylthiazole phosphate is a substrate for THI6; however, adenosine diphospho-5{beta}-ethyl-4-methylthiazole-2-carboxylic acid, the product of THI4, is not a substrate for THI6. This suggests that an unidentified enzyme is necessary to produce the substrate for THI6 from the THI4 product.« less

Aldolase as a Chirality Intersection of L-Amino Acids and D-Sugars

NASA Astrophysics Data System (ADS)

Munegumi, Toratane

2015-06-01

Aldolase plays an important role in glycolysis and gluconeogenesis to produce D-fructose-1,6-bisphosphate (D-FBP) from dihydroxyacetone phosphate (DHP) and D-glyceraldehyde-3-phosphate (D-GAP). This reaction is stereoselective and retains the D-GAP 2R configuration and yields D-FBP (with the configuration: 3S, 4S, 5R). The 3- and 4-position carbons are the newly formed chiral carbons because the 5-position carbon of D-FBP comes from the 2-position of D-GAP. Although four diastereomeric products, ( 3S, 4R, 5R), ( 3R, 4R, 5R), ( 3R, 4S, 5R), ( 3S, 4S, 5R), are expected in the nonenzymatic reaction, only the ( 3S, 4S, 5R) diastereomer (D-FBP) is obtained. Therefore, the chirality in the 3- and 4-positions is induced by the chirality of the enzyme composed of L-amino acid residues. D-Glucose-6-phosphate (D-G6P), which is generated from D-FBP in the gluconeogenesis pathway, produces D-ribose-5-phosphate (D-R5P) in the pentose phosphate pathway. D-R5P is converted to PRPP (5-phosphoribosyl-α-pyrophosphate), which is used for the de novo synthesis of nucleotides. Ribonucleic acid (RNA) uses the nucleotides as building blocks. The configurations of the 4R-carbon and of the 3S-carbon are retained. The stereochemical structure of RNA is based on 3S as well as 4R (D). The consideration above suggests that aldolase is a key enzyme that determines the 3S configuration in D-R5P. It is thus a chirality intersection between amino acids and sugars, because the sugar chirality is determined by the chiral environment of an L-amino acid protein, aldolase, to produce D-FBP.

Regional differences in brain glucose metabolism determined by imaging mass spectrometry.

PubMed

Kleinridders, André; Ferris, Heather A; Reyzer, Michelle L; Rath, Michaela; Soto, Marion; Manier, M Lisa; Spraggins, Jeffrey; Yang, Zhihong; Stanton, Robert C; Caprioli, Richard M; Kahn, C Ronald

2018-06-01

Glucose is the major energy substrate of the brain and crucial for normal brain function. In diabetes, the brain is subject to episodes of hypo- and hyperglycemia resulting in acute outcomes ranging from confusion to seizures, while chronic metabolic dysregulation puts patients at increased risk for depression and Alzheimer's disease. In the present study, we aimed to determine how glucose is metabolized in different regions of the brain using imaging mass spectrometry (IMS). To examine the relative abundance of glucose and other metabolites in the brain, mouse brain sections were subjected to imaging mass spectrometry at a resolution of 100 μm. This was correlated with immunohistochemistry, qPCR, western blotting and enzyme assays of dissected brain regions to determine the relative contributions of the glycolytic and pentose phosphate pathways to regional glucose metabolism. In brain, there are significant regional differences in glucose metabolism, with low levels of hexose bisphosphate (a glycolytic intermediate) and high levels of the pentose phosphate pathway (PPP) enzyme glucose-6-phosphate dehydrogenase (G6PD) and PPP metabolite hexose phosphate in thalamus compared to cortex. The ratio of ATP to ADP is significantly higher in white matter tracts, such as corpus callosum, compared to less myelinated areas. While the brain is able to maintain normal ratios of hexose phosphate, hexose bisphosphate, ATP, and ADP during fasting, fasting causes a large increase in cortical and hippocampal lactate. These data demonstrate the importance of direct measurement of metabolic intermediates to determine regional differences in brain glucose metabolism and illustrate the strength of imaging mass spectrometry for investigating the impact of changing metabolic states on brain function at a regional level with high resolution. Copyright © 2018 The Authors. Published by Elsevier GmbH.. All rights reserved.

Allosteric Communication Disrupted by a Small Molecule Binding to the Imidazole Glycerol Phosphate Synthase Protein-Protein Interface.

PubMed

Rivalta, Ivan; Lisi, George P; Snoeberger, Ning-Shiuan; Manley, Gregory; Loria, J Patrick; Batista, Victor S

2016-11-29

Allosteric enzymes regulate a wide range of catalytic transformations, including biosynthetic mechanisms of important human pathogens, upon binding of substrate molecules to an orthosteric (or active) site and effector ligands at distant (allosteric) sites. We find that enzymatic activity can be impaired by small molecules that bind along the allosteric pathway connecting the orthosteric and allosteric sites, without competing with endogenous ligands. Noncompetitive allosteric inhibitors disrupted allostery in the imidazole glycerol phosphate synthase (IGPS) enzyme from Thermotoga maritima as evidenced by nuclear magnetic resonance, microsecond time-scale molecular dynamics simulations, isothermal titration calorimetry, and kinetic assays. The findings are particularly relevant for the development of allosteric antibiotics, herbicides, and antifungal compounds because IGPS is absent in mammals but provides an entry point to fundamental biosynthetic pathways in plants, fungi, and bacteria.

Plastidial Glycolytic Glyceraldehyde-3-Phosphate Dehydrogenase Is an Important Determinant in the Carbon and Nitrogen Metabolism of Heterotrophic Cells in Arabidopsis1

PubMed Central

Anoman, Armand D.; Muñoz-Bertomeu, Jesús; Rosa-Téllez, Sara; Flores-Tornero, María; Serrano, Ramón; Bueso, Eduardo; Fernie, Alisdair R.; Segura, Juan; Ros, Roc

2015-01-01

This study functionally characterizes the Arabidopsis (Arabidopsis thaliana) plastidial glycolytic isoforms of glyceraldehyde-3-phosphate dehydrogenase (GAPCp) in photosynthetic and heterotrophic cells. We expressed the enzyme in gapcp double mutants (gapcp1gapcp2) under the control of photosynthetic (Rubisco small subunit RBCS2B [RBCS]) or heterotrophic (phosphate transporter PHT1.2 [PHT]) cell-specific promoters. Expression of GAPCp1 under the control of RBCS in gapcp1gapcp2 had no significant effect on the metabolite profile or growth in the aerial part (AP). GAPCp1 expression under the control of the PHT promoter clearly affected Arabidopsis development by increasing the number of lateral roots and having a major effect on AP growth and metabolite profile. Our results indicate that GAPCp1 is not functionally important in photosynthetic cells but plays a fundamental role in roots and in heterotrophic cells of the AP. Specifically, GAPCp activity may be required in root meristems and the root cap for normal primary root growth. Transcriptomic and metabolomic analyses indicate that the lack of GAPCp activity affects nitrogen and carbon metabolism as well as mineral nutrition and that glycerate and glutamine are the main metabolites responding to GAPCp activity. Thus, GAPCp could be an important metabolic connector of glycolysis with other pathways, such as the phosphorylated pathway of serine biosynthesis, the ammonium assimilation pathway, or the metabolism of γ-aminobutyrate, which in turn affect plant development. PMID:26134167

Conditional iron and pH-dependent activity of a non-enzymatic glycolysis and pentose phosphate pathway.

PubMed

Keller, Markus A; Zylstra, Andre; Castro, Cecilia; Turchyn, Alexandra V; Griffin, Julian L; Ralser, Markus

2016-01-01

Little is known about the evolutionary origins of metabolism. However, key biochemical reactions of glycolysis and the pentose phosphate pathway (PPP), ancient metabolic pathways central to the metabolic network, have non-enzymatic pendants that occur in a prebiotically plausible reaction milieu reconstituted to contain Archean sediment metal components. These non-enzymatic reactions could have given rise to the origin of glycolysis and the PPP during early evolution. Using nuclear magnetic resonance spectroscopy and high-content metabolomics that allowed us to measure several thousand reaction mixtures, we experimentally address the chemical logic of a metabolism-like network constituted from these non-enzymatic reactions. Fe(II), the dominant transition metal component of Archean oceanic sediments, has binding affinity toward metabolic sugar phosphates and drives metabolism-like reactivity acting as both catalyst and cosubstrate. Iron and pH dependencies determine a metabolism-like network topology and comediate reaction rates over several orders of magnitude so that the network adopts conditional activity. Alkaline pH triggered the activity of the non-enzymatic PPP pendant, whereas gentle acidic or neutral conditions favored non-enzymatic glycolytic reactions. Fe(II)-sensitive glycolytic and PPP-like reactions thus form a chemical network mimicking structural features of extant carbon metabolism, including topology, pH dependency, and conditional reactivity. Chemical networks that obtain structure and catalysis on the basis of transition metals found in Archean sediments are hence plausible direct precursors of cellular metabolic networks.

Conditional iron and pH-dependent activity of a non-enzymatic glycolysis and pentose phosphate pathway

PubMed Central

Keller, Markus A.; Zylstra, Andre; Castro, Cecilia; Turchyn, Alexandra V.; Griffin, Julian L.; Ralser, Markus

2016-01-01

Little is known about the evolutionary origins of metabolism. However, key biochemical reactions of glycolysis and the pentose phosphate pathway (PPP), ancient metabolic pathways central to the metabolic network, have non-enzymatic pendants that occur in a prebiotically plausible reaction milieu reconstituted to contain Archean sediment metal components. These non-enzymatic reactions could have given rise to the origin of glycolysis and the PPP during early evolution. Using nuclear magnetic resonance spectroscopy and high-content metabolomics that allowed us to measure several thousand reaction mixtures, we experimentally address the chemical logic of a metabolism-like network constituted from these non-enzymatic reactions. Fe(II), the dominant transition metal component of Archean oceanic sediments, has binding affinity toward metabolic sugar phosphates and drives metabolism-like reactivity acting as both catalyst and cosubstrate. Iron and pH dependencies determine a metabolism-like network topology and comediate reaction rates over several orders of magnitude so that the network adopts conditional activity. Alkaline pH triggered the activity of the non-enzymatic PPP pendant, whereas gentle acidic or neutral conditions favored non-enzymatic glycolytic reactions. Fe(II)-sensitive glycolytic and PPP-like reactions thus form a chemical network mimicking structural features of extant carbon metabolism, including topology, pH dependency, and conditional reactivity. Chemical networks that obtain structure and catalysis on the basis of transition metals found in Archean sediments are hence plausible direct precursors of cellular metabolic networks. PMID:26824074

Mevalonate 5-diphosphate mediates ATP binding to the mevalonate diphosphate decarboxylase from the bacterial pathogen Enterococcus faecalis

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

Chen, Chun-Liang; Mermoud, James C.; Paul, Lake N.

The mevalonate pathway produces isopentenyl diphosphate (IPP), a building block for polyisoprenoid synthesis, and is a crucial pathway for growth of the human bacterial pathogen Enterococcus faecalis. The final enzyme in this pathway, mevalonate diphosphate decarboxylase (MDD), acts on mevalonate diphosphate (MVAPP) to produce IPP while consuming ATP. This essential enzyme has been suggested as a therapeutic target for the treatment of drug-resistant bacterial infections. Here, we report functional and structural studies on the mevalonate diphosphate decarboxylase from E. faecalis (MDDEF). The MDDEF crystal structure in complex with ATP (MDDEF–ATP) revealed that the phosphate-binding loop (amino acids 97–105) is notmore » involved in ATP binding and that the phosphate tail of ATP in this structure is in an outward-facing position pointing away from the active site. This suggested that binding of MDDEF to MVAPP is necessary to guide ATP into a catalytically favorable position. Enzymology experiments show that the MDDEF performs a sequential ordered bi-substrate reaction with MVAPP as the first substrate, consistent with the isothermal titration calorimetry (ITC) experiments. On the basis of ITC results, we propose that this initial prerequisite binding of MVAPP enhances ATP binding. In summary, our findings reveal a substrate-induced substrate-binding event that occurs during the MDDEF-catalyzed reaction. The disengagement of the phosphate-binding loop concomitant with the alternative ATP-binding configuration may provide the structural basis for antimicrobial design against these pathogenic enterococci.« less

Nuclear magnetic resonance studies of the regulation of the pentose phosphate pathway

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

Bolo, N.R.

1991-11-01

The goal of this work is to investigate the potential for and limitations of in vivo nuclear magnetic resonance (NMR) spectroscopy for quantitation of glucose flux through the pentose phosphate pathway (shunt). Interest in the shunt is motivated by the possibility that its activity may be greatly increased in cancer and in the pathological states of cardiac and cerebral ischemia. The ability to dynamically monitor flux through the pentose shunt can give new knowledge about metabolism in pathological states. {sup 13}C NMR spectroscopy was used to monitor shunt activity by determination of the ratios of ({sup 13}C-4) to ({sup 13}C-5)-glutamate,more » ({sup 13}C-3) to ({sup 13}C-2)-alanine or ({sup 13}C-3) to ({sup 13}C-2)-lactate produced when ({sup 13}C-2)-glucose is infused. These methods provide measures of the effect of oxidative stresses on shunt activity in systems ranging from cell free enzyme-substrate preparations to cell suspensions and whole animals. In anaerobic cell free preparations, the fraction of glucose flux through the shunt was monitored with a time resolution of 3 minutes. This work predicts the potential for in vivo human studies of pentose phosphate pathway activity based on the mathematical simulation of the {sup 13}C fractional enrichments of C4 and C5-glutamate as a function of shunt activity and on the signal-to- noise ratio acquired in {sup 13}C NMR human studies from the current literature.« less

Nuclear magnetic resonance studies of the regulation of the pentose phosphate pathway

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

Bolo, Nicolas Robin

1991-11-01

The goal of this work is to investigate the potential for and limitations of in vivo nuclear magnetic resonance (NMR) spectroscopy for quantitation of glucose flux through the pentose phosphate pathway (shunt). Interest in the shunt is motivated by the possibility that its activity may be greatly increased in cancer and in the pathological states of cardiac and cerebral ischemia. The ability to dynamically monitor flux through the pentose shunt can give new knowledge about metabolism in pathological states. 13C NMR spectroscopy was used to monitor shunt activity by determination of the ratios of [ 13C-4] to [ 13C-5]-glutamate, [more » 13C-3] to [ 13C-2]-alanine or [ 13C-3] to [ 13C-2]-lactate produced when [ 13C-2]-glucose is infused. These methods provide measures of the effect of oxidative stresses on shunt activity in systems ranging from cell free enzyme-substrate preparations to cell suspensions and whole animals. In anaerobic cell free preparations, the fraction of glucose flux through the shunt was monitored with a time resolution of 3 minutes. This work predicts the potential for in vivo human studies of pentose phosphate pathway activity based on the mathematical simulation of the 13C fractional enrichments of C4 and C5-glutamate as a function of shunt activity and on the signal-to- noise ratio acquired in 13C NMR human studies from the current literature.« less

Respiratory metabolism in the embryonic axis of germinating pea seed exposed to cadmium.

PubMed

Smiri, Moêz; Chaoui, Abdelilah; El Ferjani, Ezzedine

2009-02-15

Seeds of pea (Pisum sativum L.) were germinated for 5d by soaking in distilled water or 5mM cadmium nitrate. The relationships among cadmium stress, germination rate, changes in respiratory enzyme activities and carbohydrates mobilization were studied. Two cell fractions were obtained from embryonic axis: (1) mitochondria, used to determine enzyme activities of citric acid cycle and electron transport chain, and (2) soluble, to measure some enzyme activities involved in fermentation and pentose phosphate pathway. Activities of malate- and succinate-dehydrogenases (MDH, SDH) and NADH- and succinate-cytochrome c reductases (NCCR, SCCR) were rapidly inhibited, while cytochrome c oxidase (CCO) was unaltered by cadmium treatment. However, this stimulated the NADPH-generating enzyme activities of the pentose phosphate pathway, glucose-6-phosphate- and 6-phosphogluconate-dehydrogenases (G6PDH, 6PGDH), as well as enzyme activity of fermentation, alcohol dehydrogenase (ADH), with concomitant inhibition in the capacity of enzyme inactivator (INADH). Moreover, Cd restricted carbohydrate mobilization in the embryonic axis. Almost no glucose and less than 7% of control fructose and total soluble sugars were available in the embryo tissues after 5d of exposure to cadmium. Cotyledonary invertase isoenzyme activity was also inhibited by Cd. The results indicate that cadmium induces disorder in the resumption of respiration in germinating pea seeds. The contribution of Cd-stimulated alternative metabolic pathways to compensate for the failure in mitochondrial respiration is discussed in relation to the delay in seed germination and embryonic axis growth.

LsrF, a coenzyme A-dependent thiolase, catalyzes the terminal step in processing the quorum sensing signal autoinducer-2

PubMed Central

Marques, João C.; Oh, Il Kyu; Ly, Daniel C.; Lamosa, Pedro; Ventura, M. Rita; Miller, Stephen T.; Xavier, Karina B.

2014-01-01

The quorum sensing signal autoinducer-2 (AI-2) regulates important bacterial behaviors, including biofilm formation and the production of virulence factors. Some bacteria, such as Escherichia coli, can quench the AI-2 signal produced by a variety of species present in the environment, and thus can influence AI-2–dependent bacterial behaviors. This process involves uptake of AI-2 via the Lsr transporter, followed by phosphorylation and consequent intracellular sequestration. Here we determine the metabolic fate of intracellular AI-2 by characterizing LsrF, the terminal protein in the Lsr AI-2 processing pathway. We identify the substrates of LsrF as 3-hydroxy-2,4-pentadione-5-phosphate (P-HPD, an isomer of AI-2-phosphate) and coenzyme A, determine the crystal structure of an LsrF catalytic mutant bound to P-HPD, and identify the reaction products. We show that LsrF catalyzes the transfer of an acetyl group from P-HPD to coenzyme A yielding dihydroxyacetone phosphate and acetyl-CoA, two key central metabolites. We further propose that LsrF, despite strong structural homology to aldolases, acts as a thiolase, an activity previously undescribed for this family of enzymes. With this work, we have fully characterized the biological pathway for AI-2 processing in E. coli, a pathway that can be used to quench AI-2 and control quorum-sensing–regulated bacterial behaviors. PMID:25225400

Understanding alternative fluxes/effluxes through comparative metabolic pathway analysis of phylum actinobacteria using a simplified approach.

PubMed

Verma, Mansi; Lal, Devi; Saxena, Anjali; Anand, Shailly; Kaur, Jasvinder; Kaur, Jaspreet; Lal, Rup

2013-12-01

Actinobacteria are known for their diverse metabolism and physiology. Some are dreadful human pathogens whereas some constitute the natural flora for human gut. Therefore, the understanding of metabolic pathways is a key feature for targeting the pathogenic bacteria without disturbing the symbiotic ones. A big challenge faced today is multiple drug resistance by Mycobacterium and other pathogens that utilize alternative fluxes/effluxes. With the availability of genome sequence, it is now feasible to conduct the comparative in silico analysis. Here we present a simplified approach to compare metabolic pathways so that the species specific enzyme may be traced and engineered for future therapeutics. The analyses of four key carbohydrate metabolic pathways, i.e., glycolysis, pyruvate metabolism, tri carboxylic acid cycle and pentose phosphate pathway suggest the presence of alternative fluxes. It was found that the upper pathway of glycolysis was highly variable in the actinobacterial genomes whereas lower glycolytic pathway was highly conserved. Likewise, pentose phosphate pathway was well conserved in contradiction to TCA cycle, which was found to be incomplete in majority of actinobacteria. The clustering based on presence and absence of genes of these metabolic pathways clearly revealed that members of different genera shared identical pathways and, therefore, provided an easy method to identify the metabolic similarities/differences between pathogenic and symbiotic organisms. The analyses could identify isoenzymes and some key enzymes that were found to be missing in some pathogenic actinobacteria. The present work defines a simple approach to explore the effluxes in four metabolic pathways within the phylum actinobacteria. The analysis clearly reflects that actinobacteria exhibit diverse routes for metabolizing substrates. The pathway comparison can help in finding the enzymes that can be used as drug targets for pathogens without effecting symbiotic organisms within the same host. This may help to prevail over the multiple drug resistance, for designing broad spectrum drugs, in food industries and other clinical research areas. © 2013.

Microbial pathways and palaeoenvironmental conditions involved in the formation of phosphorite grains, Safaga District, Egypt

NASA Astrophysics Data System (ADS)

Salama, Walid; El-Kammar, Ahmed; Saunders, Martin; Morsy, Rania; Kong, Charlie

2015-07-01

Phosphatic grains of the shallow marine phosphorite deposits of Egypt are classified as either phosphatic bioclasts preserving biological structure (e.g. skeletal fragments such as fish bones and teeth) or phosphatic peloids and intraclasts. This study describes the destructive and constructive microbial pathways represented by bioerosion of bones by endolithic cyanobacteria and accretion of phosphatic peloids by bacteria. The palaeoenvironmental conditions and post-depositional/diagenetic history of these grains have also been considered. Scanning and transmission electron microscopy showed that the phosphatic peloids under transmitted light microscopy are composed mainly of microspheres (0.5 to 2.5 μm) similar in shape and size to coccoid-like bacteria. Chemical mapping showed that these microspheres are composed of carbonate-fluorapatite (CFA) and surrounded by degraded carbonaceous matrix. These grains are suggested to be reworked from pre-existing microbial mats during transgressive-regressive cycles affecting the southern Tethyan Campanian-Maastrichtian shallow continental shelf. The bioerosion of phosphatic bones is characterized by a network of meandering microborings that penetrated inward from the bone surface by endolithic cyanobacteria. The bioerosion of bones resulted in a gradual centripetal digestion and conversion of bones into micritic phosphate peloids. The bioerosion mechanism is probably started in the acidic sheath surrounding cyanobacteria followed by supersaturation of PO4 and reprecipitation of crystalline CFA as electron dense remineralized rims. Electron microprobe microanalyses showed that the remineralized microbored areas are higher in CaO, P2O5, and F and depleted in Cl, relative to unaltered bones. A gradual demineralization of remineralized rims followed by dissolution of cyanobacterial cells is probably occurred during diagenesis and meteoric water alteration leaving behind empty microborings. Bone exposed to meteoric water alteration is lower in CaO and P2O5 and higher in F and Cl than the unaltered bones. Understanding bone bioerosion has significant implications for palaeoenvironmental and taphonomic reconstruction, archaeological applications and a regional correlation of the late Cretaceous to Palaeogene phosphogenic province extending from Middle East to North Africa.

Advanced water splitting for green hydrogen gas production through complete oxidation of starch by in vitro metabolic engineering.

PubMed

Kim, Jae-Eung; Kim, Eui-Jin; Chen, Hui; Wu, Chang-Hao; Adams, Michael W W; Zhang, Y-H Percival

2017-11-01

Starch is a natural energy storage compound and is hypothesized to be a high-energy density chemical compound or solar fuel. In contrast to industrial hydrolysis of starch to glucose, an alternative ATP-free phosphorylation of starch was designed to generate cost-effective glucose 6-phosphate by using five thermophilic enzymes (i.e., isoamylase, alpha-glucan phosphorylase, 4-α-glucanotransferase, phosphoglucomutase, and polyphosphate glucokinase). This enzymatic phosphorolysis is energetically advantageous because the energy of α-1,4-glycosidic bonds among anhydroglucose units is conserved in the form of phosphorylated glucose. Furthermore, we demonstrated an in vitro 17-thermophilic enzyme pathway that can convert all glucose units of starch, regardless of branched and linear contents, with water to hydrogen at a theoretic yield (i.e., 12 H 2 per glucose), three times of the theoretical yield from dark microbial fermentation. The use of a biomimetic electron transport chain enabled to achieve a maximum volumetric productivity of 90.2mmol of H 2 /L/h at 20g/L starch. The complete oxidation of starch to hydrogen by this in vitro synthetic (enzymatic) biosystem suggests that starch as a natural solar fuel becomes a high-density hydrogen storage compound with a gravimetric density of more than 14% H 2 -based mass and an electricity density of more than 3000Wh/kg of starch. Copyright © 2017. Published by Elsevier Inc.

The response and recovery of the Arabidopsis thaliana transcriptome to phosphate starvation.

PubMed

Woo, Jongchan; MacPherson, Cameron Ross; Liu, Jun; Wang, Huan; Kiba, Takatoshi; Hannah, Matthew A; Wang, Xiu-Jie; Bajic, Vladimir B; Chua, Nam-Hai

2012-05-03

Over application of phosphate fertilizers in modern agriculture contaminates waterways and disrupts natural ecosystems. Nevertheless, this is a common practice among farmers, especially in developing countries as abundant fertilizers are believed to boost crop yields. The study of plant phosphate metabolism and its underlying genetic pathways is key to discovering methods of efficient fertilizer usage. The work presented here describes a genome-wide resource on the molecular dynamics underpinning the response and recovery in roots and shoots of Arabidopsis thaliana to phosphate-starvation. Genome-wide profiling by micro- and tiling-arrays (accessible from GEO: GSE34004) revealed minimal overlap between root and shoot transcriptomes suggesting two independent phosphate-starvation regulons. Novel gene expression patterns were detected for over 1000 candidates and were classified as either initial, persistent, or latent responders. Comparative analysis to AtGenExpress identified cohorts of genes co-regulated across multiple stimuli. The hormone ABA displayed a dominant role in regulating many phosphate-responsive candidates. Analysis of co-regulation enabled the determination of specific versus generic members of closely related gene families with respect to phosphate-starvation. Thus, among others, we showed that PHR1-regulated members of closely related phosphate-responsive families (PHT1;1, PHT1;7-9, SPX1-3, and PHO1;H1) display greater specificity to phosphate-starvation than their more generic counterparts. Our results uncover much larger, staged responses to phosphate-starvation than previously described. To our knowledge, this work describes the most complete genome-wide data on plant nutrient stress to-date.

Phosphate toxicity: new insights into an old problem

PubMed Central

RAZZAQUE, M. Shawkat

2011-01-01

Phosphorus is an essential nutrient required for critical biological reactions that maintain the normal homoeostatic control of the cell. This element is an important component of different cellular structures, including nucleic acids and cell membranes. Adequate phosphorus balance is vital for maintaining basic cellular functions, ranging from energy metabolism to cell signalling. In addition, many intracellular pathways utilize phosphate ions for important cellular reactions; therefore, homoeostatic control of phosphate is one of the most delicate biological regulations. Impaired phosphorus balance can affect the functionality of almost every human system, including musculoskeletal and cardiovascular systems, ultimately leading to an increase in morbidity and mortality of the affected patients. Human and experimental studies have found that delicate balance among circulating factors, like vitamin D, PTH (parathyroid hormone) and FGF23 (fibroblast growth factor 23), are essential for regulation of physiological phosphate balance. Dysregulation of these factors, either alone or in combination, can induce phosphorus imbalance. Recent studies have shown that suppression of the FGF23–klotho system can lead to hyperphosphataemia with extensive tissue damage caused by phosphate toxicity. The cause and consequences of phosphate toxicity will be briefly summarized in the present review. PMID:20958267

Phosphate toxicity: new insights into an old problem.

PubMed

Razzaque, M Shawkat

2011-02-01

Phosphorus is an essential nutrient required for critical biological reactions that maintain the normal homoeostatic control of the cell. This element is an important component of different cellular structures, including nucleic acids and cell membranes. Adequate phosphorus balance is vital for maintaining basic cellular functions, ranging from energy metabolism to cell signalling. In addition, many intracellular pathways utilize phosphate ions for important cellular reactions; therefore, homoeostatic control of phosphate is one of the most delicate biological regulations. Impaired phosphorus balance can affect the functionality of almost every human system, including musculoskeletal and cardiovascular systems, ultimately leading to an increase in morbidity and mortality of the affected patients. Human and experimental studies have found that delicate balance among circulating factors, like vitamin D, PTH (parathyroid hormone) and FGF23 (fibroblast growth factor 23), are essential for regulation of physiological phosphate balance. Dysregulation of these factors, either alone or in combination, can induce phosphorus imbalance. Recent studies have shown that suppression of the FGF23-klotho system can lead to hyperphosphataemia with extensive tissue damage caused by phosphate toxicity. The cause and consequences of phosphate toxicity will be briefly summarized in the present review.

Deletion of the Glucose-6-Phosphate Dehydrogenase Gene KlZWF1 Affects both Fermentative and Respiratory Metabolism in Kluyveromyces lactis▿

PubMed Central

Saliola, Michele; Scappucci, Gina; De Maria, Ilaria; Lodi, Tiziana; Mancini, Patrizia; Falcone, Claudio

2007-01-01

In Kluyveromyces lactis, the pentose phosphate pathway is an alternative route for the dissimilation of glucose. The first enzyme of the pathway is the glucose-6-phosphate dehydrogenase (G6PDH), encoded by KlZWF1. We isolated this gene and examined its role. Like ZWF1 of Saccharomyces cerevisiae, KlZWF1 was constitutively expressed, and its deletion led to increased sensitivity to hydrogen peroxide on glucose, but unlike the case for S. cerevisiae, the Klzwf1Δ strain had a reduced biomass yield on fermentative carbon sources as well as on lactate and glycerol. In addition, the reduced yield on glucose was associated with low ethanol production and decreased oxygen consumption, indicating that this gene is required for both fermentation and respiration. On ethanol, however, the mutant showed an increased biomass yield. Moreover, on this substrate, wild-type cells showed an additional band of activity that might correspond to a dimeric form of G6PDH. The partial dimerization of the G6PDH tetramer on ethanol suggested the production of an NADPH excess that was negative for biomass yield. PMID:17085636

Inhibition of the pentose phosphate pathway by dichloroacetate unravels a missing link between aerobic glycolysis and cancer cell proliferation.

PubMed

De Preter, Géraldine; Neveu, Marie-Aline; Danhier, Pierre; Brisson, Lucie; Payen, Valéry L; Porporato, Paolo E; Jordan, Bénédicte F; Sonveaux, Pierre; Gallez, Bernard

2016-01-19

Glucose fermentation through glycolysis even in the presence of oxygen (Warburg effect) is a common feature of cancer cells increasingly considered as an enticing target in clinical development. This study aimed to analyze the link between metabolism, energy stores and proliferation rates in cancer cells. We found that cell proliferation, evaluated by DNA synthesis quantification, is correlated to glycolytic efficiency in six cancer cell lines as well as in isogenic cancer cell lines. To further investigate the link between glycolysis and proliferation, a pharmacological inhibitor of the pentose phosphate pathway (PPP) was used. We demonstrated that reduction of PPP activity decreases cancer cells proliferation, with a profound effect in Warburg-phenotype cancer cells. The crucial role of the PPP in sustaining cancer cells proliferation was confirmed using siRNAs against glucose-6-phosphate dehydrogenase, the first and rate-limiting enzyme of the PPP. In addition, we found that dichloroacetate (DCA), a new clinically tested compound, induced a switch of glycolytic cancer cells to a more oxidative phenotype and decreased proliferation. By demonstrating that DCA decreased the activity of the PPP, we provide a new mechanism by which DCA controls cancer cells proliferation.

Sources and Fates of Carbamyl Phosphate: A Labile Energy-Rich Molecule with Multiple Facets.

PubMed

Shi, Dashuang; Caldovic, Ljubica; Tuchman, Mendel

2018-06-12

Carbamyl phosphate (CP) is well-known as an essential intermediate of pyrimidine and arginine/urea biosynthesis. Chemically, CP can be easily synthesized from dihydrogen phosphate and cyanate. Enzymatically, CP can be synthesized using three different classes of enzymes: (1) ATP-grasp fold protein based carbamyl phosphate synthetase (CPS); (2) Amino-acid kinase fold carbamate kinase (CK)-like CPS (anabolic CK or aCK); and (3) Catabolic transcarbamylase. The first class of CPS can be further divided into three different types of CPS as CPS I, CPS II, and CPS III depending on the usage of ammonium or glutamine as its nitrogen source, and whether N -acetyl-glutamate is its essential co-factor. CP can donate its carbamyl group to the amino nitrogen of many important molecules including the most well-known ornithine and aspartate in the arginine/urea and pyrimidine biosynthetic pathways. CP can also donate its carbamyl group to the hydroxyl oxygen of a variety of molecules, particularly in many antibiotic biosynthetic pathways. Transfer of the carbamyl group to the nitrogen group is catalyzed by the anabolic transcarbamylase using a direct attack mechanism, while transfer of the carbamyl group to the oxygen group is catalyzed by a different class of enzymes, CmcH/NodU CTase, using a different mechanism involving a three-step reaction, decomposition of CP to carbamate and phosphate, transfer of the carbamyl group from carbamate to ATP to form carbamyladenylate and pyrophosphate, and transfer of the carbamyl group from carbamyladenylate to the oxygen group of the substrate. CP is also involved in transferring its phosphate group to ADP to generate ATP in the fermentation of many microorganisms. The reaction is catalyzed by carbamate kinase, which may be termed as catabolic CK (cCK) in order to distinguish it from CP generating CK. CP is a thermally labile molecule, easily decomposed into phosphate and cyanate, or phosphate and carbamate depending on the pH of the solution, or the presence of enzyme. Biological systems have developed several mechanisms including channeling between enzymes, increased affinity of CP to enzymes, and keeping CP in a specific conformation to protect CP from decomposition. CP is highly important for our health as both a lack of, or decreased, CP production and CP accumulation results in many disease conditions.

Correlation between root respiration and the levels of biomass and glycyrrhizic acid in Glycyrrhiza uralensis

PubMed Central

Liu, Wenlan; Sun, Zhirong; Qu, Jixu; Yang, Chunning; Zhang, Xiaomin; Wei, Xinxin

2017-01-01

The aim of the present study was to investigate the correlation between root respiration and the levels of biomass and glycyrrhizic acid in Glycyrrhiza uralensis. Root respiration was determined using a biological oxygen analyzer. Respiration-related enzymes including glucose-6-phosphate dehydrogenase plus 6-phosphogluconate dehydrogenase, phosphohexose isomerase and succinate dehydrogenase, and respiratory pathways were evaluated. Biomass was determined by a drying-weighing method. In addition, the percentage of glycyrrhizic acid was detected using high-performance liquid chromatography. The association between root respiration and the levels of biomass and glycyrrhizic acid was investigated. The glycolysis pathway (EMP), tricarboxylic acid cycle (TCA) and pentose phosphate (PPP) pathway acted concurrently in the roots of G. uralensis. Grey correlation analysis showed that TCA had the strongest correlation (correlation coefficient, 0.8003) with biomass. Starch and acetyl coenzyme A had the closest association with above-ground biomass, while soluble sugar correlated less strongly with above-ground biomass. Grey correlation analysis between biochemical pathways and the intermediates showed that pyruvic acid had the strongest correlation with EMP, while acetyl coenzyme A correlated most strongly with TCA. Among the intermediates and pathways, pyruvic acid and EMP exhibited the greatest correlation with glycyrrhizic acid, while acetyl coenzyme A and TCA correlated with glycyrrhizic acid less closely. The results of this study may aid the cultivation of G. uralensis. However, these results require verification in further studies. PMID:28962162

Correlation between root respiration and the levels of biomass and glycyrrhizic acid in Glycyrrhiza uralensis.

PubMed

Liu, Wenlan; Sun, Zhirong; Qu, Jixu; Yang, Chunning; Zhang, Xiaomin; Wei, Xinxin

2017-09-01

The aim of the present study was to investigate the correlation between root respiration and the levels of biomass and glycyrrhizic acid in Glycyrrhiza uralensis . Root respiration was determined using a biological oxygen analyzer. Respiration-related enzymes including glucose-6-phosphate dehydrogenase plus 6-phosphogluconate dehydrogenase, phosphohexose isomerase and succinate dehydrogenase, and respiratory pathways were evaluated. Biomass was determined by a drying-weighing method. In addition, the percentage of glycyrrhizic acid was detected using high-performance liquid chromatography. The association between root respiration and the levels of biomass and glycyrrhizic acid was investigated. The glycolysis pathway (EMP), tricarboxylic acid cycle (TCA) and pentose phosphate (PPP) pathway acted concurrently in the roots of G. uralensis . Grey correlation analysis showed that TCA had the strongest correlation (correlation coefficient, 0.8003) with biomass. Starch and acetyl coenzyme A had the closest association with above-ground biomass, while soluble sugar correlated less strongly with above-ground biomass. Grey correlation analysis between biochemical pathways and the intermediates showed that pyruvic acid had the strongest correlation with EMP, while acetyl coenzyme A correlated most strongly with TCA. Among the intermediates and pathways, pyruvic acid and EMP exhibited the greatest correlation with glycyrrhizic acid, while acetyl coenzyme A and TCA correlated with glycyrrhizic acid less closely. The results of this study may aid the cultivation of G. uralensis . However, these results require verification in further studies.

A family of metal-dependent phosphatases implicated in metabolite damage-control

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

Huang, Lili; Khusnutdinova, Anna; Nocek, Boguslaw

DUF89 family proteins occur widely in both prokaryotes and eukaryotes, but their functions are unknown. Here we define three DUF89 subfamilies (I, II, and III), with subfamily II being split into stand-alone proteins and proteins fused to pantothenate kinase (PanK). We demonstrated that DUF89 proteins have metal-dependent phosphatase activity against reactive phosphoesters or their damaged forms, notably sugar phosphates (subfamilies II and III), phosphopantetheine and its S-sulfonate or sulfonate (subfamily II-PanK fusions), and nucleotides (subfamily I). Genetic and comparative genomic data strongly associated DUF89 genes with phosphoester metabolism. The crystal structure of the yeast (Saccharomyces cerevisiae) subfamily III protein YMR027Wmore » revealed a novel phosphatase active site with fructose 6-phosphate and Mg2+ bound near conserved signature residues Asp254 and Asn255 that are critical for activity. These findings indicate that DUF89 proteins are previously unrecognized hydrolases whose characteristic in vivo function is to limit potentially harmful buildups of normal or damaged phosphometabolites.« less

A family of metal-dependent phosphatases implicated in metabolite damage-control

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

Huang, Lili; Shanklin, John; Khusnutdinova, Anna

DUF89 family proteins occur widely in pro- and eukaryotes but their functions are unknown. Here we define three DUF89 subfamilies (I, II, and III), subfamily II being split into standalone proteins and proteins fused to pantothenate kinase (PanK). We demonstrated that DUF89 proteins have metaldependent phosphatase activity against reactive phosphoesters or their damaged forms, notably sugar phosphates (subfamilies II and III), phosphopantetheine and its S-sulfonate or sulfonate (subfamily II-PanK fusions), and nucleotides (subfamily I). Genetic and comparative genomic data strongly associated DUF89 genes with phosphoester metabolism. The crystal structure of the yeast (Saccharomyces cerevisiae) subfamily III protein YMR027W revealed amore » novel phosphatase active site with fructose 6-phosphate and Mg 2+ bound near conserved signature residues Asp254 and Asn255 that are critical for activity. These findings indicate that DUF89 proteins are previously unrecognized hydrolases whose characteristic in vivo function is to limit potentially harmful buildups of normal or damaged phosphometabolites.« less

Arsenic species separation by IELC-ICP/OES: Arsenocholine behavior

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

Rubio, R.; Peralta, I.; Alberti, J.

1993-01-01

In the literature an increasing interest is observed in developing methods to determine arsenobetaine, arsenocholine and related compounds in sea food and in reference materials. The separation conditions and quantification of As(III), As(V), monomethylarsenate (MMA), dimethylarsinate (DMA), arsenobetaine (AsBet) and arsenocholine (AsChol) are studied by Liquid Chromatography (LC) coupled directly to an Inductively Coupled Plasma Optical Emission Spectroscopy (ICP/OES) system. The separation conditions are optimized to improve the resolution of the six arsenic species. Arsenocholine shows a particular pattern of behavior when phosphate is used as eluent: two peaks are observed in the chromatogram, thus a systematic study assaying differentmore » pH and concentration of phosphate is carried out to improve resolution and analysis time when the six arsenic compounds are analyzed in a mixture. Boric acid as mobile phase avoids the splitting of the arsenocholine peak and leads to a good separation of the six arsenic compounds. Detection limits are established for the six arsenic species.« less

A family of metal-dependent phosphatases implicated in metabolite damage-control

DOE PAGES

Huang, Lili; Shanklin, John; Khusnutdinova, Anna; ...

2016-06-20

DUF89 family proteins occur widely in pro- and eukaryotes but their functions are unknown. Here we define three DUF89 subfamilies (I, II, and III), subfamily II being split into standalone proteins and proteins fused to pantothenate kinase (PanK). We demonstrated that DUF89 proteins have metaldependent phosphatase activity against reactive phosphoesters or their damaged forms, notably sugar phosphates (subfamilies II and III), phosphopantetheine and its S-sulfonate or sulfonate (subfamily II-PanK fusions), and nucleotides (subfamily I). Genetic and comparative genomic data strongly associated DUF89 genes with phosphoester metabolism. The crystal structure of the yeast (Saccharomyces cerevisiae) subfamily III protein YMR027W revealed amore » novel phosphatase active site with fructose 6-phosphate and Mg 2+ bound near conserved signature residues Asp254 and Asn255 that are critical for activity. These findings indicate that DUF89 proteins are previously unrecognized hydrolases whose characteristic in vivo function is to limit potentially harmful buildups of normal or damaged phosphometabolites.« less

Structural and optical properties of CuO in zinc phosphate glasses and effects of gamma irradiation

NASA Astrophysics Data System (ADS)

Ouis, M. A.; ElBatal, H. A.; Abdelghany, A. M.; Hammad, Ahmed H.

2016-01-01

Collective optical and infrared measurements have been employed to investigate the state of increasing copper ions in host 0.5ZnO-0.5P2O5 glass composition. The same spectral measurements were repeated after gamma irradiation with a dose of 20 and 80 KGy. Optical absorption spectra reveal strong UV absorption due to trace ferric ions present as unavoidable impurities within the chemicals used in the preparation of the glasses. Copper containing glasses show an additional broad visible-near infrared band due to distorted octahedrally coordinated Cu2+ ions which at high CuO contents exhibit splitting to several component absorption peaks. Gamma irradiation causes several variations between the response of the base host zinc phosphate glass and effect of increasing CuO. These changes are correlated with both the formation of induced defects through suggested photochemical reactions in the UV region and some shielding effects with increasing CuO in the visible-near infrared spectrum. Infrared absorption spectra reveal repetitive vibrational bands due to phosphate groups mainly from metaphosphate units and the spectra show some variations with the increase of CuO content visualize by the increase of the intensity of the mid broad band extending in the range 800-1500 cm-1.

Characterization and relative quantification of phospholipids based on methylation and stable isotopic labeling[S

PubMed Central

Cai, Tanxi; Shu, Qingbo; Liu, Peibin; Niu, Lili; Guo, Xiaojing; Ding, Xiang; Xue, Peng; Xie, Zhensheng; Wang, Jifeng; Zhu, Nali; Wu, Peng; Niu, Lili; Yang, Fuquan

2016-01-01

Phospholipids (PLs), one of the lipid categories, are not only the primary building blocks of cellular membranes, but also can be split to produce products that function as second messengers in signal transduction and play a pivotal role in numerous cellular processes, including cell growth, survival, and motility. Here, we present an integrated novel method that combines a fast and robust TMS-diazomethane-based phosphate derivatization and isotopic labeling strategy, which enables simultaneous profiling and relative quantification of PLs from biological samples. Our results showed that phosphate methylation allows fast and sensitive identification of the six major PL classes, including their lysophospholipid counterparts, under positive ionization mode. The isotopic labeling of endogenous PLs was achieved by deuterated diazomethane, which was generated through acid-catalyzed hydrogen/deuterium (H/D) exchange and methanolysis of TMS-diazomethane during the process of phosphate derivatization. The measured H/D ratios of unlabeled and labeled PLs, which were mixed in known proportions, indicated that the isotopic labeling strategy is capable of providing relative quantitation with adequate accuracy, reproducibility, and a coefficient of variation of 9.1%, on average. This novel method offers unique advantages over existing approaches and presents a powerful tool for research of PL metabolism and signaling. PMID:26733148

Novel method for early investigation of bioactivity in different borate bio-glasses.

PubMed

Abdelghany, A M

2013-01-01

Some ternary borate glasses were prepared and corrosion behavior of such ternary borate glasses after immersion in aqueous dilute phosphate solution was studied using different immersion times. Fourier transform infrared (FTIR) absorption spectral measurements were done before and after immersion in the mentioned solution for extended times up to 2 days to justify the appearance of the characteristic FTIR bands due to calcium phosphate (hydroxyapatite (HA)) which is considered as the potential indication of bioactivity. Experimental IR data confirm the beginning of the appearance of FTIR bands at about 580 and 620 cm(-1) after 3 days and the complete resolution with its characteristic split form after 1 week and more. Deconvolution analysis technique (DAT) of the FTIR spectrum was employed to investigate the bioactivity of such ternary borate system after a short period of immersion. The corrosion behavior of such glasses is explained in relation to a suggested hydrolysis followed by direct dissolution mechanism. The ease of dissolution of all the borate glasses constituents explains the formation of calcium phosphate and conversion to crystalline hydroxyapatite within the borate glass matrix. X-ray diffraction may be used to retrace the structural changes and degree of crystallinity of the prepared glasses. Copyright © 2012 Elsevier B.V. All rights reserved.

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

Phosphate homeostasis in the yeast Saccharomyces cerevisiae, the key role of the SPX domain-containing proteins.

PubMed

Secco, David; Wang, Chuang; Shou, Huixia; Whelan, James

2012-02-17

In the yeast Saccharomyces cerevisiae, a working model for nutrient homeostasis in eukaryotes, inorganic phosphate (Pi) homeostasis is regulated by the PHO pathway, a set of phosphate starvation induced genes, acting to optimize Pi uptake and utilization. Among these, a subset of proteins containing the SPX domain has been shown to be key regulators of Pi homeostasis. In this review, we summarize the recent progresses in elucidating the mechanisms controlling Pi homeostasis in yeast, focusing on the key roles of the SPX domain-containing proteins in these processes, as well as describing the future challenges and opportunities in this fast-moving field. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Glucose-6-Phosphate Dehydrogenase Deficiency.

PubMed

Luzzatto, Lucio; Nannelli, Caterina; Notaro, Rosario

2016-04-01

G6PD is a housekeeping gene expressed in all cells. Glucose-6-phosphate dehydrogenase (G6PD) is part of the pentose phosphate pathway, and its main physiologic role is to provide NADPH. G6PD deficiency, one of the commonest inherited enzyme abnormalities in humans, arises through one of many possible mutations, most of which reduce the stability of the enzyme and its level as red cells age. G6PD-deficient persons are mostly asymptomatic, but they can develop severe jaundice during the neonatal period and acute hemolytic anemia when they ingest fava beans or when they are exposed to certain infections or drugs. G6PD deficiency is a global health issue. Copyright © 2016 Elsevier Inc. All rights reserved.

Histidinol Phosphate Phosphatase, Catalyzing the Penultimate Step of the Histidine Biosynthesis Pathway, Is Encoded by ytvP (hisJ) in Bacillus subtilis

PubMed Central

le Coq, Dominique; Fillinger, Sabine; Aymerich, Stéphane

1999-01-01

The deduced product of the Bacillus subtilis ytvP gene is similar to that of ORF13, a gene of unknown function in the Lactococcus lactis histidine biosynthesis operon. A B. subtilis ytvP mutant was auxotrophic for histidine. The only enzyme of the histidine biosynthesis pathway that remained uncharacterized in B. subtilis was histidinol phosphate phosphatase (HolPase), catalyzing the penultimate step of this pathway. HolPase activity could not be detected in crude extracts of the ytvP mutant, while purified glutathione S-transferase-YtvP fusion protein exhibited strong HolPase activity. These observations demonstrated that HolPase is encoded by ytvP in B. subtilis and led us to rename this gene hisJ. Together with the HolPase of Saccharomyces cerevisiae and the presumed HolPases of L. lactis and Schizosaccharomyces pombe, HisJ constitutes a family of related enzymes that are not homologous to the HolPases of Escherichia coli, Salmonella typhimurium, and Haemophilus influenzae. PMID:10322033

Central Role of the Trehalose Biosynthesis Pathway in the Pathogenesis of Human Fungal Infections: Opportunities and Challenges for Therapeutic Development.

PubMed

Thammahong, Arsa; Puttikamonkul, Srisombat; Perfect, John R; Brennan, Richard G; Cramer, Robert A

2017-06-01

Invasive fungal infections cause significant morbidity and mortality in part due to a limited antifungal drug arsenal. One therapeutic challenge faced by clinicians is the significant host toxicity associated with antifungal drugs. Another challenge is the fungistatic mechanism of action of some drugs. Consequently, the identification of fungus-specific drug targets essential for fitness in vivo remains a significant goal of medical mycology research. The trehalose biosynthetic pathway is found in a wide variety of organisms, including human-pathogenic fungi, but not in humans. Genes encoding proteins involved in trehalose biosynthesis are mechanistically linked to the metabolism, cell wall homeostasis, stress responses, and virulence of Candida albicans , Cryptococcus neoformans , and Aspergillus fumigatus . While there are a number of pathways for trehalose production across the tree of life, the TPS/TPP (trehalose-6-phosphate synthase/trehalose-6-phosphate phosphatase) pathway is the canonical pathway found in human-pathogenic fungi. Importantly, data suggest that proteins involved in trehalose biosynthesis play other critical roles in fungal metabolism and in vivo fitness that remain to be fully elucidated. By further defining the biology and functions of trehalose and its biosynthetic pathway components in pathogenic fungi, an opportunity exists to leverage this pathway as a potent antifungal drug target. The goal of this review is to cover the known roles of this important molecule and its associated biosynthesis-encoding genes in the human-pathogenic fungi studied to date and to employ these data to critically assess the opportunities and challenges facing development of this pathway as a therapeutic target. Copyright © 2017 American Society for Microbiology.

Molecular Clone and Expression of a NAD+-Dependent Glycerol-3-Phosphate Dehydrogenase Isozyme Gene from the Halotolerant alga Dunaliella salina

PubMed Central

Cai, Ma; He, Li-Hong; Yu, Tu-Yuan

2013-01-01

Glycerol is an important osmotically compatible solute in Dunaliella. Glycerol-3-phosphate dehydrogenase (G3PDH) is a key enzyme in the pathway of glycerol synthesis, which converts dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate. Generally, the glycerol-DHAP cycle pathway, which is driven by G3PDH, is considered as the rate-limiting enzyme to regulate the glycerol level under osmotic shocks. Considering the peculiarity in osmoregulation, the cDNA of a NAD+-dependent G3PDH was isolated from D. salina using RACE and RT-PCR approaches in this study. Results indicated that the length of the cDNA sequence of G3PDH was 2,100 bp encoding a 699 amino acid deduced polypeptide whose computational molecular weight was 76.6 kDa. Conserved domain analysis revealed that the G3PDH protein has two independent functional domains, SerB and G3PDH domains. It was predicted that the G3PDH was a nonsecretory protein and may be located in the chloroplast of D. salina. Phylogenetic analysis demonstrated that the D. salina G3PDH had a closer relationship with the G3PDHs from the Dunaliella genus than with those from other species. In addition, the cDNA was subsequently subcloned in the pET-32a(+) vector and was transformed into E. coli strain BL21 (DE3), a expression protein with 100 kDa was identified, which was consistent with the theoretical value. PMID:23626797

Regulation of phosphate transport and AMPK signal pathway by lower dietary phosphorus of broilers

PubMed Central

Miao, Zhiqiang; Feng, Yan; Zhang, Junzhen; Tian, Wenxia; Li, Jianhui; Yang, Yu

2017-01-01

Lower available P (aP) was used as a base value in nutritional strategies for mitigating P pollution by animal excreta. We hypothesized that the mechanism regulating phosphate transport under low dietary P might be related with the AMPK signal pathway. A total of 144 one-day-old Arbor Acres Plus broilers were randomly allocated to control (HP) or trial (LP) diets, containing 0.45 and 0.23% aP, respectively. Growth performance, blood, intestinal, and renal samples were tested in 21-day-old broilers. Results shown that LP decreased body weight gain and feed intake. Higher serum Ca and fructose, but lower serum P and insulin were detected in LP-fed broilers. NaPi-IIb mRNA expression in intestine and NaPi-IIa mRNA expression in kidney were higher in the LP group. AMP: ATP, p-AMPK: total AMPK, and p-ACC: total ACC ratios in the duodenal mucosa were decreased in the LP group, whereas the p-mTOR: total mTOR ratio increased. These findings suggested that the increase in phosphate transport owing to LP diet might be regulated either directly by higher mTOR activity or indirectly by the suppressive AMPK signal, with corresponding changes in blood insulin and fructose content. A novel viewpoint on the regulatory mechanism underlying phosphate transport under low dietary P conditions was revealed, which might provide theoretical guidelines for reducing P pollution by means of nutritional regulation. PMID:29296204

Vtc5, a Novel Subunit of the Vacuolar Transporter Chaperone Complex, Regulates Polyphosphate Synthesis and Phosphate Homeostasis in Yeast*

PubMed Central

Desfougères, Yann; Gerasimaitė, R̄uta; Jessen, Henning Jacob

2016-01-01

SPX domains control phosphate homeostasis in eukaryotes. Ten genes in yeast encode SPX-containing proteins, among which YDR089W is the only one of unknown function. Here, we show that YDR089W encodes a novel subunit of the vacuole transporter chaperone (VTC) complex that produces inorganic polyphosphate (polyP). The polyP synthesis transfers inorganic phosphate (Pi) from the cytosol into the acidocalcisome- and lysosome-related vacuoles of yeast, where it can be released again. It was therefore proposed for buffer changes in cytosolic Pi concentration (Thomas, M. R., and O'Shea, E. K. (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 9565–9570). Vtc5 physically interacts with the VTC complex and accelerates the accumulation of polyP synthesized by it. Deletion of VTC5 reduces polyP accumulation in vivo and in vitro. Its overexpression hyperactivates polyP production and triggers the phosphate starvation response via the PHO pathway. Because this Vtc5-induced starvation response can be reverted by shutting down polyP synthesis genetically or pharmacologically, we propose that polyP synthesis rather than Vtc5 itself is a regulator of the PHO pathway. Our observations suggest that polyP synthesis not only serves to establish a buffer for transient drops in cytosolic Pi levels but that it can actively decrease or increase the steady state of cytosolic Pi. PMID:27587415

Glucose-6-phosphate mediates activation of the carbohydrate responsive binding protein (ChREBP)

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

Li, Ming V.; Departments of Medicine and Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030; Chen, Weiqin

2010-05-07

Carbohydrate response element binding protein (ChREBP) is a Mondo family transcription factor that activates a number of glycolytic and lipogenic genes in response to glucose stimulation. We have previously reported that high glucose can activate the transcriptional activity of ChREBP independent of the protein phosphatase 2A (PP2A)-mediated increase in nuclear entry and DNA binding. Here, we found that formation of glucose-6-phosphate (G-6-P) is essential for glucose activation of ChREBP. The glucose response of GAL4-ChREBP is attenuated by D-mannoheptulose, a potent hexokinase inhibitor, as well as over-expression of glucose-6-phosphatase (G6Pase); kinetics of activation of GAL4-ChREBP can be modified by exogenously expressedmore » GCK. Further metabolism of G-6-P through the two major glucose metabolic pathways, glycolysis and pentose-phosphate pathway, is not required for activation of ChREBP; over-expression of glucose-6-phosphate dehydrogenase (G6PD) diminishes, whereas RNAi knockdown of the enzyme enhances, the glucose response of GAL4-ChREBP, respectively. Moreover, the glucose analogue 2-deoxyglucose (2-DG), which is phosphorylated by hexokinase, but not further metabolized, effectively upregulates the transcription activity of ChREBP. In addition, over-expression of phosphofructokinase (PFK) 1 and 2, synergistically diminishes the glucose response of GAL4-ChREBP. These multiple lines of evidence support the conclusion that G-6-P mediates the activation of ChREBP.« less

Glucose-6-phosphate dehydrogenase and NADPH redox regulates cardiac myocyte L-type calcium channel activity and myocardial contractile function.

PubMed

Rawat, Dhwajbahadur K; Hecker, Peter; Watanabe, Makino; Chettimada, Sukrutha; Levy, Richard J; Okada, Takao; Edwards, John G; Gupte, Sachin A

2012-01-01

We recently demonstrated that a 17-ketosteroid, epiandrosterone, attenuates L-type Ca(2+) currents (I(Ca-L)) in cardiac myocytes and inhibits myocardial contractility. Because 17-ketosteroids are known to inhibit glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme in the pentose phosphate pathway, and to reduce intracellular NADPH levels, we hypothesized that inhibition of G6PD could be a novel signaling mechanism which inhibit I(Ca-L) and, therefore, cardiac contractile function. We tested this idea by examining myocardial function in isolated hearts and Ca(2+) channel activity in isolated cardiac myocytes. Myocardial function was tested in Langendorff perfused hearts and I(Ca-L) were recorded in the whole-cell patch configuration by applying double pulses from a holding potential of -80 mV and then normalized to the peak amplitudes of control currents. 6-Aminonicotinamide, a competitive inhibitor of G6PD, increased pCO(2) and decreased pH. Additionally, 6-aminonicotinamide inhibited G6PD activity, reduced NADPH levels, attenuated peak I(Ca-L) amplitudes, and decreased left ventricular developed pressure and ±dp/dt. Finally, dialyzing NADPH into cells from the patch pipette solution attenuated the suppression of I(Ca-L) by 6-aminonicotinamide. Likewise, in G6PD-deficient mice, G6PD insufficiency in the heart decreased GSH-to-GSSG ratio, superoxide, cholesterol and acetyl CoA. In these mice, M-mode echocardiographic findings showed increased diastolic volume and end-diastolic diameter without changes in the fraction shortening. Taken together, these findings suggest that inhibiting G6PD activity and reducing NADPH levels alters metabolism and leads to inhibition of L-type Ca(2+) channel activity. Notably, this pathway may be involved in modulating myocardial contractility under physiological and pathophysiological conditions during which the pentose phosphate pathway-derived NADPH redox is modulated (e.g., ischemia-reperfusion and heart failure).

Subcellular localization of glycolytic enzymes and characterization of intermediary metabolism of Trypanosoma rangeli.

PubMed

Rondón-Mercado, Rocío; Acosta, Héctor; Cáceres, Ana J; Quiñones, Wilfredo; Concepción, Juan Luis

2017-09-01

Trypanosoma rangeli is a hemoflagellate protist that infects wild and domestic mammals as well as humans in Central and South America. Although this parasite is not pathogenic for human, it is being studied because it shares with Trypanosoma cruzi, the etiological agent of Chagas' disease, biological characteristics, geographic distribution, vectors and vertebrate hosts. Several metabolic studies have been performed with T. cruzi epimastigotes, however little is known about the metabolism of T. rangeli. In this work we present the subcellular distribution of the T. rangeli enzymes responsible for the conversion of glucose to pyruvate, as determined by epifluorescense immunomicroscopy and subcellular fractionation involving either selective membrane permeabilization with digitonin or differential and isopycnic centrifugation. We found that in T. rangeli epimastigotes the first six enzymes of the glycolytic pathway, involved in the conversion of glucose to 1,3-bisphosphoglycerate are located within glycosomes, while the last four steps occur in the cytosol. In contrast with T. cruzi, where three isoenzymes (one cytosolic and two glycosomal) of phosphoglycerate kinase are expressed simultaneously, only one enzyme with this activity is detected in T. rangeli epimastigotes, in the cytosol. Consistent with this latter result, we found enzymes involved in auxiliary pathways to glycolysis needed to maintain adenine nucleotide and redox balances within glycosomes such as phosphoenolpyruvate carboxykinase, malate dehydrogenase, fumarate reductase, pyruvate phosphate dikinase and glycerol-3-phosphate dehydrogenase. Glucokinase, galactokinase and the first enzyme of the pentose-phosphate pathway, glucose-6-phosphate dehydrogenase, were also located inside glycosomes. Furthermore, we demonstrate that T. rangeli epimastigotes growing in LIT medium only consume glucose and do not excrete ammonium; moreover, they are unable to survive in partially-depleted glucose medium. The velocity of glucose consumption is about 40% higher than that of procyclic Trypanosoma brucei, and four times faster than by T. cruzi epimastigotes under the same culture conditions. Copyright © 2017 Elsevier B.V. All rights reserved.

Regulation of the endothelin-1 transmembrane signaling pathway: the potential role of agonist-induced desensitization in the coronary artery of the rapid ventricular pacing-overdrive dog model of heart failure.

PubMed

Calderone, A; Rouleau, J L; de Champlain, J; Bélichard, P; Stewart, D J

1993-08-01

This study examined the potential role of ET-1 and the contribution of protein kinase C (PKC) in the desensitization of the ET-1 transmembrane signaling pathway in the left circumflex coronary artery (CCA) of a dog model of congestive heart failure (CHF). In the CCA of the rapid ventricular pacing-overdrive dog model of CHF, elevated plasma endothelin levels were associated with a decrease in the basal accumulation of inositol phosphates and ET-1 mediated activation of phosphatidylinositol (PI) turnover (P < 0.05). To assess whether elevated plasma ET-1 levels may have contributed to the diminished ET-1 responsiveness in the heart failure dogs, ET-1 generation of inositol phosphates was measured following a one hour pretreatment of normal coronary artery rings with 0.1 nM ET-1. As compared to non-treated rings, ET-1 pretreatment resulted in a 33% decrease of ET-1 (10 nM) production of inositol phosphates. To evaluate the role of PKC in this process, normal coronary rings pretreated for a period of one hour with the phorbol ester, phorbol 12-myristate 13-acetate (PMA, 1 microM), resulted in a similar attenuation (36%) of ET-1 production of inositol phosphates. In the presence of the protein kinase C inhibitor staurosporine, both the agonist and phorbol ester induced decreases in ET-1 mediated PI turnover were reversed. Staurosporine even potentiated (75%) ET-1 induced PI turnover despite ET-1 and PMA pretreatments. These results suggest that agonist-induced desensitization of ET-1 mediated PI turnover can occur and is at least one of the possible mechanisms contributing to the desensitization of the ET-1 transmembrane signaling pathway in the pacing-overdrive model of heart failure in the dog.

Cell-free protein synthesis energized by slowly-metabolized maltodextrin

PubMed Central

Wang, Yiran; Zhang, Y-H Percival

2009-01-01

Background Cell-free protein synthesis (CFPS) is a rapid and high throughput technology for obtaining proteins from their genes. The primary energy source ATP is regenerated from the secondary energy source through substrate phosphorylation in CFPS. Results Distinct from common secondary energy sources (e.g., phosphoenolpyruvate – PEP, glucose-6-phosphate), maltodextrin was used for energizing CFPS through substrate phosphorylation and the glycolytic pathway because (i) maltodextrin can be slowly catabolized by maltodextrin phosphorylase for continuous ATP regeneration, (ii) maltodextrin phosphorylation can recycle one phosphate per reaction for glucose-1-phosphate generation, and (iii) the maltodextrin chain-shortening reaction can produce one ATP per glucose equivalent more than glucose can. Three model proteins, esterase 2 from Alicyclobacillus acidocaldarius, green fluorescent protein, and xylose reductase from Neurospora crassa were synthesized for demonstration. Conclusion Slowly-metabolized maltodextrin as a low-cost secondary energy compound for CFPS produced higher levels of proteins than PEP, glucose, and glucose-6-phospahte. The enhancement of protein synthesis was largely attributed to better-controlled phosphate levels (recycling of inorganic phosphate) and a more homeostatic reaction environment. PMID:19558718

Involvement of 2-C-methyl-D-erythritol-4-phosphate pathway in biosynthesis of aphidicolin-like tetracyclic diterpene of Scoparia dulcis.

PubMed

Nkembo, Marguerite Kasidimoko; Lee, Jung-Bum; Nakagiri, Takeshi; Hayashi, Toshimitsu

2006-05-01

Specific inhibitors of the MVA pathway (pravastatin) and the MEP pathway (fosmidomycin) were used to interfere with the biosynthetic flux which leads to the production of aphidicolin-like diterpene in leaf organ cultures of Scoparia dulcis. Treatment of leaf organs with fosmidomycin resulted in dose dependent inhibition of chlorophylls, carotenoids, scopadulcic acid B (SDB) and phytol production, and no effect on sterol production was observed. In response to the pravastatin treatment, a significant decrease in sterol and perturbation of SDB production was observed.

Examination of Triacylglycerol Biosynthetic Pathways via De Novo Transcriptomic and Proteomic Analyses in an Unsequenced Microalga

DTIC Science & Technology

2011-10-17

analysis results. The components of the TAG biosynthetic pathway, including glycerol-3-phosphate acyl- transferase (GPAT), lyso- phosphatidic acid ...acyltransferase (LPAAT), phosphatidic acid phosphatase (PAP), lyso-phosphati- dylcholine acyltransferase (LPAT), and diacylglycerol acyltransfer- ase (DGAT...transfer to position one of G3P results in the formation of lyso- phosphatidic acid (LPA), in a reaction catalyzed by GPAT. Subsequent acyl transfer to

D-arabinose metabolism in Escherichia coli B: induction and cotransductional mapping of the L-fucose-D-arabinose pathway enzymes.

PubMed

Elsinghorst, E A; Mortlock, R P

1988-12-01

D-Arabinose is degraded by Escherichia coli B via some of the L-fucose pathway enzymes and a D-ribulokinase which is distinct from the L-fuculokinase of the L-fucose pathway. We found that L-fucose and D-arabinose acted as the apparent inducers of the enzymes needed for their degradation. These enzymes, including D-ribulokinase, appeared to be coordinately regulated, and mutants which constitutively synthesized the L-fucose enzymes also constitutively synthesized D-ribulokinase. In contrast to D-arabinose-positive mutants of E. coli K-12, in which L-fuculose-1-phosphate and D-ribulose-1-phosphate act as inducers of the L-fucose pathway, we found that these intermediates did not act as inducers in E. coli B. To further characterize the E. coli B system, some of the L-fucose-D-arabinose genes were mapped by using bacteriophage P1 transduction. A transposon Tn10 insertion near the E. coli B L-fucose regulon was used in two- and three-factor reciprocal crosses. The gene encoding D-ribulokinase, designated darK, was found to map within the L-fucose regulon, and the partial gene order was found to be Tn10-fucA-darK-fucI-fucK-thyA.

D-arabinose metabolism in Escherichia coli B: induction and cotransductional mapping of the L-fucose-D-arabinose pathway enzymes.

PubMed Central

Elsinghorst, E A; Mortlock, R P

1988-01-01

D-Arabinose is degraded by Escherichia coli B via some of the L-fucose pathway enzymes and a D-ribulokinase which is distinct from the L-fuculokinase of the L-fucose pathway. We found that L-fucose and D-arabinose acted as the apparent inducers of the enzymes needed for their degradation. These enzymes, including D-ribulokinase, appeared to be coordinately regulated, and mutants which constitutively synthesized the L-fucose enzymes also constitutively synthesized D-ribulokinase. In contrast to D-arabinose-positive mutants of E. coli K-12, in which L-fuculose-1-phosphate and D-ribulose-1-phosphate act as inducers of the L-fucose pathway, we found that these intermediates did not act as inducers in E. coli B. To further characterize the E. coli B system, some of the L-fucose-D-arabinose genes were mapped by using bacteriophage P1 transduction. A transposon Tn10 insertion near the E. coli B L-fucose regulon was used in two- and three-factor reciprocal crosses. The gene encoding D-ribulokinase, designated darK, was found to map within the L-fucose regulon, and the partial gene order was found to be Tn10-fucA-darK-fucI-fucK-thyA. PMID:3056899

Can activated sludge treatments and advanced oxidation processes remove organophosphorus flame retardants?

PubMed

Cristale, Joyce; Ramos, Dayana D; Dantas, Renato F; Machulek Junior, Amilcar; Lacorte, Silvia; Sans, Carme; Esplugas, Santiago

2016-01-01

This study aims to determine the occurrence of 10 OPFRs (including chlorinated, nonchlorinated alkyl and aryl compounds) in influent, effluent wastewaters and partitioning into sludge of 5 wastewater treatment plants (WWTP) in Catalonia (Spain). All target OPFRs were detected in the WWTPs influents, and the total concentration ranged from 3.67 µg L(-1) to 150 µg L(-1). During activated sludge treatment, most OPFRs were accumulated in the sludge at concentrations from 35.3 to 9980 ng g(-1) dw. Chlorinated compounds tris(2-chloroethyl) phosphate (TCEP), tris(2-chloroisopropyl) phosphate (TCIPP) and tris(2,3-dichloropropyl) phosphate (TDCPP) were not removed by the conventional activated sludge treatment and they were released by the effluents at approximately the same inlet concentration. On the contrary, aryl compounds tris(methylphenyl) phosphate (TMPP) and 2-ethylhexyl diphenyl phosphate (EHDP) together with alkyl tris(2-ethylhexyl) phosphate (TEHP) were not detected in any of the effluents. Advanced oxidation processes (UV/H2O2 and O3) were applied to investigate the degradability of recalcitrant OPFRs in WWTP effluents. Those detected in the effluent sample (TCEP, TCIPP, TDCPP, tributyl phosphate (TNBP), tri-iso-butyl phosphate (TIBP) and tris(2-butoxyethyl) phosphate (TBOEP)) had very low direct UV-C photolysis rates. TBOEP, TNBP and TIBP were degraded by UV/H2O2 and O3. Chlorinated compounds TCEP, TDCPP and TCIPP were the most recalcitrant OPFR to the advanced oxidation processes applied. The study provides information on the partitioning and degradability pathways of OPFR within conventional activated sludge WWTPs. Copyright © 2015 Elsevier Inc. All rights reserved.

High phosphate reduces host ability to develop arbuscular mycorrhizal symbiosis without affecting root calcium spiking responses to the fungus

PubMed Central

Balzergue, Coline; Chabaud, Mireille; Barker, David G.; Bécard, Guillaume; Rochange, Soizic F.

2013-01-01

The arbuscular mycorrhizal symbiosis associates soil fungi with the roots of the majority of plants species and represents a major source of soil phosphorus acquisition. Mycorrhizal interactions begin with an exchange of molecular signals between the two partners. A root signaling pathway is recruited, for which the perception of fungal signals triggers oscillations of intracellular calcium concentration. High phosphate availability is known to inhibit the establishment and/or persistence of this symbiosis, thereby favoring the direct, non-symbiotic uptake of phosphorus by the root system. In this study, Medicago truncatula plants were used to investigate the effects of phosphate supply on the early stages of the interaction. When plants were supplied with high phosphate fungal attachment to the roots was drastically reduced. An experimental system was designed to individually study the effects of phosphate supply on the fungus, on the roots, and on root exudates. These experiments revealed that the most important effects of high phosphate supply were on the roots themselves, which became unable to host mycorrhizal fungi even when these had been appropriately stimulated. The ability of the roots to perceive their fungal partner was then investigated by monitoring nuclear calcium spiking in response to fungal signals. This response did not appear to be affected by high phosphate supply. In conclusion, high levels of phosphate predominantly impact the plant host, but apparently not in its ability to perceive the fungal partner. PMID:24194742

Metabolic regulation of oocyte cell death through the CaMKII-mediated phosphorylation of caspase-2.

PubMed

Nutt, Leta K; Margolis, Seth S; Jensen, Mette; Herman, Catherine E; Dunphy, William G; Rathmell, Jeffrey C; Kornbluth, Sally

2005-10-07

Vertebrate female reproduction is limited by the oocyte stockpiles acquired during embryonic development. These are gradually depleted over the organism's lifetime through the process of apoptosis. The timer that triggers this cell death is yet to be identified. We used the Xenopus egg/oocyte system to examine the hypothesis that nutrient stores can regulate oocyte viability. We show that pentose-phosphate-pathway generation of NADPH is critical for oocyte survival and that the target of this regulation is caspase-2, previously shown to be required for oocyte death in mice. Pentose-phosphate-pathway-mediated inhibition of cell death was due to the inhibitory phosphorylation of caspase-2 by calcium/calmodulin-dependent protein kinase II (CaMKII). These data suggest that exhaustion of oocyte nutrients, resulting in an inability to generate NADPH, may contribute to ooctye apoptosis. These data also provide unexpected links between oocyte metabolism, CaMKII, and caspase-2.

l-Glucitol Catabolism in Stenotrophomonas maltophilia Ac

PubMed Central

Brechtel, Elke; Huwig, Alexander; Giffhorn, Friedrich

2002-01-01

The carbohydrate catabolism of the bacterium Stenotrophomonas maltophilia Ac (previously named Pseudomonas sp. strain Ac), which is known to convert the unnatural polyol l-glucitol to d-sorbose during growth on the former as the sole source of carbon and energy, was studied in detail. All enzymes operating in a pathway that channels l-glucitol via d-sorbose into compounds of the intermediary metabolism were demonstrated, and for some prominent reactions the products of conversion were identified. d-Sorbose was converted by C-3 epimerization to d-tagatose, which, in turn, was isomerized to d-galactose. d-Galactose was the initial substrate of the De Ley-Doudoroff pathway, involving reactions of NAD-dependent oxidation of d-galactose to d-galactonate, its dehydration to 2-keto-3-deoxy-d-galactonate, and its phosphorylation to 2-keto-3-deoxy-d-galactonate 6-phosphate. Finally, aldol cleavage yielded pyruvate and d-glycerate 3-phosphate as the central metabolic intermediates. PMID:11823194

OsCYCP1;1, a PHO80 homologous protein, negatively regulates phosphate starvation signaling in the roots of rice (Oryza sativa L.).

PubMed

Deng, Minjuan; Hu, Bin; Xu, Lei; Liu, Yang; Wang, Fang; Zhao, Hongyu; Wei, Xijuan; Wang, Jichao; Yi, Keke

2014-12-01

Phosphorus is one of the most essential and limiting nutrients in all living organisms, thus the organisms have evolved complicated and precise regulatory mechanisms for phosphorus acquisition, storage and homeostasis. In the budding yeast, Saccharomyces cerevisiae, the modification of PHO4 by the PHO80 and PHO85 complex is a core regulation system. However, the existence and possible functions in phosphate signaling of the homologs of the PHO80 and PHO85 components in plants has yet to be determined. Here we describe the identification of a family of seven PHO80 homologous genes in rice named OsCYCPs. Among these, the OsCYCP1;1 gene was able to partially rescue the pho80 mutant strain of yeast. The OsCYCP1;1 protein was predominantly localized in the nucleus, and was ubiquitously expressed throughout the whole plant and during the entire growth period of rice. Consistent with the negative role of PHO80 in phosphate signaling in yeast, OsCYCP1;1 expression was reduced by phosphate starvation in the roots. This reduction was dependent on PHR2, the central regulator of phosphate signaling in rice. Overexpression and suppression of the expression of OsCYCP1;1 influenced the phosphate starvation signaling response. The inducible expression of phosphate starvation inducible and phosphate transporter genes was suppressed in the OsCYCP1;1 overexpression lines and was relatively enhanced in the OsCYCP1;1 RNAi plants by phosphate starvation. Together, these results demonstrate the role of PHO80 homologs in the phosphate starvation signaling pathway in rice.

Nanobacteria: Fact or Fiction? Characteristics, Detection and Medical Importance of Novel Self-Replicating, Calcifying Nanoparticles

NASA Technical Reports Server (NTRS)

Ciftcioglu, Neva; Mckay, David S.; Mathew, Grace; Kajander, E. Olavi

2006-01-01

There is some debate in microbiology as to whether Nanobacteria (NB) are alive. This paper reviews some aspects of NB. In summary, Nanobacteria is a perfect model for studying biogenic mineralization/calcification because NB a) are self-replicating particles and have less complicated metabolic pathways b) accumulate calcium and phosphate under physiological conditions, c)produce a calcium phosphate mineral similar to bone, d) exist in physical conditions (pH, gravity, temperature, etc) that are easy to manipulate, and which can be replicated for the physiological model.

Leishmania UDP-sugar pyrophosphorylase: the missing link in galactose salvage?

PubMed

Damerow, Sebastian; Lamerz, Anne-Christin; Haselhorst, Thomas; Führing, Jana; Zarnovican, Patricia; von Itzstein, Mark; Routier, Françoise H

2010-01-08

The Leishmania parasite glycocalyx is rich in galactose-containing glycoconjugates that are synthesized by specific glycosyltransferases that use UDP-galactose as a glycosyl donor. UDP-galactose biosynthesis is thought to be predominantly a de novo process involving epimerization of the abundant nucleotide sugar UDP-glucose by the UDP-glucose 4-epimerase, although galactose salvage from the environment has been demonstrated for Leishmania major. Here, we present the characterization of an L. major UDP-sugar pyrophosphorylase able to reversibly activate galactose 1-phosphate into UDP-galactose thus proving the existence of the Isselbacher salvage pathway in this parasite. The ordered bisubstrate mechanism and high affinity of the enzyme for UTP seem to favor the synthesis of nucleotide sugar rather than their pyrophosphorolysis. Although L. major UDP-sugar pyrophosphorylase preferentially activates galactose 1-phosphate and glucose 1-phosphate, the enzyme is able to act on a variety of hexose 1-phosphates as well as pentose 1-phosphates but not hexosamine 1-phosphates and hence presents a broad in vitro specificity. The newly identified enzyme exhibits a low but significant homology with UDP-glucose pyrophosphorylases and conserved in particular is the pyrophosphorylase consensus sequence and residues involved in nucleotide and phosphate binding. Saturation transfer difference NMR spectroscopy experiments confirm the importance of these moieties for substrate binding. The described leishmanial enzyme is closely related to plant UDP-sugar pyrophosphorylases and presents a similar substrate specificity suggesting their common origin.

Modeling and experimental design for metabolic flux analysis of lysine-producing Corynebacteria by mass spectrometry.

PubMed

Wittmann, C; Heinzle, E

2001-04-01

Experimental design of (13)C-tracer studies for metabolic flux analysis with mass spectrometric determination of labeling patterns was performed for the central metabolism of Corynebacterium glutamicum comprising various flux scenarios. Ratio measurement of mass isotopomer pools of Corynebacterium products lysine, alanine, and trehalose is sufficient to quantify the flux partitioning ratios (i) between glycolysis and pentose phosphate pathways (Phi(PPP)), (ii) between the split pathways in the lysine biosynthesis (Phi(DH)), (iii) at the pyruvate node (Phi(PC)), and reversibilities of (iv) glucose 6-phosphate isomerase (zeta(PGI)), (v) at the pyruvate node (zeta(PC/PEPCK)), and (vi) of transaldolase and transketolases in the PPP. Weighted sensitivities for flux parameters were derived from partial derivatives to quantitatively evaluate experimental approaches and predict precision for estimated flux parameters. Deviation of intensity ratios from ideal values of 1 was used as weighting function. Weighted flux sensitivities can be used to identify optimal type and degree of tracer labeling or potential intensity ratios to be measured. Experimental design for lysine-producing strain C. glutamicum MH 20-22B (Marx et al., Biotechnol. Bioeng. 49, 111-129, 1996) and various potential mutants with different alterations in the flux pattern showed that specific tracer labelings are optimal to quantify a certain flux parameter uninfluenced by the overall flux situation. Identified substrates of choice are [1-(13)C]glucose for the estimation of Phi(PPP) and zeta(PGI) and a 1 : 1 mixture of [U-(12)C/U-(13)C]glucose for the determination of zeta(PC/PEPCK). Phi(PC) can be quantified by feeding [4-(13)C]glucose or [U-(12)C/U-(13)C]glucose (1 : 1), whereas Phi(DH) is accessible via [4-(13)C]glucose. The sensitivity for the quantification of a certain flux parameter can be influenced by superposition through other flux parameters in the network, but substrate and measured mass isotopomers of choice remain the same. In special cases, reduced labeling degree of the tracer substrate can increase the precision of flux analysis. Enhanced precision and flux information can be achieved via multiply labeled substrates. The presented approach can be applied for effective experimental design of (13)C tracer studies for metabolic flux analysis. Intensity ratios of other products such as glutamate, valine, phenylalanine, and riboflavin also sensitively reflect flux parameters, which underlines the great potential of mass spectrometry for flux analysis. Copyright 2001 Academic Press.

Bacillus stearothermophilus sporulation response to different composition media.

PubMed

Penna, T C; Machoshvili, I A; Taqueda, M E; Ferraz, C A

1998-01-01

To evaluate the effectiveness of 11 commonly used ingredients to improve Bacillus stearothermophilus ATCC 7953 sporulation, with high spore yields in a short period of incubation, 32 composition media were set up by a fractional factorial 2IV11-6 design at two levels: D-glucose (0.018-0.25%), L-glutamic acid (0.040-0.10%), yeast extract (0.050-0.40%), peptone (0.30-0.50%), sodium chloride (0.001-1.0%), magnesium sulfate (0.001-0.20%), ammonium phosphate (0.010-0.035%), potassium phosphate monobasic (0.050-0.25%), calcium chloride (0.001-0.05%), ferrous sulfate (0.0003-0.002%), manganese sulfate (0.001-0.50%). The largest variation on Log10 CFU response took place due to sodium chloride main effect, by changing it from low to high levels. Magnesium sulfate, calcium chloride, and ferrous sulfate were split and exerted no detectable main effect influence on sporulation. Setting up two 16 runs for sodium chloride effect, in each of which the remainder levels were kept constant, other components contribution was studied. At low sodium chloride, best average 7.25 Log10 CFU yielded by fastening yeast extract and peptone at high level, and remainders at low level. Considering high level of sodium chloride, peptone, yeast extract and ammonium phosphate kept at high level and remainders at low level confirmed the best sporulation yield. Adjusted models evidenced a strong influence of joint yeast/peptone effect, associated to ammonium phosphate contributing positively. The reduced incubation period from 15 days to 3-6 days at 62 degrees C was attained for all 32 experimental runs.

Crystal Structure and Substrate Specificity of D-Galactose-6-Phosphate Isomerase Complexed with Substrates

PubMed Central

Lee, Jung-Kul; Pan, Cheol-Ho

2013-01-01

D-Galactose-6-phosphate isomerase from Lactobacillus rhamnosus (LacAB; EC 5.3.1.26), which is encoded by the tagatose-6-phosphate pathway gene cluster (lacABCD), catalyzes the isomerization of D-galactose-6-phosphate to D-tagatose-6-phosphate during lactose catabolism and is used to produce rare sugars as low-calorie natural sweeteners. The crystal structures of LacAB and its complex with D-tagatose-6-phosphate revealed that LacAB is a homotetramer of LacA and LacB subunits, with a structure similar to that of ribose-5-phosphate isomerase (Rpi). Structurally, LacAB belongs to the RpiB/LacAB superfamily, having a Rossmann-like αβα sandwich fold as has been identified in pentose phosphate isomerase and hexose phosphate isomerase. In contrast to other family members, the LacB subunit also has a unique α7 helix in its C-terminus. One active site is distinctly located at the interface between LacA and LacB, whereas two active sites are present in RpiB. In the structure of the product complex, the phosphate group of D-tagatose-6-phosphate is bound to three arginine residues, including Arg-39, producing a different substrate orientation than that in RpiB, where the substrate binds at Asp-43. Due to the proximity of the Arg-134 residue and backbone Cα of the α6 helix in LacA to the last Asp-172 residue of LacB with a hydrogen bond, a six-carbon sugar-phosphate can bind in the larger pocket of LacAB, compared with RpiB. His-96 in the active site is important for ring opening and substrate orientation, and Cys-65 is essential for the isomerization activity of the enzyme. Two rare sugar substrates, D-psicose and D-ribulose, show optimal binding in the LacAB-substrate complex. These findings were supported by the results of LacA activity assays. PMID:24015281

Effect of submarine groundwater discharge containing phosphate on coral calcification

NASA Astrophysics Data System (ADS)

Yasumoto, J.; Yasumoto, K.; Iijima, M.; Nozaki, M.; Asai, K.; Yasumoto, M. H.

2017-12-01

It is well known that the anthropogenic eutrophication enriched with various substances including phosphate in coastal waters has resulted in coral degradation. However, to the best of our knowledge, the phosphate threshold value to inhibit the coral calcification has been unclear, due to the unknown mechanisms involved in the inhibition of the calcification by phosphate. In island regions, groundwater is one of the most important clues to transport the nutrients contained in livestock or agricultural wastewaters. However, the actual conditions of coastal pollution with such nutrients have not been understood because of unperceived submarine groundwater discharge (SGD). In this study, to quantify of extremely rapid and localized SGD from Ryukyu limestone aquifer, we investigated the rate and concentration of phosphate of SGD using automated seepage mater in Yoron Island, which is located southern part of Japan. And, to elucidate the inhibition mechanisms for phosphate against coral calcification, we examined its effect on the bottom skeleton formation in primary polyps of Acropora digitifera by using the fluorescence derivatizing reagent having phosphate group (FITC-AA). As a result, the SGD was found to contain 1 to 2 µM of phosphate as much as the concentration in the coastal ground water under agricultural land. Moreover, the amount of phosphate contained in the surface layers of bottom calcareous sands close to the region of SGD were about 5 µmol/g. When the primary polyps were treated with 50 µM of FITC-AA, the bottom skeleton of the primary polyps showed the fluorescence from FITC-AA within a few minutes, suggesting the phosphate binding. Furthermore, when the polyps were treated with 10 µM of FITC-AA, irregular patterns of the elongated skeleton were observed. These results led us to conclude that phosphate is transported via a paracellular pathway to the subcalicoblastic extracellular calcifying medium. These results indicate that the phosphate adsorbed to the bottom sand may also inhibit coral skeleton formation.

Multiplex detection of protein-protein interactions using a next generation luciferase reporter.

PubMed

Verhoef, Lisette G G C; Mattioli, Michela; Ricci, Fernanda; Li, Yao-Cheng; Wade, Mark

2016-02-01

Cell-based assays of protein-protein interactions (PPIs) using split reporter proteins can be used to identify PPI agonists and antagonists. Generally, such assays measure one PPI at a time, and thus counterscreens for on-target activity must be run in parallel or at a subsequent stage; this increases both the cost and time during screening. Split luciferase systems offer advantages over those that use split fluorescent proteins (FPs). This is since split luciferase offers a greater signal:noise ratio and, unlike split FPs, the PPI can be reversed upon small molecule treatment. While multiplexed PPI assays using luciferase have been reported, they suffer from low signal:noise and require fairly complex spectral deconvolution during analysis. Furthermore, the luciferase enzymes used are large, which limits the range of PPIs that can be interrogated due to steric hindrance from the split luciferase fragments. Here, we report a multiplexed PPI assay based on split luciferases from Photinus pyralis (firefly luciferase, FLUC) and the deep-sea shrimp, Oplophorus gracilirostris (NanoLuc, NLUC). Specifically, we show that the binding of the p53 tumor suppressor to its two major negative regulators, MDM2 and MDM4, can be simultaneously measured within the same sample, without the requirement for complex filters or deconvolution. We provide chemical and genetic validation of this system using MDM2-targeted small molecules and mutagenesis, respectively. Combined with the superior signal:noise and smaller size of split NanoLuc, this multiplexed PPI assay format can be exploited to study the induction or disruption of pairwise interactions that are prominent in many cell signaling pathways. Copyright © 2015 Elsevier B.V. All rights reserved.

ATP-binding Cassette (ABC) Transport System Solute-binding Protein-guided Identification of Novel d-Altritol and Galactitol Catabolic Pathways in Agrobacterium tumefaciens C58*

PubMed Central

Wichelecki, Daniel J.; Vetting, Matthew W.; Chou, Liyushang; Al-Obaidi, Nawar; Bouvier, Jason T.; Almo, Steven C.; Gerlt, John A.

2015-01-01

Innovations in the discovery of the functions of uncharacterized proteins/enzymes have become increasingly important as advances in sequencing technology flood protein databases with an exponentially growing number of open reading frames. This study documents one such innovation developed by the Enzyme Function Initiative (EFI; U54GM093342), the use of solute-binding proteins for transport systems to identify novel metabolic pathways. In a previous study, this strategy was applied to the tripartite ATP-independent periplasmic transporters. Here, we apply this strategy to the ATP-binding cassette transporters and report the discovery of novel catabolic pathways for d-altritol and galactitol in Agrobacterium tumefaciens C58. These efforts resulted in the description of three novel enzymatic reactions as follows: 1) oxidation of d-altritol to d-tagatose via a dehydrogenase in Pfam family PF00107, a previously unknown reaction; 2) phosphorylation of d-tagatose to d-tagatose 6-phosphate via a kinase in Pfam family PF00294, a previously orphan EC number; and 3) epimerization of d-tagatose 6-phosphate C-4 to d-fructose 6-phosphate via a member of Pfam family PF08013, another previously unknown reaction. The epimerization reaction catalyzed by a member of PF08013 is especially noteworthy, because the functions of members of PF08013 have been unknown. These discoveries were assisted by the following two synergistic bioinformatics web tools made available by the Enzyme Function Initiative: the EFI-Enzyme Similarity Tool and the EFI-Genome Neighborhood Tool. PMID:26472925

Bisphosphonate Inhibitors Reveal a Large Elasticity of Plastidic Isoprenoid Synthesis Pathway in Isoprene-Emitting Hybrid Aspen1

PubMed Central

2015-01-01

Recently, a feedback inhibition of the chloroplastic 1-deoxy-d-xylulose 5-phosphate (DXP)/2-C-methyl-d-erythritol 4-phosphate (MEP) pathway of isoprenoid synthesis by end products dimethylallyl diphosphate (DMADP) and isopentenyl diphosphate (IDP) was postulated, but the extent to which DMADP and IDP can build up is not known. We used bisphosphonate inhibitors, alendronate and zoledronate, that inhibit the consumption of DMADP and IDP by prenyltransferases to gain insight into the extent of end product accumulation and possible feedback inhibition in isoprene-emitting hybrid aspen (Populus tremula × Populus tremuloides). A kinetic method based on dark release of isoprene emission at the expense of substrate pools accumulated in light was used to estimate the in vivo pool sizes of DMADP and upstream metabolites. Feeding with fosmidomycin, an inhibitor of DXP reductoisomerase, alone or in combination with bisphosphonates was used to inhibit carbon input into DXP/MEP pathway or both input and output. We observed a major increase in pathway intermediates, 3- to 4-fold, upstream of DMADP in bisphosphonate-inhibited leaves, but the DMADP pool was enhanced much less, 1.3- to 1.5-fold. In combined fosmidomycin/bisphosphonate treatment, pathway intermediates accumulated, reflecting cytosolic flux of intermediates that can be important under strong metabolic pull in physiological conditions. The data suggested that metabolites accumulated upstream of DMADP consist of phosphorylated intermediates and IDP. Slow conversion of the huge pools of intermediates to DMADP was limited by reductive energy supply. These data indicate that the DXP/MEP pathway is extremely elastic, and the presence of a significant pool of phosphorylated intermediates provides an important valve for fine tuning the pathway flux. PMID:25926480

Quantitative Analysis of Energy Metabolic Pathways in MCF-7 Breast Cancer Cells by Selected Reaction Monitoring Assay*

PubMed Central

Drabovich, Andrei P.; Pavlou, Maria P.; Dimitromanolakis, Apostolos; Diamandis, Eleftherios P.

2012-01-01

To investigate the quantitative response of energy metabolic pathways in human MCF-7 breast cancer cells to hypoxia, glucose deprivation, and estradiol stimulation, we developed a targeted proteomics assay for accurate quantification of protein expression in glycolysis/gluconeogenesis, TCA cycle, and pentose phosphate pathways. Cell growth conditions were selected to roughly mimic the exposure of cells in the cancer tissue to the intermittent hypoxia, glucose deprivation, and hormonal stimulation. Targeted proteomics assay allowed for reproducible quantification of 76 proteins in four different growth conditions after 24 and 48 h of perturbation. Differential expression of a number of control and metabolic pathway proteins in response to the change of growth conditions was found. Elevated expression of the majority of glycolytic enzymes was observed in hypoxia. Cancer cells, as opposed to near-normal MCF-10A cells, exhibited significantly increased expression of key energy metabolic pathway enzymes (FBP1, IDH2, and G6PD) that are known to redirect cellular metabolism and increase carbon flux through the pentose phosphate pathway. Our quantitative proteomic protocol is based on a mass spectrometry-compatible acid-labile detergent and is described in detail. Optimized parameters of a multiplex selected reaction monitoring (SRM) assay for 76 proteins, 134 proteotypic peptides, and 401 transitions are included and can be downloaded and used with any SRM-compatible mass spectrometer. The presented workflow is an integrated tool for hypothesis-driven studies of mammalian cells as well as functional studies of proteins, and can greatly complement experimental methods in systems biology, metabolic engineering, and metabolic transformation of cancer cells. PMID:22535206

Targeting the Nonmevalonate Pathway in Burkholderia cenocepacia Increases Susceptibility to Certain β-Lactam Antibiotics.

PubMed

Sass, Andrea; Everaert, Annelien; Van Acker, Heleen; Van den Driessche, Freija; Coenye, Tom

2018-05-01

The nonmevalonate pathway is the sole pathway for isoprenoid biosynthesis in Burkholderia cenocepacia and is possibly a novel target for the development of antibacterial chemotherapy. The goals of the present study were to evaluate the essentiality of dxr , the second gene of the nonmevalonate pathway, in B. cenocepacia and to determine whether interfering with the nonmevalonate pathway increases susceptibility toward antibiotics. To this end, a rhamnose-inducible conditional dxr knockdown mutant of B. cenocepacia strain K56-2 ( B. cenocepacia K56-2 dxr ) was constructed, using a plasmid which enables the delivery of a rhamnose-inducible promoter in the chromosome. Expression of dxr is essential for bacterial growth; the growth defect observed in the dxr mutant could be complemented by expressing dxr in trans under the control of a constitutive promoter, but not by providing 2- C -methyl-d-erythritol-4-phosphate, the reaction product of DXR (1-deoxy-d-xylulose 5-phosphate reductoisomerase). B. cenocepacia K56-2 dxr showed markedly increased susceptibility to the β-lactam antibiotics aztreonam, ceftazidime, and cefotaxime, while susceptibility to other antibiotics was not (or was much less) affected; this increased susceptibility could also be complemented by in trans expression of dxr A similarly increased susceptibility was observed when antibiotics were combined with FR900098, a known DXR inhibitor. Our data confirm that the nonmevalonate pathway is essential in B. cenocepacia and suggest that combining potent DXR inhibitors with selected β-lactam antibiotics is a useful strategy to combat B. cenocepacia infections. Copyright © 2018 American Society for Microbiology.

Tilapia (Oreochromis mossambicus) brain cells respond to hyperosmotic challenge by inducing myo-inositol biosynthesis

PubMed Central

Gardell, Alison M.; Yang, Jun; Sacchi, Romina; Fangue, Nann A.; Hammock, Bruce D.; Kültz, Dietmar

2013-01-01

SUMMARY This study aimed to determine the regulation of the de novo myo-inositol biosynthetic (MIB) pathway in Mozambique tilapia (Oreochromis mossambicus) brain following acute (25 ppt) and chronic (30, 60 and 90 ppt) salinity acclimations. The MIB pathway plays an important role in accumulating the compatible osmolyte, myo-inositol, in cells in response to hyperosmotic challenge and consists of two enzymes, myo-inositol phosphate synthase and inositol monophosphatase. In tilapia brain, MIB enzyme transcriptional regulation was found to robustly increase in a time (acute acclimation) or dose (chronic acclimation) dependent manner. Blood plasma osmolality and Na+ and Cl− concentrations were also measured and significantly increased in response to both acute and chronic salinity challenges. Interestingly, highly significant positive correlations were found between MIB enzyme mRNA and blood plasma osmolality in both acute and chronic salinity acclimations. Additionally, a mass spectrometry assay was established and used to quantify total myo-inositol concentration in tilapia brain, which closely mirrored the hyperosmotic MIB pathway induction. Thus, myo-inositol is a major compatible osmolyte that is accumulated in brain cells when exposed to acute and chronic hyperosmotic challenge. These data show that the MIB pathway is highly induced in response to environmental salinity challenge in tilapia brain and that this induction is likely prompted by increases in blood plasma osmolality. Because the MIB pathway uses glucose-6-phosphate as a substrate and large amounts of myo-inositol are being synthesized, our data also illustrate that the MIB pathway likely contributes to the high energetic demand posed by salinity challenge. PMID:24072790

Anaerobic phosphate release from activated sludge with enhanced biological phosphorus removal. A possible mechanism of intracellular pH control

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

Bond, P.L.; Keller, J.; Blackall, L.L.

The biochemical mechanisms of the wastewater treatment process known as enhanced biological phosphorus removal (EBPR) are presently described in a metabolic model. The authors investigated details of the EBPR model to determine the nature of the anaerobic phosphate release and how this may be metabolically associated with polyhydroxyalkanoate (PHA) formation. Iodoacetate, an inhibitor of glycolysis, was found to inhibit the anaerobic formation of PHA and phosphate release, supporting the pathways proposed in the EBPR metabolic model. In the metabolic model, it is proposed that polyphosphate degradation provides energy for the microorganisms in anaerobic regions of these treatment systems. Other investigationsmore » have shown that anaerobic phosphate release depends on the extracellular pH. The authors observed that when the intracellular pH of EBPR sludge was raised, substantial anaerobic phosphate release was caused without volatile fatty acid (VFA) uptake. Acidification of the sludge inhibited anaerobic phosphate release even in the presence of VFA. from these observations, the authors postulate that an additional possible role of anaerobic polyphosphate degradation in EBPR is for intracellular pH control. Intracellular pH control may be a metabolic feature of EBPR, not previously considered, that could have some use in the control and optimization of EBPR.« less

The PhoBR two-component system regulates antibiotic biosynthesis in Serratia in response to phosphate

PubMed Central

2009-01-01

Background Secondary metabolism in Serratia sp. ATCC 39006 (Serratia 39006) is controlled via a complex network of regulators, including a LuxIR-type (SmaIR) quorum sensing (QS) system. Here we investigate the molecular mechanism by which phosphate limitation controls biosynthesis of two antibiotic secondary metabolites, prodigiosin and carbapenem, in Serratia 39006. Results We demonstrate that a mutation in the high affinity phosphate transporter pstSCAB-phoU, believed to mimic low phosphate conditions, causes upregulation of secondary metabolism and QS in Serratia 39006, via the PhoBR two-component system. Phosphate limitation also activated secondary metabolism and QS in Serratia 39006. In addition, a pstS mutation resulted in upregulation of rap. Rap, a putative SlyA/MarR-family transcriptional regulator, shares similarity with the global regulator RovA (regulator of virulence) from Yersina spp. and is an activator of secondary metabolism in Serratia 39006. We demonstrate that expression of rap, pigA-O (encoding the prodigiosin biosynthetic operon) and smaI are controlled via PhoBR in Serratia 39006. Conclusion Phosphate limitation regulates secondary metabolism in Serratia 39006 via multiple inter-linked pathways, incorporating transcriptional control mediated by three important global regulators, PhoB, SmaR and Rap. PMID:19476633

Determination of the fluxes in the central metabolism of Corynebacterium glutamicum by nuclear magnetic resonance spectroscopy combined with metabolite balancing.

PubMed

Marx, A; de Graaf, A A; Wiechert, W; Eggeling, L; Sahm, H

1996-01-20

To determine the in vivo fluxes of the central metabolism we have developed a comprehensive approach exclusively based on the fundamental enzyme reactions known to be present, the fate of the carbon atoms of individual reactions, and the metabolite balance of the culture. No information on the energy balance is required, nor information on enzyme activities, or the directionalities of reactions. Our approach combines the power of (1)H-detected (13)C nuclear magnetic resonance spectroscopy to follow individual carbons with the simplicity of establishing carbon balances of bacterial cultures. We grew a lysine-producing strain of Corynebacterium glutamicum to the metabolic and isotopic steady state with [1-(13)C]glucose and determined the fractional enrichments in 27 carbon atoms of 11 amino acids isolated from the cell. Since precursor metabolites of the central metabolism are incorporated in an exactly defined manner in the carbon skeleton of amino acids, the fractional enrichments in carbons of precursor metabolites (oxaloacetate, glyceraldehyde 3-phosphate, erythrose 4-phosphate, etc.) became directly accessible. A concise and generally applicable mathematical model was established using matrix calculus to express all metabolite mass and carbon labeling balances. An appropriate all-purpose software for the iterative solution of the equations is supplied. Applying this comprehensive methodology to C. glutamicum, all major fluxes within the central metabolism were determined. The result is that the flux through the pentose phosphate pathway is 66.4% (relative to the glucose input flux of 1.49 mmol/g dry weight h), that of entry into the tricarboxylic acid cycle 62.2%, and the contribution of the succinylase pathway of lysine synthesis 13.7%. Due to the large amount and high quality of measured data in vivo exchange reactions could also be quantitated with particularly high exchange rates within the pentose phosphate pathway for the ribose 5-phosphate transketolase reaction. Moreover, the total net flux of the anaplerotic reactions was quantitated as 38.0%. Most importantly, we found that in vivo one component within these anaplerotic reactions is a back flux from the carbon 4 units of the tricarboxylic acid cycle to the carbon 3 units of glycolysis of 30.6%. (c) 1996 John Wiley & Sons, Inc.

REGULATION OF METABOLISM, IN VIVO,

DTIC Science & Technology

The concentrations of 35 critical metabolites, including glycogen, trehalose , components of the glycolytic pathway and citric acid cycle, amino acids...sites of regulation: the phosphorylation of fructose-6-phosphate; the cleavage of trehalose ; and the phosphorolysis of glycogen were established in

Plastidic Phosphoglucose Isomerase Is an Important Determinant of Starch Accumulation in Mesophyll Cells, Growth, Photosynthetic Capacity, and Biosynthesis of Plastidic Cytokinins in Arabidopsis

PubMed Central

De Diego, Nuria; Muñoz, Francisco J.; Baroja-Fernández, Edurne; Li, Jun; Ricarte-Bermejo, Adriana; Baslam, Marouane; Aranjuelo, Iker; Almagro, Goizeder; Humplík, Jan F.; Novák, Ondřej; Spíchal, Lukáš; Doležal, Karel; Pozueta-Romero, Javier

2015-01-01

Phosphoglucose isomerase (PGI) catalyzes the reversible isomerization of glucose-6-phosphate and fructose-6-phosphate. It is involved in glycolysis and in the regeneration of glucose-6-P molecules in the oxidative pentose phosphate pathway (OPPP). In chloroplasts of illuminated mesophyll cells PGI also connects the Calvin-Benson cycle with the starch biosynthetic pathway. In this work we isolated pgi1-3, a mutant totally lacking pPGI activity as a consequence of aberrant intron splicing of the pPGI encoding gene, PGI1. Starch content in pgi1-3 source leaves was ca. 10-15% of that of wild type (WT) leaves, which was similar to that of leaves of pgi1-2, a T-DNA insertion pPGI null mutant. Starch deficiency of pgi1 leaves could be reverted by the introduction of a sex1 null mutation impeding β-amylolytic starch breakdown. Although previous studies showed that starch granules of pgi1-2 leaves are restricted to both bundle sheath cells adjacent to the mesophyll and stomata guard cells, microscopy analyses carried out in this work revealed the presence of starch granules in the chloroplasts of pgi1-2 and pgi1-3 mesophyll cells. RT-PCR analyses showed high expression levels of plastidic and extra-plastidic β-amylase encoding genes in pgi1 leaves, which was accompanied by increased β-amylase activity. Both pgi1-2 and pgi1-3 mutants displayed slow growth and reduced photosynthetic capacity phenotypes even under continuous light conditions. Metabolic analyses revealed that the adenylate energy charge and the NAD(P)H/NAD(P) ratios in pgi1 leaves were lower than those of WT leaves. These analyses also revealed that the content of plastidic 2-C-methyl-D-erythritol 4-phosphate (MEP)-pathway derived cytokinins (CKs) in pgi1 leaves were exceedingly lower than in WT leaves. Noteworthy, exogenous application of CKs largely reverted the low starch content phenotype of pgi1 leaves. The overall data show that pPGI is an important determinant of photosynthesis, energy status, growth and starch accumulation in mesophyll cells likely as a consequence of its involvement in the production of OPPP/glycolysis intermediates necessary for the synthesis of plastidic MEP-pathway derived hormones such as CKs. PMID:25811607

Novel Fusion Protein Approach for Efficient High-Throughput Screening of Small Molecule–Mediating Protein-Protein Interactions in Cells and Living Animals

PubMed Central

Paulmurugan, Ramasamy; Gambhir, Sanjiv S.

2014-01-01

Networks of protein interactions execute many different intracellular pathways. Small molecules either synthesized within the cell or obtained from the external environment mediate many of these protein-protein interactions. The study of these small molecule–mediated protein-protein interactions is important in understanding abnormal signal transduction pathways in a variety of disorders, as well as in optimizing the process of drug development and validation. In this study, we evaluated the rapamycin-mediated interaction of the human proteins FK506-binding protein (FKBP12) rapamycin-binding domain (FRB) and FKBP12 by constructing a fusion of these proteins with a split-Renilla luciferase or a split enhanced green fluorescent protein (split-EGFP) such that complementation of the reporter fragments occurs in the presence of rapamycin. Different linker peptides in the fusion protein were evaluated for the efficient maintenance of complemented reporter activity. This system was studied in both cell culture and xenografts in living animals. We found that peptide linkers with two or four EAAAR repeat showed higher protein-protein interaction–mediated signal with lower background signal compared with having no linker or linkers with amino acid sequences GGGGSGGGGS, ACGSLSCGSF, and ACGSLSCGS-FACGSLSCGSF. A 9 ± 2-fold increase in signal intensity both in cell culture and in living mice was seen compared with a system that expresses both reporter fragments and the interacting proteins separately. In this fusion system, rapamycin induced heterodimerization of the FRB and FKBP12 moieties occurred rapidly even at very lower concentrations (0.00001 nmol/L) of rapamycin. For a similar fusion system employing split-EGFP, flow cytometry analysis showed significant level of rapamycin-induced complementation. PMID:16103094

Novel fusion protein approach for efficient high-throughput screening of small molecule-mediating protein-protein interactions in cells and living animals.

PubMed

Paulmurugan, Ramasamy; Gambhir, Sanjiv S

2005-08-15

Networks of protein interactions execute many different intracellular pathways. Small molecules either synthesized within the cell or obtained from the external environment mediate many of these protein-protein interactions. The study of these small molecule-mediated protein-protein interactions is important in understanding abnormal signal transduction pathways in a variety of disorders, as well as in optimizing the process of drug development and validation. In this study, we evaluated the rapamycin-mediated interaction of the human proteins FK506-binding protein (FKBP12) rapamycin-binding domain (FRB) and FKBP12 by constructing a fusion of these proteins with a split-Renilla luciferase or a split enhanced green fluorescent protein (split-EGFP) such that complementation of the reporter fragments occurs in the presence of rapamycin. Different linker peptides in the fusion protein were evaluated for the efficient maintenance of complemented reporter activity. This system was studied in both cell culture and xenografts in living animals. We found that peptide linkers with two or four EAAAR repeat showed higher protein-protein interaction-mediated signal with lower background signal compared with having no linker or linkers with amino acid sequences GGGGSGGGGS, ACGSLSCGSF, and ACGSLSCGSFACGSLSCGSF. A 9 +/- 2-fold increase in signal intensity both in cell culture and in living mice was seen compared with a system that expresses both reporter fragments and the interacting proteins separately. In this fusion system, rapamycin induced heterodimerization of the FRB and FKBP12 moieties occurred rapidly even at very lower concentrations (0.00001 nmol/L) of rapamycin. For a similar fusion system employing split-EGFP, flow cytometry analysis showed significant level of rapamycin-induced complementation.

Ab-initio study of dilute nitride substitutional and split-interstitial impurities in gallium antimonide (N-GaSb)

NASA Astrophysics Data System (ADS)

Jadaun, Priyamvada; Nair, Hari P.; Bank, Seth R.; Banerjee, Sanjay K.

2012-02-01

We present an ab-initio density functinal theory study of dilute-nitride GaSb. Adding dilute quantities of nitrogen causes rapid reduction in bandgap of GaSb (˜300 meV for 2% N). Due to this rapid reduction in bandgap, dilute-nitrides provide a pathway for extending the emission of GaSb based type-I diode lasers into the mid-infrared wavelength region (3-5 micron). In this study we look at the effect of substitutional N impurity on the electronic properties of our system and compare it with the band-anticrossing model, a phenomenological model, which has been used to explain giant band bowing observed in dilute-nitride alloys. We also study the effect of Sb-N split interstitials which are known to be non-radiative recombination centers. Furthermore we also discuss the stability of the Sb-N split interstitial relative to substitutional nitrogen to determine if the split interstitials can be annihilated using post-growth annealing to improve the radiative lifetime of the material which essential for laser operation.

Split luciferase complementation assay for the analysis of G protein-coupled receptor ligand response in Saccharomyces cerevisiae.

PubMed

Fukutani, Yosuke; Ishii, Jun; Kondo, Akihiko; Ozawa, Takeaki; Matsunami, Hiroaki; Yohda, Masafumi

2017-06-01

The budding yeast Saccharomyces cerevisiae is equipped with G protein-coupled receptors (GPCR). Because the yeast GPCR signaling mechanism is partly similar to that of the mammalian system, S. cerevisiae can be used for a host of mammalian GPCR expression and ligand-mediated activation assays. However, currently available yeast systems require several hours to observe the responses because they depend on the expression of reporter genes. In this study, we attempted to develop a simple GPCR assay system using split luciferase and β-arrestin, which are independent of the endogenous S. cerevisiae GPCR signaling pathways. We applied the split luciferase complementation assay method to S. cerevisiae and found that it can be used to analyze the ligand response of the human somatostatin receptor in S. cerevisiae. On the contrary, the response of the pheromone receptor Ste2 was not observed by the assay. Thus, the split luciferase complementation should be free from the effect of the endogenous GPCR signaling. Biotechnol. Bioeng. 2017;114: 1354-1361. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Diiridium Bimetallic Complexes Function as a Redox Switch To Directly Split Carbonate into Carbon Monoxide and Oxygen.

PubMed

Chen, Tsun-Ren; Wu, Fang-Siou; Lee, Hsiu-Pen; Chen, Kelvin H-C

2016-03-23

A pair of diiridium bimetallic complexes exhibit a special type of oxidation-reduction reaction that could directly split carbonate into carbon monoxide and molecular oxygen via a low-energy pathway needing no sacrificial reagent. One of the bimetallic complexes, Ir(III)(μ-Cl)2Ir(III), can catch carbonato group from carbonate and reduce it to CO. The second complex, the rare bimetallic complex Ir(IV)(μ-oxo)2Ir(IV), can react with chlorine to release O2 by the oxidation of oxygen ions with synergistic oxidative effect of iridium ions and chlorine atoms. The activation energy needed for the key reaction is quite low (∼20 kJ/mol), which is far less than the dissociation energy of the C═O bond in CO2 (∼750 kJ/mol). These diiridium bimetallic complexes could be applied as a redox switch to split carbonate or combined with well-known processes in the chemical industry to build up a catalytic system to directly split CO2 into CO and O2.

Phosphine from rocks: mechanically driven phosphate reduction?

PubMed

Glindemann, Dietmar; Edwards, Marc; Morgenstern, Peter

2005-11-01

Natural rock and mineral samples released trace amounts of phosphine during dissolution in mineral acid. An order of magnitude more phosphine (average 1982 ng PH3 kg rock and maximum 6673 ng PH3/kg rock) is released from pulverized rock samples (basalt, gneiss, granite, clay, quartzitic pebbles, or marble). Phosphine was correlated to hardness and mechanical pulverization energy of the rocks. The yield of PH3 ranged from 0 to 0.01% of the total P content of the dissolved rock. Strong circumstantial evidence was gathered for reduction of phosphate in the rock via mechanochemical or "tribochemical" weathering at quartz and calcite/marble inclusions. Artificial reproduction of this mechanism by rubbing quartz rods coated with apatite-phosphate to the point of visible triboluminescence, led to detection of more than 70 000 ng/kg PH3 in the apatite. This reaction pathway may be considered a mechano-chemical analogue of phosphate reduction from lightning or electrical discharges and may contribute to phosphine production via tectonic forces and processing of rocks.

Homo-D-lactic acid fermentation from arabinose by redirection of the phosphoketolase pathway to the pentose phosphate pathway in L-lactate dehydrogenase gene-deficient Lactobacillus plantarum.

PubMed

Okano, Kenji; Yoshida, Shogo; Tanaka, Tsutomu; Ogino, Chiaki; Fukuda, Hideki; Kondo, Akihiko

2009-08-01

Optically pure d-lactic acid fermentation from arabinose was achieved by using the Lactobacillus plantarum NCIMB 8826 strain whose l-lactate dehydrogenase gene was deficient and whose phosphoketolase gene was substituted with a heterologous transketolase gene. After 27 h of fermentation, 38.6 g/liter of d-lactic acid was produced from 50 g/liter of arabinose.

Efficient electrolyzer for CO2 splitting in neutral water using earth-abundant materials.

PubMed

Tatin, Arnaud; Comminges, Clément; Kokoh, Boniface; Costentin, Cyrille; Robert, Marc; Savéant, Jean-Michel

2016-05-17

Low-cost, efficient CO2-to-CO+O2 electrochemical splitting is a key step for liquid-fuel production for renewable energy storage and use of CO2 as a feedstock for chemicals. Heterogeneous catalysts for cathodic CO2-to-CO associated with an O2-evolving anodic reaction in high-energy-efficiency cells are not yet available. An iron porphyrin immobilized into a conductive Nafion/carbon powder layer is a stable cathode producing CO in pH neutral water with 90% faradaic efficiency. It is coupled with a water oxidation phosphate cobalt oxide anode in a home-made electrolyzer by means of a Nafion membrane. Current densities of approximately 1 mA/cm(2) over 30-h electrolysis are achieved at a 2.5-V cell voltage, splitting CO2 and H2O into CO and O2 with a 50% energy efficiency. Remarkably, CO2 reduction outweighs the concurrent water reduction. The setup does not prevent high-efficiency proton transport through the Nafion membrane separator: The ohmic drop loss is only 0.1 V and the pH remains stable. These results demonstrate the possibility to set up an efficient, low-voltage, electrochemical cell that converts CO2 into CO and O2 by associating a cathodic-supported molecular catalyst based on an abundant transition metal with a cheap, easy-to-prepare anodic catalyst oxidizing water into O2.

Critical importance of the de novo pyrimidine biosynthesis pathway for Trypanosoma cruzi growth in the mammalian host cell cytoplasm

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

Hashimoto, Muneaki, E-mail: muneaki@juntendo.ac.jp; Morales, Jorge; Fukai, Yoshihisa

2012-01-20

Highlights: Black-Right-Pointing-Pointer We established Trypanosoma cruzi lacking the gene for carbamoyl phosphate synthetase II. Black-Right-Pointing-Pointer Disruption of the cpsII gene significantly reduced the growth of epimastigotes. Black-Right-Pointing-Pointer In particular, the CPSII-null mutant severely retarded intracellular growth. Black-Right-Pointing-Pointer The de novo pyrimidine pathway is critical for the parasite growth in the host cell. -- Abstract: The intracellular parasitic protist Trypanosoma cruzi is the causative agent of Chagas disease in Latin America. In general, pyrimidine nucleotides are supplied by both de novo biosynthesis and salvage pathways. While epimastigotes-an insect form-possess both activities, amastigotes-an intracellular replicating form of T. cruzi-are unable to mediatemore » the uptake of pyrimidine. However, the requirement of de novo pyrimidine biosynthesis for parasite growth and survival has not yet been elucidated. Carbamoyl-phosphate synthetase II (CPSII) is the first and rate-limiting enzyme of the de novo biosynthetic pathway, and increased CPSII activity is associated with the rapid proliferation of tumor cells. In the present study, we showed that disruption of the T. cruzicpsII gene significantly reduced parasite growth. In particular, the growth of amastigotes lacking the cpsII gene was severely suppressed. Thus, the de novo pyrimidine pathway is important for proliferation of T. cruzi in the host cell cytoplasm and represents a promising target for chemotherapy against Chagas disease.« less

UV light selectively coinduces supply pathways from primary metabolism and flavonoid secondary product formation in parsley

PubMed Central

Logemann, Elke; Tavernaro, Annette; Schulz, Wolfgang; Somssich, Imre E.; Hahlbrock, Klaus

2000-01-01

The UV light-induced synthesis of UV-protective flavonoids diverts substantial amounts of substrates from primary metabolism into secondary product formation and thus causes major perturbations of the cellular homeostasis. Results from this study show that the mRNAs encoding representative enzymes from various supply pathways are coinduced in UV-irradiated parsley cells (Petroselinum crispum) with two mRNAs of flavonoid glycoside biosynthesis, encoding phenylalanine ammonia-lyase and chalcone synthase. Strong induction was observed for mRNAs encoding glucose 6-phosphate dehydrogenase (carbohydrate metabolism, providing substrates for the shikimate pathway), 3-deoxyarabinoheptulosonate 7-phosphate synthase (shikimate pathway, yielding phenylalanine), and acyl-CoA oxidase (fatty acid degradation, yielding acetyl-CoA), and moderate induction for an mRNA encoding S-adenosyl-homocysteine hydrolase (activated methyl cycle, yielding S-adenosyl-methionine for B-ring methylation). Ten arbitrarily selected mRNAs representing various unrelated metabolic activities remained unaffected. Comparative analysis of acyl-CoA oxidase and chalcone synthase with respect to mRNA expression modes and gene promoter structure and function revealed close similarities. These results indicate a fine-tuned regulatory network integrating those functionally related pathways of primary and secondary metabolism that are specifically required for protective adaptation to UV irradiation. Although the response of parsley cells to UV light is considerably broader than previously assumed, it contrasts greatly with the extensive metabolic reprogramming observed previously in elicitor-treated or fungus-infected cells. PMID:10677554

Nitrifier-induced denitrification is an important source of soil nitrous oxide and can be inhibited by a nitrification inhibitor 3,4-dimethylpyrazole phosphate.

PubMed

Shi, Xiuzhen; Hu, Hang-Wei; Zhu-Barker, Xia; Hayden, Helen; Wang, Juntao; Suter, Helen; Chen, Deli; He, Ji-Zheng

2017-12-01

Soil ecosystem represents the largest contributor to global nitrous oxide (N 2 O) production, which is regulated by a wide variety of microbial communities in multiple biological pathways. A mechanistic understanding of these N 2 O production biological pathways in complex soil environment is essential for improving model performance and developing innovative mitigation strategies. Here, combined approaches of the 15 N- 18 O labelling technique, transcriptome analysis, and Illumina MiSeq sequencing were used to identify the relative contributions of four N 2 O pathways including nitrification, nitrifier-induced denitrification (nitrifier denitrification and nitrification-coupled denitrification) and heterotrophic denitrification in six soils (alkaline vs. acid soils). In alkaline soils, nitrification and nitrifier-induced denitrification were the dominant pathways of N 2 O production, and application of the nitrification inhibitor 3,4-dimethylpyrazole phosphate (DMPP) significantly reduced the N 2 O production from these pathways; this is probably due to the observed reduction in the expression of the amoA gene in ammonia-oxidizing bacteria (AOB) in the DMPP-amended treatments. In acid soils, however, heterotrophic denitrification was the main source for N 2 O production, and was not impacted by the application of DMPP. Our results provide robust evidence that the nitrification inhibitor DMPP can inhibit the N 2 O production from nitrifier-induced denitrification, a potential significant source of N 2 O production in agricultural soils. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

Library of synthetic transcriptional AND gates built with split T7 RNA polymerase mutants

PubMed Central

Shis, David L.; Bennett, Matthew R.

2013-01-01

The construction of synthetic gene circuits relies on our ability to engineer regulatory architectures that are orthogonal to the host’s native regulatory pathways. However, as synthetic gene circuits become larger and more complicated, we are limited by the small number of parts, especially transcription factors, that work well in the context of the circuit. The current repertoire of transcription factors consists of a limited selection of activators and repressors, making the implementation of transcriptional logic a complicated and component-intensive process. To address this, we modified bacteriophage T7 RNA polymerase (T7 RNAP) to create a library of transcriptional AND gates for use in Escherichia coli by first splitting the protein and then mutating the DNA recognition domain of the C-terminal fragment to alter its promoter specificity. We first demonstrate that split T7 RNAP is active in vivo and compare it with full-length enzyme. We then create a library of mutant split T7 RNAPs that have a range of activities when used in combination with a complimentary set of altered T7-specific promoters. Finally, we assay the two-input function of both wild-type and mutant split T7 RNAPs and find that regulated expression of the N- and C-terminal fragments of the split T7 RNAPs creates AND logic in each case. This work demonstrates that mutant split T7 RNAP can be used as a transcriptional AND gate and introduces a unique library of components for use in synthetic gene circuits. PMID:23479654

Library of synthetic transcriptional AND gates built with split T7 RNA polymerase mutants.

PubMed

Shis, David L; Bennett, Matthew R

2013-03-26

The construction of synthetic gene circuits relies on our ability to engineer regulatory architectures that are orthogonal to the host's native regulatory pathways. However, as synthetic gene circuits become larger and more complicated, we are limited by the small number of parts, especially transcription factors, that work well in the context of the circuit. The current repertoire of transcription factors consists of a limited selection of activators and repressors, making the implementation of transcriptional logic a complicated and component-intensive process. To address this, we modified bacteriophage T7 RNA polymerase (T7 RNAP) to create a library of transcriptional AND gates for use in Escherichia coli by first splitting the protein and then mutating the DNA recognition domain of the C-terminal fragment to alter its promoter specificity. We first demonstrate that split T7 RNAP is active in vivo and compare it with full-length enzyme. We then create a library of mutant split T7 RNAPs that have a range of activities when used in combination with a complimentary set of altered T7-specific promoters. Finally, we assay the two-input function of both wild-type and mutant split T7 RNAPs and find that regulated expression of the N- and C-terminal fragments of the split T7 RNAPs creates AND logic in each case. This work demonstrates that mutant split T7 RNAP can be used as a transcriptional AND gate and introduces a unique library of components for use in synthetic gene circuits.

Alterations of hippocampal glucose metabolism by even versus uneven medium chain triglycerides

PubMed Central

McDonald, Tanya S; Tan, Kah Ni; Hodson, Mark P; Borges, Karin

2014-01-01

Medium chain triglycerides (MCTs) are used to treat neurologic disorders with metabolic impairments, including childhood epilepsy and early Alzheimer's disease. However, the metabolic effects of MCTs in the brain are still unclear. Here, we studied the effects of feeding even and uneven MCTs on brain glucose metabolism in the mouse. Adult mice were fed 35% (calories) of trioctanoin or triheptanoin (the triglycerides of octanoate or heptanoate, respectively) or a matching control diet for 3 weeks. Enzymatic assays and targeted metabolomics by liquid chromatography tandem mass spectrometry were used to quantify metabolites in extracts from the hippocampal formations (HFs). Both oils increased the levels of β-hydroxybutyrate, but no other significant metabolic alterations were observed after triheptanoin feeding. The levels of glucose 6-phosphate and fructose 6-phosphate were increased in the HF of mice fed trioctanoin, whereas levels of metabolites further downstream in the glycolytic pathway and the pentose phosphate pathway were reduced. This indicates that trioctanoin reduces glucose utilization because of a decrease in phosphofructokinase activity. Trioctanoin and triheptanoin showed similar anticonvulsant effects in the 6 Hz seizure model, but it remains unknown to what extent the anticonvulsant mechanism(s) are shared. In conclusion, triheptanoin unlike trioctanoin appears to not alter glucose metabolism in the healthy brain. PMID:24169853

Alterations of hippocampal glucose metabolism by even versus uneven medium chain triglycerides.

PubMed

McDonald, Tanya S; Tan, Kah Ni; Hodson, Mark P; Borges, Karin

2014-01-01

Medium chain triglycerides (MCTs) are used to treat neurologic disorders with metabolic impairments, including childhood epilepsy and early Alzheimer's disease. However, the metabolic effects of MCTs in the brain are still unclear. Here, we studied the effects of feeding even and uneven MCTs on brain glucose metabolism in the mouse. Adult mice were fed 35% (calories) of trioctanoin or triheptanoin (the triglycerides of octanoate or heptanoate, respectively) or a matching control diet for 3 weeks. Enzymatic assays and targeted metabolomics by liquid chromatography tandem mass spectrometry were used to quantify metabolites in extracts from the hippocampal formations (HFs). Both oils increased the levels of β-hydroxybutyrate, but no other significant metabolic alterations were observed after triheptanoin feeding. The levels of glucose 6-phosphate and fructose 6-phosphate were increased in the HF of mice fed trioctanoin, whereas levels of metabolites further downstream in the glycolytic pathway and the pentose phosphate pathway were reduced. This indicates that trioctanoin reduces glucose utilization because of a decrease in phosphofructokinase activity. Trioctanoin and triheptanoin showed similar anticonvulsant effects in the 6 Hz seizure model, but it remains unknown to what extent the anticonvulsant mechanism(s) are shared. In conclusion, triheptanoin unlike trioctanoin appears to not alter glucose metabolism in the healthy brain.

Fructose 6-phosphate phosphoketolase activity in wild-type strains of Lactobacillus, isolated from the intestinal tract of pigs.

PubMed

Bolado-Martínez, E; Acedo-Félix, E; Peregrino-Uriarte, A B; Yepiz-Plascencia, G

2012-01-01

Phosphoketolases are key enzymes of the phosphoketolase pathway of heterofermentative lactic acid bacteria, which include lactobacilli. In heterofermentative lactobacilli xylulose 5-phosphate phosphoketolase (X5PPK) is the main enzyme of the phosphoketolase pathway. However, activity of fructose 6-phosphate phosphoketolase (F6PPK) has always been considered absent in lactic acid bacteria. In this study, the F6PPK activity was detected in 24 porcine wild-type strains of Lactobacillus reuteri and Lactobacillus mucosae, but not in the Lactobacillus salivarius or in L. reuteri ATCC strains. The activity of F6PPK increased after treatment of the culture at low-pH and diminished after porcine bile-salts stress conditions in wild-type strains of L. reuteri. Colorimetric quantification at 505 nm allowed to differentiate between microbial strains with low activity and without the activity of F6PPK. Additionally, activity of F6PPK and the X5PPK gene expression levels were evaluated by real time PCR, under stress and nonstress conditions, in 3 L. reuteri strains. Although an exact correlation, between enzyme activity and gene expression was not obtained, it remains possible that the xpk gene codes for a phosphoketolase with dual substrate, at least in the analyzed strains of L. reuteri.

Regulatory functions of trehalose-6-phosphate synthase in the chitin biosynthesis pathway in Tribolium castaneum (Coleoptera: Tenebrionidae) revealed by RNA interference.

PubMed

Chen, Q W; Jin, S; Zhang, L; Shen, Q D; Wei, P; Wei, Z M; Wang, S G; Tang, B

2018-06-01

RNA interference (RNAi) is a very effective technique for studying gene function and may be an efficient method for controlling pests. Trehalose-6-phosphate synthase (TPS), which plays a key role in the synthesis of trehalose and insect development, was cloned in Tribolium castaneum (Herbst) (TcTPS) and the putative functions were studied using RNAi via the injection of double-stranded RNA (dsRNA) corresponding to conserved TPS and trehalose-6-phosphate phosphatase domains. Expression analyses show that TcTPS is expressed higher in the fat body, while quantitative real-time polymerase chain reaction results show that the expression of four trehalase isoforms was significantly suppressed by dsTPS injection. Additionally, the expression of six chitin synthesis-related genes, such as hexokinase 2 and glutamine-fructose-6-phosphate aminotransferase, was suppressed at 48 and 72 h post-dsTPS-1 and dsTPS-2 RNA injection, which were two dsTPS fragments that had been designed for two different locations in TcTPS open reading frame, and that trehalose content and trehalase 1 activity decreased significantly at 72 h post-dsRNA injection. Furthermore, T. castaneum injected with dsTPS-1 and dsTPS-2 RNA displayed significantly lower levels of chitin and could not complete the molting process from larvae to pupae, revealing abnormal molting phenotypes. These results demonstrate that silencing TPS gene leads to molting deformities and high mortality rates via regulation of gene expression in the chitin biosynthetic pathway, and may be a promising approach for pest control in the future.

Adjustment of Trehalose Metabolism in Wine Saccharomyces cerevisiae Strains To Modify Ethanol Yields

PubMed Central

Rossouw, D.; Heyns, E. H.; Setati, M. E.; Bosch, S.

2013-01-01

The ability of Saccharomyces cerevisiae to efficiently produce high levels of ethanol through glycolysis has been the focus of much scientific and industrial activity. Despite the accumulated knowledge regarding glycolysis, the modification of flux through this pathway to modify ethanol yields has proved difficult. Here, we report on the systematic screening of 66 strains with deletion mutations of genes encoding enzymes involved in central carbohydrate metabolism for altered ethanol yields. Five of these strains showing the most prominent changes in carbon flux were selected for further investigation. The genes were representative of trehalose biosynthesis (TPS1, encoding trehalose-6-phosphate synthase), central glycolysis (TDH3, encoding glyceraldehyde-3-phosphate dehydrogenase), the oxidative pentose phosphate pathway (ZWF1, encoding glucose-6-phosphate dehydrogenase), and the tricarboxylic acid (TCA) cycle (ACO1 and ACO2, encoding aconitase isoforms 1 and 2). Two strains exhibited lower ethanol yields than the wild type (tps1Δ and tdh3Δ), while the remaining three showed higher ethanol yields. To validate these findings in an industrial yeast strain, the TPS1 gene was selected as a good candidate for genetic modification to alter flux to ethanol during alcoholic fermentation in wine. Using low-strength promoters active at different stages of fermentation, the expression of the TPS1 gene was slightly upregulated, resulting in a decrease in ethanol production and an increase in trehalose biosynthesis during fermentation. Thus, the mutant screening approach was successful in terms of identifying target genes for genetic modification in commercial yeast strains with the aim of producing lower-ethanol wines. PMID:23793638

The missing link in plant histidine biosynthesis: Arabidopsis myoinositol monophosphatase-like2 encodes a functional histidinol-phosphate phosphatase.

PubMed

Petersen, Lindsay N; Marineo, Sandra; Mandalà, Salvatore; Davids, Faezah; Sewell, Bryan T; Ingle, Robert A

2010-03-01

Histidine (His) plays a critical role in plant growth and development, both as one of the standard amino acids in proteins, and as a metal-binding ligand. While genes encoding seven of the eight enzymes in the pathway of His biosynthesis have been characterized from a number of plant species, the identity of the enzyme catalyzing the dephosphorylation of histidinol-phosphate to histidinol has remained elusive. Recently, members of a novel family of histidinol-phosphate phosphatase proteins, displaying significant sequence similarity to known myoinositol monophosphatases (IMPs) have been identified from several Actinobacteria. Here we demonstrate that a member of the IMP family from Arabidopsis (Arabidopsis thaliana), myoinositol monophosphatase-like2 (IMPL2; encoded by At4g39120), has histidinol-phosphate phosphatase activity. Heterologous expression of IMPL2, but not the related IMPL1 protein, was sufficient to rescue the His auxotrophy of a Streptomyces coelicolor hisN mutant. Homozygous null impl2 Arabidopsis mutants displayed embryonic lethality, which could be rescued by supplying plants heterozygous for null impl2 alleles with His. In common with the previously characterized HISN genes from Arabidopsis, IMPL2 was expressed in all plant tissues and throughout development, and an IMPL2:green fluorescent protein fusion protein was targeted to the plastid, where His biosynthesis occurs in plants. Our data demonstrate that IMPL2 is the HISN7 gene product, and suggest a lack of genetic redundancy at this metabolic step in Arabidopsis, which is characteristic of the His biosynthetic pathway.

A Bacterial Glucanotransferase Can Replace the Complex Maltose Metabolism Required for Starch to Sucrose Conversion in Leaves at Night*

PubMed Central

Ruzanski, Christian; Smirnova, Julia; Rejzek, Martin; Cockburn, Darrell; Pedersen, Henriette L.; Pike, Marilyn; Willats, William G. T.; Svensson, Birte; Steup, Martin; Ebenhöh, Oliver; Smith, Alison M.; Field, Robert A.

2013-01-01

Controlled conversion of leaf starch to sucrose at night is essential for the normal growth of Arabidopsis. The conversion involves the cytosolic metabolism of maltose to hexose phosphates via an unusual, multidomain protein with 4-glucanotransferase activity, DPE2, believed to transfer glucosyl moieties to a complex heteroglycan prior to their conversion to hexose phosphate via a cytosolic phosphorylase. The significance of this complex pathway is unclear; conversion of maltose to hexose phosphate in bacteria proceeds via a more typical 4-glucanotransferase that does not require a heteroglycan acceptor. It has recently been suggested that DPE2 generates a heterogeneous series of terminal glucan chains on the heteroglycan that acts as a “glucosyl buffer” to ensure a constant rate of sucrose synthesis in the leaf at night. Alternatively, DPE2 and/or the heteroglycan may have specific properties important for their function in the plant. To distinguish between these ideas, we compared the properties of DPE2 with those of the Escherichia coli glucanotransferase MalQ. We found that MalQ cannot use the plant heteroglycan as an acceptor for glucosyl transfer. However, experimental and modeling approaches suggested that it can potentially generate a glucosyl buffer between maltose and hexose phosphate because, unlike DPE2, it can generate polydisperse malto-oligosaccharides from maltose. Consistent with this suggestion, MalQ is capable of restoring an essentially wild-type phenotype when expressed in mutant Arabidopsis plants lacking DPE2. In light of these findings, we discuss the possible evolutionary origins of the complex DPE2-heteroglycan pathway. PMID:23950181

De novo transcriptome sequencing in Bixa orellana to identify genes involved in methylerythritol phosphate, carotenoid and bixin biosynthesis

DOE PAGES

Cárdenas-Conejo, Yair; Carballo-Uicab, Víctor; Lieberman, Meric; ...

2015-10-28

Bixin or annatto is a commercially important natural orange-red pigment derived from lycopene that is produced and stored in seeds of Bixa orellana L. An enzymatic pathway for bixin biosynthesis was inferred from homology of putative proteins encoded by differentially expressed seed cDNAs. Some activities were later validated in a heterologous system. Nevertheless, much of the pathway remains to be clarified. For example, it is essential to identify the methylerythritol phosphate (MEP) and carotenoid pathways genes. In order to investigate the MEP, carotenoid, and bixin pathways genes, total RNA from young leaves and two different developmental stages of seeds frommore » B. orellana were used for the construction of indexed mRNA libraries, sequenced on the Illumina HiSeq 2500 platform and assembled de novo using Velvet, CLC Genomics Workbench and CAP3 software. A total of 52,549 contigs were obtained with average length of 1,924 bp. Two phylogenetic analyses of inferred proteins, in one case encoded by thirteen general, single-copy cDNAs, in the other from carotenoid and MEP cDNAs, indicated that B. orellana is closely related to sister Malvales species cacao and cotton. Using homology, we identified 7 and 14 core gene products from the MEP and carotenoid pathways, respectively. Surprisingly, previously defined bixin pathway cDNAs were not present in our transcriptome. Here we propose a new set of gene products involved in bixin pathway. In conclusion, the identification and qRT-PCR quantification of cDNAs involved in annatto production suggest a hypothetical model for bixin biosynthesis that involve coordinated activation of some MEP, carotenoid and bixin pathway genes. These findings provide a better understanding of the mechanisms regulating these pathways and will facilitate the genetic improvement of B. orellana.« less

De novo transcriptome sequencing in Bixa orellana to identify genes involved in methylerythritol phosphate, carotenoid and bixin biosynthesis

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

Cárdenas-Conejo, Yair; Carballo-Uicab, Víctor; Lieberman, Meric

Bixin or annatto is a commercially important natural orange-red pigment derived from lycopene that is produced and stored in seeds of Bixa orellana L. An enzymatic pathway for bixin biosynthesis was inferred from homology of putative proteins encoded by differentially expressed seed cDNAs. Some activities were later validated in a heterologous system. Nevertheless, much of the pathway remains to be clarified. For example, it is essential to identify the methylerythritol phosphate (MEP) and carotenoid pathways genes. In order to investigate the MEP, carotenoid, and bixin pathways genes, total RNA from young leaves and two different developmental stages of seeds frommore » B. orellana were used for the construction of indexed mRNA libraries, sequenced on the Illumina HiSeq 2500 platform and assembled de novo using Velvet, CLC Genomics Workbench and CAP3 software. A total of 52,549 contigs were obtained with average length of 1,924 bp. Two phylogenetic analyses of inferred proteins, in one case encoded by thirteen general, single-copy cDNAs, in the other from carotenoid and MEP cDNAs, indicated that B. orellana is closely related to sister Malvales species cacao and cotton. Using homology, we identified 7 and 14 core gene products from the MEP and carotenoid pathways, respectively. Surprisingly, previously defined bixin pathway cDNAs were not present in our transcriptome. Here we propose a new set of gene products involved in bixin pathway. In conclusion, the identification and qRT-PCR quantification of cDNAs involved in annatto production suggest a hypothetical model for bixin biosynthesis that involve coordinated activation of some MEP, carotenoid and bixin pathway genes. These findings provide a better understanding of the mechanisms regulating these pathways and will facilitate the genetic improvement of B. orellana.« less

Proteomic analysis of pancreatic cancer stem cells: Functional role of fatty acid synthesis and mevalonate pathways.

PubMed

Brandi, Jessica; Dando, Ilaria; Pozza, Elisa Dalla; Biondani, Giulia; Jenkins, Rosalind; Elliott, Victoria; Park, Kevin; Fanelli, Giuseppina; Zolla, Lello; Costello, Eithne; Scarpa, Aldo; Cecconi, Daniela; Palmieri, Marta

2017-01-06

Recently, we have shown that the secretome of pancreatic cancer stem cells (CSCs) is characterized by proteins that participate in cancer differentiation, invasion, and metastasis. However, the differentially expressed intracellular proteins that lead to the specific characteristics of pancreatic CSCs have not yet been identified, and as a consequence the deranged metabolic pathways are yet to be elucidated. To identify the modulated proteins of pancreatic CSCs, iTRAQ-based proteomic analysis was performed to compare the proteome of Panc1 CSCs and Panc1 parental cells, identifying 230 modulated proteins. Pathway analysis revealed activation of glycolysis, the pentose phosphate pathway, the pyruvate-malate cycle, and lipid metabolism as well as downregulation of the Krebs cycle, the splicesome and non-homologous end joining. These findings were supported by metabolomics and immunoblotting analysis. It was also found that inhibition of fatty acid synthase by cerulenin and of mevalonate pathways by atorvastatin have a greater anti-proliferative effect on cancer stem cells than parental cells. Taken together, these results clarify some important aspects of the metabolic network signature of pancreatic cancer stem cells, shedding light on key and novel therapeutic targets and suggesting that fatty acid synthesis and mevalonate pathways play a key role in ensuring their viability. To better understand the altered metabolic pathways of pancreatic cancer stem cells (CSCs), a comprehensive proteomic analysis and metabolite profiling investigation of Panc1 and Panc1 CSCs were carried out. The findings obtained indicate that Panc1 CSCs are characterized by upregulation of glycolysis, pentose phosphate pathway, pyruvate-malate cycle, and lipid metabolism and by downregulation of Krebs cycle, spliceosome and non-homologous end joining. Moreover, fatty acid synthesis and mevalonate pathways are shown to play a critical contribution to the survival of pancreatic cancer stem cells. This study is helpful for broadening the knowledge of pancreatic cancer stem cells and could accelerate the development of novel therapeutic strategies. Copyright © 2016 Elsevier B.V. All rights reserved.

Energy distribution analysis in boosted HCCI-like / LTGC engines – Understanding the trade-offs to maximize the thermal efficiency

DOE PAGES

Dernotte, Jeremie; Dec, John E.; Ji, Chunsheng

2015-04-14

A detailed understanding of the various factors affecting the trends in gross-indicated thermal efficiency with changes in key operating parameters has been carried out, applied to a one-liter displacement single-cylinder boosted Low-Temperature Gasoline Combustion (LTGC) engine. This work systematically investigates how the supplied fuel energy splits into the following four energy pathways: gross-indicated thermal efficiency, combustion inefficiency, heat transfer and exhaust losses, and how this split changes with operating conditions. Additional analysis is performed to determine the influence of variations in the ratio of specific heat capacities (γ) and the effective expansion ratio, related to the combustion-phasing retard (CA50), onmore » the energy split. Heat transfer and exhaust losses are computed using multiple standard cycle analysis techniques. Furthermore, the various methods are evaluated in order to validate the trends.« less

Hund’s rule in superatoms with transition metal impurities

PubMed Central

Medel, Victor M.; Reveles, Jose Ulises; Khanna, Shiv N.; Chauhan, Vikas; Sen, Prasenjit; Castleman, A. Welford

2011-01-01

The quantum states in metal clusters bunch into supershells with associated orbitals having shapes resembling those in atoms, giving rise to the concept that selected clusters could mimic the characteristics of atoms and be classified as superatoms. Unlike atoms, the superatom orbitals span over multiple atoms and the filling of orbitals does not usually exhibit Hund’s rule seen in atoms. Here, we demonstrate the possibility of enhancing exchange splitting in superatom shells via a composite cluster of a central transition metal and surrounding nearly free electron metal atoms. The transition metal d states hybridize with superatom D states and result in enhanced splitting between the majority and minority sets where the moment and the splitting can be controlled by the nature of the central atom. We demonstrate these findings through studies on TMMgn clusters where TM is a 3d atom. The clusters exhibit Hund’s filling, opening the pathway to superatoms with magnetic shells. PMID:21646542

Hund's rule in superatoms with transition metal impurities.

PubMed

Medel, Victor M; Reveles, Jose Ulises; Khanna, Shiv N; Chauhan, Vikas; Sen, Prasenjit; Castleman, A Welford

2011-06-21

The quantum states in metal clusters bunch into supershells with associated orbitals having shapes resembling those in atoms, giving rise to the concept that selected clusters could mimic the characteristics of atoms and be classified as superatoms. Unlike atoms, the superatom orbitals span over multiple atoms and the filling of orbitals does not usually exhibit Hund's rule seen in atoms. Here, we demonstrate the possibility of enhancing exchange splitting in superatom shells via a composite cluster of a central transition metal and surrounding nearly free electron metal atoms. The transition metal d states hybridize with superatom D states and result in enhanced splitting between the majority and minority sets where the moment and the splitting can be controlled by the nature of the central atom. We demonstrate these findings through studies on TMMg(n) clusters where TM is a 3d atom. The clusters exhibit Hund's filling, opening the pathway to superatoms with magnetic shells.

Charge transport through split photoelectrodes in dye-sensitized solar cells

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

Fakharuddin, Azhar; Ahmed, Irfan; Yusoff, Mashitah M.

2014-04-28

Charge transport and recombination are relatively ignored parameters while upscaling dye-sensitized solar cells (DSCs). Enhanced photovoltaic parameters are anticipated by merely widening the devices physical dimensions, viz., thickness and area as evident from the device design adopted in reported large area DSCs. These strip designs lead to ≤50% loss in photocurrent compared to the high efficiency lab scale devices. Herein, we report that the key to achieving higher current density (J{sub SC}) is optimized diffusion volume rather than the increased photoelectrode area because kinetics of the devices is strongly influenced by the varied choices of diffusion pathways upon increasing themore » electrode area. For a given electrode area and thickness, we altered the photoelectrode design by splitting the electrode into multiple fractions to restrict the electron diffusion pathways. We observed a correlation between the device physical dimensions and its charge collection efficiency via current-voltage and impedance spectroscopy measurements. The modified electrode designs showed >50% increased J{sub SC} due to shorter transport time, higher recombination resistance and enhanced charge collection efficiency compared to the conventional ones despite their similar active volume (∼3.36 × 10{sup −4} cm{sup 3}). A detailed charge transport characteristic of the split devices and their comparison with single electrode configuration is described in this article.« less

Redirection of metabolite biosynthesis from hydroxybenzoates to volatile terpenoids in green hairy roots of Daucus carota.

PubMed

Mukherjee, Chiranjit; Samanta, Tanmoy; Mitra, Adinpunya

2016-02-01

A metabolic shift in green hairy root cultures of carrot from phenylpropanoid/benzenoid biosynthesis toward volatile isoprenoids was observed when compared with the metabolite profile of normal hairy root cultures. Hairy roots cultures of Daucus carota turned green under continuous illumination, while the content of the major phenolic compound p-hydroxybenzoic acid (p-HBA) was reduced to half as compared to normal hairy roots cultured in darkness. p-Hydroxybenzaldehyde dehydrogenase (HBD) activity was suppressed in the green hairy roots. However, comparative volatile analysis of 14-day-old green hairy roots revealed higher monoterpene and sesquiterpene contents than found in normal hairy roots. Methyl salicylate content was higher in normal hairy roots than in green ones. Application of clomazone, an inhibitor of 1-deoxy-D-xylulose 5-phosphate synthase (DXS), reduced the amount of total monoterpenes and sesquiterpenes in green hairy roots compared to normal hairy roots. However, methyl salicylate content was enhanced in both green and normal hairy roots treated with clomazone as compared to their respective controls. Because methyl-erythritol 4-phosphate (MEP) and phenylpropanoid pathways, respectively, contribute to the formation of monoterpenes and phenolic acids biosynthesis, the activities of enzymes regulating those pathways were measured in terms of their in vitro activities, in both green and normal hairy root cultures. These key enzymes were 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR), an early regulatory enzyme of the MEP pathway, pyruvate kinase (PK), an enzyme of primary metabolism related to the MEP pathway, shikimate dehydrogenase (SKDH) which is involved in biosynthesis of aromatic amino acids, and phenylalanine ammonia-lyase (PAL) that catalyzes the first step of phenylpropanoid biosynthesis. Activities of DXR and PK were higher in green hairy roots as compared to normal ones, whereas the opposite trend was observed for SKDH and PAL activities. Gene expression analysis of DXR and PAL showed trends similar to those for the respective enzyme activities. Based on these observations, we suggest a possible redirection of metabolites from the primary metabolism toward isoprenoid biosynthesis, limiting the phenolic biosynthetic pathway in green hairy roots grown under continuous light.

Magnesium Inhibits Wnt/β-Catenin Activity and Reverses the Osteogenic Transformation of Vascular Smooth Muscle Cells

PubMed Central

Montes de Oca, Addy; Guerrero, Fatima; Martinez-Moreno, Julio M.; Madueño, Juan A.; Herencia, Carmen; Peralta, Alan; Almaden, Yolanda; Lopez, Ignacio; Aguilera-Tejero, Escolastico; Gundlach, Kristina; Büchel, Janine; Peter, Mirjam E.; Passlick-Deetjen, Jutta; Rodriguez, Mariano; Muñoz-Castañeda, Juan R.

2014-01-01

Magnesium reduces vascular smooth muscle cell (VSMC) calcification in vitro but the mechanism has not been revealed so far. This work used only slightly increased magnesium levels and aimed at determining: a) whether inhibition of magnesium transport into the cell influences VSMC calcification, b) whether Wnt/β-catenin signaling, a key mediator of osteogenic differentiation, is modified by magnesium and c) whether magnesium can influence already established vascular calcification. Human VSMC incubated with high phosphate (3.3 mM) and moderately elevated magnesium (1.4 mM) significantly reduced VSMC calcification and expression of the osteogenic transcription factors Cbfa-1 and osterix, and up-regulated expression of the natural calcification inhibitors matrix Gla protein (MGP) and osteoprotegerin (OPG). The protective effects of magnesium on calcification and expression of osteogenic markers were no longer observed in VSMC cultured with an inhibitor of cellular magnesium transport (2-aminoethoxy-diphenylborate [2-APB]). High phosphate induced activation of Wnt/β-catenin pathway as demonstrated by the translocation of β-catenin into the nucleus, increased expression of the frizzled-3 gene, and downregulation of Dkk-1 gene, a specific antagonist of the Wnt/β-catenin signaling pathway. The addition of magnesium however inhibited phosphate-induced activation of Wnt/β-catenin signaling pathway. Furthermore, TRPM7 silencing using siRNA resulted in activation of Wnt/β-catenin signaling pathway. Additional experiments were performed to test the ability of magnesium to halt the progression of already established VSMC calcification in vitro. The delayed addition of magnesium decreased calcium content, down-regulated Cbfa-1 and osterix and up-regulated MGP and OPG, when compared with a control group. This effect was not observed when 2-APB was added. In conclusion, magnesium transport through the cell membrane is important to inhibit VSMC calcification in vitro. Inhibition of Wnt/β-catenin by magnesium is one potential intracellular mechanism by which this anti-calcifying effect is achieved. PMID:24586847

Phosphate limitation promotes unsaturated fatty acids and arachidonic acid biosynthesis by microalgae Porphyridium purpureum.

PubMed

Su, Gaomin; Jiao, Kailin; Li, Zheng; Guo, Xiaoyi; Chang, Jingyu; Ndikubwimana, Theoneste; Sun, Yong; Zeng, Xianhai; Lu, Yinghua; Lin, Lu

2016-07-01

Polyunsaturated fatty acids (PUFAs) are highly appreciated on their nutritive value for human health and aquaculture. P. purpureum, one of the red microalgae acknowledged as a promising accumulator of ARA, was chosen as the target algae in the present research. Effects of sodium bicarbonate (0.04-1.2 g/L), temperature (25, 30 and 33 °C) and phosphate (0.00-0.14 g/L) on biomass yield, total fatty acids (TFA) and arachidonic acid (ARA) accumulation were investigated systemically. NaHCO3 dose of 0.8 g/L and moderate temperature of 30 °C were preferred. In addition, TFA and ARA production were significantly enhanced by an appropriate concentration of phosphate, and the highest TFA yield of 666.38 mg/L and ARA yield of 159.74 mg/L were obtained at a phosphate concentration of 0.035 g/L. Interestingly, with phosphate concentration continuing to fall, UFA/TFA and ARA/EPA ratios were increased accordingly, suggesting that phosphate limitation promoted unsaturated fatty acids and arachidonic acid biosynthesis. Low concentration of phosphate may be favored to increase the enzymatic activities of ∆6-desaturase, which played a key role in catalyzing the conversion of C16:0 to C18:2, and thus the selectivity of UFA increased. Meanwhile, the increase of ARA selectivity could be attributed to ω6 pathway promotion and ∆17-desaturase activity inhibition with phosphate limitation. Phosphate limitation strategy enhanced unsaturated fatty acids and ARA biosynthesis in P. purpureum, and can be applied in commercial scale manufacturing and commercialization of ARA.

Conversion of D-ribulose 5-phosphate to D-xylulose 5-phosphate : new insights from structural and biochemical studies on human RPE.

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

Liang, W.; Ouyang, S.; Shaw, N.

2011-02-01

The pentose phosphate pathway (PPP) confers protection against oxidative stress by supplying NADPH necessary for the regeneration of glutathione, which detoxifies H{sub 2}O{sub 2} into H{sub 2}O and O{sub 2}. RPE functions in the PPP, catalyzing the reversible conversion of D-ribulose 5-phosphate to D-xylulose 5-phosphate and is an important enzyme for cellular response against oxidative stress. Here, using structural, biochemical, and functional studies, we show that human D-ribulose 5-phosphate 3-epimerase (hRPE) uses Fe{sup 2+} for catalysis. Structures of the binary complexes of hRPE with D-ribulose 5-phosphate and D-xylulose 5-phosphate provide the first detailed molecular insights into the binding mode ofmore » physiological ligands and reveal an octahedrally coordinated Fe{sup 2+} ion buried deep inside the active site. Human RPE folds into a typical ({beta}/{alpha}){sub 8} triosephosphate isomerase (TIM) barrel with a loop regulating access to the active site. Two aspartic acids are well positioned to carry out the proton transfers in an acid-base type of reaction mechanism. Interestingly, mutating Ser-10 to alanine almost abolished the enzymatic activity, while L12A and M72A mutations resulted in an almost 50% decrease in the activity. The binary complexes of hRPE reported here will aid in the design of small molecules for modulating the activity of the enzyme and altering flux through the PPP.« less

HemX is required for production of 2-ketogluconate, the predominant organic anion required for inorganic phosphate solubilization by Burkholderia sp. Ha185.

PubMed

Hsu, Pei-Chun Lisa; Condron, Leo; O'Callaghan, Maureen; Hurst, Mark R H

2015-12-01

The bacterium Burkholderia sp. Ha185 readily solubilizes inorganic phosphate by releasing the low molecular weight organic anion, 2-ketogluconate. Using random transposon mutagenesis and in silico analysis, a mutation that caused almost complete abolition of phosphate solubilization was located within hemX, which is part of the hem operon. Burkholderia sp. Ha185 HemX is a multidomain protein, predicted to encode a bifunctional uroporphyrinogen-III synthetase/uroporphyrin-III C-methyltransferase, which has not previously been implicated in phosphate solubilization. Complementation of hemX restored the ability of the mutant to solubilize phosphate in both plate and liquid cultures. Based on a combination of organic-anion profiling, quantitative polymerase chain reaction and in silico analyses, hemX was confirmed to be solely responsible for hydroxyapatite solubilization in Burkholderia sp. Ha185. It is proposed that the biosynthesis of a yet to be determined redox cofactor by HemX is the main pathway for generating 2-ketogluconate via a haem-dependent gluconate 2-dehydrogenase in Burkholderia sp. Ha185. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

Solution NMR studies of Chlorella virus DNA ligase-adenylate.

PubMed

Piserchio, Andrea; Nair, Pravin A; Shuman, Stewart; Ghose, Ranajeet

2010-01-15

DNA ligases are essential guardians of genome integrity by virtue of their ability to recognize and seal 3'-OH/5'-phosphate nicks in duplex DNA. The substrate binding and three chemical steps of the ligation pathway are coupled to global and local changes in ligase structure, involving both massive protein domain movements and subtle remodeling of atomic contacts in the active site. Here we applied solution NMR spectroscopy to study the conformational dynamics of the Chlorella virus DNA ligase (ChVLig), a minimized eukaryal ATP-dependent ligase consisting of nucleotidyltransferase, OB, and latch domains. Our analysis of backbone (15)N spin relaxation and (15)N,(1)H residual dipolar couplings of the covalent ChVLig-AMP intermediate revealed conformational sampling on fast (picosecond to nanosecond) and slow timescales (microsecond to millisecond), indicative of interdomain and intradomain flexibility. We identified local and global changes in ChVLig-AMP structure and dynamics induced by phosphate. In particular, the chemical shift perturbations elicited by phosphate were clustered in the peptide motifs that comprise the active site. We hypothesize that phosphate anion mimics some of the conformational transitions that occur when ligase-adenylate interacts with the nick 5'-phosphate. Copyright 2009 Elsevier Ltd. All rights reserved.

Probing de novo sphingolipid metabolism in mammalian cells utilizing mass spectrometry.

PubMed

Snider, Justin M; Snider, Ashley J; Obeid, Lina M; Luberto, Chiara; Hannun, Yusuf A

2018-06-01

Sphingolipids constitute a dynamic metabolic network that interconnects several bioactive molecules, including ceramide (Cer), sphingosine (Sph), Sph 1-phosphate, and Cer 1-phosphate. The interconversion of these metabolites is controlled by a cohort of at least 40 enzymes, many of which respond to endogenous or exogenous stimuli. Typical probing of the sphingolipid pathway relies on sphingolipid mass levels or determination of the activity of individual enzymes. Either approach is unable to provide a complete analysis of flux through sphingolipid metabolism, which, given the interconnectivity of the sphingolipid pathway, is critical information to identify nodes of regulation. Here, we present a one-step in situ assay that comprehensively probes the flux through de novo sphingolipid synthesis, post serine palmitoyltransferase, by monitoring the incorporation and metabolism of the 17 carbon dihydrosphingosine precursor with LC/MS. Pulse labeling and analysis of precursor metabolism identified sequential well-defined phases of sphingolipid synthesis, corresponding to the activity of different enzymes in the pathway, further confirmed by the use of specific inhibitors and modulators of sphingolipid metabolism. This work establishes precursor pulse labeling as a practical tool for comprehensively studying metabolic flux through de novo sphingolipid synthesis and complex sphingolipid generation.

Isoprenoid-Based Biofuels: Homologous Expression and Heterologous Expression in Prokaryotes.

PubMed

Phulara, Suresh Chandra; Chaturvedi, Preeti; Gupta, Pratima

2016-10-01

Enthusiasm for mining advanced biofuels from microbial hosts has increased remarkably in recent years. Isoprenoids are one of the highly diverse groups of secondary metabolites and are foreseen as an alternative to petroleum-based fuels. Most of the prokaryotes synthesize their isoprenoid backbone via the deoxyxylulose-5-phosphate pathway from glyceraldehyde-3-phosphate and pyruvate, whereas eukaryotes synthesize isoprenoids via the mevalonate pathway from acetyl coenzyme A (acetyl-CoA). Microorganisms do not accumulate isoprenoids in large quantities naturally, which restricts their application for fuel purposes. Various metabolic engineering efforts have been utilized to overcome the limitations associated with their natural and nonnatural production. The introduction of heterologous pathways/genes and overexpression of endogenous/homologous genes have shown a remarkable increase in isoprenoid yield and substrate utilization in microbial hosts. Such modifications in the hosts' genomes have enabled researchers to develop commercially competent microbial strains for isoprenoid-based biofuel production utilizing a vast array of substrates. The present minireview briefly discusses the recent advancement in metabolic engineering efforts in prokaryotic hosts for the production of isoprenoid-based biofuels, with an emphasis on endogenous, homologous, and heterologous expression strategies. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Isoprenoid-Based Biofuels: Homologous Expression and Heterologous Expression in Prokaryotes

PubMed Central

Phulara, Suresh Chandra; Chaturvedi, Preeti

2016-01-01

Enthusiasm for mining advanced biofuels from microbial hosts has increased remarkably in recent years. Isoprenoids are one of the highly diverse groups of secondary metabolites and are foreseen as an alternative to petroleum-based fuels. Most of the prokaryotes synthesize their isoprenoid backbone via the deoxyxylulose-5-phosphate pathway from glyceraldehyde-3-phosphate and pyruvate, whereas eukaryotes synthesize isoprenoids via the mevalonate pathway from acetyl coenzyme A (acetyl-CoA). Microorganisms do not accumulate isoprenoids in large quantities naturally, which restricts their application for fuel purposes. Various metabolic engineering efforts have been utilized to overcome the limitations associated with their natural and nonnatural production. The introduction of heterologous pathways/genes and overexpression of endogenous/homologous genes have shown a remarkable increase in isoprenoid yield and substrate utilization in microbial hosts. Such modifications in the hosts' genomes have enabled researchers to develop commercially competent microbial strains for isoprenoid-based biofuel production utilizing a vast array of substrates. The present minireview briefly discusses the recent advancement in metabolic engineering efforts in prokaryotic hosts for the production of isoprenoid-based biofuels, with an emphasis on endogenous, homologous, and heterologous expression strategies. PMID:27422837

Phosphorus acquisition efficiency in arbuscular mycorrhizal maize is correlated with the abundance of root-external hyphae and the accumulation of transcripts encoding PHT1 phosphate transporters.

PubMed

Sawers, Ruairidh J H; Svane, Simon F; Quan, Clement; Grønlund, Mette; Wozniak, Barbara; Gebreselassie, Mesfin-Nigussie; González-Muñoz, Eliécer; Chávez Montes, Ricardo A; Baxter, Ivan; Goudet, Jerome; Jakobsen, Iver; Paszkowski, Uta

2017-04-01

Plant interactions with arbuscular mycorrhizal fungi have long attracted interest for their potential to promote more efficient use of mineral resources in agriculture. Their use, however, remains limited by a lack of understanding of the processes that determine the outcome of the symbiosis. In this study, the impact of host genotype on growth response to mycorrhizal inoculation was investigated in a panel of diverse maize lines. A panel of 30 maize lines was evaluated with and without inoculation with arbuscular mycorrhizal fungi. The line Oh43 was identified to show superior response and, along with five other reference lines, was characterized in greater detail in a split-compartment system, using 33 P to quantify mycorrhizal phosphorus uptake. Changes in relative growth indicated variation in host capacity to profit from the symbiosis. Shoot phosphate content, abundance of root-internal and -external fungal structures, mycorrhizal phosphorus uptake, and accumulation of transcripts encoding plant PHT1 family phosphate transporters varied among lines. Superior response in Oh43 is correlated with extensive development of root-external hyphae, accumulation of specific Pht1 transcripts and high phosphorus uptake by mycorrhizal plants. The data indicate that host genetic factors influence fungal growth strategy with an impact on plant performance. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

Randomized, Observer-blind, Split-face Compatibility Study with Clindamycin Phosphate 1.2%/Benzoyl Peroxide 3.75% gel and Facial Foundation Makeup

PubMed Central

Pillai, Radhakrishnan

2015-01-01

Background: Cosmetic compatibility in the treatment of acne is an important issue significantly impacting quality of life, but often overlooked, as dermatologists commonly recommended avoidance of cosmetic foundations when treating adult female patients. Fixed combinations of clindamycin/benzoyl peroxide are widely used in the treatment of acne, but little is known about the impact of their concomitant use with facial foundation. Objective: To assess the compatibility of clindamycin phosphate 1. 2%/benzoyl peroxide 3. 75% gel with foundation makeup for up to six hours after application. Methods: Twenty-nine female subjects applied makeup to their face after randomly applying clindamycin phosphate 1. 2%/benzoyl peroxide 3. 75% gel to one side of the face. Investigator and subject self- assessment included facial skin attributes, facial tolerability, and cosmetic compatibility post-application and at Hour 6; as well as cutaneous tolerability. Results: No statistical difference was noted between the treated and untreated side of the face in terms of coverage, blotchiness, appearance, skin tone, or visual smoothness. Tolerability was excellent, with no erythema, edema, dryness, and peeling post-makeup application. For both the treated and untreated side, there was a slight lack of improvement in cosmetic appearance six hours post-makeup application. Conclusion: Clindamycin/benzoyl peroxide 3. 75% gel was shown to have excellent cosmetic compatibility with facial foundation. PMID:26430488

Randomized, Observer-blind, Split-face Compatibility Study with Clindamycin Phosphate 1.2%/Benzoyl Peroxide 3.75% gel and Facial Foundation Makeup.

PubMed

Bhatia, Neal; Pillai, Radhakrishnan

2015-09-01

Cosmetic compatibility in the treatment of acne is an important issue significantly impacting quality of life, but often overlooked, as dermatologists commonly recommended avoidance of cosmetic foundations when treating adult female patients. Fixed combinations of clindamycin/benzoyl peroxide are widely used in the treatment of acne, but little is known about the impact of their concomitant use with facial foundation. To assess the compatibility of clindamycin phosphate 1. 2%/benzoyl peroxide 3. 75% gel with foundation makeup for up to six hours after application. Twenty-nine female subjects applied makeup to their face after randomly applying clindamycin phosphate 1. 2%/benzoyl peroxide 3. 75% gel to one side of the face. Investigator and subject self- assessment included facial skin attributes, facial tolerability, and cosmetic compatibility post-application and at Hour 6; as well as cutaneous tolerability. No statistical difference was noted between the treated and untreated side of the face in terms of coverage, blotchiness, appearance, skin tone, or visual smoothness. Tolerability was excellent, with no erythema, edema, dryness, and peeling post-makeup application. For both the treated and untreated side, there was a slight lack of improvement in cosmetic appearance six hours post-makeup application. Clindamycin/benzoyl peroxide 3. 75% gel was shown to have excellent cosmetic compatibility with facial foundation.

Mediator- and co-catalyst-free direct Z-scheme composites of Bi2WO6-Cu3P for solar-water splitting.

PubMed

Rauf, Ali; Ma, Ming; Kim, Sungsoon; Sher Shah, Md Selim Arif; Chung, Chan-Hwa; Park, Jong Hyeok; Yoo, Pil J

2018-02-08

Exploring new single, active photocatalysts for solar-water splitting is highly desirable to expedite current research on solar-chemical energy conversion. In particular, Z-scheme-based composites (ZBCs) have attracted extensive attention due to their unique charge transfer pathway, broader redox range, and stronger redox power compared to conventional heterostructures. In the present report, we have for the first time explored Cu 3 P, a new, single photocatalyst for solar-water splitting applications. Moreover, a novel ZBC system composed of Bi 2 WO 6 -Cu 3 P was designed employing a simple method of ball-milling complexation. The synthesized materials were examined and further investigated through various microscopic, spectroscopic, and surface area characterization methods, which have confirmed the successful hybridization between Bi 2 WO 6 and Cu 3 P and the formation of a ZBC system that shows the ideal position of energy levels for solar-water splitting. Notably, the ZBC composed of Bi 2 WO 6 -Cu 3 P is a mediator- and co-catalyst-free photocatalyst system. The improved photocatalytic efficiency obtained with this system compared to other ZBC systems assisted by mediators and co-catalysts establishes the critical importance of interfacial solid-solid contact and the well-balanced position of energy levels for solar-water splitting. The promising solar-water splitting under optimum composition conditions highlighted the relationship between effective charge separation and composition.

Glutathione-related enzymes and the eye.

PubMed

Ganea, Elena; Harding, John J

2006-01-01

Glutathione and the related enzymes belong to the defence system protecting the eye against chemical and oxidative stress. This review focuses on GSH and two key enzymes, glutathione reductase and glucose-6-phosphate dehydrogenase in lens, cornea, and retina. Lens contains a high concentration of reduced glutathione, which maintains the thiol groups in the reduced form. These contribute to lens complete transparency as well as to the transparent and refractive properties of the mammalian cornea, which are essential for proper image formation on the retina. In cornea, gluthatione also plays an important role in maintaining normal hydration level, and in protecting cellular membrane integrity. In retina, glutathione is distributed in the different types of retinal cells. Intracellular enzyme, glutathione reductase, involved in reducing the oxidized glutathione has been found at highest activity in human and primate lenses, as compared to other species. Besides the enzymes directly involved in maintaining the normal redox status of the cell, glucose-6-phosphate dehydrogenase which catalyzes the first reaction of the pentose phosphate pathway, plays a key role in protection of the eye against reactive oxygen species. Cornea has a high activity of the pentose phosphate pathway and glucose-6-phosphate dehydrogenase activity. Glycation, the non-enzymic reaction between a free amino group in proteins and a reducing sugar, slowly inactivates gluthathione-related and other enzymes. In addition, glutathione can be also glycated. The presence of glutathione, and of the related enzymes has been also reported in other parts of the eye, such as ciliary body and trabecular meshwork, suggesting that the same enzyme systems are present in all tissues of the eye to generate NADPH and to maintain gluthatione in the reduced form. Changes of glutathione and related enzymes activity in lens, cornea, retina and other eye tissues, occur with ageing, cataract, diabetes, irradiation and administration of some drugs.

Characterization of Recombinant UDP- and ADP-Glucose Pyrophosphorylases and Glycogen Synthase To Elucidate Glucose-1-Phosphate Partitioning into Oligo- and Polysaccharides in Streptomyces coelicolor

PubMed Central

Asención Diez, Matías D.; Peirú, Salvador; Demonte, Ana M.; Gramajo, Hugo

2012-01-01

Streptomyces coelicolor exhibits a major secondary metabolism, deriving important amounts of glucose to synthesize pigmented antibiotics. Understanding the pathways occurring in the bacterium with respect to synthesis of oligo- and polysaccharides is of relevance to determine a plausible scenario for the partitioning of glucose-1-phosphate into different metabolic fates. We report the molecular cloning of the genes coding for UDP- and ADP-glucose pyrophosphorylases as well as for glycogen synthase from genomic DNA of S. coelicolor A3(2). Each gene was heterologously expressed in Escherichia coli cells to produce and purify to electrophoretic homogeneity the respective enzymes. UDP-glucose pyrophosphorylase (UDP-Glc PPase) was characterized as a dimer exhibiting a relatively high Vmax in catalyzing UDP-glucose synthesis (270 units/mg) and with respect to dTDP-glucose (94 units/mg). ADP-glucose pyrophosphorylase (ADP-Glc PPase) was found to be tetrameric in structure and specific in utilizing ATP as a substrate, reaching similar activities in the directions of ADP-glucose synthesis or pyrophosphorolysis (Vmax of 0.15 and 0.27 units/mg, respectively). Glycogen synthase was arranged as a dimer and exhibited specificity in the use of ADP-glucose to elongate α-1,4-glucan chains in the polysaccharide. ADP-Glc PPase was the only of the three enzymes exhibiting sensitivity to allosteric regulation by different metabolites. Mannose-6-phosphate, phosphoenolpyruvate, fructose-6-phosphate, and glucose-6-phosphate behaved as major activators, whereas NADPH was a main inhibitor of ADP-Glc PPase. The results support a metabolic picture where glycogen synthesis occurs via ADP-glucose in S. coelicolor, with the pathway being strictly regulated in connection with other routes involved with oligo- and polysaccharides, as well as with antibiotic synthesis in the bacterium. PMID:22210767

The potential of species-specific tagatose-6-phosphate (T6P) pathway in Lactobacillus casei group for galactose reduction in fermented dairy foods.

PubMed

Wu, Qinglong; Shah, Nagendra P

2017-04-01

Residual lactose and galactose in fermented dairy foods leads to several industrial and health concerns. There is very little information pertaining to manufacture of fermented dairy foods that are low in lactose and galactose. In the present study, comparative genomic survey demonstrated the constant presence of chromosome-encoded tagatose-6-phosphate (T6P) pathway in Lactobacillus casei group. Lactose/galactose utilization tests and β-galactosidase assay suggest that PTS Gal system, PTS Lac system and T6P pathway are major contributors for lactose/galactose catabolism in this group of organisms. In addition, it was found than lactose catabolism by Lb. casei group accumulated very limited galactose in the MRS-lactose medium and in reconstituted skim milk, whereas Streptococcus thermophilus and Lb. delbrueckii subsp. bulgaricus (Lb. bulgaricus) strains secreted high amount of galactose extracellularly. Moreover, co-culturing Lb. casei group with Str. thermophilus showed significant reduction in galactose content, while co-culturing Lb. casei group with Lb. bulgaricus showed significant reduction in lactose content but significant increase in galactose content in milk. Overall, the present study highlighted the potential of Lb. casei group for reducing galactose accumulation in fermented milks due to its species-specific T6P pathway. Copyright © 2016 Elsevier Ltd. All rights reserved.

High salt diet induces metabolic alterations in multiple biological processes of Dahl salt-sensitive rats.

PubMed

Wang, Yanjun; Liu, Xiangyang; Zhang, Chen; Wang, Zhengjun

2018-06-01

High salt induced renal disease is a condition resulting from the interactions of genetic and dietary factors causing multiple complications. To understand the metabolic alterations associated with renal disease, we comprehensively analyzed the metabonomic changes induced by high salt intake in Dahl salt-sensitive (SS) rats using GC-MS technology and biochemical analyses. Physiological features, serum chemistry, and histopathological data were obtained as complementary information. Our results showed that high salt (HS) intake for 16 weeks caused significant metabolic alterations in both the renal medulla and cortex involving a variety pathways involved in the metabolism of organic acids, amino acids, fatty acids, and purines. In addition, HS enhanced glycolysis (hexokinase, phosphofructokinase and pyruvate kinase) and amino acid metabolism and suppressed the TCA (citrate synthase and aconitase) cycle. Finally, HS intake caused up-regulation of the pentose phosphate pathway (glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase), the ratio of NADPH/NADP + , NADPH oxidase activity and ROS production, suggesting that increased oxidative stress was associated with an altered PPP pathway. The metabolic pathways identified may serve as potential targets for the treatment of renal damage. Our findings provide comprehensive biochemical details about the metabolic responses to a high salt diet, which may contribute to the understanding of renal disease and salt-induced hypertension in SS rats. Copyright © 2018. Published by Elsevier Inc.

Biotechnology to harness the benefits of dietary phenolics; focus on Lamiaceae.

PubMed

Shetty, K

1997-09-01

Phytochemicals from herbs and fermented legumes are excellent dietary sources of phenolic metabolites. These phenolics have importance not only as food preservatives but increasingly have therapeutic and pharmaceutical applications. The long-term research objecitves of the food biotechnology program at the University of Massachusetts are to elucidate the molecular and physiological mechanisms associated with synthesis of important health-related, therapeutic phenolic metabolites in food-related plants and fermented plant foods. Current efforts focus on elucidation of the role of the proline-linked pentose phosphate pathway in regulating the synthesis of anti-inflammatory compound, rosmarinic acid (RA). Specific aims of the current research efforts are: (i) To develop novel tissue culture-based selection techniques to isolate high RA-producing, shoot-based clonal lines from genetically heterogeneous, cross-pollinating species in the family Lamiaceae; (ii) To target genetically uniform, regenerated shoot-based clonal lines for: (a) preliminary characterization of key enzymes associated with the pentose phosphate pathway and linked to RA synthesis; (b) development of genetic transformation techniques for subsequent engineering of metabolic pathways associated with RA synthesis. These research objectives have substantial implications for harnessing the genetic and biochemical potential of genetically heterogeneous, food-related medicinal plant species. The success of this research also provides novel methods and strategies to gain access to metabolic pathways of pharmaceutically important metabolites from ginger, curcuma, chili peppers, melon or other food-related species with novel phenolics.

Maxillary sinus floor augmentation using a beta-tricalcium phosphate (Cerasorb) alone compared to autogenous bone grafts.

PubMed

Zijderveld, Steven A; Zerbo, Ilara R; van den Bergh, Johan P A; Schulten, Engelbert A J M; ten Bruggenkate, Chris M

2005-01-01

A prospective human clinical study was conducted to determine the clinical and histologic bone formation ability of 2 graft materials, a beta-tricalcium phosphate (Cerasorb; Curasan, Kleinostheim, Germany) and autogenous chin bone, in maxillary sinus floor elevation surgery. Ten healthy patients underwent a bilateral (n = 6) or unilateral (n = 4) maxillary sinus floor elevation procedure under local anesthesia. In each case, residual posterior maxillary bone height was between 4 and 8 mm. In cases of bilateral sinus floor elevation, the original bone was augmented with a split-mouth design with 100% beta-tricalcium phosphate on the test side and 100% chin bone on the contralateral control side. The unilateral cases were augmented with 100% beta-tricalcium phosphate. After a healing period of 6 months, ITI full body screw-type implants (Straumann, Waldenburg, Switzerland) were placed. At the time of implant surgery, biopsy samples were removed with a 3.5-mm trephine drill. Sixteen sinus floor elevations were performed. Forty-one implants were placed, 26 on the test side and 15 on the control side. The clinical characteristics at the time of implantation differed, especially regarding clinical appearance and drilling resistance. The increase in height was examined radiographically prior to implantation and was found to be sufficient in all cases. After a mean of nearly 1 year of follow-up, no implant losses or failures had occurred. The promising clinical results of the present study and the lack of implant failures are probably mainly the result of requiring an original bone height of at least 4 mm at the implant location. Although autogenous bone grafting is still the gold standard, according to the clinical results, the preimplantation sinus floor elevation procedure used, which involved a limited volume of beta-tricalcium phosphate, appeared to be a clinically reliable procedure in this patient population.

slalom encodes an adenosine 3′-phosphate 5′-phosphosulfate transporter essential for development in Drosophila

PubMed Central

Lüders, Florian; Segawa, Hiroaki; Stein, David; Selva, Erica M.; Perrimon, Norbert; Turco, Salvatore J.; Häcker, Udo

2003-01-01

Sulfation of all macromolecules entering the secretory pathway in higher organisms occurs in the Golgi and requires the high-energy sulfate donor adenosine 3′-phosphate 5′-phosphosulfate. Here we report the first molecular identification of a gene that encodes a transmembrane protein required to transport adenosine 3′-phosphate 5′-phosphosulfate from the cytosol into the Golgi lumen. Mutations in this gene, which we call slalom, display defects in Wg and Hh signaling, which are likely due to the lack of sulfation of glycos aminoglycans by the sulfotransferase sulfateless. Analysis of mosaic mutant ovaries shows that sll function is also essential for dorsal–ventral axis determination, suggesting that sll transports the sulfate donor required for sulfotransferase activity of the dorsal–ventral determinant pipe. PMID:12853478

Introducing the Hero Complex and the Mythic Iconic Pathway of Problem Gambling

ERIC Educational Resources Information Center

Nixon, Gary; Solowoniuk, Jason

2009-01-01

Early research into the motivations behind problem gambling reflected separate paradigms of thought splitting our understanding of the gambler into divergent categories. However, over the past 25 years, problem gambling is now best understood to arise from biological, environmental, social, and psychological processes, and is now encapsulated…

A nucleobase-centered coarse-grained representation for structure prediction of RNA motifs.

PubMed

Poblete, Simón; Bottaro, Sandro; Bussi, Giovanni

2018-02-28

We introduce the SPlit-and-conQueR (SPQR) model, a coarse-grained (CG) representation of RNA designed for structure prediction and refinement. In our approach, the representation of a nucleotide consists of a point particle for the phosphate group and an anisotropic particle for the nucleoside. The interactions are, in principle, knowledge-based potentials inspired by the $\\mathcal {E}$SCORE function, a base-centered scoring function. However, a special treatment is given to base-pairing interactions and certain geometrical conformations which are lost in a raw knowledge-based model. This results in a representation able to describe planar canonical and non-canonical base pairs and base-phosphate interactions and to distinguish sugar puckers and glycosidic torsion conformations. The model is applied to the folding of several structures, including duplexes with internal loops of non-canonical base pairs, tetraloops, junctions and a pseudoknot. For the majority of these systems, experimental structures are correctly predicted at the level of individual contacts. We also propose a method for efficiently reintroducing atomistic detail from the CG representation.

Fabrication of carbonate apatite block based on internal dissolution-precipitation reaction of dicalcium phosphate and calcium carbonate.

PubMed

Daitou, Fumikazu; Maruta, Michito; Kawachi, Giichiro; Tsuru, Kanji; Matsuya, Shigeki; Terada, Yoshihiro; Ishikawa, Kunio

2010-05-01

In this study, we investigated a novel method for fabrication of carbonate apatite block without ionic movement between precursor and solution by using precursor that includes all constituent ions of carbonate apatite. A powder mixture prepared from dicalcium phosphate anhydrous and calcite at appropriate Ca/P ratios (1.5, 1.67, and 1.8) was used as starting material. For preparation of specimens, the slurry made from the powder mixture and distilled water was packed in a split stainless steel mold and heat - treated, ranging from 60 degrees C to 100 degrees C up to 48 hours at 100% humidity. It appeared that carbonate apatite could be obtained above 70 degrees C and monophasic carbonate apatite could be obtained from the powder mixture at Ca/P ratio of 1.67. Carbonate content of the specimen was about 5-7%. Diametral tensile strength of the carbonate apatite blocks slightly decreased with increasing treatment temperature. The decrease in diametral tensile strength is thought to be related to the crystal size of the carbonate apatite formed.

Analysis of Metabolic Pathways and Fluxes in a Newly Discovered Thermophilic and Ethanol-Tolerant Geobacillus Strain

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

Tang, Yinjie J.; Sapra, Rajat; Joyner, Dominique

2009-01-20

A recently discovered thermophilic bacterium, Geobacillus thermoglucosidasius M10EXG, ferments a range of C5 (e.g., xylose) and C6 sugars (e.g., glucose) and istolerant to high ethanol concentrations (10percent, v/v). We have investigated the central metabolism of this bacterium using both in vitro enzyme assays and 13C-based flux analysis to provide insights into the physiological properties of this extremophile and explore its metabolism for bio-ethanol or other bioprocess applications. Our findings show that glucose metabolism in G. thermoglucosidasius M10EXG proceeds via glycolysis, the pentose phosphate pathway, and the TCA cycle; the Entner?Doudoroff pathway and transhydrogenase activity were not detected. Anaplerotic reactions (includingmore » the glyoxylate shunt, pyruvate carboxylase, and phosphoenolpyruvate carboxykinase) were active, but fluxes through those pathways could not be accuratelydetermined using amino acid labeling. When growth conditions were switched from aerobic to micro-aerobic conditions, fluxes (based on a normalized glucose uptake rate of 100 units (g DCW)-1 h-1) through the TCA cycle and oxidative pentose phosphate pathway were reduced from 64+-3 to 25+-2 and from 30+-2 to 19+-2, respectively. The carbon flux under micro-aerobic growth was directed formate. Under fully anerobic conditions, G. thermoglucosidasius M10EXG used a mixed acid fermentation process and exhibited a maximum ethanol yield of 0.38+-0.07 mol mol-1 glucose. In silico flux balance modeling demonstrates that lactate and acetate production from G. thermoglucosidasius M10EXG reduces the maximum ethanol yieldby approximately threefold, thus indicating that both pathways should be modified to maximize ethanol production.« less

Systematic engineering of the central metabolism in Escherichia coli for effective production of n-butanol.

PubMed

Saini, Mukesh; Li, Si-Yu; Wang, Ze Win; Chiang, Chung-Jen; Chao, Yun-Peng

2016-01-01

Microbes have been extensively explored for production of environment-friendly fuels and chemicals. The microbial fermentation pathways leading to these commodities usually involve many redox reactions. This makes the fermentative production of highly reduced products challenging, because there is a limited NADH output from glucose catabolism. Microbial production of n-butanol apparently represents one typical example. In this study, we addressed the issue by adjustment of the intracellular redox state in Escherichia coli. This was initiated with strain BuT-8 which carries the clostridial CoA-dependent synthetic pathway. Three metabolite nodes in the central metabolism of the strain were targeted for engineering. First, the pyruvate node was manipulated by enhancement of pyruvate decarboxylation in the oxidative pathway. Subsequently, the pentose phosphate (PP) pathway was amplified at the glucose-6-phosphate (G6P) node. The pathway for G6P isomerization was further blocked to force the glycolytic flux through the PP pathway. It resulted in a growth defect, and the cell growth was later recovered by limiting the tricarboxylic acid cycle at the acetyl-CoA node. Finally, the resulting strain exhibited a high NADH level and enabled production of 6.1 g/L n-butanol with a yield of 0.31 g/g-glucose and a productivity of 0.21 g/L/h. The production efficiency of fermentative products in microbes strongly depends on the intracellular redox state. This work illustrates the flexibility of pyruvate, G6P, and acetyl-CoA nodes at the junction of the central metabolism for engineering. In principle, high production of reduced products of interest can be achieved by individual or coordinated modulation of these metabolite nodes.

Astroglial pentose phosphate pathway rates in response to high-glucose environments

PubMed Central

Takahashi, Shinichi; Izawa, Yoshikane; Suzuki, Norihiro

2012-01-01

ROS (reactive oxygen species) play an essential role in the pathophysiology of diabetes, stroke and neurodegenerative disorders. Hyperglycaemia associated with diabetes enhances ROS production and causes oxidative stress in vascular endothelial cells, but adverse effects of either acute or chronic high-glucose environments on brain parenchymal cells remain unclear. The PPP (pentose phosphate pathway) and GSH participate in a major defence mechanism against ROS in brain, and we explored the role and regulation of the astroglial PPP in response to acute and chronic high-glucose environments. PPP activity was measured in cultured neurons and astroglia by determining the difference in rate of 14CO2 production from [1-14C]glucose and [6-14C]glucose. ROS production, mainly H2O2, and GSH were also assessed. Acutely elevated glucose concentrations in the culture media increased PPP activity and GSH level in astroglia, decreasing ROS production. Chronically elevated glucose environments also induced PPP activation. Immunohistochemical analyses revealed that chronic high-glucose environments induced ER (endoplasmic reticulum) stress (presumably through increased hexosamine biosynthetic pathway flux). Nuclear translocation of Nrf2 (nuclear factor-erythroid 2 p45 subunit-related factor 2), which regulates G6PDH (glyceraldehyde-6-phosphate dehydrogenase) by enhancing transcription, was also observed in association with BiP (immunoglobulin heavy-chain-binding protein) expression. Acute and chronic high-glucose environments activated the PPP in astroglia, preventing ROS elevation. Therefore a rapid decrease in glucose level seems to enhance ROS toxicity, perhaps contributing to neural damage when insulin levels given to diabetic patients are not properly calibrated and plasma glucose levels are not adequately maintained. These findings may also explain the lack of evidence for clinical benefits from strict glycaemic control during the acute phase of stroke. PMID:22300409

Characterization of the Plasmodium falciparum and P. berghei glycerol 3-phosphate acyltransferase involved in FASII fatty acid utilization in the malaria parasite apicoplast.

PubMed

Shears, Melanie J; MacRae, James I; Mollard, Vanessa; Goodman, Christopher D; Sturm, Angelika; Orchard, Lindsey M; Llinás, Manuel; McConville, Malcolm J; Botté, Cyrille Y; McFadden, Geoffrey I

2017-01-01

Malaria parasites can synthesize fatty acids via a type II fatty acid synthesis (FASII) pathway located in their apicoplast. The FASII pathway has been pursued as an anti-malarial drug target, but surprisingly little is known about its role in lipid metabolism. Here we characterize the apicoplast glycerol 3-phosphate acyltransferase that acts immediately downstream of FASII in human (Plasmodium falciparum) and rodent (Plasmodium berghei) malaria parasites and investigate how this enzyme contributes to incorporating FASII fatty acids into precursors for membrane lipid synthesis. Apicoplast targeting of the P. falciparum and P. berghei enzymes are confirmed by fusion of the N-terminal targeting sequence to GFP and 3' tagging of the full length protein. Activity of the P. falciparum enzyme is demonstrated by complementation in mutant bacteria, and critical residues in the putative active site identified by site-directed mutagenesis. Genetic disruption of the P. falciparum enzyme demonstrates it is dispensable in blood stage parasites, even in conditions known to induce FASII activity. Disruption of the P. berghei enzyme demonstrates it is dispensable in blood and mosquito stage parasites, and only essential for development in the late liver stage, consistent with the requirement for FASII in rodent malaria models. However, the P. berghei mutant liver stage phenotype is found to only partially phenocopy loss of FASII, suggesting newly made fatty acids can take multiple pathways out of the apicoplast and so giving new insight into the role of FASII and apicoplast glycerol 3-phosphate acyltransferase in malaria parasites. © 2016 The Authors Cellular Microbiology Published by John Wiley & Sons Ltd.

Metabolic effect of TAp63α: enhanced glycolysis and pentose phosphate pathway, resulting in increased antioxidant defense

PubMed Central

D'Alessandro, Angelo; Amelio, Ivano; Berkers, Celia R.; Antonov, Alexey; Vousden, Karen H.; Melino, Gerry; Zolla, Lello

2014-01-01

TAp63α is a member of the p53 family, which plays a central role in epithelial cancers. Recently, a role has emerged for p53 family members in cancer metabolic modulation. In order to assess whether TAp63α plays a role in cancer metabolism, we exploited p53-null osteosarcoma Tet-On Saos-2 cells, in which the expression of TAp63α was dependent on doxycycline supplementation to the medium. Metabolomics labeling experiments were performed by incubating the cells in 13C-glucose or 13C15N-glutamine-labeled culture media, as to monitor metabolic fluxes upon induced expression of TAp63α. Induced expression of TAp63α resulted in cell cycle arrest at the G1 phase. From a metabolic standpoint, expression of Tap63α promoted glycolysis and the pentose phosphate pathway, which was uncoupled from nucleotide biosynthesis, albeit prevented oxidative stress in the form of oxidized glutathione. Double 13C-glucose and 13C15N-glutamine metabolic labeling confirmed that induced expression of TAp63α corresponded to a decreased flux of pyruvate to the Krebs cycle and decreased utilization of glutamine for catabolic purposes in the TCA cycle. Results were not conclusive in relation to anabolic utilization of labeled glutamine, since it is unclear to what extent the observed minor TAp63α-dependent increases of glutamine-derived labeling in palmitate could be tied to increased rates of reductive carboxylation and de novo synthesis of fatty acids. Finally, bioinformatics elaborations highlighted a link between patient survival rates and the co-expression of p63 and rate limiting enzymes of the pentose phosphate pathway, G6PD and PGD. PMID:25229745

²H enrichment distribution of hepatic glycogen from ²H₂O reveals the contribution of dietary fructose to glycogen synthesis.

PubMed

Delgado, Teresa C; Martins, Fátima O; Carvalho, Filipa; Gonçalves, Ana; Scott, Donald K; O'Doherty, Robert; Macedo, M Paula; Jones, John G

2013-02-15

Dietary fructose can benefit or hinder glycemic control, depending on the quantity consumed, and these contrasting effects are reflected by alterations in postprandial hepatic glycogen synthesis. Recently, we showed that ²H enrichment of glycogen positions 5 and 2 from deuterated water (²H₂O) informs direct and indirect pathway contributions to glycogenesis in naturally feeding rats. Inclusion of position 6(S) ²H enrichment data allows indirect pathway sources to be further resolved into triose phosphate and Krebs cycle precursors. This analysis was applied to six rats that had fed on standard chow (SC) and six rats that had fed on SC plus 35% sucrose in their drinking water (HS). After 2 wk, hepatic glycogenesis sources during overnight feeding were determined by ²H₂O administration and postmortem analysis of glycogen ²H enrichment at the conclusion of the dark period. Net overnight hepatic glycogenesis was similar between SC and HS rodents. Whereas direct pathway contributions were similar (403 ± 71 μmol/g dry wt HS vs. 578 ± 76 μmol/g dry wt SC), triose phosphate contributions were significantly higher for HS compared with SC (382 ± 61 vs. 87 ± 24 μmol/g dry wt, P < 0.01) and Krebs cycle inputs lower for HS compared with SC (110 ± 9 vs. 197 ± 32 μmol/g dry wt, P < 0.05). Analysis of plasma glucose ²H enrichments at the end of the feeding period also revealed a significantly higher fractional contribution of triose phosphate to plasma glucose levels in HS vs. SC. Hence, the ²H enrichment distributions of hepatic glycogen and glucose from ²H₂O inform the contribution of dietary fructose to hepatic glycogen and glucose synthesis.

Structure of 3,4-dihydroxy-2-butanone 4-phosphate synthase from Methanococcus jannaschii in complex with divalent metal ions and the substrate ribulose 5-phosphate: implications for the catalytic mechanism.

PubMed

Steinbacher, Stefan; Schiffmann, Susanne; Richter, Gerald; Huber, Robert; Bacher, Adelbert; Fischer, Markus

2003-10-24

Skeletal rearrangements of carbohydrates are crucial for many biosynthetic pathways. In riboflavin biosynthesis ribulose 5-phosphate is converted into 3,4-dihydroxy-2-butanone 4-phosphate while its C4 atom is released as formate in a sequence of metal-dependent reactions. Here, we present the crystal structure of Methanococcus jannaschii 3,4-dihydroxy-2-butanone 4-phosphate synthase in complex with the substrate ribulose 5-phosphate at a dimetal center presumably consisting of non-catalytic zinc and calcium ions at 1.7-A resolution. The carbonyl group (O2) and two out of three free hydroxyl groups (OH3 and OH4) of the substrate are metal-coordinated. We correlate previous mutational studies on this enzyme with the present structural results. Residues of the first coordination sphere involved in metal binding are indispensable for catalytic activity. Only Glu-185 of the second coordination sphere cannot be replaced without complete loss of activity. It contacts the C3 hydrogen atom directly and probably initiates enediol formation in concert with both metal ions to start the reaction sequence. Mechanistic similarities to Rubisco acting on the similar substrate ribulose 1,5-diphosphate in carbon dioxide fixation as well as other carbohydrate (reducto-) isomerases are discussed.

Can activated sludge treatments and advanced oxidation processes remove organophosphorus flame retardants?

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

Cristale, Joyce; Ramos, Dayana D.; Dantas, Renato F.

2016-01-15

This study aims to determine the occurrence of 10 OPFRs (including chlorinated, nonchlorinated alkyl and aryl compounds) in influent, effluent wastewaters and partitioning into sludge of 5 wastewater treatment plants (WWTP) in Catalonia (Spain). All target OPFRs were detected in the WWTPs influents, and the total concentration ranged from 3.67 µg L{sup −1} to 150 µg L{sup −1}. During activated sludge treatment, most OPFRs were accumulated in the sludge at concentrations from 35.3 to 9980 ng g{sup −1} dw. Chlorinated compounds tris(2-chloroethyl) phosphate (TCEP), tris(2-chloroisopropyl) phosphate (TCIPP) and tris(2,3-dichloropropyl) phosphate (TDCPP) were not removed by the conventional activated sludge treatmentmore » and they were released by the effluents at approximately the same inlet concentration. On the contrary, aryl compounds tris(methylphenyl) phosphate (TMPP) and 2-ethylhexyl diphenyl phosphate (EHDP) together with alkyl tris(2-ethylhexyl) phosphate (TEHP) were not detected in any of the effluents. Advanced oxidation processes (UV/H{sub 2}O{sub 2} and O{sub 3}) were applied to investigate the degradability of recalcitrant OPFRs in WWTP effluents. Those detected in the effluent sample (TCEP, TCIPP, TDCPP, tributyl phosphate (TNBP), tri-iso-butyl phosphate (TIBP) and tris(2-butoxyethyl) phosphate (TBOEP)) had very low direct UV-C photolysis rates. TBOEP, TNBP and TIBP were degraded by UV/H{sub 2}O{sub 2} and O{sub 3}. Chlorinated compounds TCEP, TDCPP and TCIPP were the most recalcitrant OPFR to the advanced oxidation processes applied. The study provides information on the partitioning and degradability pathways of OPFR within conventional activated sludge WWTPs. - Highlights: • OPFRs were detected in wastewater and sludge of all studied WWTPs. • Alkyl and chloroalkyl phosphates were present in secondary treatment effluents. • TBOEP, TNBP and TIBP were degraded by UV/H{sub 2}O{sub 2} and O{sub 3} treatment. • TCEP, TCIPP and TDCPP were resistant to both secondary and tertiary treatment.« less

6-Phosphogluconate dehydrogenase links oxidative PPP, lipogenesis and tumour growth by inhibiting LKB1-AMPK signalling.

PubMed

Lin, Ruiting; Elf, Shannon; Shan, Changliang; Kang, Hee-Bum; Ji, Quanjiang; Zhou, Lu; Hitosugi, Taro; Zhang, Liang; Zhang, Shuai; Seo, Jae Ho; Xie, Jianxin; Tucker, Meghan; Gu, Ting-Lei; Sudderth, Jessica; Jiang, Lei; Mitsche, Matthew; DeBerardinis, Ralph J; Wu, Shaoxiong; Li, Yuancheng; Mao, Hui; Chen, Peng R; Wang, Dongsheng; Chen, Georgia Zhuo; Hurwitz, Selwyn J; Lonial, Sagar; Arellano, Martha L; Khoury, Hanna J; Khuri, Fadlo R; Lee, Benjamin H; Lei, Qunying; Brat, Daniel J; Ye, Keqiang; Boggon, Titus J; He, Chuan; Kang, Sumin; Fan, Jun; Chen, Jing

2015-11-01

The oxidative pentose phosphate pathway (PPP) contributes to tumour growth, but the precise contribution of 6-phosphogluconate dehydrogenase (6PGD), the third enzyme in this pathway, to tumorigenesis remains unclear. We found that suppression of 6PGD decreased lipogenesis and RNA biosynthesis and elevated ROS levels in cancer cells, attenuating cell proliferation and tumour growth. 6PGD-mediated production of ribulose-5-phosphate (Ru-5-P) inhibits AMPK activation by disrupting the active LKB1 complex, thereby activating acetyl-CoA carboxylase 1 and lipogenesis. Ru-5-P and NADPH are thought to be precursors in RNA biosynthesis and lipogenesis, respectively; thus, our findings provide an additional link between the oxidative PPP and lipogenesis through Ru-5-P-dependent inhibition of LKB1-AMPK signalling. Moreover, we identified and developed 6PGD inhibitors, physcion and its derivative S3, that effectively inhibited 6PGD, cancer cell proliferation and tumour growth in nude mice xenografts without obvious toxicity, suggesting that 6PGD could be an anticancer target.

A DNA 3′-phosphatase functions in active DNA demethylation in Arabidopsis

PubMed Central

Martínez-Macías, María Isabel; Qian, Weiqiang; Miki, Daisuke; Pontes, Olga; Liu, Yunhua; Tang, Kai; Liu, Renyi; Morales-Ruiz, Teresa; Ariza, Rafael R.; Roldán-Arjona, Teresa; Zhu, Jian-Kang

2012-01-01

SUMMARY DNA methylation is an important epigenetic mark established by the combined actions of methylation and demethylation reactions. Plants use a base excision repair pathway for active DNA demethylation. After 5-methylcytosine removal, the Arabidopsis DNA glycosylase/lyase ROS1 incises the DNA backbone and part of the product has a single-nucleotide gap flanked by 3′- and 5′-phosphate termini. Here we show that the DNA phosphatase ZDP removes the blocking 3′-phosphate, allowing subsequent DNA polymerization and ligation steps needed to complete the repair reactions. ZDP and ROS1 interact in vitro and co-localize in vivo in nucleoplasmic foci. Extracts from zdp mutant plants are unable to complete DNA demethylation in vitro, and the mutations cause DNA hypermethylation and transcriptional silencing of a reporter gene. Genome-wide methylation analysis in zdp mutant plants identified hundreds of hypermethylated endogenous loci. Our results show that ZDP functions downstream of ROS1 in one branch of the active DNA demethylation pathway. PMID:22325353

Epithelial Mesenchymal Transition Induces Aberrant Glycosylation through Hexosamine Biosynthetic Pathway Activation.

PubMed

Lucena, Miguel C; Carvalho-Cruz, Patricia; Donadio, Joana L; Oliveira, Isadora A; de Queiroz, Rafaela M; Marinho-Carvalho, Monica M; Sola-Penna, Mauro; de Paula, Iron F; Gondim, Katia C; McComb, Mark E; Costello, Catherine E; Whelan, Stephen A; Todeschini, Adriane R; Dias, Wagner B

2016-06-17

Deregulated cellular metabolism is a hallmark of tumors. Cancer cells increase glucose and glutamine flux to provide energy needs and macromolecular synthesis demands. Several studies have been focused on the importance of glycolysis and pentose phosphate pathway. However, a neglected but very important branch of glucose metabolism is the hexosamine biosynthesis pathway (HBP). The HBP is a branch of the glucose metabolic pathway that consumes ∼2-5% of the total glucose, generating UDP-GlcNAc as the end product. UDP-GlcNAc is the donor substrate used in multiple glycosylation reactions. Thus, HBP links the altered metabolism with aberrant glycosylation providing a mechanism for cancer cells to sense and respond to microenvironment changes. Here, we investigate the changes of glucose metabolism during epithelial mesenchymal transition (EMT) and the role of O-GlcNAcylation in this process. We show that A549 cells increase glucose uptake during EMT, but instead of increasing the glycolysis and pentose phosphate pathway, the glucose is shunted through the HBP. The activation of HBP induces an aberrant cell surface glycosylation and O-GlcNAcylation. The cell surface glycans display an increase of sialylation α2-6, poly-LacNAc, and fucosylation, all known epitopes found in different tumor models. In addition, modulation of O-GlcNAc levels was demonstrated to be important during the EMT process. Taken together, our results indicate that EMT is an applicable model to study metabolic and glycophenotype changes during carcinogenesis, suggesting that cell glycosylation senses metabolic changes and modulates cell plasticity. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Biosynthesis of isoprene in Escherichia coli via methylerythritol phosphate (MEP) pathway.

PubMed

Zhao, Yaru; Yang, Jianming; Qin, Bo; Li, Yonghao; Sun, Yuanzhang; Su, Sizheng; Xian, Mo

2011-06-01

Isoprene is an aviation fuel of high quality and an important polymer building block in the synthetic chemistry industry. In light of high oil prices, sustained availability, and environmental concerns, isoprene from renewable materials is contemplated as a substitute for petroleum-based product. Escherichia coli with advantages over other wild microorganisms, is considered as a powerful host for biofuels and chemicals. Here, we constructed a synthetic pathway of isoprene in E. coli by introducing an isoprene synthase (ispS) gene from Populus nigra, which catalyzes the conversion of dimethylallyl diphosphate (DMAPP) to isoprene. To improve the isoprene production, we overexpressed the native 1-deoxy-D: -xylulose-5-phosphate (DXP) synthase gene (dxs) and DXP reductoisomerase gene (dxr) in E. coli, which catalyzed the first step and the second step of MEP pathway, respectively. The fed-batch fermentation results showed that overexpression of DXS is helpful for the improvement of isoprene production. Surprisingly, heterologous expression of dxs and dxr from Bacillus subtilis in the E. coli expressing ispS resulted in a 2.3-fold enhancement of isoprene production (from 94 to 314 mg/L). The promising results showed that dxs and dxr from B. subtilis functioned more efficiently on the enhancement of isoprene production than native ones. This could be caused by the consequence of great difference in protein structures of the two original DXSs. It could be practical to produce isoprene in E. coli via MEP pathway through metabolic engineering. This work provides an alternative way for production of isoprene by engineered E. coli via MEP pathway through metabolic engineering.

Improved production of homo-D-lactic acid via xylose fermentation by introduction of xylose assimilation genes and redirection of the phosphoketolase pathway to the pentose phosphate pathway in L-Lactate dehydrogenase gene-deficient Lactobacillus plantarum.

PubMed

Okano, Kenji; Yoshida, Shogo; Yamada, Ryosuke; Tanaka, Tsutomu; Ogino, Chiaki; Fukuda, Hideki; Kondo, Akihiko

2009-12-01

The production of optically pure d-lactic acid via xylose fermentation was achieved by using a Lactobacillus plantarum NCIMB 8826 strain whose l-lactate dehydrogenase gene was deficient and whose phosphoketolase genes were replaced with a heterologous transketolase gene. After 60 h of fermentation, 41.2 g/liter of d-lactic acid was produced from 50 g/liter of xylose.

Native fluorescence spectroscopy of blood plasma of rats with experimental diabetes: identifying fingerprints of glucose-related metabolic pathways

NASA Astrophysics Data System (ADS)

Shirshin, Evgeny; Cherkasova, Olga; Tikhonova, Tatiana; Berlovskaya, Elena; Priezzhev, Alexander; Fadeev, Victor

2015-05-01

We present the results of a native fluorescence spectroscopy study of blood plasma of rats with experimental diabetes. It was shown that the fluorescence emission band shape at 320 nm excitation is the most indicative of hyperglycemia in the blood plasma samples. We provide the interpretation of this fact based on the changes in reduced nicotinamide adenine dinucleotide phosphate concentration due to glucose-related metabolic pathways and protein fluorescent cross-linking formation following nonenzymatic glycation.

A highly specific l-galactose-1-phosphate phosphatase on the path to ascorbate biosynthesis

PubMed Central

Laing, William A.; Bulley, Sean; Wright, Michele; Cooney, Janine; Jensen, Dwayne; Barraclough, Di; MacRae, Elspeth

2004-01-01

Ascorbate is a critical compound in plants and animals. Humans are unable to synthesize ascorbate, and their main source of this essential vitamin are plants. However, the pathway of synthesis in plants is yet to be established, and several unknown enzymes are only postulated to exist. We describe a specific l-galactose-1-phosphate (l-gal-1-P) phosphatase that we partially purified from young kiwifruit (Actinidia deliciosa) berries. The enzyme had a native molecular mass of ≈65 kDa, was completely dependent on Mg2+ for activity and was very specific in its ability to hydrolyze l-gal-1-P. The activity had a pH optimum of 7.0, a KM(l-gal-1-P) of 20–40 μM and a Ka(Mg2+) of 0.2 mM. The activity was inhibited by Mg2+ at concentrations >2 mM. The enzyme from Arabidopsis thaliana shoots showed similar properties to the kiwifruit enzyme. The Arabidopsis thaliana enzyme preparation was digested with trypsin, and proteins present were identified by using liquid chromatography–MS. One of 24 proteins present in our preparation was an Arabidopsis thaliana protein, At3g02870, annotated myo-inositol-1-phosphate phosphatase in GenBank, that matched the characteristics of the purified l-gal-1-phosphate phosphatase. We then expressed a kiwifruit homologue of this gene in Escherichia coli and found that it showed 14-fold higher maximum velocity for l-gal-1-P than myo-inositol-1-P. The expressed enzyme showed very similar properties to the enzyme purified from kiwifruit and Arabidopsis, except that its KM(l-gal-1-P) and Ka(Mg2+) were higher in the expressed enzyme. The data are discussed in terms of the pathway to ascorbate biosynthesis in plants PMID:15550539

Glucosamine Inhibits Decidualization of Human Endometrial Stromal Cells and Decreases Litter Sizes in Mice1

PubMed Central

Tsai, Jui-He; Schulte, Maureen; O'Neill, Kathleen; Chi, Maggie M.-Y.; Frolova, Antonina I.; Moley, Kelle H.

2013-01-01

ABSTRACT Embryo implantation in the uterus depends on decidualization of the endometrial stromal cells (ESCs), and glucose utilization via the pentose phosphate pathway is critical in this process. We hypothesized that the amino sugar glucosamine may block the pentose phosphate pathway via inhibition of the rate-limiting enzyme glucose-6-phosphate dehydrogenase in ESCs and therefore impair decidualization and embryo implantation, thus preventing pregnancy. Both human primary and immortalized ESCs were decidualized in vitro in the presence of 0, 2.5, or 5 mM glucosamine for 9 days. Viability assays demonstrated that glucosamine was well tolerated by human ESCs. Exposure of human ESCs to glucosamine resulted in significant decreases in the activity and expression of glucose-6-phosphate dehydrogenase and in the mRNA expression of the decidual markers prolactin, somatostatin, interleukin-15, and left-right determination factor 2. In mouse ESCs, expression of the decidual marker Prp decreased upon addition of glucosamine. In comparison with control mice, glucosamine-treated mice showed weak artificial deciduoma formation along the stimulated uterine horn. In a complementary in vivo experiment, a 60-day-release glucosamine (15, 150, or 1500 μg) or placebo pellet was implanted in a single uterine horn of mice. Mice with a glucosamine pellet delivered fewer live pups per litter than those with a control pellet, and pup number returned to normal after the end of the pellet-active period. In conclusion, glucosamine is a nonhormonal inhibitor of decidualization of both human and mouse ESCs and of pregnancy in mice. Our data indicate the potential for development of glucosamine as a novel, reversible, nonhormonal contraceptive. PMID:23718985

Primary Metabolism during Biosynthesis of Secondary Wall Polymers of Protoxylem Vessel Elements1[OPEN

PubMed Central

Morisaki, Keiko; Sawada, Yuji; Sano, Ryosuke; Yamamoto, Atsushi; Kurata, Tetsuya; Suzuki, Shiro; Matsuda, Mami; Hasunuma, Tomohisa; Hirai, Masami Yokota

2016-01-01

Xylem vessels, the water-conducting cells in vascular plants, undergo characteristic secondary wall deposition and programmed cell death. These processes are regulated by the VASCULAR-RELATED NAC-DOMAIN (VND) transcription factors. Here, to identify changes in metabolism that occur during protoxylem vessel element differentiation, we subjected tobacco (Nicotiana tabacum) BY-2 suspension culture cells carrying an inducible VND7 system to liquid chromatography-mass spectrometry-based wide-target metabolome analysis and transcriptome analysis. Time-course data for 128 metabolites showed dynamic changes in metabolites related to amino acid biosynthesis. The concentration of glyceraldehyde 3-phosphate, an important intermediate of the glycolysis pathway, immediately decreased in the initial stages of cell differentiation. As cell differentiation progressed, specific amino acids accumulated, including the shikimate-related amino acids and the translocatable nitrogen-rich amino acid arginine. Transcriptome data indicated that cell differentiation involved the active up-regulation of genes encoding the enzymes catalyzing fructose 6-phosphate biosynthesis from glyceraldehyde 3-phosphate, phosphoenolpyruvate biosynthesis from oxaloacetate, and phenylalanine biosynthesis, which includes shikimate pathway enzymes. Concomitantly, active changes in the amount of fructose 6-phosphate and phosphoenolpyruvate were detected during cell differentiation. Taken together, our results show that protoxylem vessel element differentiation is associated with changes in primary metabolism, which could facilitate the production of polysaccharides and lignin monomers and, thus, promote the formation of the secondary cell wall. Also, these metabolic shifts correlate with the active transcriptional regulation of specific enzyme genes. Therefore, our observations indicate that primary metabolism is actively regulated during protoxylem vessel element differentiation to alter the cell’s metabolic activity for the biosynthesis of secondary wall polymers. PMID:27600813

Opposite Effects of Dihydrosphingosine 1-Phosphate and Sphingosine 1-Phosphate on Transforming Growth Factor-β/Smad Signaling Are Mediated through the PTEN/PPM1A-dependent Pathway*S⃞

PubMed Central

Bu, Shizhong; Kapanadze, Bagrat; Hsu, Tien; Trojanowska, Maria

2008-01-01

Transforming growth factor-β (TGF-β) is an important regulator of physiological connective tissue biosynthesis and plays a central role in pathological tissue fibrosis. Previous studies have established that a biologically active lipid mediator, sphingosine 1-phosphate (S1P), mimics some of the profibrotic functions of TGF-β through cross-activation of Smad signaling. Here we report that another product of sphingosine kinase, dihydrosphingosine 1-phosphate (dhS1P), has an opposite role in the regulation of TGF-β signaling. In contrast to S1P, dhS1P inhibits TGF-β-induced Smad2/3 phosphorylation and up-regulation of collagen synthesis. The effects of dhS1P require a lipid phosphatase, PTEN, a key modulator of cell growth and survival. dhS1P stimulates phosphorylation of the C-terminal domain of PTEN and its subsequent translocation into the nucleus. We demonstrate a novel function of nuclear PTEN as a co-factor of the Smad2/3 phosphatase, PPM1A. Complex formation of PTEN with PPM1A does not require the lipid phosphatase activity but depends on phosphorylation of the serine/threonine residues located in the C-terminal domain of PTEN. Upon complex formation with PTEN, PPM1A is protected from degradation induced by the TGF-β signaling. Consequently, overexpression of PTEN abrogates TGF-β-induced Smad2/3 phosphorylation. This study establishes a novel role for nuclear PTEN in the stabilization of PPM1A. PTEN-mediated cross-talk between the sphingolipid and TGF-β signaling pathways may play an important role in physiological and pathological TGF-β signaling. PMID:18482992

Opposite effects of dihydrosphingosine 1-phosphate and sphingosine 1-phosphate on transforming growth factor-beta/Smad signaling are mediated through the PTEN/PPM1A-dependent pathway.

PubMed

Bu, Shizhong; Kapanadze, Bagrat; Hsu, Tien; Trojanowska, Maria

2008-07-11

Transforming growth factor-beta (TGF-beta) is an important regulator of physiological connective tissue biosynthesis and plays a central role in pathological tissue fibrosis. Previous studies have established that a biologically active lipid mediator, sphingosine 1-phosphate (S1P), mimics some of the profibrotic functions of TGF-beta through cross-activation of Smad signaling. Here we report that another product of sphingosine kinase, dihydrosphingosine 1-phosphate (dhS1P), has an opposite role in the regulation of TGF-beta signaling. In contrast to S1P, dhS1P inhibits TGF-beta-induced Smad2/3 phosphorylation and up-regulation of collagen synthesis. The effects of dhS1P require a lipid phosphatase, PTEN, a key modulator of cell growth and survival. dhS1P stimulates phosphorylation of the C-terminal domain of PTEN and its subsequent translocation into the nucleus. We demonstrate a novel function of nuclear PTEN as a co-factor of the Smad2/3 phosphatase, PPM1A. Complex formation of PTEN with PPM1A does not require the lipid phosphatase activity but depends on phosphorylation of the serine/threonine residues located in the C-terminal domain of PTEN. Upon complex formation with PTEN, PPM1A is protected from degradation induced by the TGF-beta signaling. Consequently, overexpression of PTEN abrogates TGF-beta-induced Smad2/3 phosphorylation. This study establishes a novel role for nuclear PTEN in the stabilization of PPM1A. PTEN-mediated cross-talk between the sphingolipid and TGF-beta signaling pathways may play an important role in physiological and pathological TGF-beta signaling.

Simultaneous removal of ammonium and phosphate by alkaline-activated and lanthanum-impregnated zeolite.

PubMed

He, Yinhai; Lin, Hai; Dong, Yingbo; Liu, Quanli; Wang, Liang

2016-12-01

Simultaneous ammonium and phosphate removal characteristics and mechanism, as well as the major influencing factors, such as pH, temperature and co-existing ions, onto NaOH-activated and lanthanum-impregnated zeolite (NLZ) were investigated. The phosphate adsorption increases from 0.2 mg g -1 for natural zeolite up to 8.96 mg g -1 for NLZ, while only a slight decrease on the ammonium adsorption capacity from 23.9 mg g -1 for NaOH-activated zeolite to 21.2 mg g -1 for NLZ was observed. The ammonium and phosphate adsorption showed little pH dependence in the range from pH 3 to 7, while it decreased sharply with the pH increased above pH 7. Adsorption of ammonium and phosphate could be well described by the pseudo-second-order model and the process was mainly governed by intra-particle diffusion. The Langmuir and Freundlich model can be acceptably applied to fit the experimental data, which suggested that adsorption was caused by both the monolayer and homogeneous coverage at specific and equal affinity sites available NLZ. The underlying mechanism for the specific adsorption of phosphate by NLZ was revealed with the aid of SEM-EDS, XPS, and FTIR analysis, and the formation of (LaO)(OH)PO 2 was verified to be the dominant pathway for selective phosphate adsorption by lanthanum-impregnated zeolite. While the removal mechanism of ammonium could be well interpreted by SEM-EDS, FTIR and ICP analysis, and ion-exchange was expected to be the main removal process for ammonium. The results indicate that NLZ could efficiently and simultaneously remove low concentration of ammonium and phosphate from contaminated waters. Copyright © 2016 Elsevier Ltd. All rights reserved.

Vitamin D receptor-independent FGF23 actions in regulating phosphate and vitamin D metabolism.

PubMed

Shimada, Takashi; Yamazaki, Yuji; Takahashi, Motoo; Hasegawa, Hisashi; Urakawa, Itaru; Oshima, Takeshi; Ono, Kaori; Kakitani, Makoto; Tomizuka, Kazuma; Fujita, Toshiro; Fukumoto, Seiji; Yamashita, Takeyoshi

2005-11-01

FGF23 suppresses both serum phosphate and 1,25-dihydroxyvitamin D [1,25D] levels in vivo. Because 1,25D itself is a potent regulator of phosphate metabolism, it has remained unclear whether FGF23-induced changes in phosphate metabolism were caused by a 1,25D-independent mechanism. To address this issue, we intravenously administered recombinant FGF23 to vitamin D receptor (VDR) null (KO) mice as a rapid bolus injection and evaluated the early effects of FGF23. Administration of recombinant FGF23 further decreased the serum phosphate level in VDR KO mice, accompanied by a reduction in renal sodium-phosphate cotransporter type IIa (NaPi2a) protein abundance and a reduced renal 25-hydroxyvitamin D-1alpha-hydroxylase (1alphaOHase) mRNA level. Thus FGF23-induced changes in NaPi2a and 1alphaOHase expression are independent of the 1,25D/VDR system. However, 24-hydroxylase (24OHase) mRNA expression remained undetectable by the treatment with FGF23. We also analyzed the regulatory mechanism for FGF23 expression. The serum FGF23 level was almost undetectable in VDR KO mice, whereas dietary calcium supplementation significantly increased circulatory levels of FGF23 and its mRNA abundance in bone. This finding indicates that calcium is another determinant of FGF23 production that occurs independently of the VDR-mediated mechanism. In contrast, dietary phosphate supplementation failed to induce FGF23 expression in the absence of VDR, whereas marked elevation in circulatory FGF23 was observed in wild-type mice fed with a high-phosphate diet. Taken together, FGF23 works, at least in part, in a VDR-independent manner, and FGF23 production is also regulated by multiple mechanisms involving VDR-independent pathways.

A novel theoretical probe of the SrTiO3 surface under water-splitting conditions

NASA Astrophysics Data System (ADS)

Letchworth-Weaver, Kendra; Gunceler, Deniz; Arias, Tomás; Plaza, Manuel; Huang, Xin; Brock, Joel; Rodriguez-López, Joaquin; Abruña, Hector

2014-03-01

Understanding the reaction mechanisms required to generate hydrogen fuel by photoelectrolysis of water is essential to energy conversion research. These reaction pathways are strongly influenced by the geometry and electronic structure of the electrode surface under water-splitting conditions. Electrochemical microscopy has demonstrated that biasing a SrTiO3 (001) surface can lead to an increase in water-splitting activity. In operando X-ray reflectivity measurements at the Cornell High Energy Synchrotron Source (CHESS) correlate this increase in activity to a significant reorganization in the surface structure but are unable to determine the exact nature of this change. Joint Density-Functional Theory (JDFT), a rigorous yet computationally efficient alternative to molecular dynamics, provides a quantum-mechanical description of an electrode surface in contact with an aqueous environment, and a microscopically detailed description of the interfacial liquid structure. Our JDFT calculations determine the structure of the activated SrTiO3 surface and explore why it is correlated with higher activity for water splitting. With no empirical parameters whatsoever, we predict the X-ray crystal truncation rods for SrTiO3, finding excellent agreement with experiment. Funded by the Energy Materials Center at Cornell (EMC2).

Plasmonic resonance in planer split ring trimer

NASA Astrophysics Data System (ADS)

Xu, Haiqing; Li, Hongjian; Xiao, Gang

2014-12-01

We have numerically investigated the plasmon properties supported by asymmetry planer split ring trimer structures. We investigate the modification of gap distance, thickness and gap width on the transmission properties of the weak coupling model (g is larger than or equal to 120 nm, d=48 nm, t is larger than 30 nm, w1=200 nm, and w2=40 nm), as the coupling becomes weaker, the first peak sharply attenuates, the second peak slightly decreases, the transmission dip in the near-infrared region becomes shallow, and they are very sensitive to the gap distance between two small split ring pairs and the thickness and gap width of the big split ring. We also study the change of gap distance on the strong coupling model (g is smaller than or equal to 40 nm, d=24 nm, t=10 nm, w1=80 nm, and w2=20 nm), there exists a new Fano resonance peak, the strongest peak in visible region becomes symmetry, while the peak in near-infrared region becomes asymmetry. The resonator design strategy opens up a rich pathway for the implementation of optimized optical properties for specific applications.

Novel Spray Dried Glycerol 2-Phosphate Cross-Linked Chitosan Microparticulate Vaginal Delivery System—Development, Characterization and Cytotoxicity Studies

PubMed Central

Szymańska, Emilia; Szekalska, Marta; Czarnomysy, Robert; Lavrič, Zoran; Srčič, Stane; Miltyk, Wojciech; Winnicka, Katarzyna

2016-01-01

Chitosan microparticulate delivery systems containing clotrimazole were prepared by a spray drying technique using glycerol 2-phosphate as an ion cross-linker. The impact of a cross-linking ratio on microparticle characteristics was evaluated. Drug-free and drug-loaded unmodified or ion cross-linked chitosan microparticles were examined for the in vitro cytotoxicity in VK2/E6E7 human vaginal epithelial cells. The presence of glycerol 2-phosphate influenced drug loading and encapsulation efficacy in chitosan microparticles. By increasing the cross-linking ratio, the microparticles with lower diameter, moisture content and smoother surface were observed. Mucoadhesive studies displayed that all formulations possessed mucoadhesive properties. The in vitro release profile of clotrimazole was found to alter considerably by changing the glycerol 2-phosphate/chitosan ratio. Results from cytotoxicity studies showed occurrence of apoptotic cells in the presence of chitosan and ion cross-linked chitosan microparticles, followed by a loss of membrane potential suggesting that cell death might go through the mitochondrial apoptotic pathway. PMID:27690062

Analysis of organophosphate hydraulic fluids in U.S. Air force base soils

PubMed

David; Seiber

1999-04-01

Tri-aryl and tri-alkyl organophosphates (TAPs) have been used extensively as flame-retardant hydraulic fluids and fluid additives in commercial and military aircraft. Up to 80% of the consumption of these fluids has been estimated to be lost to unrecovered leakage. Tri-aryl phosphate components of these fluids are resistant to volatilization and solubilization in water, thus, their primary environmental fate pathway is sorption to soils. Environmental audits of military air bases generally do not include quantification of these compounds in soils. We have determined the presence and extent of TAP contamination in soil samples from several U.S. Air Force bases. Soils were collected, extracted, and analyzed using GC/FPD and GC/MS. Tricresyl phosphate was the most frequently found TAP in soil, ranging from 0.02 to 130 ppm. Other TAPs in soils included triphenyl phosphate and isopropylated triphenyl phosphate. Observations are made regarding the distribution, typical concentrations, persistence, and need for further testing of TAPs in soils at military installations. Additionally, GC and mass spectral data for these TAPs are presented, along with methods for their extraction, sample clean-up, and quantification.

Evaluation of synthase and hemisynthase activities of glucosamine-6-phosphate synthase by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

PubMed

Gaucher-Wieczorek, Florence; Guérineau, Vincent; Touboul, David; Thétiot-Laurent, Sophie; Pelissier, Franck; Badet-Denisot, Marie-Ange; Badet, Bernard; Durand, Philippe

2014-08-01

Glucosamine-6-phosphate synthase (GlmS, EC 2.6.1.16) catalyzes the first and rate-limiting step in the hexosamine biosynthetic pathway, leading to the synthesis of uridine-5'-diphospho-N-acetyl-D-glucosamine, the major building block for the edification of peptidoglycan in bacteria, chitin in fungi, and glycoproteins in mammals. This bisubstrate enzyme converts D-fructose-6-phosphate (Fru-6P) and L-glutamine (Gln) into D-glucosamine-6-phosphate (GlcN-6P) and L-glutamate (Glu), respectively. We previously demonstrated that matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) allows determination of the kinetic parameters of the synthase activity. We propose here to refine the experimental protocol to quantify Glu and GlcN-6P, allowing determination of both hemisynthase and synthase parameters from a single assay kinetic experiment, while avoiding interferences encountered in other assays. It is the first time that MALDI-MS is used to survey the activity of a bisubstrate enzyme. Copyright © 2014 Elsevier Inc. All rights reserved.

Regulatory insights into the production of UDP-N-acetylglucosamine by Lactobacillus casei

PubMed Central

Rodríguez-Díaz, Jesús; Rubio-del-Campo, Antonio; Yebra, María J.

2012-01-01

UDP-N-acetylglucosamine (UDP-GlcNAc) is an important sugar nucleotide used as a precursor of cell wall components in bacteria, and as a substrate in the synthesis of oligosaccharides in eukaryotes. In bacteria UDP-GlcNAc is synthesized from the glycolytic intermediate D-fructose-6-phosphate (fructose-6P) by four successive reactions catalyzed by three enzymes: glucosamine-6-phosphate synthase (GlmS), phosphoglucosamine mutase (GlmM) and the bi-functional enzyme glucosamine-1-phosphate acetyltransferase/ N-acetylglucosamine-1-phosphate uridyltransferase (GlmU). We have previously reported a metabolic engineering strategy in Lactobacillus casei directed to increase the intracellular levels of UDP-GlcNAc by homologous overexpression of the genes glmS, glmM and glmU. One of the most remarkable features regarding the production of UDP-GlcNAc in L. casei was to find multiple regulation points on its biosynthetic pathway: (1) regulation by the NagB enzyme, (2) glmS RNA specific degradation through the possible participation of a glmS riboswitch mechanism, (3) regulation of the GlmU activity probably by end product inhibition and (4) transcription of glmU. PMID:22825354

Bone morphogenetic protein Smads signaling in mesenchymal stem cells affected by osteoinductive calcium phosphate ceramics.

PubMed

Tang, Zhurong; Wang, Zhe; Qing, Fangzhu; Ni, Yilu; Fan, Yujiang; Tan, Yanfei; Zhang, Xingdong

2015-03-01

Porous calcium phosphate ceramics (CaP ceramics) could induce ectopic bone formation which was regulated by various signal molecules. In this work, bone marrow mesenchymal stem cells (MSCs) were cultured on the surface of osteoinductive hydroxyapatite (HA) and biphasic calcium phosphate (BCP) ceramics in comparison with control (culture plate) for up to 14 days to detect the signal molecules which might be affected by the CaP ceramics. Without adding osteogenic factors, MSCs cultured on HA and BCP both expressed higher Runx2, Osterix, collagen type I, osteopontin, bone sialoprotein, and osteocalcin at various stages compared with control, thus confirmed the osteoblastic differentiation of MSCs. Later study demonstrated the messenger RNA level of bone morphogenetic protein 2 (BMP2) and BMP4 were also significantly enhanced by HA and BCP. Furthermore, Smad1, 4, 5, and Dlx5, the main molecules in the BMP/Smads signaling pathway, were upregulated by HA and BCP. Moreover, the higher expression of Smads and BMP2, 4 in BCP over HA, corresponded to the better performance of BCP in stimulating in vitro osteoblastic differentiation of MSCs. This was in accordance with the better osteoinductivity of BCP over HA in vivo. Altogether, these results implied that the CaP ceramics may initiate the osteoblastic differentiation of MSCs by influencing the expression of molecules in BMP/Smads pathway. © 2014 Wiley Periodicals, Inc.

Rice ingestion is a major pathway for human exposure to organophosphate flame retardants (OPFRs) in China.

PubMed

Zhang, Xingli; Zou, Wei; Mu, Li; Chen, Yuming; Ren, Chaoxiu; Hu, Xiangang; Zhou, Qixing

2016-11-15

Although organophosphate flame retardants (OPFRs) have been shown to accumulate in abiotic and biotic environmental compartments, data about OPFRs concentrations in various foods are limited and are none in humans through diets. In this work, the concentrations of 6 typical OPFRs were investigated in 50 rice samples, 75 commonly consumed foods and 45 human hair samples from China. The dietary intakes of OPFRs for adult people via food ingestion were estimated. The concentrations of ΣOPFRs in foods ranged from 0.004ng/g to 287ng/g. OPFRs were detected in 53.3% of the human hair samples. The highest OPFRs concentrations were found in rice and vegetables. Tri(2-chloroethyl)phosphate(TCEP), tris(2-chloroisopropyl)phosphate(TCIPP), and tri(2-ethyltexyl)phosphate(TEHP) were predominant in all food samples. OPFRs concentrations in foods were not significantly affected by the packaging materials. The mean dietary intakes of ΣOPFRs for adult males and females were 539 and 601ng/kg body weight/day, respectively. The greatest contribution to these values is from rice, accounting for approximately 60% of the total intake, particularly from rice protein. Rice ingestion was considered a potential major pathway for human exposure to OPFRs, and regional differences in the levels of OPFRs in foods and dietary differences should be given more attention in the future. Copyright © 2016 Elsevier B.V. All rights reserved.

Impact of expression of EMP enzymes on glucose metabolism in Zymomonas mobilis.

PubMed

Chen, Rachel Ruizhen; Agrawal, Manoj; Mao, Zichao

2013-06-01

Zymomonas mobilis is the only known microorganism that utilizes the Entner-Doudoroff (ED) pathway anaerobically. In this work, we investigated whether the overexpression of a phosphofructokinase (PFK), the only missing Embden-Meyerhof-Parnas (EMP) pathway enzyme, could establish the pathway in this organism. Introduction of a pyrophosphate-dependent PFK, along with co-expression of homologous fructose-1,6-bisphosphate aldolase and triosephosphate isomerase, did not result in an EMP flux to any appreciable level. However, the metabolism of glucose was impacted significantly. Eight percent of glucose was metabolized to form a new metabolite, dihydroxyacetone. Reducing flux through the ED pathway by as much as 40 % through antisense of a key enzyme, ED aldolase, did not result in a fully functional EMP pathway, suggesting that the ED pathway, especially the lower arm, downstream from glyceraldehyde-3-phosphate, is very rigid, possibly due to redox balance.

Reconstruction and Evaluation of the Synthetic Bacterial MEP Pathway in Saccharomyces cerevisiae

PubMed Central

Partow, Siavash; Siewers, Verena; Daviet, Laurent; Schalk, Michel; Nielsen, Jens

2012-01-01

Isoprenoids, which are a large group of natural and chemical compounds with a variety of applications as e.g. fragrances, pharmaceuticals and potential biofuels, are produced via two different metabolic pathways, the mevalonate (MVA) pathway and the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. Here, we attempted to replace the endogenous MVA pathway in Saccharomyces cerevisiae by a synthetic bacterial MEP pathway integrated into the genome to benefit from its superior properties in terms of energy consumption and productivity at defined growth conditions. It was shown that the growth of a MVA pathway deficient S. cerevisiae strain could not be restored by the heterologous MEP pathway even when accompanied by the co-expression of genes erpA, hISCA1 and CpIscA involved in the Fe-S trafficking routes leading to maturation of IspG and IspH and E. coli genes fldA and fpr encoding flavodoxin and flavodoxin reductase believed to be responsible for electron transfer to IspG and IspH. PMID:23285068

Porous Structured Ni–Fe–P Nanocubes Derived from a Prussian Blue Analogue as an Electrocatalyst for Efficient Overall Water Splitting

DOE PAGES

Xuan, Cuijuan; Wang, Jie; Xia, Weiwei; ...

2017-07-18

Exploring nonprecious metal electrocatalysts to replace the noble metal-based catalysts for full water electrocatalysis is still an ongoing challenge. In this work, porous structured ternary nickel–iron–phosphide (Ni–Fe–P) nanocubes were synthesized through one-step phosphidation of a Ni–Fe-based Prussian blue analogue. The Ni–Fe–P nanocubes exhibit a rough and loose porous structure on their surface under suitable phosphating temperature, which is favorable for the mass transfer and oxygen diffusion during the electrocatalysis process. As a result, Ni–Fe–P obtained at 350 °C with poorer crystallinity offers more unsaturated atoms as active sites to expedite the absorption of reactants. Additionally, the introduction of nickel improvedmore » the electronic structure and then reduced the charge-transfer resistance, which would result in a faster electron transport and an enhancement of the intrinsic electrocatalytic activities. Benefiting from the unique porous nanocubes and the chemical composition, the Ni–Fe–P nanocubes exhibit excellent hydrogen evolution reaction and oxygen evolution reaction activities in alkaline medium, with low overpotentials of 182 and 271 mV for delivering a current density of 10 mA cm–2, respectively. Moreover, the Ni–Fe–P nanocubes show outstanding stability for sustained water splitting in the two-electrode alkaline electrolyzer. Furthermore, this work not only provides a facile approach for designing bifunctional electrocatalysts but also further extends the application of metal–organic frameworks in overall water splitting.« less

Porous Structured Ni–Fe–P Nanocubes Derived from a Prussian Blue Analogue as an Electrocatalyst for Efficient Overall Water Splitting

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

Xuan, Cuijuan; Wang, Jie; Xia, Weiwei

Exploring nonprecious metal electrocatalysts to replace the noble metal-based catalysts for full water electrocatalysis is still an ongoing challenge. In this work, porous structured ternary nickel–iron–phosphide (Ni–Fe–P) nanocubes were synthesized through one-step phosphidation of a Ni–Fe-based Prussian blue analogue. The Ni–Fe–P nanocubes exhibit a rough and loose porous structure on their surface under suitable phosphating temperature, which is favorable for the mass transfer and oxygen diffusion during the electrocatalysis process. As a result, Ni–Fe–P obtained at 350 °C with poorer crystallinity offers more unsaturated atoms as active sites to expedite the absorption of reactants. Additionally, the introduction of nickel improvedmore » the electronic structure and then reduced the charge-transfer resistance, which would result in a faster electron transport and an enhancement of the intrinsic electrocatalytic activities. Benefiting from the unique porous nanocubes and the chemical composition, the Ni–Fe–P nanocubes exhibit excellent hydrogen evolution reaction and oxygen evolution reaction activities in alkaline medium, with low overpotentials of 182 and 271 mV for delivering a current density of 10 mA cm–2, respectively. Moreover, the Ni–Fe–P nanocubes show outstanding stability for sustained water splitting in the two-electrode alkaline electrolyzer. Furthermore, this work not only provides a facile approach for designing bifunctional electrocatalysts but also further extends the application of metal–organic frameworks in overall water splitting.« less

Inositol phosphates in the duckweed Spirodela polyrhiza L.

PubMed Central

Brearley, C A; Hanke, D E

1996-01-01

We have undertaken an analysis of the inositol phosphates of Spirodela polyrhiza at a developmental stage when massive accumulation of InsP6 indicates that a large net synthesis is occurring. We have identified Ins3P, Ins(1,4)P2, Ins(3,4)P2 and possibly Ins(4,6)P2, Ins(3,4,6)P3, Ins(3,4,5,6)P4, Ins (1,3,4,5,6)P5, D- and/or L-Ins(1,2,4,5,6)P5 and InsP6 and revealed the likely presence of a second InsP3 with chromatographic properties similar to Ins(1,4,5)P3. The higher inositol phosphates identified show no obvious direct link to pathways of metabolism of second messengers purported to operate in higher plants, nor do they resemble the immediate products of plant phytase action on InsP6. PMID:8660286

Inhibition effects of some metal ions on the rat liver 6-phosphogluconate dehydrogenase

NASA Astrophysics Data System (ADS)

Adem, Şevki; Kayhan, Naciye

2016-04-01

6-phosphogluconate dehydrogenase is an enzyme in the pentose phosphate path. The main functions of the pathway are the manufacture of the reduced coenzyme NADPH and the formation of ribose 5-phosphate for nucleic acid synthesis and nucleotide. Both NADPH and ribose 5-phosphate involve a critical biochemical process. Metals have been recognized as important toxic agents for living for a long time. It has been considered that they lead to in the emergence of many diseases. To evaluate whether metals is effect towards rat liver 6PGD, we apply various concentrations of metals and enzyme inhibition was analyzed using enzyme activity assays. The IC50 values of Pb+2, Cr+3, Co+2, Ni+2, Cd+2, and Va+2, metals on rat liver 6PGD were calculated as 138,138, 169, 214, 280, and 350 µM, respectively.

Inorganic phosphate deprivation causes tRNA nuclear accumulation via retrograde transport in Saccharomyces cerevisiae.

PubMed

Hurto, Rebecca L; Tong, Amy Hin Yan; Boone, Charles; Hopper, Anita K

2007-06-01

Nuclear export of tRNA is an essential eukaryotic function, yet the one known yeast tRNA nuclear exporter, Los1, is nonessential. Moreover recent studies have shown that tRNAs can move retrograde from the cytosol to the nucleus by an undefined process. Therefore, additional gene products involved in tRNA nucleus-cytosol dynamics have yet to be identified. Synthetic genetic array (SGA) analysis was employed to identify proteins involved in Los1-independent tRNA transport and in regulating tRNA nucleus-cytosol distribution. These studies uncovered synthetic interactions between los1Delta and pho88Delta involved in inorganic phopsphate uptake. Further analysis revealed that inorganic phosphate deprivation causes transient, temperature-dependent nuclear accumulation of mature cytoplasmic tRNA within nuclei via a Mtr10- and retrograde-dependent pathway, providing a novel connection between tRNA subcellular dynamics and phosphate availability.

Inorganic Phosphate Deprivation Causes tRNA Nuclear Accumulation via Retrograde Transport in Saccharomyces cerevisiae

PubMed Central

Hurto, Rebecca L.; Tong, Amy Hin Yan; Boone, Charles; Hopper, Anita K.

2007-01-01

Nuclear export of tRNA is an essential eukaryotic function, yet the one known yeast tRNA nuclear exporter, Los1, is nonessential. Moreover recent studies have shown that tRNAs can move retrograde from the cytosol to the nucleus by an undefined process. Therefore, additional gene products involved in tRNA nucleus–cytosol dynamics have yet to be identified. Synthetic genetic array (SGA) analysis was employed to identify proteins involved in Los1-independent tRNA transport and in regulating tRNA nucleus–cytosol distribution. These studies uncovered synthetic interactions between los1Δ and pho88Δ involved in inorganic phopshate uptake. Further analysis revealed that inorganic phosphate deprivation causes transient, temperature-dependent nuclear accumulation of mature cytoplasmic tRNA within nuclei via a Mtr10- and retrograde-dependent pathway, providing a novel connection between tRNA subcellular dynamics and phosphate availability. PMID:17409072

Urinary metabolites of organophosphate esters in children in South China: Concentrations, profiles and estimated daily intake.

PubMed

Chen, Yi; Fang, Jianzhang; Ren, Lu; Fan, Ruifang; Zhang, Jianqing; Liu, Guihua; Zhou, Li; Chen, Dingyan; Yu, Yingxin; Lu, Shaoyou

2018-04-01

Organophosphate esters (OPEs) are widely used in household products as flame retardants or plasticizers and have become ubiquitous pollutants in environmental media. However, little is known about OPE metabolites in humans, especially in children. In this study, eight OPE metabolites were measured in 411 urine samples collected from 6 to 14-year-old children in South China. Bis(2-chloroethyl) phosphate (BCEP), bis(1-chloro-2-propyl) phosphate (BCIPP) and diphenyl phosphate (DPHP) were the dominant OPE metabolites, and their median concentrations were 1.04, 0.15 and 0.28 μg/L, respectively. The levels of urinary OPE metabolites in the present study were much lower than those in participants from other countries, with the exception of BCEP, suggesting widespread exposure to tris(2-chlorethyl) phosphate (TCEP, the parent chemical of BCEP) in South China. No significant difference in the concentrations of any of the OPE metabolites was observed between males and females (p > .05). Significant negative correlations were observed between age and BCEP, BCIPP, bis(1,3-dichloro-2-propyl) phosphate (BDCIPP), di-o-cresyl phosphate (DoCP) and di-p-cresyl phosphate (DpCP) (DCP), or DPHP (p < .05). Pearson correlation coefficients between urinary OPE metabolites indicated multiple sources and OPE exposure pathways in children. The estimated daily intake suggested that children in South China have a relatively high exposure level to TCEP. To the best of our knowledge, this is the first study to report the urinary levels of OPE metabolites in Chinese children. Copyright © 2018 Elsevier Ltd. All rights reserved.

Strigolactones

PubMed Central

Foo, Eloise; Yoneyama, Kaori; Hugill, Cassandra; Quittenden, Laura J.; Reid, James B.

2013-01-01

As the newest plant hormone, strigolactone research is undergoing an exciting expansion. In less than five years, roles for strigolactones have been defined in shoot branching, secondary growth, root growth and nodulation, to add to the growing understanding of their role in arbuscular mycorrhizae and parasitic weed interactions.1 Strigolactones are particularly fascinating as signaling molecules as they can act both inside the plant as an endogenous hormone and in the soil as a rhizosphere signal.2-4 Our recent research has highlighted such a dual role for strigolactones, potentially acting as both an endogenous and exogenous signal for arbuscular mycorrhizal development.5 There is also significant interest in examining strigolactones as putative regulators of responses to environmental stimuli, especially the response to nutrient availability, given the strong regulation of strigolactone production by nitrate and phosphate observed in many species.5,6 In particular, the potential for strigolactones to mediate the ecologically important response of mycorrhizal colonization to phosphate has been widely discussed. However, using a mutant approach we found that strigolactones are not essential for phosphate regulation of mycorrhizal colonization or nodulation.5 This is consistent with the relatively mild impairment of phosphate control of seedling root growth observed in Arabidopsis strigolactone mutants.7 This contrasts with the major role for strigolactones in phosphate control of shoot branching of rice and Arabidopsis8,9 and indicates that the integration of strigolactones into our understanding of nutrient response will be complex. New data presented here, along with the recent discovery of phosphate specific CLE peptides,10 indicates a potential role for PsNARK, a component of the autoregulation of nodulation pathway, in phosphate control of nodulation. PMID:23299321

Uranium Biominerals Precipitated by an Environmental Isolate of Serratia under Anaerobic Conditions.

PubMed

Newsome, Laura; Morris, Katherine; Lloyd, Jonathan R

2015-01-01

Stimulating the microbially-mediated precipitation of uranium biominerals may be used to treat groundwater contamination at nuclear sites. The majority of studies to date have focussed on the reductive precipitation of uranium as U(IV) by U(VI)- and Fe(III)-reducing bacteria such as Geobacter and Shewanella species, although other mechanisms of uranium removal from solution can occur, including the precipitation of uranyl phosphates via bacterial phosphatase activity. Here we present the results of uranium biomineralisation experiments using an isolate of Serratia obtained from a sediment sample representative of the Sellafield nuclear site, UK. When supplied with glycerol phosphate, this Serratia strain was able to precipitate 1 mM of soluble U(VI) as uranyl phosphate minerals from the autunite group, under anaerobic and fermentative conditions. Under phosphate-limited anaerobic conditions and with glycerol as the electron donor, non-growing Serratia cells could precipitate 0.5 mM of uranium supplied as soluble U(VI), via reduction to nano-crystalline U(IV) uraninite. Some evidence for the reduction of solid phase uranyl(VI) phosphate was also observed. This study highlights the potential for Serratia and related species to play a role in the bioremediation of uranium contamination, via a range of different metabolic pathways, dependent on culturing or in situ conditions.

Uranium Biominerals Precipitated by an Environmental Isolate of Serratia under Anaerobic Conditions

PubMed Central

Newsome, Laura; Morris, Katherine; Lloyd, Jonathan. R.

2015-01-01

Stimulating the microbially-mediated precipitation of uranium biominerals may be used to treat groundwater contamination at nuclear sites. The majority of studies to date have focussed on the reductive precipitation of uranium as U(IV) by U(VI)- and Fe(III)-reducing bacteria such as Geobacter and Shewanella species, although other mechanisms of uranium removal from solution can occur, including the precipitation of uranyl phosphates via bacterial phosphatase activity. Here we present the results of uranium biomineralisation experiments using an isolate of Serratia obtained from a sediment sample representative of the Sellafield nuclear site, UK. When supplied with glycerol phosphate, this Serratia strain was able to precipitate 1 mM of soluble U(VI) as uranyl phosphate minerals from the autunite group, under anaerobic and fermentative conditions. Under phosphate-limited anaerobic conditions and with glycerol as the electron donor, non-growing Serratia cells could precipitate 0.5 mM of uranium supplied as soluble U(VI), via reduction to nano-crystalline U(IV) uraninite. Some evidence for the reduction of solid phase uranyl(VI) phosphate was also observed. This study highlights the potential for Serratia and related species to play a role in the bioremediation of uranium contamination, via a range of different metabolic pathways, dependent on culturing or in situ conditions. PMID:26132209

Exploring reaction pathways for O-GlcNAc transferase catalysis. A string method study.

PubMed

Kumari, Manju; Kozmon, Stanislav; Kulhánek, Petr; Štepán, Jakub; Tvaroška, Igor; Koča, Jaroslav

2015-03-26

The inverting O-GlcNAc glycosyltransferase (OGT) is an important post-translation enzyme, which catalyzes the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine (UDP-GlcNAc) to the hydroxyl group of the Ser/Thr of cytoplasmic, nuclear, and mitochondrial proteins. In the past, three different catalytic bases were proposed for the reaction: His498, α-phosphate, and Asp554. In this study, we used hybrid quantum mechanics/molecular mechanics (QM/MM) Car-Parrinello molecular dynamics to investigate reaction paths using α-phosphate and Asp554 as the catalytic bases. The string method was used to calculate the free-energy reaction profiles of the tested mechanisms. During the investigations, an additional mechanism was observed. In this mechanism, a proton is transferred to α-phosphate via a water molecule. Our calculations show that the mechanism with α-phosphate acting as the base is favorable. This reaction has a rate-limiting free-energy barrier of 23.5 kcal/mol, whereas reactions utilizing Asp554 and water-assisted α-phosphate have barriers of 41.7 and 40.9 kcal/mol, respectively. Our simulations provide a new insight into the catalysis of OGT and may thus guide rational drug design of transition-state analogue inhibitors with potential therapeutic use.

FGF23 and Klotho: the new cornerstones of phosphate/calcium metabolism

PubMed Central

Bacchetta, Justine; Cochat, Pierre; Salusky, Isidro B

2014-01-01

Since its first description as a phosphaturic agent in the early 2000’s, the Fibroblast Growth Factor 23 (FGF23) has rapidly become the third key player of phosphate/calcium metabolism with the two ‘old’ PTH and vitamin D. FGF23 is a protein synthesized by osteocytes that acts mainly as a phosphaturic factor and a suppressor of 1α hydroxylase activity in the kidney. It inhibits the expression of type IIa and IIc sodium-phosphate cotransporters on the apical membrane of proximal tubular cells, thus leading to an inhibition of phosphate reabsorption. Moreover, it also inhibits the 1α hydroxylase activity. These two renal pathways account together for the hypophosphatemic effect of FGF23, but FGF23 has also been recently described as an inhibiting factor for PTH synthesis. Its exact role in bone remains to be defined. A transmembrane protein, Klotho, is an essential cofactor for FGF23 biological activity, but it can also act by itself for calcium and PTH regulation. This paper gives an overview of these recent data of phosphate/calcium physiology, as well as a description of clinical conditions associated with FGF23 deregulation (genetic diseases and chronic kidney disease). As a conclusion, future therapeutic consequences of the FGF23/Klotho axis are discussed. PMID:21497493

Oxidation of ethane by an Acremonium species.

PubMed Central

Davies, J S; Wellman, A M; Zajic, J E

1976-01-01

Ethane oxidation was studied in ethane-grown resting cells (mycelia) of an Acremonium sp. and in cell-free preparations of such mycelia. From resting cell experiments evidence was found for a pathway of ethane oxidation via ethanol, acetaldehyde, and acetic acid. In vitro studies indicated that ethane-oxidizing activity in such mycelia occurred predominantly in the microsomal fraction of crude homogenates. Microsomal preparations were inactive in the absence of added coenzyme. Marked stimulation of activity was obtained in such preparations with reduced nicotinamide adenine dinucleotide phosphate and to a much lesser degree with nicotinamide adenine dinucleotide phosphate. Ethane oxidation was inhibited by sodium azide and carbon monoxide. PMID:9900

Nature of phosphorus limitation in the ultraoligotrophic eastern Mediterranean.

PubMed

Thingstad, T F; Krom, M D; Mantoura, R F C; Flaten, G A F; Groom, S; Herut, B; Kress, N; Law, C S; Pasternak, A; Pitta, P; Psarra, S; Rassoulzadegan, F; Tanaka, T; Tselepides, A; Wassmann, P; Woodward, E M S; Riser, C Wexels; Zodiatis, G; Zohary, T

2005-08-12

Phosphate addition to surface waters of the ultraoligotrophic, phosphorus-starved eastern Mediterranean in a Lagrangian experiment caused unexpected ecosystem responses. The system exhibited a decline in chlorophyll and an increase in bacterial production and copepod egg abundance. Although nitrogen and phosphorus colimitation hindered phytoplankton growth, phosphorous may have been transferred through the microbial food web to copepods via two, not mutually exclusive, pathways: (i) bypass of the phytoplankton compartment by phosphorus uptake in heterotrophic bacteria and (ii) tunnelling, whereby phosphate luxury consumption rapidly shifts the stoichiometric composition of copepod prey. Copepods may thus be coupled to lower trophic levels through interactions not usually considered.

Nature of Phosphorus Limitation in the Ultraoligotrophic Eastern Mediterranean

NASA Astrophysics Data System (ADS)

Thingstad, T. F.; Krom, M. D.; Mantoura, R. F. C.; Flaten, G. A. F.; Groom, S.; Herut, B.; Kress, N.; Law, C. S.; Pasternak, A.; Pitta, P.; Psarra, S.; Rassoulzadegan, F.; Tanaka, T.; Tselepides, A.; Wassmann, P.; Woodward, E. M. S.; Riser, C. Wexels; Zodiatis, G.; Zohary, T.

2005-08-01

Phosphate addition to surface waters of the ultraoligotrophic, phosphorus-starved eastern Mediterranean in a Lagrangian experiment caused unexpected ecosystem responses. The system exhibited a decline in chlorophyll and an increase in bacterial production and copepod egg abundance. Although nitrogen and phosphorus colimitation hindered phytoplankton growth, phosphorous may have been transferred through the microbial food web to copepods via two, not mutually exclusive, pathways: (i) bypass of the phytoplankton compartment by phosphorus uptake in heterotrophic bacteria and (ii) tunnelling, whereby phosphate luxury consumption rapidly shifts the stoichiometric composition of copepod prey. Copepods may thus be coupled to lower trophic levels through interactions not usually considered.

Transcriptome analysis of thermogenic Arum concinnatum reveals the molecular components of floral scent production

PubMed Central

Onda, Yoshihiko; Mochida, Keiichi; Yoshida, Takuhiro; Sakurai, Tetsuya; Seymour, Roger S.; Umekawa, Yui; Pirintsos, Stergios Arg; Shinozaki, Kazuo; Ito, Kikukatsu

2015-01-01

Several plant species can generate enough heat to increase their internal floral temperature above ambient temperature. Among thermogenic plants, Arum concinnatum shows the highest respiration activity during thermogenesis. However, an overall understanding of the genes related to plant thermogenesis has not yet been achieved. In this study, we performed de novo transcriptome analysis of flower organs in A. concinnatum. The de novo transcriptome assembly represented, in total, 158,490 non-redundant transcripts, and 53,315 of those showed significant homology with known genes. To explore genes associated with thermogenesis, we filtered 1266 transcripts that showed a significant correlation between expression pattern and the temperature trend of each sample. We confirmed five putative alternative oxidase transcripts were included in filtered transcripts as expected. An enrichment analysis of the Gene Ontology terms for the filtered transcripts suggested over-representation of genes involved in 1-deoxy-d-xylulose-5-phosphate synthase (DXS) activity. The expression profiles of DXS transcripts in the methyl-d-erythritol 4-phosphate (MEP) pathway were significantly correlated with thermogenic levels. Our results suggest that the MEP pathway is the main biosynthesis route for producing scent monoterpenes. To our knowledge, this is the first report describing the candidate pathway and the key enzyme for floral scent production in thermogenic plants. PMID:25736477

Sphingosine 1-Phosphate (S1P) Receptors 1 and 2 Coordinately Induce Mesenchymal Cell Migration through S1P Activation of Complementary Kinase Pathways*

PubMed Central

Quint, Patrick; Ruan, Ming; Pederson, Larry; Kassem, Moustapha; Westendorf, Jennifer J.; Khosla, Sundeep; Oursler, Merry Jo

2013-01-01

Normal bone turnover requires tight coupling of bone resorption and bone formation to preserve bone quantity and structure. With aging and during several pathological conditions, this coupling breaks down, leading to either net bone loss or excess bone formation. To preserve or restore normal bone metabolism, it is crucial to determine the mechanisms by which osteoclasts and osteoblast precursors interact and contribute to coupling. We showed that osteoclasts produce the chemokine sphingosine 1-phosphate (S1P), which stimulates osteoblast migration. Thus, osteoclast-derived S1P may recruit osteoblasts to sites of bone resorption as an initial step in replacing lost bone. In this study we investigated the mechanisms by which S1P stimulates mesenchymal (skeletal) cell chemotaxis. S1P treatment of mesenchymal (skeletal) cells activated RhoA GTPase, but this small G protein did not contribute to migration. Rather, two S1P receptors, S1PR1 and S1PR2, coordinately promoted migration through activation of the JAK/STAT3 and FAK/PI3K/AKT signaling pathways, respectively. These data demonstrate that the chemokine S1P couples bone formation to bone resorption through activation of kinase signaling pathways. PMID:23300082

Positive selection in glycolysis among Australasian stick insects

PubMed Central

2013-01-01

Background The glycolytic pathway is central to cellular energy production. Selection on individual enzymes within glycolysis, particularly phosphoglucose isomerase (Pgi), has been associated with metabolic performance in numerous organisms. Nonetheless, how whole energy-producing pathways evolve to allow organisms to thrive in different environments and adopt new lifestyles remains little explored. The Lanceocercata radiation of Australasian stick insects includes transitions from tropical to temperate climates, lowland to alpine habitats, and winged to wingless forms. This permits a broad investigation to determine which steps within glycolysis and what sites within enzymes are the targets of positive selection. To address these questions we obtained transcript sequences from seven core glycolysis enzymes, including two Pgi paralogues, from 29 Lanceocercata species. Results Using maximum likelihood methods a signature of positive selection was inferred in two core glycolysis enzymes. Pgi and Glyceraldehyde 3-phosphate dehydrogenase (Gaphd) genes both encode enzymes linking glycolysis to the pentose phosphate pathway. Positive selection among Pgi paralogues and orthologues predominately targets amino acids with residues exposed to the protein’s surface, where changes in physical properties may alter enzyme performance. Conclusion Our results suggest that, for Lancerocercata stick insects, adaptation to new stressful lifestyles requires a balance between maintaining cellular energy production, efficiently exploiting different energy storage pools and compensating for stress-induced oxidative damage. PMID:24079656

Surface, Bulk, and Interface: Rational Design of Hematite Architecture toward Efficient Photo-Electrochemical Water Splitting.

PubMed

Li, Chengcheng; Luo, Zhibin; Wang, Tuo; Gong, Jinlong

2018-05-11

Collecting and storing solar energy to hydrogen fuel through a photo-electrochemical (PEC) cell provides a clean and renewable pathway for future energy demands. Having earth-abundance, low biotoxicity, robustness, and an ideal n-type band position, hematite (α-Fe 2 O 3 ), the most common natural form of iron oxide, has occupied the research hotspot for decades. Here, a close look into recent progress of hematite photoanodes for PEC water splitting is provided. Effective approaches are introduced, such as cocatalysts loading and surface passivation layer deposition, to improve the hematite surface reaction in thermodynamics and kinetics. Second, typical methods for enhancing light absorption and accelerating charge transport in hematite bulk are reviewed, concentrating upon doping and nanostructuring. Third, the back contact between hematite and substrate, which affects interface states and electron transfer, is deliberated. In addition, perspectives on the key challenges and future prospects for the development of hematite photoelectrodes for PEC water splitting are given. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Identification of the PhoB Regulon and Role of PhoU in the Phosphate Starvation Response of Caulobacter crescentus.

PubMed

Lubin, Emma A; Henry, Jonathan T; Fiebig, Aretha; Crosson, Sean; Laub, Michael T

2016-01-01

An ability to sense and respond to changes in extracellular phosphate is critical for the survival of most bacteria. For Caulobacter crescentus, which typically lives in phosphate-limited environments, this process is especially crucial. Like many bacteria, Caulobacter responds to phosphate limitation through a conserved two-component signaling pathway called PhoR-PhoB, but the direct regulon of PhoB in this organism is unknown. Here we used chromatin immunoprecipitation-DNA sequencing (ChIP-Seq) to map the global binding patterns of the phosphate-responsive transcriptional regulator PhoB under phosphate-limited and -replete conditions. Combined with genome-wide expression profiling, our work demonstrates that PhoB is induced to regulate nearly 50 genes under phosphate-starved conditions. The PhoB regulon is comprised primarily of genes known or predicted to help Caulobacter scavenge for and import inorganic phosphate, including 15 different membrane transporters. We also investigated the regulatory role of PhoU, a widely conserved protein proposed to coordinate phosphate import with expression of the PhoB regulon by directly modulating the histidine kinase PhoR. However, our studies show that it likely does not play such a role in Caulobacter, as PhoU depletion has no significant effect on PhoB-dependent gene expression. Instead, cells lacking PhoU exhibit striking accumulation of large polyphosphate granules, suggesting that PhoU participates in controlling intracellular phosphate metabolism. The transcription factor PhoB is widely conserved throughout the bacterial kingdom, where it helps organisms respond to phosphate limitation by driving the expression of a battery of genes. Most of what is known about PhoB and its target genes is derived from studies of Escherichia coli. Our work documents the PhoB regulon in Caulobacter crescentus, and comparison to the regulon in E. coli reveals significant differences, highlighting the evolutionary plasticity of transcriptional responses driven by highly conserved transcription factors. We also demonstrated that the conserved protein PhoU, which is implicated in bacterial persistence, does not regulate PhoB activity, as previously suggested. Instead, our results favor a model in which PhoU affects intracellular phosphate accumulation, possibly through the high-affinity phosphate transporter. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Phylogenetic analysis of genes involved in mycosporine-like amino acid biosynthesis in symbiotic dinoflagellates.

PubMed

Rosic, Nedeljka N

2012-04-01

Mycosporine-like amino acids (MAAs) are multifunctional secondary metabolites involved in photoprotection in many marine organisms. As well as having broad ultraviolet (UV) absorption spectra (310-362 nm), these biological sunscreens are also involved in the prevention of oxidative stress. More than 20 different MAAs have been discovered so far, characterized by distinctive chemical structures and a broad ecological distribution. Additionally, UV-screening MAA metabolites have been investigated and used in biotechnology and cosmetics. The biosynthesis of MAAs has been suggested to occur via either the shikimate or pentose phosphate pathways. Despite their wide distribution in marine and freshwater species and also the commercial application in cosmetic products, there are still a number of uncertainties regarding the genetic, biochemical, and evolutionary origin of MAAs. Here, using a transcriptome-mining approach, we identify the gene counterparts from the shikimate or pentose phosphate pathway involved in MAA biosynthesis within the sequences of the reef-building coral symbiotic dinoflagellates (genus Symbiodinium). We also report the highly similar sequences of genes from the proposed MAA biosynthetic pathway involved in the metabolism of 4-deoxygadusol (direct MAA precursor) in various Symbiodinium strains confirming their algal origin and conserved nature. Finally, we reveal the separate identity of two O-methyltransferase genes, possibly involved in MAA biosynthesis, as well as nonribosomal peptide synthetase and adenosine triphosphate grasp homologs in symbiotic dinoflagellates. This study provides a biochemical and phylogenetic overview of the genes from the proposed MAA biosynthetic pathway with a focus on coral endosymbionts.

Combined metabonomic and quantitative real-time PCR analyses reveal systems metabolic changes in Jurkat T-cells treated with HIV-1 Tat protein.

PubMed

Liao, Wenting; Tan, Guangguo; Zhu, Zhenyu; Chen, Qiuli; Lou, Ziyang; Dong, Xin; Zhang, Wei; Pan, Wei; Chai, Yifeng

2012-11-02

HIV-1 Tat protein is released by infected cells and can affect bystander uninfected T cells and induce numerous biological responses which contribute to its pathogenesis. To elucidate the complex pathogenic mechanism, we conducted a comprehensive investigation on Tat protein-related extracellular and intracellular metabolic changes in Jurkat T-cells using combined gas chromatography-mass spectrometry (GC-MS), reversed-phase liquid chromatography-mass spectrometry (RPLC-MS) and a hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS)-based metabonomics approach. Quantitative real-time PCR (qRT-PCR) analyses were further employed to measure expressions of several relevant enzymes together with perturbed metabolic pathways. Combined metabonomic and qRT-PCR analyses revealed that HIV-1 Tat caused significant and comprehensive metabolic changes, as represented by significant changes of 37 metabolites and 10 relevant enzymes in HIV-1 Tat-treated cells. Using MetaboAnalyst 2.0, it was found that 11 pathways (Impact-value >0.10) among the regulated pathways were acutely perturbed, including sphingolipid metabolism, glycine, serine and threonine metabolism, pyruvate metabolism, inositol phosphate metabolism, arginine and proline metabolism, citrate cycle, phenylalanine metabolism, tryptophan metabolism, pentose phosphate pathway, glycerophospholipid metabolism, glycolysis or gluconeogenesis. These results provide metabolic evidence of the complex pathogenic mechanism of HIV-1 Tat protein as a "viral toxin", and would help obligate Tat protein as "an important target" for therapeutic intervention and vaccine development.

Sugar Allocation to Metabolic Pathways is Tightly Regulated and Affects the Virulence of Streptococcus mutans

PubMed Central

Kawada-Matsuo, Miki; Oogai, Yuichi; Komatsuzawa, Hitoshi

2016-01-01

Bacteria take up and metabolize sugar as a carbohydrate source for survival. Most bacteria can utilize many sugars, including glucose, sucrose, and galactose, as well as amino sugars, such as glucosamine and N-acetylglucosamine. After entering the cytoplasm, the sugars are mainly allocated to the glycolysis pathway (energy production) and to various bacterial component biosynthesis pathways, including the cell wall, nucleic acids and amino acids. Sugars are also utilized to produce several virulence factors, such as capsule and lipoteichoic acid. Glutamine-fructose-6-phosphate aminotransferase (GlmS) and glucosamine-6-phosphate deaminase (NagB) have crucial roles in sugar distribution to the glycolysis pathway and to cell wall biosynthesis. In Streptococcus mutans, a cariogenic pathogen, the expression levels of glmS and nagB are coordinately regulated in response to the presence or absence of amino sugars. In addition, the disruption of this regulation affects the virulence of S. mutans. The expression of nagB and glmS is regulated by NagR in S. mutans, but the precise mechanism underlying glmS regulation is not clear. In Staphylococcus aureus and Bacillus subtilis, the mRNA of glmS has ribozyme activity and undergoes self-degradation at the mRNA level. However, there is no ribozyme activity region on glmS mRNA in S. mutans. In this review article, we summarize the sugar distribution, particularly the coordinated regulation of GlmS and NagB expression, and its relationship with the virulence of S. mutans. PMID:28036052

CcpN Controls Central Carbon Fluxes in Bacillus subtilis▿ ‡

PubMed Central

Tännler, Simon; Fischer, Eliane; Le Coq, Dominique; Doan, Thierry; Jamet, Emmanuel; Sauer, Uwe; Aymerich, Stéphane

2008-01-01

The transcriptional regulator CcpN of Bacillus subtilis has been recently characterized as a repressor of two gluconeogenic genes, gapB and pckA, and of a small noncoding regulatory RNA, sr1, involved in arginine catabolism. Deletion of ccpN impairs growth on glucose and strongly alters the distribution of intracellular fluxes, rerouting the main glucose catabolism from glycolysis to the pentose phosphate (PP) pathway. Using transcriptome analysis, we show that during growth on glucose, gapB and pckA are the only protein-coding genes directly repressed by CcpN. By quantifying intracellular fluxes in deletion mutants, we demonstrate that derepression of pckA under glycolytic condition causes the growth defect observed in the ccpN mutant due to extensive futile cycling through the pyruvate carboxylase, phosphoenolpyruvate carboxykinase, and pyruvate kinase. Beyond ATP dissipation via this cycle, PckA activity causes a drain on tricarboxylic acid cycle intermediates, which we show to be the main reason for the reduced growth of a ccpN mutant. The high flux through the PP pathway in the ccpN mutant is modulated by the flux through the alternative glyceraldehyde-3-phosphate dehydrogenases, GapA and GapB. Strongly increased concentrations of intermediates in upper glycolysis indicate that GapB overexpression causes a metabolic jamming of this pathway and, consequently, increases the relative flux through the PP pathway. In contrast, derepression of sr1, the third known target of CcpN, plays only a marginal role in ccpN mutant phenotypes. PMID:18586936

The Putative Mevalonate Diphosphate Decarboxylase from Picrophilus torridus Is in Reality a Mevalonate-3-Kinase with High Potential for Bioproduction of Isobutene

PubMed Central

Hall, Stephen J.; Eastham, Graham; Licence, Peter; Stephens, Gill

2015-01-01

Mevalonate diphosphate decarboxylase (MVD) is an ATP-dependent enzyme that catalyzes the phosphorylation/decarboxylation of (R)-mevalonate-5-diphosphate to isopentenyl pyrophosphate in the mevalonate (MVA) pathway. MVD is a key enzyme in engineered metabolic pathways for bioproduction of isobutene, since it catalyzes the conversion of 3-hydroxyisovalerate (3-HIV) to isobutene, an important platform chemical. The putative homologue from Picrophilus torridus has been identified as a highly efficient variant in a number of patents, but its detailed characterization has not been reported. In this study, we have successfully purified and characterized the putative MVD from P. torridus. We discovered that it is not a decarboxylase per se but an ATP-dependent enzyme, mevalonate-3-kinase (M3K), which catalyzes the phosphorylation of MVA to mevalonate-3-phosphate. The enzyme's potential in isobutene formation is due to the conversion of 3-HIV to an unstable 3-phosphate intermediate that undergoes consequent spontaneous decarboxylation to form isobutene. Isobutene production rates were as high as 507 pmol min−1 g cells−1 using Escherichia coli cells expressing the enzyme and 2,880 pmol min−1 mg protein−1 with the purified histidine-tagged enzyme, significantly higher than reported previously. M3K is a key enzyme of the novel MVA pathway discovered very recently in Thermoplasma acidophilum. We suggest that P. torridus metabolizes MVA by the same pathway. PMID:25636853

Possible Association of Ferrous Phosphates and Ferric Sulfates in S-rich Soil on Mars

NASA Astrophysics Data System (ADS)

Mao, J.; Schroeder, C.; Haderlein, S.

2012-12-01

NASA Mars Exploration Rover (MER) Spirit explored Gusev Crater to look for signs of ancient aqueous activity, assess past environmental conditions and suitability for life. Spirit excavated light-toned, S-rich soils at several locations. These are likely of hydrothermal, possibly fumarolic origin. At a location dubbed Paso Robles the light-toned soil was also rich in P - a signature from surrounding rock. While S is mainly bound in ferric hydrated sulfates [1], the mineralogy of P is ill-constrained [2]. P is a key element for life and its mineralogy constrains its availability. Ferrous phases observed in Paso Robles Mössbauer spectra may represent olivine and pyroxene from surrounding basaltic soil [1] or ferrous phosphate minerals [3]. Phosphate is well-known to complex and stabilize Fe 2+ against oxidation to Fe 3+ . Schröder et al. [3] proposed a formation pathway of ferrous phosphate/ferric sulfate associations: sulfuric acid reacts with basalt containing apatite, forming CaSO4 and phosphoric acid. The phosphoric and/or excess sulfuric acid reacts with olivine, forming Fe2+-phosphate and sulfate. The phosphate is less soluble and precipitates. Ferrous sulfate remains in solution and is oxidized as pH increases. To verify this pathway, we dissolved Fe2+-chloride and Na-phosphate salts in sulfuric acid inside an anoxic glovebox. The solution was titrated to pH 6 by adding NaOH when a first precipitate formed, which was ferrous phosphate according to Mössbauer spectroscopy (MB). At that point the solution was removed from the glovebox and allowed to evaporate in the presence of atmospheric oxygen, leading to the oxidation of Fe2+. The evaporation rate was controlled by keeping the suspensions at different temperatures; pH was monitored during the evaporation process. The final precipitates were analyzed by MB and X-Ray Fluorescence (XRF), comparable to MER MB and Alpha Particle X-ray Spectrometer instrument datasets, and complementary techniques such as X-ray diffraction. Fourier Transform Infrared spectroscopy measurements to compare to MER miniature thermal emission spectrometer data are planned. We observed differences depending on the heat source during evaporation. The closest match to Martian data on the basis of Mössbauer spectra was achieved with a suspension evaporated at 80°C on a hot plate, i.e. heated from below with a temperature gradient in the bottle. The Fe2+/FeT ratio matched, and ferrous phases were all phosphate. When heated in a water bath, i.e. without a temperature gradient in the bottle, Fe2+/FeT ratios increased and ferrous sulfates precipitated also. These results indicate that the Martian light-toned S-rich deposits formed by evaporation on the surface where temperature gradients would be expected rather than underground. They confirm that ferrous phosphate/ferric sulfate associations are possible on Mars and could be preserved in the oxygen-free Martian atmosphere. References: [1] Morris et al., J.Geophys. Res. 111 (2006) E02S13; [2] Ming et al., J. Geophys. Res. 111 (2006) E02S12; [3] Schröder et al., GSA Annual Meeting 2008, Paper No. 171-3.

Involvement of Vitamin B6 Biosynthesis Pathways in the Insecticidal Activity of Photorhabdus luminescens.

PubMed

Sato, Kazuki; Yoshiga, Toyoshi; Hasegawa, Koichi

2016-06-15

Photorhabdus luminescens is a Gram-negative entomopathogenic bacterium which symbiotically associates with the entomopathogenic nematode Heterorhabditis bacteriophora P. luminescens is highly virulent to many insects and nonsymbiotic nematodes, including Caenorhabditis elegans To understand the virulence mechanisms of P. luminescens, we obtained virulence-deficient and -attenuated mutants against C. elegans through a transposon-mutagenized library. From the genetic screening, we identified the pdxB gene, encoding erythronate-4-phosphate dehydrogenase, as required for de novo vitamin B6 biosynthesis. Mutation in pdxB caused growth deficiency of P. luminescens in nutrient-poor medium, which was restored under nutrient-rich conditions or by supplementation with pyridoxal 5'-phosphate (PLP), an active form of vitamin B6 Supplementation with three other B6 vitamers (pyridoxal, pyridoxine, and pyridoxamine) also restored the growth of the pdxB mutant, suggesting the existence of a salvage pathway for vitamin B6 biosynthesis in P. luminescens Moreover, supplementation with PLP restored the virulence-deficient phenotype against C. elegans Combining these results with the fact that pdxB mutation also caused attenuation of insecticidal activity, we concluded that the production of appropriate amounts of vitamin B6 is critical for P. luminescens pathogenicity. The Gram-negative entomopathogenic bacterium Photorhabdus luminescens symbiotically associates with the entomopathogenic nematode Heterorhabditis bacteriophora P. luminescens is highly virulent to many insects and nonsymbiotic nematodes, including Caenorhabditis elegans We have obtained several virulence-deficient and -attenuated P. luminescens mutants against C. elegans through genetic screening. From the genetic analysis, we present the vitamin B6 biosynthetic pathways in P. luminescens that are important for its insecticidal activity. Mutation in pdxB, encoding erythronate-4-phosphate dehydrogenase and required for the de novo vitamin B6 biosynthesis pathway, caused virulence deficiency against C. elegans and growth deficiency of P. luminescens in nutrient-poor medium. Because such phenotypes were restored under nutrient-rich conditions or by supplementation with B6 vitamers, we showed the presence of the two vitamin B6 synthetic pathways (de novo and salvage) in P. luminescens and also showed that the ability to produce an appropriate amount of vitamin B6 is critical for P. luminescens pathogenicity. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Enzymological Basis for Growth Inhibition by l-Phenylalanine in the Cyanobacterium Synechocystis sp. 29108

PubMed Central

Hall, Geraldine C.; Jensen, Roy A.

1980-01-01

The pattern of allosteric control in the biosynthetic pathway for aromatic amino acids provides a basis to explain vulnerability to growth inhibition by l-phenylalanine (0.2 mM or greater) in the unicellular cyanobacterium Synechocystis sp. 29108. We attribute growth inhibition to the hypersensitivity of 3-deoxy-d-arabinoheptulosonate 7-phosphate synthase to feedback inhibition by l-phenylalanine. Hyperregulation of this initial enzyme of aromatic biosynthesis depletes the supply of precursors needed for biosynthesis of l-tyrosine and l-tryptophan. Consistent with this mechanism is the total reversal of phenylalanine inhibition by a combination of tyrosine and tryptophan. Inhibited cultures also contained decreased levels of phycocyanin pigments, a characteristic previously correlated with amino acid starvation in cyanobacteria. l-Phenylalanine is a potent noncompetitive inhibitor (with both substrates) of 3-deoxy-d-arabinoheptulosonate 7-phosphate synthase, whereas l-tyrosine is a very weak inhibitor. Prephenate dehydratase also displays allosteric sensitivity to phenylalanine (inhibition) and to tyrosine (activation). Both 2-fluoro and 4-fluoro derivatives of phenylalanine were potent analog antimetabolites, and these were used in addition to l-phenylalanine as selective agents for resistant mutants. Mutants were isolated which excreted both phenylalanine and tyrosine, the consequence of an altered 3-deoxy-d-arabinoheptulosonate 7-phosphate synthase no longer sensitive to feedback inhibition. Simultaneous insensitivity to l-tyrosine suggests that l-tyrosine acts as a weak analog mimic of l-phenylalanine at a common binding site. Prephenate dehydratase in the regulatory mutants was unaltered. Surprisingly, in view of the lack of regulation in the tyrosine branchlet of the pathway, such mutants excrete more phenylalanine than tyrosine, indicating that l-tyrosine activation dominates l-phenylalanine inhibition of prephenate dehydratase in vivo. In mutant Phe r19 the loss in allosteric sensitivity of 3-deoxy-d-arabinoheptulosonate 7-phosphate synthase was accompanied by a threefold increase in specific activity. This could suggest that existence of a modest degree of repression control (autogenous) over 3-deoxy-d-arabinoheptulosonate synthase, although other explanations are possible. Specific activities of chorismate mutase, prephenate dehydratase, shikimate/nicotinamide adenine dinucleotide phosphate dehydrogenase, and arogenate/nicotinamide adenine dinucleotide phosphate dehydrogenase in mutant Phe r19 were identical with those of the wild type. PMID:6108316

Chronically increased glucose uptake by adipose tissue leads to lactate production and improved insulin sensitivity rather than obesity in the mouse.

PubMed

Muñoz, S; Franckhauser, S; Elias, I; Ferré, T; Hidalgo, A; Monteys, A M; Molas, M; Cerdán, S; Pujol, A; Ruberte, J; Bosch, F

2010-11-01

In adipocytes, triacylglycerol synthesis depends on the formation of glycerol 3-phosphate, which originates either from glucose, through glycolysis, or from lactate, through glyceroneogenesis. However, glucose is traditionally viewed as the main precursor of the glycerol backbone and thus, enhanced glucose uptake would be expected to result in increased triacylglycerol synthesis and contribute to obesity. To further explore this issue, we generated a mouse model with chronically increased glucose uptake in adipose tissue by expressing Gck, which encodes the glucokinase enzyme. Here we show that the production of high levels of glucokinase led to increased adipose tissue glucose uptake and lactate production, improved glucose tolerance and higher whole-body and skeletal muscle insulin sensitivity. There was no parallel increase in glycerol 3-phosphate synthesis in vivo, fat accumulation or obesity. Moreover, at high glucose concentrations, in cultured fat cells overproducing glucokinase, glycerol 3-phosphate synthesis from pyruvate decreased, while glyceroneogenesis increased in fat cells overproducing hexokinase II. These findings indicate that the absence of glucokinase inhibition by glucose 6-phosphate probably led to increased glycolysis and blocked glyceroneogenesis in the mouse model. Furthermore, this study suggests that under physiological conditions, when blood glucose increases, glyceroneogenesis may prevail over glycolysis for triacylglycerol formation because of the inhibition of hexokinase II by glucose 6-phosphate. Together these results point to the indirect pathway (glucose to lactate to glycerol 3-phosphate) being key for fat deposition in adipose tissue.

The impact of phosphate scarcity on pharmaceutical protein production in S. cerevisiae: linking transcriptomic insights to phenotypic responses

PubMed Central

2011-01-01

Background The adaptation of unicellular organisms like Saccharomyces cerevisiae to alternating nutrient availability is of great fundamental and applied interest, as understanding how eukaryotic cells respond to variations in their nutrient supply has implications spanning from physiological insights to biotechnological applications. Results The impact of a step-wise restricted supply of phosphate on the physiological state of S. cerevisiae cells producing human Insulin was studied. The focus was to determine the changes within the global gene expression of cells being cultured to an industrially relevant high cell density of 33 g/l cell dry weight and under six distinct phosphate concentrations, ranging from 33 mM (unlimited) to 2.6 mM (limited). An increased flux through the secretory pathway, being induced by the PHO circuit during low Pi supplementation, proved to enhance the secretory production of the heterologous protein. The re-distribution of the carbon flux from biomass formation towards increased glycerol production under low phosphate led to increased transcript levels of the insulin gene, which was under the regulation of the TPI1 promoter. Conclusions Our study underlines the dynamic character of adaptive responses of cells towards a change in their nutrient access. The gradual decrease of the phosphate supply resulted in a step-wise modulated phenotypic response, thereby alternating the specific productivity and the secretory flux. Our work emphasizes the importance of reduced phosphate supply for improved secretory production of heterologous proteins. PMID:22151908

Integration of P, S, Fe, and Zn nutrition signals in Arabidopsis thaliana: potential involvement of PHOSPHATE STARVATION RESPONSE 1 (PHR1)

PubMed Central

Briat, Jean-François; Rouached, Hatem; Tissot, Nicolas; Gaymard, Frédéric; Dubos, Christian

2015-01-01

Phosphate and sulfate are essential macro-elements for plant growth and development, and deficiencies in these mineral elements alter many metabolic functions. Nutritional constraints are not restricted to macro-elements. Essential metals such as zinc and iron have their homeostasis strictly genetically controlled, and deficiency or excess of these micro-elements can generate major physiological disorders, also impacting plant growth and development. Phosphate and sulfate on one hand, and zinc and iron on the other hand, are known to interact. These interactions have been partly described at the molecular and physiological levels, and are reviewed here. Furthermore the two macro-elements phosphate and sulfate not only interact between themselves but also influence zinc and iron nutrition. These intricated nutritional cross-talks are presented. The responses of plants to phosphorus, sulfur, zinc, or iron deficiencies have been widely studied considering each element separately, and some molecular actors of these regulations have been characterized in detail. Although some scarce reports have started to examine the interaction of these mineral elements two by two, a more complex analysis of the interactions and cross-talks between the signaling pathways integrating the homeostasis of these various elements is still lacking. However, a MYB-like transcription factor, PHOSPHATE STARVATION RESPONSE 1, emerges as a common regulator of phosphate, sulfate, zinc, and iron homeostasis, and its role as a potential general integrator for the control of mineral nutrition is discussed. PMID:25972885

Mechanism of RNA 2′,3′-cyclic phosphate end healing by T4 polynucleotide kinase–phosphatase

PubMed Central

Das, Ushati; Shuman, Stewart

2013-01-01

T4 polynucleotide kinase–phosphatase (Pnkp) exemplifies a family of enzymes with 5′-kinase and 3′-phosphatase activities that function in nucleic acid repair. The polynucleotide 3′-phosphatase reaction is executed by the Pnkp C-terminal domain, which belongs to the DxDxT acylphosphatase superfamily. The 3′-phosphatase reaction entails formation and hydrolysis of a covalent enzyme-(Asp165)-phosphate intermediate, driven by general acid–base catalyst Asp167. We report that Pnkp also has RNA 2′-phosphatase activity that requires Asp165 and Asp167. The physiological substrate for Pnkp phosphatase is an RNA 2′,3′-cyclic phosphate end (RNA > p), but the pathway of cyclic phosphate removal and its enzymic requirements are undefined. Here we find that Pnkp reactivity with RNA > p requires Asp165, but not Asp167. Whereas wild-type Pnkp transforms RNA > p to RNAOH, mutant D167N converts RNA > p to RNA 3′-phosphate, which it sequesters in the phosphatase active site. In support of the intermediacy of an RNA phosphomonoester, the reaction of mutant S211A with RNA > p results in transient accumulation of RNAp en route to RNAOH. Our results suggest that healing of 2′,3′-cyclic phosphate ends is a four-step processive reaction: RNA > p + Pnkp → RNA-(3′-phosphoaspartyl)-Pnkp → RNA3′p + Pnkp → RNAOH + phosphoaspartyl-Pnkp → Pi + Pnkp. PMID:23118482

13C based proteinogenic amino acid (PAA) and metabolic flux ratio analysis of Lactococcus lactis reveals changes in pentose phosphate (PP) pathway in response to agitation and temperature related stresses.

PubMed

Azizan, Kamalrul Azlan; Ressom, Habtom W; Mendoza, Eduardo R; Baharum, Syarul Nataqain

2017-01-01

Lactococcus lactis subsp. cremoris MG1363 is an important starter culture for dairy fermentation. During industrial fermentations, L. lactis is constantly exposed to stresses that affect the growth and performance of the bacterium. Although the response of L. lactis to several stresses has been described, the adaptation mechanisms at the level of in vivo fluxes have seldom been described. To gain insights into cellular metabolism, 13 C metabolic flux analysis and gas chromatography mass spectrometry (GC-MS) were used to measure the flux ratios of active pathways in the central metabolism of L. lactis when subjected to three conditions varying in temperature (30°C, 37°C) and agitation (with and without agitation at 150 rpm). Collectively, the concentrations of proteinogenic amino acids (PAAs) and free fatty acids (FAAs) were compared, and Pearson correlation analysis ( r ) was calculated to measure the pairwise relationship between PAAs. Branched chain and aromatic amino acids, threonine, serine, lysine and histidine were correlated strongly, suggesting changes in flux regulation in glycolysis, the pentose phosphate (PP) pathway, malic enzyme and anaplerotic reaction catalysed by pyruvate carboxylase (pycA). Flux ratio analysis revealed that glucose was mainly converted by glycolysis, highlighting the stability of L. lactis' central carbon metabolism despite different conditions. Higher flux ratios through oxaloacetate (OAA) from pyruvate (PYR) reaction in all conditions suggested the activation of pyruvate carboxylate (pycA) in L. lactis , in response to acid stress during exponential phase. Subsequently, more significant flux ratio differences were seen through the oxidative and non-oxidative pentose phosphate (PP) pathways, malic enzyme, and serine and C1 metabolism, suggesting NADPH requirements in response to environmental stimuli. These reactions could play an important role in optimization strategies for metabolic engineering in L. lactis . Overall, the integration of systematic analysis of amino acids and flux ratio analysis provides a systems-level understanding of how L. lactis regulates central metabolism under various conditions.

Metabolic engineering of an ATP-neutral Embden-Meyerhof-Parnas pathway in Corynebacterium glutamicum: growth restoration by an adaptive point mutation in NADH dehydrogenase.

PubMed

Komati Reddy, Gajendar; Lindner, Steffen N; Wendisch, Volker F

2015-03-01

Corynebacterium glutamicum uses the Embden-Meyerhof-Parnas pathway of glycolysis and gains 2 mol of ATP per mol of glucose by substrate-level phosphorylation (SLP). To engineer glycolysis without net ATP formation by SLP, endogenous phosphorylating NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was replaced by nonphosphorylating NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (GapN) from Clostridium acetobutylicum, which irreversibly converts glyceraldehyde-3-phosphate (GAP) to 3-phosphoglycerate (3-PG) without generating ATP. As shown recently (S. Takeno, R. Murata, R. Kobayashi, S. Mitsuhashi, and M. Ikeda, Appl Environ Microbiol 76:7154-7160, 2010, http://dx.doi.org/10.1128/AEM.01464-10), this ATP-neutral, NADPH-generating glycolytic pathway did not allow for the growth of Corynebacterium glutamicum with glucose as the sole carbon source unless hitherto unknown suppressor mutations occurred; however, these mutations were not disclosed. In the present study, a suppressor mutation was identified, and it was shown that heterologous expression of udhA encoding soluble transhydrogenase from Escherichia coli partly restored growth, suggesting that growth was inhibited by NADPH accumulation. Moreover, genome sequence analysis of second-site suppressor mutants that were able to grow faster with glucose revealed a single point mutation in the gene of non-proton-pumping NADH:ubiquinone oxidoreductase (NDH-II) leading to the amino acid change D213G, which was shared by these suppressor mutants. Since related NDH-II enzymes accepting NADPH as the substrate possess asparagine or glutamine residues at this position, D213G, D213N, and D213Q variants of C. glutamicum NDH-II were constructed and were shown to oxidize NADPH in addition to NADH. Taking these findings together, ATP-neutral glycolysis by the replacement of endogenous NAD-dependent GAPDH with NADP-dependent GapN became possible via oxidation of NADPH formed in this pathway by mutant NADPH-accepting NDH-II(D213G) and thus by coupling to electron transport phosphorylation (ETP). Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Pantomime to visual presentation of objects: left hand dyspraxia in patients with complete callosotomy.

PubMed

Lausberg, Hedda; Cruz, Robyn F; Kita, Sotaro; Zaidel, Eran; Ptito, Alain

2003-02-01

Investigations of left hand praxis in imitation and object use in patients with callosal disconnection have yielded divergent results, inducing a debate between two theoretical positions. Whereas Liepmann suggested that the left hemisphere is motor dominant, others maintain that both hemispheres have equal motor competences and propose that left hand apraxia in patients with callosal disconnection is secondary to left hemispheric specialization for language or other task modalities. The present study aims to gain further insight into the motor competence of the right hemisphere by investigating pantomime of object use in split-brain patients. Three patients with complete callosotomy and, as control groups, five patients with partial callosotomy and nine healthy subjects were examined for their ability to pantomime object use to visual object presentation and demonstrate object manipulation. In each condition, 11 objects were presented to the subjects who pantomimed or demonstrated the object use with either hand. In addition, six object pairs were presented to test bimanual coordination. Two independent raters evaluated the videotaped movement demonstrations. While object use demonstrations were perfect in all three groups, the split-brain patients displayed apraxic errors only with their left hands in the pantomime condition. The movement analysis of concept and execution errors included the examination of ipsilateral versus contralateral motor control. As the right hand/left hemisphere performances demonstrated retrieval of the correct movement concepts, concept errors by the left hand were taken as evidence for right hemisphere control. Several types of execution errors reflected a lack of distal motor control indicating the use of ipsilateral pathways. While one split-brain patient controlled his left hand predominantly by ipsilateral pathways in the pantomime condition, the error profile in the other two split-brain patients suggested that the right hemisphere controlled their left hands. In the object use condition, in all three split-brain patients fine-graded distal movements in the left hand indicated right hemispheric control. Our data show left hand apraxia in split-brain patients is not limited to verbal commands, but also occurs in pantomime to visual presentation of objects. As the demonstration with object in hand was unimpaired in either hand, both hemispheres must contain movement concepts for object use. However, the disconnected right hemisphere is impaired in retrieving the movement concept in response to visual object presentation, presumably because of a deficit in associating perceptual object representation with the movement concepts.

HIV-1 Vpr modulates macrophage metabolic pathways: a SILAC-based quantitative analysis.

PubMed

Barrero, Carlos A; Datta, Prasun K; Sen, Satarupa; Deshmane, Satish; Amini, Shohreh; Khalili, Kamel; Merali, Salim

2013-01-01

Human immunodeficiency virus type 1 encoded viral protein Vpr is essential for infection of macrophages by HIV-1. Furthermore, these macrophages are resistant to cell death and are viral reservoir. However, the impact of Vpr on the macrophage proteome is yet to be comprehended. The goal of the present study was to use a stable-isotope labeling by amino acids in cell culture (SILAC) coupled with mass spectrometry-based proteomics approach to characterize the Vpr response in macrophages. Cultured human monocytic cells, U937, were differentiated into macrophages and transduced with adenovirus construct harboring the Vpr gene. More than 600 proteins were quantified in SILAC coupled with LC-MS/MS approach, among which 136 were significantly altered upon Vpr overexpression in macrophages. Quantified proteins were selected and clustered by biological functions, pathway and network analysis using Ingenuity computational pathway analysis. The proteomic data illustrating increase in abundance of enzymes in the glycolytic pathway (pentose phosphate and pyruvate metabolism) was further validated by western blot analysis. In addition, the proteomic data demonstrate down regulation of some key mitochondrial enzymes such as glutamate dehydrogenase 2 (GLUD2), adenylate kinase 2 (AK2) and transketolase (TKT). Based on these observations we postulate that HIV-1 hijacks the macrophage glucose metabolism pathway via the Vpr-hypoxia inducible factor 1 alpha (HIF-1 alpha) axis to induce expression of hexokinase (HK), glucose-6-phosphate dehyrogenase (G6PD) and pyruvate kinase muscle type 2 (PKM2) that facilitates viral replication and biogenesis, and long-term survival of macrophages. Furthermore, dysregulation of mitochondrial glutamate metabolism in macrophages can contribute to neurodegeneration via neuroexcitotoxic mechanisms in the context of NeuroAIDS.

Metabolic characterization and transformation of the non-dairy Lactococcus lactis strain KF147, for production of ethanol from xylose.

PubMed

Petersen, Kia Vest; Liu, Jianming; Chen, Jun; Martinussen, Jan; Jensen, Peter Ruhdal; Solem, Christian

2017-08-01

The non-dairy lactic acid bacterium Lactococcus lactis KF147 can utilize xylose as the sole energy source. To assess whether KF147 could serve as a platform organism for converting second generation sugars into useful chemicals, the authors characterized growth and product formation for KF147 when grown on xylose. In a defined medium KF147 was found to co-metabolize xylose and arginine, resulting in bi-phasic growth. Especially at low xylose concentrations, arginine significantly improved growth rate. To facilitate further studies of the xylose metabolism, the authors eliminated arginine catabolism by deleting the arcA gene encoding the arginine deiminase. The fermentation product profile suggested two routes for xylose degradation, the phosphoketolase pathway and the pentose phosphate pathway. Inactivation of the phosphoketolase pathway redirected the entire flux through the pentose phosphate pathway whereas over-expression of phosphoketolase increased the flux through the phosphoketolase pathway. In general, significant amounts of the mixed-acid products, including lactate, formate, acetate and ethanol, were formed irrespective of xylose concentrations. To demonstrate the potential of KF147 for converting xylose into useful chemicals the authors chose to redirect metabolism towards ethanol production. A synthetic promoter library was used to drive the expression of codon-optimized versions of the Zymomonas mobilis genes encoding pyruvate decarboxylase and alcohol dehydrogenase, and the outcome was a strain producing ethanol as the sole fermentation product with a high yield corresponding to 83% of the theoretical maximum. The results clearly indicate the great potential of using the more metabolically diverse non-dairy L. lactis strains for bio-production based on xylose containing feedstocks. Copyright © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Review: Metabolic Control of Immune System Activation in Rheumatic Diseases.

PubMed

Perl, Andras

2017-12-01

Metabolic pathways mediate lineage specification within the immune system through the regulation of glucose utilization, a process that generates energy in the form of ATP and synthesis of amino acids, nucleotides, and lipids to enable cell growth, proliferation, and survival. CD4+ T cells, a proinflammatory cell subset, preferentially produce ATP through glycolysis, whereas cells with an antiinflammatory lineage, such as memory and regulatory T cells, favor mitochondrial ATP generation. In conditions of metabolic stress or a shortage of nutrients, cells rely on autophagy to secure amino acids and other substrates, while survival depends on the sparing of mitochondria and maintenance of a reducing environment. The pentose phosphate pathway acts as a key gatekeeper of inflammation by supplying ribose-5-phosphate for cell proliferation and NADPH for antioxidant defenses. Increased lysosomal catabolism, accumulation of branched amino acids, glutamine, kynurenine, and histidine, and depletion of glutathione and cysteine activate the mechanistic target of rapamycin (mTOR), an arbiter of lineage development within the innate and adaptive immune systems. Mapping the impact of susceptibility genes to metabolic pathways allows for better understanding and therapeutic targeting of disease-specific expansion of proinflammatory cells. Therapeutic approaches aimed at glutathione depletion and mTOR pathway activation appear to be safe and effective for treating lupus, while an opposing intervention may be of benefit in rheumatoid arthritis. Environmental sources of origin for metabolites within immune cells may include microbiota and plants. Thus, a better understanding of the pathways of immunometabolism could provide new insights into the pathogenesis and treatment of the rheumatic diseases. © 2017 The Authors. Arthritis & Rheumatology published by Wiley Periodicals, Inc. on behalf of American College of Rheumatology.

Metabolic fate of glucose and candidate signaling and excess-fuel detoxification pathways in pancreatic β-cells

PubMed Central

Mugabo, Yves; Zhao, Shangang; Lamontagne, Julien; Al-Mass, Anfal; Peyot, Marie-Line; Corkey, Barbara E.; Joly, Erik; Madiraju, S. R. Murthy; Prentki, Marc

2017-01-01

Glucose metabolism promotes insulin secretion in β-cells via metabolic coupling factors that are incompletely defined. Moreover, chronically elevated glucose causes β-cell dysfunction, but little is known about how cells handle excess fuels to avoid toxicity. Here we sought to determine which among the candidate pathways and coupling factors best correlates with glucose-stimulated insulin secretion (GSIS), define the fate of glucose in the β-cell, and identify pathways possibly involved in excess-fuel detoxification. We exposed isolated rat islets for 1 h to increasing glucose concentrations and measured various pathways and metabolites. Glucose oxidation, oxygen consumption, and ATP production correlated well with GSIS and saturated at 16 mm glucose. However, glucose utilization, glycerol release, triglyceride and glycogen contents, free fatty acid (FFA) content and release, and cholesterol and cholesterol esters increased linearly up to 25 mm glucose. Besides being oxidized, glucose was mainly metabolized via glycerol production and release and lipid synthesis (particularly FFA, triglycerides, and cholesterol), whereas glycogen production was comparatively low. Using targeted metabolomics in INS-1(832/13) cells, we found that several metabolites correlated well with GSIS, in particular some Krebs cycle intermediates, malonyl-CoA, and lower ADP levels. Glucose dose-dependently increased the dihydroxyacetone phosphate/glycerol 3-phosphate ratio in INS-1(832/13) cells, indicating a more oxidized state of NAD in the cytosol upon glucose stimulation. Overall, the data support a role for accelerated oxidative mitochondrial metabolism, anaplerosis, and malonyl-CoA/lipid signaling in β-cell metabolic signaling and suggest that a decrease in ADP levels is important in GSIS. The results also suggest that excess-fuel detoxification pathways in β-cells possibly comprise glycerol and FFA formation and release extracellularly and the diversion of glucose carbons to triglycerides and cholesterol esters. PMID:28280244

Updates to a 13C metabolic flux analysis model for evaluating energy metabolism in cultured cerebellar granule neurons from neonatal rats.

PubMed

Jekabsons, Mika B; Gebril, Hoda M; Wang, Yan-Hong; Avula, Bharathi; Khan, Ikhlas A

2017-10-01

A hexose phosphate recycling model previously developed to infer fluxes through the major glucose consuming pathways in cultured cerebellar granule neurons (CGNs) from neonatal rats metabolizing [1,2- 13 C 2 ]glucose was revised by considering reverse flux through the non-oxidative pentose phosphate pathway (PPP) and symmetrical succinate oxidation within the tricarboxylic acid (TCA) cycle. The model adjusts three flux ratios to effect 13 C distribution in the hexose, pentose, and triose phosphate pools, and in TCA cycle malate to minimize the error between predicted and measured 13 C labeling in exported lactate (i.e., unlabeled, single-, double-, and triple-labeled; M, M1, M2, and M3, respectively). Inclusion of reverse non-oxidative PPP flux substantially increased the number of calculations but ultimately had relatively minor effects on the labeling of glycolytic metabolites. From the error-minimized solution in which the predicted M-M3 lactate differed by 0.49% from that measured by liquid chromatography-triple quadrupole mass spectrometry, the neurons exhibited negligible forward non-oxidative PPP flux. Thus, no glucose was used by the pentose cycle despite explicit consideration of hexose phosphate recycling. Mitochondria consumed only 16% of glucose while 45% was exported as lactate by aerobic glycolysis. The remaining 39% of glucose was shunted to pentose phosphates presumably for de novo nucleotide synthesis, but the proportion metabolized through the oxidative PPP vs. the reverse non-oxidative PPP could not be determined. The lactate exported as M1 (2.5%) and M3 (1.2%) was attributed to malic enzyme, which was responsible for 7.8% of pyruvate production (vs. 92.2% by glycolysis). The updated model is more broadly applicable to different cell types by considering bi-directional flux through the non-oxidative PPP. Its application to cultured neurons utilizing glucose as the sole exogenous substrate has demonstrated substantial oxygen-independent glucose utilization by aerobic glycolysis as well as the oxidative PPP and/or reverse non-oxidative PPP, but negligible glucose consumption by the pentose cycle. Copyright © 2017 Elsevier Ltd. All rights reserved.

Glucose-6-phosphate dehydrogenase is a regulator of vascular smooth muscle contraction.

PubMed

Gupte, Rakhee S; Ata, Hirotaka; Rawat, Dhawjbahadur; Abe, Madoka; Taylor, Mark S; Ochi, Rikuo; Gupte, Sachin A

2011-02-15

Glucose-6-phosphate dehydrogenase (G6PD) is the rate-limiting enzyme in the pentose phosphate pathway and a major source of nicotinamide adenine dinucleotide phosphate reduced (NADPH), which regulates numerous enzymatic (including glutathione reductase and NADPH oxidase that, respectively, generates reduced glutathione and reactive oxygen species) reactions involved in various cellular actions, yet its physiological function is seldom investigated. We, however, recently showed that inhibiting G6PD causes precontracted coronary artery (CA) to relax in an endothelium-derived relaxing factor- and second messenger-independent manner. Here we assessed the role of G6PD in regulating CA contractility. Treating bovine CAs for 20 min with potassium chloride (KCl; 30 mM), amphotericin B (50 μM), or U46619 (100 nM) significantly (p < 0.05) increased both G6PD activity and glucose flux through the pentose phosphate pathway. The effect was Ca(2+) independent, and there was a corresponding increase in protein kinase C (PKC) activity. Activation of G6PD by KCl was blocked by the PKCδ inhibitor rottlerin (10 μM) or by knocking down PKCδ expression using siRNA. Phorbol 12, 13-dibutyrate (10 μM), a PKC activator, significantly increased G6PD phosphorylation and activity, whereas single (S210A, T266A) and double (S210A/T266A) mutations at sites flanking the G6PD active site significantly inhibited phosphorylation, shifted the isoelectric point, and reduced enzyme activity. Knocking down G6PD decreased NADPH and reactive oxygen species generation, and reduced KCl-evoked increases in [Ca(2+)](i) and myosin light chain phosphorylation, thereby reducing CA contractility. Similarly, aortas from G6PD-deficient mice developed less KCl/phorbol 12, 13-dibutyrate-evoked force than those from their wild-type littermates. Conversely, overexpression of G6PD augmented KCl-evoked increases in [Ca(2+)](i), thereby augmenting CA contraction. Our findings demonstrate that G6PD activity and NADPH is increased in activated CA in a PKCδ-dependent manner and that G6PD modulates Ca(2+) entry and CA contractions evoked by membrane depolarization.

Dynamics of Monoterpene Formation in Spike Lavender Plants.

PubMed

Mendoza-Poudereux, Isabel; Kutzner, Erika; Huber, Claudia; Segura, Juan; Arrillaga, Isabel; Eisenreich, Wolfgang

2017-12-19

The metabolic cross-talk between the mevalonate (MVA) and the methylerythritol phosphate (MEP) pathways was analyzed in spike lavender ( Lavandula latifolia Med) on the basis of 13 CO₂-labelling experiments using wildtype and transgenic plants overexpressing the 3-hydroxy-3-methylglutaryl CoA reductase (HMGR), the first and key enzyme of the MVA pathway. The plants were labelled in the presence of 13 CO₂ in a gas chamber for controlled pulse and chase periods of time. GC/MS and NMR analysis of 1,8-cineole and camphor, the major monoterpenes present in their essential oil, indicated that the C5-precursors, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) of both monoterpenes are predominantly biosynthesized via the MEP pathway. Surprisingly, overexpression of HMGR did not have significant impact upon the crosstalk between the MVA and MEP pathways indicating that the MEP route is the preferred pathway for the synthesis of C5 monoterpene precursors in spike lavender.

New insights into electrocatalytic ozone generation via splitting of water over PbO2 electrode: A DFT study

NASA Astrophysics Data System (ADS)

Gibson, Gregory; Morgan, Ashley; Hu, P.; Lin, Wen-Feng

2016-06-01

The viable mechanisms for O3 generation via the electrocatalytic splitting of H2O over β-PbO2 catalyst were identified through Density Functional Theory calculations. H2O adsorbed onto the surface was oxidized to form OH then O; the latter reacted with a surface bridging O to form O2 which in turn reacted with another surface O to form O3. The final step of the mechanisms occurs via an Eley-Rideal style interaction where surface O2 desorbs and then attacks the surface bridging oxygen, forming O3. A different reaction pathway via an O3H intermediate was found less favoured both thermodynamically and kinetically.

Asymmetric adiabatic couplers for fully-integrated broadband quantum-polarization state preparation.

PubMed

Chung, Hung-Pin; Huang, Kuang-Hsu; Wang, Kai; Yang, Sung-Lin; Yang, Shih-Yuan; Sung, Chun-I; Solntsev, Alexander S; Sukhorukov, Andrey A; Neshev, Dragomir N; Chen, Yen-Hung

2017-12-04

Spontaneous parametric down-conversion (SPDC) is a widely used method to generate entangled photons, enabling a range of applications from secure communication to tests of quantum physics. Integrating SPDC on a chip provides interferometric stability, allows to reduce a physical footprint, and opens a pathway to true scalability. However, dealing with different photon polarizations and wavelengths on a chip presents a number of challenging problems. In this work, we demonstrate an on-chip polarization beam-splitter based on z-cut titanium-diffused lithium niobate asymmetric adiabatic couplers (AAC) designed for integration with a type-II SPDC source. Our experimental measurements reveal unique polarization beam-splitting regime with the ability to tune the splitting ratios based on wavelength. In particular, we measured a splitting ratio of 17 dB over broadband regions (>60 nm) for both H- and V-polarized lights and a specific 50%/50% splitting ratio for a cross-polarized photon pair from the AAC. The results show that such a system can be used for preparing different quantum polarization-path states that are controllable by changing the phase-matching conditions in the SPDC over a broad band. Furthermore, we propose a fully integrated electro-optically tunable type-II SPDC polarization-path-entangled state preparation circuit on a single lithium niobate photonic chip.

In silico profiling of Escherichia coli and Saccharomyces cerevisiae as terpenoid factories

PubMed Central

2013-01-01

Background Heterologous microbial production of rare plant terpenoids of medicinal or industrial interest is attracting more and more attention but terpenoid yields are still low. Escherichia coli and Saccharomyces cerevisiae are the most widely used heterologous hosts; a direct comparison of both hosts based on experimental data is difficult though. Hence, the terpenoid pathways of E. coli (via 1-deoxy-D-xylulose 5-phosphate, DXP) and S. cerevisiae (via mevalonate, MVA), the impact of the respective hosts metabolism as well as the impact of different carbon sources were compared in silico by means of elementary mode analysis. The focus was set on the yield of isopentenyl diphosphate (IPP), the general terpenoid precursor, to identify new metabolic engineering strategies for an enhanced terpenoid yield. Results Starting from the respective precursor metabolites of the terpenoid pathways (pyruvate and glyceraldehyde-3-phosphate for the DXP pathway and acetyl-CoA for the MVA pathway) and considering only carbon stoichiometry, the two terpenoid pathways are identical with respect to carbon yield. However, with glucose as substrate, the MVA pathway has a lower potential to supply terpenoids in high yields than the DXP pathway if the formation of the required precursors is taken into account, due to the carbon loss in the formation of acetyl-CoA. This maximum yield is further reduced in both hosts when the required energy and reduction equivalents are considered. Moreover, the choice of carbon source (glucose, xylose, ethanol or glycerol) has an effect on terpenoid yield with non-fermentable carbon sources being more promising. Both hosts have deficiencies in energy and redox equivalents for high yield terpenoid production leading to new overexpression strategies (heterologous enzymes/pathways) for an enhanced terpenoid yield. Finally, several knockout strategies are identified using constrained minimal cut sets enforcing a coupling of growth to a terpenoid yield which is higher than any yield published in scientific literature so far. Conclusions This study provides for the first time a comprehensive and detailed in silico comparison of the most prominent heterologous hosts E. coli and S. cerevisiae as terpenoid factories giving an overview on several promising metabolic engineering strategies paving the way for an enhanced terpenoid yield. PMID:24059635

A comparative assessment of enamel mineral content and Streptococcus mutans population between conventional composites and composites containing nano amorphous calcium phosphate in fixed orthodontic patients: a split-mouth randomized clinical trial.

PubMed

Jahanbin, Arezoo; Farzanegan, Fahimeh; Atai, Mohammad; Jamehdar, Saeed Amel; Golfakhrabadi, Parvaneh; Shafaee, Hooman

2017-02-01

The aim of this 'split-mouth design' trial was to evaluate the effect of the nano amorphous calcium phosphate (NACP) containing composite on enamel mineral contents and streptococcus mutans population in fixed orthodontic patients. Randomized, prospective, single-center controlled trial. Twenty-four patients between the ages of 13-18 years participated in this study. The control and test sides were randomly selected by a coin toss (1:1 ratio). On the control side orthodontic brackets were bonded on the buccal surfaces of upper premolars and laterals using an orthodontic composite (Transbond XT), and on the study side NACP-containing composite was used. Outcome measures were the mineral content around the brackets and S.mutans count. The later were calculated in the plaque around the brackets by real-time PCR at 3 months, and 6 months after the initiation of treatment. All stages of the study were blind using coding system. Paired t-test and repeated measurements were used for data analysis. In the third and sixth month, the bacterial population was significantly lower in the study side than the control side (P = 0.01 and 0.000).The mineral content of the study side was significantly higher than the controls, 6 months after brocket bonding (P = 0.004). There were no significant differences between the premolars and lateral teeth for all measurements. This research was performed in a single-center by one experienced clinician. NACP-containing composites have the potential to inhibit mineral content loss and S.mutans colonization around orthodontic brackets during fixed orthodontic treatments. This trial was not registered. The protocol was not published before trial commencement. © The Author 2016. Published by Oxford University Press on behalf of the European Orthodontic Society. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Arabidopsis plants deficient in plastidial glyceraldehyde-3-phosphate dehydrogenase show alterations in abscisic acid (ABA) signal transduction: interaction between ABA and primary metabolism

PubMed Central

Muñoz-Bertomeu, Jesús; Bermúdez, María Angeles; Segura, Juan; Ros, Roc

2011-01-01

Abscisic acid (ABA) controls plant development and regulates plant responses to environmental stresses. A role for ABA in sugar regulation of plant development has also been well documented although the molecular mechanisms connecting the hormone with sugar signal transduction pathways are not well understood. In this work it is shown that Arabidopsis thaliana mutants deficient in plastidial glycolytic glyceraldehyde-3-phosphate dehydrogenase (gapcp1gapcp2) are ABA insensitive in growth, stomatal closure, and germination assays. The ABA levels of gapcp1gapcp2 were normal, suggesting that the ABA signal transduction pathway is impaired in the mutants. ABA modified gapcp1gapcp2 gene expression, but the mutant response to the hormone differed from that observed in wild-type plants. The gene expression of the transcription factor ABI4, involved in both sugar and ABA signalling, was altered in gapcp1gapcp2, suggesting that their ABA insensitivity is mediated, at least partially, through this transcriptional regulator. Serine supplementation was able partly to restore the ABA sensitivity of gapcp1gapcp2, indicating that amino acid homeostasis and/or serine metabolism may also be important determinants in the connections of ABA with primary metabolism. Overall, these studies provide new insights into the links between plant primary metabolism and ABA signalling, and demonstrate the importance of plastidial glycolytic glyceraldehyde-3-phosphate dehydrogenase in these interactions. PMID:21068209

Glucose-6-phosphate dehydrogenase, NADPH, and cell survival.

PubMed

Stanton, Robert C

2012-05-01

Glucose-6-phosphate dehydrogenase (G6PD) is the rate-limiting enzyme of the pentose phosphate pathway. Many scientists think that the roles and regulation of G6PD in physiology and pathophysiology have been well established as the enzyme was first identified 80 years ago. And that G6PD has been extensively studied especially with respect to G6PD deficiency and its association with hemolysis, and with respect to the role G6PD plays in lipid metabolism. But there has been a growing understanding of the central importance of G6PD to cellular physiology as it is a major source of NADPH that is required by many essential cellular systems including the antioxidant pathways, nitric oxide synthase, NADPH oxidase, cytochrome p450 system, and others. Indeed G6PD is essential for cell survival. It has also become evident that G6PD is highly regulated by many signals that affect transcription, post-translation, intracellular location, and interactions with other protein. Pathophysiologic roles for G6PD have also been identified in such disease processes as diabetes, aldosterone-induced endothelial dysfunction, cancer, and others. It is now clear that G6PD is under complex regulatory control and of central importance to many cellular processes. In this review the biochemistry, regulatory signals, physiologic roles, and pathophysiologic roles for G6PD that have been elucidated over the past 20 years are discussed. Copyright © 2012 Wiley Periodicals, Inc.

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.

Formation of xylitol and xylitol-5-phosphate and its impact on growth of d-xylose-utilizing Corynebacterium glutamicum strains.

PubMed

Radek, Andreas; Müller, Moritz-Fabian; Gätgens, Jochem; Eggeling, Lothar; Krumbach, Karin; Marienhagen, Jan; Noack, Stephan

2016-08-10

Wild-type Corynebacterium glutamicum has no endogenous metabolic activity for utilizing the lignocellulosic pentose d-xylose for cell growth. Therefore, two different engineering approaches have been pursued resulting in platform strains harbouring a functional version of either the Isomerase (ISO) or the Weimberg (WMB) pathway for d-xylose assimilation. In a previous study we found for C. glutamicum WMB by-product formation of xylitol during growth on d-xylose and speculated that the observed lower growth rates are due to the growth inhibiting effect of this compound. Based on a detailed phenotyping of the ISO, WMB and the wild-type strain of C. glutamicum, we here show that this organism has a natural capability to synthesize xylitol from d-xylose under aerobic cultivation conditions. We furthermore observed the intracellular accumulation of xylitol-5-phosphate as a result of the intracellular phosphorylation of xylitol, which was particularly pronounced in the C. glutamicum ISO strain. Interestingly, low amounts of supplemented xylitol strongly inhibit growth of this strain on d-xylose, d-glucose and d-arabitol. These findings demonstrate that xylitol is a suitable substrate of the endogenous xylulokinase (XK, encoded by xylB) and its overexpression in the ISO strain leads to a significant phosphorylation of xylitol in C. glutamicum. Therefore, in order to circumvent cytotoxicity by xylitol-5-phosphate, the WMB pathway represents an interesting alternative route for engineering C. glutamicum towards efficient d-xylose utilization. Copyright © 2016 Elsevier B.V. All rights reserved.

Prevention of hepatocarcinogenesis and increased susceptibility to acetaminophen-induced liver failure in transaldolase-deficient mice by N-acetylcysteine

PubMed Central

Hanczko, Robert; Fernandez, David R.; Doherty, Edward; Qian, Yueming; Vas, Gyorgy; Niland, Brian; Telarico, Tiffany; Garba, Adinoyi; Banerjee, Sanjay; Middleton, Frank A.; Barrett, Donna; Barcza, Maureen; Banki, Katalin; Landas, Steve K.; Perl, Andras

2009-01-01

Although oxidative stress has been implicated in acute acetaminophen-induced liver failure and in chronic liver cirrhosis and hepatocellular carcinoma (HCC), no common underlying metabolic pathway has been identified. Recent case reports suggest a link between the pentose phosphate pathway (PPP) enzyme transaldolase (TAL; encoded by TALDO1) and liver failure in children. Here, we show that Taldo1–/– and Taldo1+/– mice spontaneously developed HCC, and Taldo1–/– mice had increased susceptibility to acetaminophen-induced liver failure. Oxidative stress in Taldo1–/– livers was characterized by the accumulation of sedoheptulose 7-phosphate, failure to recycle ribose 5-phosphate for the oxidative PPP, depleted NADPH and glutathione levels, and increased production of lipid hydroperoxides. Furthermore, we found evidence of hepatic mitochondrial dysfunction, as indicated by loss of transmembrane potential, diminished mitochondrial mass, and reduced ATP/ADP ratio. Reduced β-catenin phosphorylation and enhanced c-Jun expression in Taldo1–/– livers reflected adaptation to oxidative stress. Taldo1–/– hepatocytes were resistant to CD95/Fas-mediated apoptosis in vitro and in vivo. Remarkably, lifelong administration of the potent antioxidant N-acetylcysteine (NAC) prevented acetaminophen-induced liver failure, restored Fas-dependent hepatocyte apoptosis, and blocked hepatocarcinogenesis in Taldo1–/– mice. These data reveal a protective role for the TAL-mediated branch of the PPP against hepatocarcinogenesis and identify NAC as a promising treatment for liver disease in TAL deficiency. PMID:19436114

Engineering of N-acetylglucosamine metabolism for improved antibiotic production in Streptomyces coelicolor A3(2) and an unsuspected role of NagA in glucosamine metabolism.

PubMed

Świątek, Magdalena A; Urem, Mia; Tenconi, Elodie; Rigali, Sébastien; van Wezel, Gilles P

2012-01-01

N-acetylglucosamine (GlcNAc), the monomer of chitin and constituent of bacterial peptidoglycan, is a preferred carbon and nitrogen source for streptomycetes. Recent studies have revealed new functions of GlcNAc in nutrient signaling of bacteria. Exposure to GlcNAc activates development and antibiotic production of Streptomyces coelicolor under poor growth conditions (famine) and blocks these processes under rich conditions (feast). Glucosamine-6-phosphate (GlcN-6P) is a key molecule in this signaling pathway and acts as an allosteric effector of a pleiotropic transcriptional repressor DasR, the regulon of which includes the GlcNAc metabolic enzymes N-actetylglucosamine-6-phosphate (GlcNAc-6P) deacetylase (NagA) and GlcN-6P deaminase (NagB). Intracellular accumulation of GlcNAc-6P and GlcN-6P enhanced production of the pigmented antibiotic actinorhodin. When the nagB mutant was challenged with GlcNAc or GlcN, spontaneous second-site mutations that relieved the toxicity of the accumulated sugar phosphates were obtained. Surprisingly, deletion of nagA also relieved toxicity of GlcN, indicating novel linkage between the GlcN and GlcNAc utilization pathways. The strongly enhanced antibiotic production observed for many suppressor mutants shows the potential of the modulation of GlcNAc and GlcN metabolism as a metabolic engineering tool toward the improvement of antibiotic productivity or even the discovery of novel compounds.

The role of transporters in supplying energy to plant plastids.

PubMed

Flügge, Ulf-Ingo; Häusler, Rainer E; Ludewig, Frank; Gierth, Markus

2011-04-01

The energy status of plant cells strongly depends on the energy metabolism in chloroplasts and mitochondria, which are capable of generating ATP either by photosynthetic or oxidative phosphorylation, respectively. Another energy-rich metabolite inside plastids is the glycolytic intermediate phosphoenolpyruvate (PEP). However, chloroplasts and most non-green plastids lack the ability to generate PEP via a complete glycolytic pathway. Hence, PEP import mediated by the plastidic PEP/phosphate translocator or PEP provided by the plastidic enolase are vital for plant growth and development. In contrast to chloroplasts, metabolism in non-green plastids (amyloplasts) of starch-storing tissues strongly depends on both the import of ATP mediated by the plastidic nucleotide transporter NTT and of carbon (glucose 6-phosphate, Glc6P) mediated by the plastidic Glc6P/phosphate translocator (GPT). Both transporters have been shown to co-limit starch biosynthesis in potato plants. In addition, non-photosynthetic plastids as well as chloroplasts during the night rely on the import of energy in the form of ATP via the NTT. During energy starvation such as prolonged darkness, chloroplasts strongly depend on the supply of ATP which can be provided by lipid respiration, a process involving chloroplasts, peroxisomes, and mitochondria and the transport of intermediates, i.e. fatty acids, ATP, citrate, and oxaloacetate across their membranes. The role of transporters involved in the provision of energy-rich metabolites and in pathways supplying plastids with metabolic energy is summarized here.

Synthesis of 5-hydroxymethylfurural from carbohydrates using large-pore mesoporous tin phosphate.

PubMed

Dutta, Arghya; Gupta, Dinesh; Patra, Astam K; Saha, Basudeb; Bhaumik, Asim

2014-03-01

A large-pore mesoporous tin phosphate (LPSnP-1) material has been synthesized hydrothermally by using Pluronic P123 as the structure-directing agent. The material is composed of aggregated nanoparticles of 10-15 nm in diameter and has a BET surface area of 216 m(2)  g(-1) with an average pore diameter of 10.4 nm. This pore diameter is twice as large as that of mesoporous tin phosphate materials synthesized through the surfactant-templating pathways reported previously. LPSnP-1 shows excellent catalytic activity for the conversion of fructose, glucose, sucrose, cellobiose, and cellulose to 5-hydroxymethylfurfural (HMF) in a water/methyl isobutyl ketone biphasic solvent to give maximum yields of HMF of 77, 50, 51, 39, and 32 mol %, respectively, under microwave-assisted heating at 423 K. Under comparable reaction conditions, LPSnP-1 gives 12 % more HMF yield than a small-pore mesoporous tin phosphate catalyst that has an identical framework composition. This confirms the beneficial role of large mesopores and nanoscale particle morphology in catalytic reactions that involve bulky natural carbohydrate molecules. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

From soil to plant, the journey of P through trophic relationships and ectomycorrhizal association

PubMed Central

Becquer, Adeline; Trap, Jean; Irshad, Usman; Ali, Muhammad A.; Claude, Plassard

2014-01-01

Phosphorus (P) is essential for plant growth and productivity. It is one of the most limiting macronutrients in soil because it is mainly present as unavailable, bound P whereas plants can only use unbound, inorganic phosphate (Pi), which is found in very low concentrations in soil solution. Some ectomycorrhizal fungi are able to release organic compounds (organic anions or phosphatases) to mobilize unavailable P. Recent studies suggest that bacteria play a major role in the mineralization of nutrients such as P through trophic relationships as they can produce specific phosphatases such as phytases to degrade phytate, the main form of soil organic P. Bacteria are also more effective than other microorganisms or plants at immobilizing free Pi. Therefore, bacterial grazing by grazers, such as nematodes, could release Pi locked in bacterial biomass. Free Pi may be taken up by ectomycorrhizal fungus by specific phosphate transporters and transferred to the plant by mechanisms that have not yet been identified. This mini-review aims to follow the phosphate pathway to understand the ecological and molecular mechanisms responsible for transfer of phosphate from the soil to the plant, to improve plant P nutrition. PMID:25360140

Crystallization and preliminary X-ray analysis of the isomerase domain of glucosamine-6-phosphate synthase from Candida albicans

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

Olchowy, Jaroslaw; Jedrzejczak, Robert; Milewski, Slawomir

2005-11-01

The isomerase domain of glucosamine-6-phosphate synthase from C. albicans has been crystallized and X-ray diffraction data have been collected. Preliminary analysis of the data reveals the oligomeric structure of the eukaryotic synthase to be a ‘dimer’ of prokaryotic-like dimers. Glucosamine-6-phosphate synthase (EC 2.6.1.16) catalyses the first and practically irreversible step in the hexosamine metabolism pathway, the end product of which, uridine 5′-diphospho-N-acetyl d-glucosamine, is an essential substrate for assembly of the cell wall. The isomerase domain, consisting of residues 346–712 (42 kDa), of glucosamine-6-phosphate synthase from Candida albicans has been crystallized. X-ray analysis revealed that the crystals belonged to spacemore » group I4, with unit-cell parameters a = b = 149, c = 103 Å. Diffraction data were collected to 3.8 Å. Preliminary results from molecular replacement using the homologous bacterial monomer reveal that the asymmetric unit contains two monomers that resemble a bacterial dimer. The crystal lattice consists of pairs of such symmetry-related dimers forming elongated tetramers.« less

Mesoporous Phosphate Heterostructures: Synthesis and Application on Adsorption and Catalysis

NASA Astrophysics Data System (ADS)

Moreno-Tost, Ramón; Jiménez-Jiménez, José; Infantes-Molina, Antonia; Cavalcante, Celio L.; Azevedo, Diana C. S.; Soriano, María Dolores; López Nieto, José Manuel; Jiménez-López, Antonio; Rodríguez-Castellón, Enrique

Porous phosphate heterostructures (PPHs) are solids formed by a layered metal(IV) phosphate expanded with silica galleries obtained by combining the two main strategies for obtaining mesoporous materials [pillared layered structures (PLS') and MCM-41]. The different synthetic pathways for obtaining mesoporous phosphate structures with silica galleries with Zr- or Ti-doped silica, the study of their structural, textural and acid properties, its functionalisation with different organic substances such as propionitrile, 3-aminopropyl triethoxysilane, (3-mercaptopropyl)trimethoxysilane, vinyltrimethoxysilane, phenyltriethoxysilane and 3-(triethoxysilyl)propionitrile are discussed. The preparation of metal-supported catalysts and their application in gas separation, adsorption and catalysis are reviewed. Specifically, the use of Cu- and Fe-exchanged PPH for the adsorption of benzothiophene and the separation of propane/propene is the main application as adsorbent. The hydrotreating of aromatic hydrocarbons using ruthenium-impregnated catalysts via hydrogenation and hydrogenolysis/hydrocracking for the production of clean diesel fuels, the selective catalytic reduction of NO from stationary and mobile sources by using Cu-PPH with 1, 3 and 7 wt% of Cu and the selective oxidation of hydrogen sulphide to sulphur with vanadium-containing PPH are the three catalytic reactions of environmental interest studied.

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.

Two novel DNA variants associated with glucose-6-phosphate dehydrogenase deficiency found in Argentine pediatric patients.

PubMed

Chaves, Alejandro; Eberle, Silvia Eandi; Defelipe, Lucas; Pepe, Carolina; Milanesio, Berenice; Aguirre, Fernando; Fernandez, Diego; Turjanski, Adrian; Feliú-Torres, Aurora

2016-07-01

The enzyme glucose-6-phosphate dehydrogenase (G6PD) catalyses the first step in the pentose phosphate pathway, producing nicotinamide adenine dinucleotide phosphate (NADPH). NADPH plays a crucial role in preventing oxidative damage to proteins and other molecules in cells, mostly red blood cells. G6PD deficiency has an x-linked pattern of inheritance in which hemizygous males are deficient, while females may or may not be deficient depending on the number of affected alleles. We report two novel DNA variants in the G6PD gene detected in two male probands with chronic nonspherocytic hemolytic anemia (CNSHA), who were referred for hematological evaluation. Probands and their relatives underwent clinical, biochemical, and molecular assessment. Two novel DNA variants, c.995C>T and c.1226C>A, were found in this study. At the protein level, they produce the substitution of Ser332Phe and Pro409Gln, respectively. These DNA variants were analyzed in the female relatives of probands for genetic counseling. The novel DNA variants were classified as class I based on the clinical, biochemical, and molecular evaluations performed. Copyright © 2016 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

A nucleobase-centered coarse-grained representation for structure prediction of RNA motifs

PubMed Central

Poblete, Simón; Bottaro, Sandro; Bussi, Giovanni

2018-01-01

Abstract We introduce the SPlit-and-conQueR (SPQR) model, a coarse-grained (CG) representation of RNA designed for structure prediction and refinement. In our approach, the representation of a nucleotide consists of a point particle for the phosphate group and an anisotropic particle for the nucleoside. The interactions are, in principle, knowledge-based potentials inspired by the \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{upgreek} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} }{}$\\mathcal {E}$\\end{document}SCORE function, a base-centered scoring function. However, a special treatment is given to base-pairing interactions and certain geometrical conformations which are lost in a raw knowledge-based model. This results in a representation able to describe planar canonical and non-canonical base pairs and base–phosphate interactions and to distinguish sugar puckers and glycosidic torsion conformations. The model is applied to the folding of several structures, including duplexes with internal loops of non-canonical base pairs, tetraloops, junctions and a pseudoknot. For the majority of these systems, experimental structures are correctly predicted at the level of individual contacts. We also propose a method for efficiently reintroducing atomistic detail from the CG representation. PMID:29272539

Keratinocyte-driven contraction of reconstructed human skin.

PubMed

Chakrabarty, K H; Heaton, M; Dalley, A J; Dawson, R A; Freedlander, E; Khaw, P T; Mac Neil, S

2001-01-01

We have previously reported that reconstructed human skin, using deepidermized acellular sterilized dermis and allogeneic keratinocytes and fibroblasts, significantly contracts in vitro. Contracture of split skin grafts in burns injuries remains a serious problem and this in vitro model provides an opportunity to study keratinocyte/mesenchymal cell interactions and cell interactions with extracted normal human dermis. The aim of this study was to investigate the nature of this in vitro contraction and explore several approaches to prevent or reduce contraction. Three different methodologies for sterilization of the dermal matrix were examined: glycerol, ethylene oxide and a combination of glycerol and ethylene oxide. While the nature of the sterilization technique influenced the extent of contraction and thinner dermal matrices contracted proportionately more than thicker matrices, in all cases contraction was driven by the keratinocytes with relatively little influence from the fibroblasts. The contraction of the underlying dermis did not represent any change in tissue mass but rather a reorganization of the dermis which was rapidly reversed (within minutes) when the epidermal layer was removed. Pharmacological approaches to block contraction showed forskolin and mannose-6-phosphate to be ineffective and ascorbic acid-2-phosphate to exacerbate contraction. However, Galardin, a matrix metalloproteinase inhibitor and keratinocyte conditioned media, both inhibited contraction.

Automated Gravimetric Calibration to Optimize the Accuracy and Precision of TECAN Freedom EVO Liquid Handler

PubMed Central

Bessemans, Laurent; Jully, Vanessa; de Raikem, Caroline; Albanese, Mathieu; Moniotte, Nicolas; Silversmet, Pascal; Lemoine, Dominique

2016-01-01

High-throughput screening technologies are increasingly integrated into the formulation development process of biopharmaceuticals. The performance of liquid handling systems is dependent on the ability to deliver accurate and precise volumes of specific reagents to ensure process quality. We have developed an automated gravimetric calibration procedure to adjust the accuracy and evaluate the precision of the TECAN Freedom EVO liquid handling system. Volumes from 3 to 900 µL using calibrated syringes and fixed tips were evaluated with various solutions, including aluminum hydroxide and phosphate adjuvants, β-casein, sucrose, sodium chloride, and phosphate-buffered saline. The methodology to set up liquid class pipetting parameters for each solution was to split the process in three steps: (1) screening of predefined liquid class, including different pipetting parameters; (2) adjustment of accuracy parameters based on a calibration curve; and (3) confirmation of the adjustment. The run of appropriate pipetting scripts, data acquisition, and reports until the creation of a new liquid class in EVOware was fully automated. The calibration and confirmation of the robotic system was simple, efficient, and precise and could accelerate data acquisition for a wide range of biopharmaceutical applications. PMID:26905719

Accumulation of sphingoid bases and sphingoid base 1-phosphates: A possible mechanism for Fusarium verticillioides corn-seedling disease

USDA-ARS?s Scientific Manuscript database

Sphingolipids are important structural components of membranes involved in signaling pathways that regulate cell growth and death. Fumonisins (FB) are water soluble mycotoxins produced by F. verticillioides, which is parasitic to corn. FBs are inhibitors of ceramide synthase (CS), a key enzyme in sp...

Metal insertion into NiFe-hydrogenases.

PubMed

Blokesch, M; Paschos, A; Theodoratou, E; Bauer, A; Hube, M; Huth, S; Böck, A

2002-08-01

The synthesis and the insertion of the metallocentre of NiFe-hydrogenases is a complex process, in which seven maturation enzymes plus ATP, GTP and carbamoyl phosphate are involved. The review summarizes what is known about the properties and activities of these auxiliary proteins, and postulates a pathway along which maturation may take place.

Saccharomyces cerevisiae engineered for xylose metabolism requires gluconeogenesis and the oxidative branch of the pentose phosphate pathway for aerobic xylose assimilation

USDA-ARS?s Scientific Manuscript database

Saccharomyces strains engineered to ferment xylose using Scheffersomyces stipitis xylose reductase (XR) and xylitol dehydrogenase (XDH) genes appear to be limited by metabolic imbalances due to differing cofactor specificities of XR and XDH. The S. stipitis XR, which uses nicotinamide adenine dinucl...

Fission Yeast Model Study for Dissection of TSC Pathway

DTIC Science & Technology

2010-04-01

prepared as follows. A total of 1010 cells were incubated at 37! for 1 hr in spheroplasts buffer [50 mm citrate–phosphate (pH 5.6) and 1.2 m sorbitol ...potassium acetate, and 0.1 m sorbitol ] containing 0.4 mm phenylmethyl- sulfonyl fluoride and 13 protease inhibitor cocktail (Nacalai Tesque) and downed

Identification of Arabidopsis GPAT9 (At5g60620) as an essential gene involved in Triacylglycerol Biosynthesis

USDA-ARS?s Scientific Manuscript database

The first step in the biosynthesis of nearly all plant membrane phospholipids and storage triacylglycerols is catalyzed by a glycerol-3-phosphate acyltransferase (GPAT). The requirement for an endoplasmic reticulum (ER) localized GPAT for both of these critical metabolic pathways was recognized more...

Interactions between calcium precipitation and the polyphosphate-accumulating bacteria metabolism.

PubMed

Barat, R; Montoya, T; Borrás, L; Ferrer, J; Seco, A

2008-07-01

A sequencing batch reactor that is operated for biological phosphorus removal has been operated under different influent calcium concentrations to study the precipitation process and the possible effects of phosphorus precipitation in the biological phosphorus removal process. Four experiments were carried out under different influent calcium concentrations ranging from 10 to 90 g Ca m(-3). The experimental results and the equilibrium study, which are based on the saturation index calculation, confirm that the process controlling the calcium behaviour is the calcium phosphate precipitation. This precipitation takes place at two stages: initially, precipitation of the amorphous calcium phosphate, and later crystallization of hydroxyapatite. Also the accumulation of phosphorus precipitated was observed when the influent calcium concentration was increased. In all the experiments, the influent wastewater ratio P/COD was kept constant. It has been observed that, at high calcium concentration, the ratio between phosphate release and acetate uptake (P(rel)/Ac(uptake)) decreases. Changes in the polyphosphate-accumulating organism (PAO) population and in the glycogen-accumulating organism (GAO) population during the experimental period were ruled out by means of fluorescence in situ hybridization. These results could suggest that PAO are able to change their metabolic pathways based on external conditions, such as influent calcium concentration. The accumulation of phosphorus precipitated as calcium phosphate at high influent calcium concentration throughout the experimental period confirmed that phosphate precipitation is a process that can affect the PAO metabolism.

Phosphoribosyl diphosphate synthetase-independent NAD de novo synthesis in Escherichia coli: a new phenotype of phosphate regulon mutants.

PubMed Central

Hove-Jensen, B

1996-01-01

Phosphoribosyl diphosphate-lacking (delta prs) mutant strains of Escherichia coli require NAD, guanosine, uridine, histidine, and tryptophan for growth. NAD is required by phosphoribosyl diphosphate-lacking mutants because of lack of one of the substrates for the quinolinate phosphoribosyltransferase reaction, an enzyme of the NAD de novo pathway. Several NAD-independent mutants of a host from which prs had been deleted were isolated; all of them were shown to have lesions in the pstSCAB-phoU operon, in which mutations lead to derepression of the Pho regulon. In addition NAD-independent growth was dependent on a functional quinolinate phosphoribosyltransferase. The prs suppressor mutations led to the synthesis of a new phosphoryl compound that may act as a precursor for a new NAD biosynthetic pathway. This compound may be synthesized by the product of an unknown phosphate starvation-inducible gene of the Pho regulon because the ability of pst or phoU mutations to suppress the NAD requirement requires PhoB, the transcriptional activator of the Pho regulon. PMID:8550505

Further increased production of free fatty acids by overexpressing a predicted transketolase gene of the pentose phosphate pathway in Aspergillus oryzae faaA disruptant.

PubMed

Tamano, Koichi; Miura, Ai

2016-09-01

Free fatty acids are useful as source materials for the production of biodiesel fuel and various chemicals such as pharmaceuticals and dietary supplements. Previously, we attained a 9.2-fold increase in free fatty acid productivity by disrupting a predicted acyl-CoA synthetase gene (faaA, AO090011000642) in Aspergillus oryzae. In this study, we achieved further increase in the productivity by overexpressing a predicted transketolase gene of the pentose phosphate pathway in the faaA disruptant. The A. oryzae genome is predicted to have three transketolase genes and overexpression of AO090023000345, one of the three genes, resulted in phenotypic change and further increase (corresponding to an increased production of 0.38 mmol/g dry cell weight) in free fatty acids at 1.4-fold compared to the faaA disruptant. Additionally, the biomass of hyphae increased at 1.2-fold by the overexpression. As a result, free fatty acid production yield per liter of liquid culture increased at 1.7-fold by the overexpression.