pck catalyzed reaction  (Millipore)


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    Structured Review

    Millipore pck catalyzed reaction
    Activities of <t>Pck</t> mutants. (A) Dependence of the gluconeogenic reaction velocities of Pck mutants on GTP concentration. (B) Dependence of the anaplerotic reaction velocities of Pck mutants on GDP. The assays were performed as described in Materials and Methods. The concentrations of individual components were as follows: 2 mM PEP and 2 U/ml MDH for the anaplerotic reaction; 0.3 mM <t>OAA,</t> 10 U/ml LDH, and 3 U/ml PK for the gluconeogenic reaction.
    Pck Catalyzed Reaction, supplied by Millipore, used in various techniques. Bioz Stars score: 89/100, based on 980 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 89 stars, based on 980 article reviews
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    pck catalyzed reaction - by Bioz Stars, 2020-09
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    Images

    1) Product Images from "Structural and Functional Studies of Phosphoenolpyruvate Carboxykinase from Mycobacterium tuberculosis"

    Article Title: Structural and Functional Studies of Phosphoenolpyruvate Carboxykinase from Mycobacterium tuberculosis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0120682

    Activities of Pck mutants. (A) Dependence of the gluconeogenic reaction velocities of Pck mutants on GTP concentration. (B) Dependence of the anaplerotic reaction velocities of Pck mutants on GDP. The assays were performed as described in Materials and Methods. The concentrations of individual components were as follows: 2 mM PEP and 2 U/ml MDH for the anaplerotic reaction; 0.3 mM OAA, 10 U/ml LDH, and 3 U/ml PK for the gluconeogenic reaction.
    Figure Legend Snippet: Activities of Pck mutants. (A) Dependence of the gluconeogenic reaction velocities of Pck mutants on GTP concentration. (B) Dependence of the anaplerotic reaction velocities of Pck mutants on GDP. The assays were performed as described in Materials and Methods. The concentrations of individual components were as follows: 2 mM PEP and 2 U/ml MDH for the anaplerotic reaction; 0.3 mM OAA, 10 U/ml LDH, and 3 U/ml PK for the gluconeogenic reaction.

    Techniques Used: Concentration Assay

    2) Product Images from "Crystal structure of a Pseudomonas malonate decarboxylase holoenzyme hetero-tetramer"

    Article Title: Crystal structure of a Pseudomonas malonate decarboxylase holoenzyme hetero-tetramer

    Journal: Nature Communications

    doi: 10.1038/s41467-017-00233-z

    Interactions among the subunits in the MDC hetero-tetramer. a Interactions between MdcA (domain AC, orange ) and MdcC ( light blue ) in MDC. b Interactions between MdcC ( light blue ) and MdcE ( green ) in MDC. c Interactions between MdcA (domain AC, orange ) and MdcE ( green ) in MDC. MdcD ( cyan ) makes a small contribution to this interface. d Gel filtration profiles showing that the MDC hetero-tetramer can be formed by mixing its subunits. MdcACDE: the hetero-tetramer purified from co-expressing the four subunits; MdcA+C+DE: mixture of purified MdcA, MdcC, and MdcD–MdcE subunits. e Gel filtration profiles showing that the sub-complexes of MDC are not stable, with the exception of MdcD–MdcE. f Nickel pull-down experiments showing the effects of mutations in the MDC interface on hetero-tetramer formation. Only MdcE carries a His tag. The deletion mutations abolished the complex, as no MdcA nor MdcC was pulled down, while most of the single-site mutations had essentially no effect
    Figure Legend Snippet: Interactions among the subunits in the MDC hetero-tetramer. a Interactions between MdcA (domain AC, orange ) and MdcC ( light blue ) in MDC. b Interactions between MdcC ( light blue ) and MdcE ( green ) in MDC. c Interactions between MdcA (domain AC, orange ) and MdcE ( green ) in MDC. MdcD ( cyan ) makes a small contribution to this interface. d Gel filtration profiles showing that the MDC hetero-tetramer can be formed by mixing its subunits. MdcACDE: the hetero-tetramer purified from co-expressing the four subunits; MdcA+C+DE: mixture of purified MdcA, MdcC, and MdcD–MdcE subunits. e Gel filtration profiles showing that the sub-complexes of MDC are not stable, with the exception of MdcD–MdcE. f Nickel pull-down experiments showing the effects of mutations in the MDC interface on hetero-tetramer formation. Only MdcE carries a His tag. The deletion mutations abolished the complex, as no MdcA nor MdcC was pulled down, while most of the single-site mutations had essentially no effect

    Techniques Used: Filtration, Purification, Expressing

    Structure of the MdcD–MdcE complex. a Schematic drawing of the structure of the P. aeruginosa MdcD–MdcE complex. MdcD is shown in cyan , and MdcE in green . The secondary structure elements are named following the convention of yeast ACC CT domain. The active site is indicated with the red asterisk . b Overlay of the structures of MdcD ( cyan ) and MdcE ( green ). The β-strands in the β–β–α superhelix are mostly superposed. All structure figures were produced with the program PyMOL ( www.pymol.org )
    Figure Legend Snippet: Structure of the MdcD–MdcE complex. a Schematic drawing of the structure of the P. aeruginosa MdcD–MdcE complex. MdcD is shown in cyan , and MdcE in green . The secondary structure elements are named following the convention of yeast ACC CT domain. The active site is indicated with the red asterisk . b Overlay of the structures of MdcD ( cyan ) and MdcE ( green ). The β-strands in the β–β–α superhelix are mostly superposed. All structure figures were produced with the program PyMOL ( www.pymol.org )

    Techniques Used: Produced

    Impact of mutations in MDC holoenzyme subunits on growth with malonate as the sole carbon source. Individual deletion of genes for the MdcA, MdcC, or MdcE subunit in P. aeruginosa abolished growth on malonate. Insertion of the coding sequence for the MdcC subunit at the mdcC deletion site restored growth. Deletion of MdcA residues 519–554 (required for MDC hetero-tetramer formation) and the R314E mutation in MdcA (involved in recognition of malonate in the active site) also abolished growth. Finally, Escherichia coli , which is not able to grow on malonate as a sole carbon source, served as a negative control. Attempts at deleting the MdcD subunit were not successful. Results from one representative experiment from three biological replicates are shown
    Figure Legend Snippet: Impact of mutations in MDC holoenzyme subunits on growth with malonate as the sole carbon source. Individual deletion of genes for the MdcA, MdcC, or MdcE subunit in P. aeruginosa abolished growth on malonate. Insertion of the coding sequence for the MdcC subunit at the mdcC deletion site restored growth. Deletion of MdcA residues 519–554 (required for MDC hetero-tetramer formation) and the R314E mutation in MdcA (involved in recognition of malonate in the active site) also abolished growth. Finally, Escherichia coli , which is not able to grow on malonate as a sole carbon source, served as a negative control. Attempts at deleting the MdcD subunit were not successful. Results from one representative experiment from three biological replicates are shown

    Techniques Used: Sequencing, Mutagenesis, Negative Control

    Structure of the MDC hetero-tetramer. a Schematic drawing of the structure of the MDC hetero-tetramer. The two domains of MdcA, AN and AC, are shown in yellow and orange , respectively. MdcC is shown in light blue , MdcD in cyan , and MdcE in green . The side chain of Ser25 in MdcC, the site of attachment of the prosthetic group, is shown as a stick model. A malonate bound in the active site of MdcA is shown as a sphere model ( black ) and labeled Mal. The bound position of CoA is also indicated ( gray ). The two active sites are indicated with the red asterisks , and the direct distance between them indicated with the red arrow . b Structure of the MDC hetero-tetramer, after 90° rotation around the vertical axis from a . c Structure of MdcA. d Structure of MdcC. e Molecular surface of MdcC colored based on sequence conservation 50 . Purple : highly conserved, cyan: poorly conserved. The view is related to that of d after a 90° rotation around the vertical axis
    Figure Legend Snippet: Structure of the MDC hetero-tetramer. a Schematic drawing of the structure of the MDC hetero-tetramer. The two domains of MdcA, AN and AC, are shown in yellow and orange , respectively. MdcC is shown in light blue , MdcD in cyan , and MdcE in green . The side chain of Ser25 in MdcC, the site of attachment of the prosthetic group, is shown as a stick model. A malonate bound in the active site of MdcA is shown as a sphere model ( black ) and labeled Mal. The bound position of CoA is also indicated ( gray ). The two active sites are indicated with the red asterisks , and the direct distance between them indicated with the red arrow . b Structure of the MDC hetero-tetramer, after 90° rotation around the vertical axis from a . c Structure of MdcA. d Structure of MdcC. e Molecular surface of MdcC colored based on sequence conservation 50 . Purple : highly conserved, cyan: poorly conserved. The view is related to that of d after a 90° rotation around the vertical axis

    Techniques Used: Labeling, Sequencing

    Reactions catalyzed by the malonate decarboxylase (MDC) system. a Chemical structure of the 2′-(5′′-phosphoribosyl)-3′-dephospho-CoA prosthetic group for the acyl-carrier protein (MdcC) in malonate decarboxylase. b Malonate decarboxylase contains two distinct active sites. MdcA is an acetyl-ACP:malonate ACP transferase and converts free malonate to malonyl-ACP, which is then decarboxylated by MdcD–MdcE. The prosthetic group of the MdcC is indicated with the wavy lines. MdcB, MdcG and MdcH are involved in activating the ACP for catalysis (shown in gray )
    Figure Legend Snippet: Reactions catalyzed by the malonate decarboxylase (MDC) system. a Chemical structure of the 2′-(5′′-phosphoribosyl)-3′-dephospho-CoA prosthetic group for the acyl-carrier protein (MdcC) in malonate decarboxylase. b Malonate decarboxylase contains two distinct active sites. MdcA is an acetyl-ACP:malonate ACP transferase and converts free malonate to malonyl-ACP, which is then decarboxylated by MdcD–MdcE. The prosthetic group of the MdcC is indicated with the wavy lines. MdcB, MdcG and MdcH are involved in activating the ACP for catalysis (shown in gray )

    Techniques Used:

    The active site of MdcD–MdcE. a Simulated-annealing omit F o − F c electron density map at 3.0 Å resolution for CoA in the active site of MdcD–MdcE, contoured at 3 σ . b Residues in the active site of MdcD–MdcE. Interactions between CoA ( gray ) and MdcD–MdcE are shown. Hydrogen-bonding interactions are indicated with dashed lines ( red ). The possible binding site for the malonyl group is indicated with the red arrow. c MdcD–MdcE demonstrates cooperative behavior towards malonyl-CoA. The initial velocity data are fitted to the Hill equation to obtain the kinetic parameters ( blue curve ). The Michael–Menten equation does not produce as good a fit to the data ( red curve ). Data from one representative set of experiments are shown
    Figure Legend Snippet: The active site of MdcD–MdcE. a Simulated-annealing omit F o − F c electron density map at 3.0 Å resolution for CoA in the active site of MdcD–MdcE, contoured at 3 σ . b Residues in the active site of MdcD–MdcE. Interactions between CoA ( gray ) and MdcD–MdcE are shown. Hydrogen-bonding interactions are indicated with dashed lines ( red ). The possible binding site for the malonyl group is indicated with the red arrow. c MdcD–MdcE demonstrates cooperative behavior towards malonyl-CoA. The initial velocity data are fitted to the Hill equation to obtain the kinetic parameters ( blue curve ). The Michael–Menten equation does not produce as good a fit to the data ( red curve ). Data from one representative set of experiments are shown

    Techniques Used: Binding Assay

    3) Product Images from "Characterization of Pseudomonas aeruginosa Exoenzyme S as a Bifunctional Enzyme in J774A.1 Macrophages "

    Article Title: Characterization of Pseudomonas aeruginosa Exoenzyme S as a Bifunctional Enzyme in J774A.1 Macrophages

    Journal: Infection and Immunity

    doi: 10.1128/IAI.71.9.5296-5305.2003

    Analysis of J774A.1 cell morphology by SEM. J774A.1 cells were grown on Thermanox coverslips and cocultured for 2.5 h with the indicated ExoS-expressing PA103ΔUT strain. Cells were fixed with 2% cacodylate glutaraldehyde, followed by 2% osmium tetroxide. Samples were rinsed, dehydrated with ethanol, and then incubated with hexamethyldisilazane until dry, mounted, sputter coated with gold palladium, and examined with a JEOL 5410 scanning electron microscope. Representative pictures of the predominant phenotype associated with each mutant are shown. Control cells (0) showed normal macrophage morphology. Cells cocultured with PA103ΔUT expressing ExoS showed decreased lamellipodia and membrane ruffling (70.5%; 12 of 17 cells) and decreased filopodia (58.8%; 10 of 17 cells). Cells cocultured with the R146A-GAP mutant showed enhanced lamellipodia and ruffles (100%; 10 of 10 cells) and no filopodia (70%; 7 of 10 cells). Cells cocultured with the E379A/E381A-ADPRT mutant showed no lamellipodia and ruffles (60%; 6 of 10 cells) and restored filopodia (70%; 7 of 10 cells). Cells cocultured with the R146A/E379A/E381A-GAP/ADPRT mutant showed restored lamellipodia, membrane ruffles, and filopodia (100%; nine of nine cells). Arrowheads identify lamellipodia, and arrows identify filopodia. Bar, 5 μm.
    Figure Legend Snippet: Analysis of J774A.1 cell morphology by SEM. J774A.1 cells were grown on Thermanox coverslips and cocultured for 2.5 h with the indicated ExoS-expressing PA103ΔUT strain. Cells were fixed with 2% cacodylate glutaraldehyde, followed by 2% osmium tetroxide. Samples were rinsed, dehydrated with ethanol, and then incubated with hexamethyldisilazane until dry, mounted, sputter coated with gold palladium, and examined with a JEOL 5410 scanning electron microscope. Representative pictures of the predominant phenotype associated with each mutant are shown. Control cells (0) showed normal macrophage morphology. Cells cocultured with PA103ΔUT expressing ExoS showed decreased lamellipodia and membrane ruffling (70.5%; 12 of 17 cells) and decreased filopodia (58.8%; 10 of 17 cells). Cells cocultured with the R146A-GAP mutant showed enhanced lamellipodia and ruffles (100%; 10 of 10 cells) and no filopodia (70%; 7 of 10 cells). Cells cocultured with the E379A/E381A-ADPRT mutant showed no lamellipodia and ruffles (60%; 6 of 10 cells) and restored filopodia (70%; 7 of 10 cells). Cells cocultured with the R146A/E379A/E381A-GAP/ADPRT mutant showed restored lamellipodia, membrane ruffles, and filopodia (100%; nine of nine cells). Arrowheads identify lamellipodia, and arrows identify filopodia. Bar, 5 μm.

    Techniques Used: Expressing, Incubation, Microscopy, Mutagenesis

    4) Product Images from "Genetically encoded fluorescent indicator for imaging NAD+/NADH ratio changes in different cellular compartments"

    Article Title: Genetically encoded fluorescent indicator for imaging NAD+/NADH ratio changes in different cellular compartments

    Journal: Biochimica et biophysica acta

    doi: 10.1016/j.bbagen.2013.11.018

    A) Diagram of the RexYFP structure. RexYFP consists of cpYFP (yellow) integrated between residues 79 and 80 of T-Rex (blue) via short polypeptide linkers SAG and GT (red). The diagram shows mutations in the structure of RexYFP (numbers in parentheses indicate the position for EYFP). B) Fluorescence spectra of RexYFP. Excitation spectrum has a maximum at 490 nm. Emission spectrum has a maximum at 516 nm. C) Excitation spectrum of RexYFP (250 nM) in Tris–HCl (pH 7.5) with 150 mM NaCl and 10 mM MgCl 2 upon addition of NADH (50, 250, 1000 nM) to the probe. Emission was measured at 530 nm. D) Dependence of RexYFP signal on concentrations of various nucleotides (NAD + , NADH, NADPH, ATP) in range of concentration from 10 nM to 50 μM in the probe (Tris–HCl (pH 7.5), 150 mM NaCl, 10 mM MgCl 2 ). The RexYFP signal is expressed as 1/F490. Plotted line for each type of nucleotide is the result of five independent experiments.
    Figure Legend Snippet: A) Diagram of the RexYFP structure. RexYFP consists of cpYFP (yellow) integrated between residues 79 and 80 of T-Rex (blue) via short polypeptide linkers SAG and GT (red). The diagram shows mutations in the structure of RexYFP (numbers in parentheses indicate the position for EYFP). B) Fluorescence spectra of RexYFP. Excitation spectrum has a maximum at 490 nm. Emission spectrum has a maximum at 516 nm. C) Excitation spectrum of RexYFP (250 nM) in Tris–HCl (pH 7.5) with 150 mM NaCl and 10 mM MgCl 2 upon addition of NADH (50, 250, 1000 nM) to the probe. Emission was measured at 530 nm. D) Dependence of RexYFP signal on concentrations of various nucleotides (NAD + , NADH, NADPH, ATP) in range of concentration from 10 nM to 50 μM in the probe (Tris–HCl (pH 7.5), 150 mM NaCl, 10 mM MgCl 2 ). The RexYFP signal is expressed as 1/F490. Plotted line for each type of nucleotide is the result of five independent experiments.

    Techniques Used: Fluorescence, Concentration Assay

    5) Product Images from "Decreased NAA in Gray Matter is Correlated with Decreased Availability of Acetate in White Matter in Postmortem Multiple Sclerosis Cortex"

    Article Title: Decreased NAA in Gray Matter is Correlated with Decreased Availability of Acetate in White Matter in Postmortem Multiple Sclerosis Cortex

    Journal: Neurochemical research

    doi: 10.1007/s11064-013-1151-8

    Treatment with the electron transport chain inhibitor antimycin A reduced NAA levels in SH-SY5Y cells without inducing neuronal cell loss or degeneration of neurites. a Representative HPLC chromatogram showing NAA peak retention time of 5.10 min with
    Figure Legend Snippet: Treatment with the electron transport chain inhibitor antimycin A reduced NAA levels in SH-SY5Y cells without inducing neuronal cell loss or degeneration of neurites. a Representative HPLC chromatogram showing NAA peak retention time of 5.10 min with

    Techniques Used: High Performance Liquid Chromatography

    The effect of antimycin A on respiration and levels of the NAA substrates, l -aspartate and acetyl-CoA, was determined in SH-SY5Y cells. OCR and ECAR were measured simultaneously in SH-SY5Y neuroblastoma cells before and after antimycin A treatment. a
    Figure Legend Snippet: The effect of antimycin A on respiration and levels of the NAA substrates, l -aspartate and acetyl-CoA, was determined in SH-SY5Y cells. OCR and ECAR were measured simultaneously in SH-SY5Y neuroblastoma cells before and after antimycin A treatment. a

    Techniques Used:

    6) Product Images from "Methanol fermentation increases the production of NAD(P)H-dependent chemicals in synthetic methylotrophic Escherichia coli"

    Article Title: Methanol fermentation increases the production of NAD(P)H-dependent chemicals in synthetic methylotrophic Escherichia coli

    Journal: Biotechnology for Biofuels

    doi: 10.1186/s13068-019-1356-4

    Methanol bioconversion for improved lysine synthesis in synthetic methylotrophic E. coli . Enzymes required for the assimilation of methanol into central metabolism are shown in blue: MDH methanol dehydrogenase, HPS 3-hexulose-6-phosphate synthase, and PHI 6-phospho-3-hexuloisomerase. The genes overexpressed in the lysine biosynthetic pathway are shown in green. The cofactor generation pathway was reconstructed by expressing POS5 from S. cerevisiae to convert extra NADH and generate NADPH, which is shown in red
    Figure Legend Snippet: Methanol bioconversion for improved lysine synthesis in synthetic methylotrophic E. coli . Enzymes required for the assimilation of methanol into central metabolism are shown in blue: MDH methanol dehydrogenase, HPS 3-hexulose-6-phosphate synthase, and PHI 6-phospho-3-hexuloisomerase. The genes overexpressed in the lysine biosynthetic pathway are shown in green. The cofactor generation pathway was reconstructed by expressing POS5 from S. cerevisiae to convert extra NADH and generate NADPH, which is shown in red

    Techniques Used: Expressing

    Improved lysine production in synthetic methylotrophic E. coli . a Methylotrophic E. coli was engineered for the bioconversion of methanol to improve lysine production. The extra NADH from methanol consumption was engineered to generate NADPH for lysine production. b Lysine production in the strains BL21/ΔfrmA-ML and BL21/ΔfrmA-ML-POS5 cultivated with and without methanol. c Methanol consumption in the strains BL21/ΔfrmA-ML and BL21/ΔfrmA-ML-POS5. d The intracellular NADH pools in the strains BL21/ΔfrmA-ML and BL21/ΔfrmA-ML-POS5 cultivated with and without methanol. e The intracellular NADPH pools in the strains BL21/ΔfrmA-ML-POS5 cultivated with 55 mM glucose and 50 mM methanol. f Lysine production from 13 C-methanol in the strains BL21/ΔfrmA-Mdh2-Hps-Phi, BL21/ΔfrmA-ML, and BL21/ΔfrmA-ML-POS5. Error bars indicate standard error of the mean ( n = 3)
    Figure Legend Snippet: Improved lysine production in synthetic methylotrophic E. coli . a Methylotrophic E. coli was engineered for the bioconversion of methanol to improve lysine production. The extra NADH from methanol consumption was engineered to generate NADPH for lysine production. b Lysine production in the strains BL21/ΔfrmA-ML and BL21/ΔfrmA-ML-POS5 cultivated with and without methanol. c Methanol consumption in the strains BL21/ΔfrmA-ML and BL21/ΔfrmA-ML-POS5. d The intracellular NADH pools in the strains BL21/ΔfrmA-ML and BL21/ΔfrmA-ML-POS5 cultivated with and without methanol. e The intracellular NADPH pools in the strains BL21/ΔfrmA-ML-POS5 cultivated with 55 mM glucose and 50 mM methanol. f Lysine production from 13 C-methanol in the strains BL21/ΔfrmA-Mdh2-Hps-Phi, BL21/ΔfrmA-ML, and BL21/ΔfrmA-ML-POS5. Error bars indicate standard error of the mean ( n = 3)

    Techniques Used:

    7) Product Images from "Methanol fermentation increases the production of NAD(P)H-dependent chemicals in synthetic methylotrophic Escherichia coli"

    Article Title: Methanol fermentation increases the production of NAD(P)H-dependent chemicals in synthetic methylotrophic Escherichia coli

    Journal: Biotechnology for Biofuels

    doi: 10.1186/s13068-019-1356-4

    Methanol bioconversion for improved lysine synthesis in synthetic methylotrophic E. coli . Enzymes required for the assimilation of methanol into central metabolism are shown in blue: MDH methanol dehydrogenase, HPS 3-hexulose-6-phosphate synthase, and PHI 6-phospho-3-hexuloisomerase. The genes overexpressed in the lysine biosynthetic pathway are shown in green. The cofactor generation pathway was reconstructed by expressing POS5 from S. cerevisiae to convert extra NADH and generate NADPH, which is shown in red
    Figure Legend Snippet: Methanol bioconversion for improved lysine synthesis in synthetic methylotrophic E. coli . Enzymes required for the assimilation of methanol into central metabolism are shown in blue: MDH methanol dehydrogenase, HPS 3-hexulose-6-phosphate synthase, and PHI 6-phospho-3-hexuloisomerase. The genes overexpressed in the lysine biosynthetic pathway are shown in green. The cofactor generation pathway was reconstructed by expressing POS5 from S. cerevisiae to convert extra NADH and generate NADPH, which is shown in red

    Techniques Used: Expressing

    Improved lysine production in synthetic methylotrophic E. coli . a Methylotrophic E. coli was engineered for the bioconversion of methanol to improve lysine production. The extra NADH from methanol consumption was engineered to generate NADPH for lysine production. b Lysine production in the strains BL21/ΔfrmA-ML and BL21/ΔfrmA-ML-POS5 cultivated with and without methanol. c Methanol consumption in the strains BL21/ΔfrmA-ML and BL21/ΔfrmA-ML-POS5. d The intracellular NADH pools in the strains BL21/ΔfrmA-ML and BL21/ΔfrmA-ML-POS5 cultivated with and without methanol. e The intracellular NADPH pools in the strains BL21/ΔfrmA-ML-POS5 cultivated with 55 mM glucose and 50 mM methanol. f Lysine production from 13 C-methanol in the strains BL21/ΔfrmA-Mdh2-Hps-Phi, BL21/ΔfrmA-ML, and BL21/ΔfrmA-ML-POS5. Error bars indicate standard error of the mean ( n = 3)
    Figure Legend Snippet: Improved lysine production in synthetic methylotrophic E. coli . a Methylotrophic E. coli was engineered for the bioconversion of methanol to improve lysine production. The extra NADH from methanol consumption was engineered to generate NADPH for lysine production. b Lysine production in the strains BL21/ΔfrmA-ML and BL21/ΔfrmA-ML-POS5 cultivated with and without methanol. c Methanol consumption in the strains BL21/ΔfrmA-ML and BL21/ΔfrmA-ML-POS5. d The intracellular NADH pools in the strains BL21/ΔfrmA-ML and BL21/ΔfrmA-ML-POS5 cultivated with and without methanol. e The intracellular NADPH pools in the strains BL21/ΔfrmA-ML-POS5 cultivated with 55 mM glucose and 50 mM methanol. f Lysine production from 13 C-methanol in the strains BL21/ΔfrmA-Mdh2-Hps-Phi, BL21/ΔfrmA-ML, and BL21/ΔfrmA-ML-POS5. Error bars indicate standard error of the mean ( n = 3)

    Techniques Used:

    8) Product Images from "Methanol fermentation increases the production of NAD(P)H-dependent chemicals in synthetic methylotrophic Escherichia coli"

    Article Title: Methanol fermentation increases the production of NAD(P)H-dependent chemicals in synthetic methylotrophic Escherichia coli

    Journal: Biotechnology for Biofuels

    doi: 10.1186/s13068-019-1356-4

    Improved lysine production in synthetic methylotrophic E. coli . a Methylotrophic E. coli was engineered for the bioconversion of methanol to improve lysine production. The extra NADH from methanol consumption was engineered to generate NADPH for lysine production. b Lysine production in the strains BL21/ΔfrmA-ML and BL21/ΔfrmA-ML-POS5 cultivated with and without methanol. c Methanol consumption in the strains BL21/ΔfrmA-ML and BL21/ΔfrmA-ML-POS5. d The intracellular NADH pools in the strains BL21/ΔfrmA-ML and BL21/ΔfrmA-ML-POS5 cultivated with and without methanol. e The intracellular NADPH pools in the strains BL21/ΔfrmA-ML-POS5 cultivated with 55 mM glucose and 50 mM methanol. f Lysine production from 13 C-methanol in the strains BL21/ΔfrmA-Mdh2-Hps-Phi, BL21/ΔfrmA-ML, and BL21/ΔfrmA-ML-POS5. Error bars indicate standard error of the mean ( n = 3)
    Figure Legend Snippet: Improved lysine production in synthetic methylotrophic E. coli . a Methylotrophic E. coli was engineered for the bioconversion of methanol to improve lysine production. The extra NADH from methanol consumption was engineered to generate NADPH for lysine production. b Lysine production in the strains BL21/ΔfrmA-ML and BL21/ΔfrmA-ML-POS5 cultivated with and without methanol. c Methanol consumption in the strains BL21/ΔfrmA-ML and BL21/ΔfrmA-ML-POS5. d The intracellular NADH pools in the strains BL21/ΔfrmA-ML and BL21/ΔfrmA-ML-POS5 cultivated with and without methanol. e The intracellular NADPH pools in the strains BL21/ΔfrmA-ML-POS5 cultivated with 55 mM glucose and 50 mM methanol. f Lysine production from 13 C-methanol in the strains BL21/ΔfrmA-Mdh2-Hps-Phi, BL21/ΔfrmA-ML, and BL21/ΔfrmA-ML-POS5. Error bars indicate standard error of the mean ( n = 3)

    Techniques Used:

    Methanol bioconversion for improved lysine synthesis in synthetic methylotrophic E. coli . Enzymes required for the assimilation of methanol into central metabolism are shown in blue: MDH methanol dehydrogenase, HPS 3-hexulose-6-phosphate synthase, and PHI 6-phospho-3-hexuloisomerase. The genes overexpressed in the lysine biosynthetic pathway are shown in green. The cofactor generation pathway was reconstructed by expressing POS5 from S. cerevisiae to convert extra NADH and generate NADPH, which is shown in red
    Figure Legend Snippet: Methanol bioconversion for improved lysine synthesis in synthetic methylotrophic E. coli . Enzymes required for the assimilation of methanol into central metabolism are shown in blue: MDH methanol dehydrogenase, HPS 3-hexulose-6-phosphate synthase, and PHI 6-phospho-3-hexuloisomerase. The genes overexpressed in the lysine biosynthetic pathway are shown in green. The cofactor generation pathway was reconstructed by expressing POS5 from S. cerevisiae to convert extra NADH and generate NADPH, which is shown in red

    Techniques Used: Expressing

    9) Product Images from "Novel Listerial Glycerol Dehydrogenase- and Phosphoenolpyruvate-Dependent Dihydroxyacetone Kinase System Connected to the Pentose Phosphate Pathway"

    Article Title: Novel Listerial Glycerol Dehydrogenase- and Phosphoenolpyruvate-Dependent Dihydroxyacetone Kinase System Connected to the Pentose Phosphate Pathway

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00801-12

    Spectrophotometric assays of Dha phosphorylation. Spectrophotometric assays were carried out at room temperature by monitoring the decrease in the amount of NADH at an A 340 over a time period of 60 min by using a Kontron Bio-Tek spectrophotometer. All samples consisted of 700-μl reaction mixtures containing 75 mM Tris-HCl (pH 7.4), 6 mM PEP, 1 mM NADH, 6 mM Dha, 10 mM MgCl 2 , and 1 U of glycerol-3-P dehydrogenase. The following five proteins were assumed to be required for Dha phosphorylation: EI, HPr, DhaK-2, DhaL-2, and DhaM-2 (EIIA Dha ). Samples lacking any one of the above-mentioned five proteins were prepared in duplicates and preincubated for 10 min before the lacking protein was added to one of the two samples. The decrease of the A 340 was monitored for both samples. All complemented samples exhibited a more or less rapid decrease of the A 340 . For the control assays, we show only the sample lacking DhaK-2 (−DhaK-2), because a similar straight line was obtained for all other “noncomplemented” samples. These results confirm that all five bacterial proteins are necessary for Dha phosphorylation.
    Figure Legend Snippet: Spectrophotometric assays of Dha phosphorylation. Spectrophotometric assays were carried out at room temperature by monitoring the decrease in the amount of NADH at an A 340 over a time period of 60 min by using a Kontron Bio-Tek spectrophotometer. All samples consisted of 700-μl reaction mixtures containing 75 mM Tris-HCl (pH 7.4), 6 mM PEP, 1 mM NADH, 6 mM Dha, 10 mM MgCl 2 , and 1 U of glycerol-3-P dehydrogenase. The following five proteins were assumed to be required for Dha phosphorylation: EI, HPr, DhaK-2, DhaL-2, and DhaM-2 (EIIA Dha ). Samples lacking any one of the above-mentioned five proteins were prepared in duplicates and preincubated for 10 min before the lacking protein was added to one of the two samples. The decrease of the A 340 was monitored for both samples. All complemented samples exhibited a more or less rapid decrease of the A 340 . For the control assays, we show only the sample lacking DhaK-2 (−DhaK-2), because a similar straight line was obtained for all other “noncomplemented” samples. These results confirm that all five bacterial proteins are necessary for Dha phosphorylation.

    Techniques Used: Spectrophotometry

    10) Product Images from "Homozygous NMNAT2 mutation in sisters with polyneuropathy and erythromelalgia"

    Article Title: Homozygous NMNAT2 mutation in sisters with polyneuropathy and erythromelalgia

    Journal: bioRxiv

    doi: 10.1101/610907

    Temperature studies of WT and T94M NMNAT2. Data represent mean ± SEM of n experiments as indicated. (A) Apo-enzymes’ stability at different temperatures. Buffered enzyme solutions (40 μg/ml hNMNAT2 WT or 30 μg/ml T94M mutant in 50 mM HEPES/NaOH buffer, pH 7.5, 1 mM TCEP, 20 % glycerol) were pre-incubated for 1 hour at the indicated temperatures, then assayed at 37 °C. Values (n = 6) are referred to the untreated enzyme kept at +4 °C (arbitrary 100%). (B) Apo-enzymes’ stability at 37 °C. Enzyme solutions were incubated and collected at the indicated time intervals, then assayed at 37 °C. Values (n = 4) are referred to that of time zero (arbitrary 100%). T test p values (*) for T94M vs WT: 0.013 at 8 min, 0.010 at 14 min, 0.013 at 20 min, and 0.014 at 28 min. (C) Enzymes’ stability at 37 °C in the presence of substrates. Enzyme solutions supplied with 100 μM both NMN and ATP were treated and assayed as in panel B. (D) Optimal temperature. Enzyme rates were measured after warming the assay mixtures at the indicated temperatures. Values (n = 4) are referred to the relative maximal rate observed for each enzyme (arbitrary 100%). After this assay, the mixtures at 47 °C were rapidly cooled down to 37 °C then new NMNAT2 aliquots were added and rates measured again, demonstrating full recovery of the original activity. This excludes any effect caused by heating on the ancillary enzyme ADH.
    Figure Legend Snippet: Temperature studies of WT and T94M NMNAT2. Data represent mean ± SEM of n experiments as indicated. (A) Apo-enzymes’ stability at different temperatures. Buffered enzyme solutions (40 μg/ml hNMNAT2 WT or 30 μg/ml T94M mutant in 50 mM HEPES/NaOH buffer, pH 7.5, 1 mM TCEP, 20 % glycerol) were pre-incubated for 1 hour at the indicated temperatures, then assayed at 37 °C. Values (n = 6) are referred to the untreated enzyme kept at +4 °C (arbitrary 100%). (B) Apo-enzymes’ stability at 37 °C. Enzyme solutions were incubated and collected at the indicated time intervals, then assayed at 37 °C. Values (n = 4) are referred to that of time zero (arbitrary 100%). T test p values (*) for T94M vs WT: 0.013 at 8 min, 0.010 at 14 min, 0.013 at 20 min, and 0.014 at 28 min. (C) Enzymes’ stability at 37 °C in the presence of substrates. Enzyme solutions supplied with 100 μM both NMN and ATP were treated and assayed as in panel B. (D) Optimal temperature. Enzyme rates were measured after warming the assay mixtures at the indicated temperatures. Values (n = 4) are referred to the relative maximal rate observed for each enzyme (arbitrary 100%). After this assay, the mixtures at 47 °C were rapidly cooled down to 37 °C then new NMNAT2 aliquots were added and rates measured again, demonstrating full recovery of the original activity. This excludes any effect caused by heating on the ancillary enzyme ADH.

    Techniques Used: Mutagenesis, Incubation, Activity Assay

    11) Product Images from "Methanol-Essential Growth of Corynebacterium glutamicum: Adaptive Laboratory Evolution Overcomes Limitation due to Methanethiol Assimilation Pathway"

    Article Title: Methanol-Essential Growth of Corynebacterium glutamicum: Adaptive Laboratory Evolution Overcomes Limitation due to Methanethiol Assimilation Pathway

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms21103617

    The ribulose monophosphate pathway (RuMP) implemented in  C. glutamicum . ( A ) Δ rpe  and ( B ) Δ rpi  concepts for methanol-dependent complementation of two metabolic cut-offs of the pentose phosphate pathway in  C. glutamicum , respectively. Substrates in grey boxes: MeOH, methanol; metabolites in black boxes: E4P, erythrose 4-phosphate; F6P, fructose 6-phosphate; FA, formaldehyde; GAP, glyceraldehyde 3-phosphate; Hu6P, hexulose 6-phosphate; R5P, ribose 5-phosphate; Ru5P, ribulose 5-phosphate; S7P, sedoheptulose 7-phosphate; Xul, xylulose; Xu5P, xylulose 5-phosphate; interconnected pathways, violet boxes: PPP, pentose phosphate pathway; native or homologous overexpression of genes in orange circles:  rpe , ribulose 5-phosphate epimerase;  rpi , ribose 5-phosphate isomerase;  tal , transaldolase;  tkt , transketolase;  xylA , xylose isomerase;  xylB , xylulokinase; heterologous overexpression of  xylA  gene (xylose isomerase) from  X. campestris  in green circle; heterologous overexpression of RuMP pathway genes from  B. subtilis  in blue circles:  hxlA , 3-hexulose 6-phosphate synthase;  hxlB , 6-phospho 3-hexulose isomerase; heterologous overexpression of  mdh  gene (methanol dehydrogenase) from  B. methanolicus  in pink circle; red arrows, knocked out reactions; green arrows, complementing reactions.
    Figure Legend Snippet: The ribulose monophosphate pathway (RuMP) implemented in C. glutamicum . ( A ) Δ rpe and ( B ) Δ rpi concepts for methanol-dependent complementation of two metabolic cut-offs of the pentose phosphate pathway in C. glutamicum , respectively. Substrates in grey boxes: MeOH, methanol; metabolites in black boxes: E4P, erythrose 4-phosphate; F6P, fructose 6-phosphate; FA, formaldehyde; GAP, glyceraldehyde 3-phosphate; Hu6P, hexulose 6-phosphate; R5P, ribose 5-phosphate; Ru5P, ribulose 5-phosphate; S7P, sedoheptulose 7-phosphate; Xul, xylulose; Xu5P, xylulose 5-phosphate; interconnected pathways, violet boxes: PPP, pentose phosphate pathway; native or homologous overexpression of genes in orange circles: rpe , ribulose 5-phosphate epimerase; rpi , ribose 5-phosphate isomerase; tal , transaldolase; tkt , transketolase; xylA , xylose isomerase; xylB , xylulokinase; heterologous overexpression of xylA gene (xylose isomerase) from X. campestris in green circle; heterologous overexpression of RuMP pathway genes from B. subtilis in blue circles: hxlA , 3-hexulose 6-phosphate synthase; hxlB , 6-phospho 3-hexulose isomerase; heterologous overexpression of mdh gene (methanol dehydrogenase) from B. methanolicus in pink circle; red arrows, knocked out reactions; green arrows, complementing reactions.

    Techniques Used: Over Expression

    12) Product Images from "Studies on the catalytic domains of multiple JmjC oxygenases using peptide substrates"

    Article Title: Studies on the catalytic domains of multiple JmjC oxygenases using peptide substrates

    Journal: Epigenetics

    doi: 10.4161/15592294.2014.983381

    JmjC Oxygenases MINA53, NO66 and JMJD5 do not catalyze demethylation of histone peptides. In addition to putative demethylation activities, MINA53 and NO66 have been characterized as hydroxylases acting on ribosomal proteins Rpl27a and Rpl8 respectively. Hydroxylation activities were observed for MINA53 and NO66, acting on Rpl27a and Rpl8 peptide fragments respectively ( A and C ); no demethylation was observed with methylated histone peptides ( B , D and E ). Prime-substrate uncoupled turnover of 2OG by JMJD5 (residues 1–416) was observed in a [ 14 C]-labeled 2OG assay, which was dependent on the presence of iron(II) and inhibited by the broad-spectrum 2OG oxygenase inhibitor 2,4-pyridinedicarboxylic acid (2,4 PDA) ( F ). However, demethylation of an H3K36me2 histone peptide was not observed ( G ). Control reactions without added protein are in red.
    Figure Legend Snippet: JmjC Oxygenases MINA53, NO66 and JMJD5 do not catalyze demethylation of histone peptides. In addition to putative demethylation activities, MINA53 and NO66 have been characterized as hydroxylases acting on ribosomal proteins Rpl27a and Rpl8 respectively. Hydroxylation activities were observed for MINA53 and NO66, acting on Rpl27a and Rpl8 peptide fragments respectively ( A and C ); no demethylation was observed with methylated histone peptides ( B , D and E ). Prime-substrate uncoupled turnover of 2OG by JMJD5 (residues 1–416) was observed in a [ 14 C]-labeled 2OG assay, which was dependent on the presence of iron(II) and inhibited by the broad-spectrum 2OG oxygenase inhibitor 2,4-pyridinedicarboxylic acid (2,4 PDA) ( F ). However, demethylation of an H3K36me2 histone peptide was not observed ( G ). Control reactions without added protein are in red.

    Techniques Used: Methylation, Labeling

    13) Product Images from "ADP-Dependent Kinases From the Archaeal Order Methanosarcinales Adapt to Salt by a Non-canonical Evolutionarily Conserved Strategy"

    Article Title: ADP-Dependent Kinases From the Archaeal Order Methanosarcinales Adapt to Salt by a Non-canonical Evolutionarily Conserved Strategy

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.01305

    Effect of KCl, NaCl and glycine betaine on the activity of ADP-PFK/GK from Methanosarcinales . Effect of KCl or NaCl on the: (A) MevePFK/GK (halophilic), (B) MmazPFK/GK (non-halophilic), and (C) ancMsPFK/GK (ancestor) activity in the absence and presence of 1 M glycine betaine. (D) Activity of: MevePFK/GK, MmazPFK/GK and ancMsPFK/GK as a function of glycine betaine concentration in the absence of salt. In all cases, activity determinations were made at saturating substrate concentrations. Activity was expressed as the percentage of that obtained in the absence of both salt and glycine betaine.
    Figure Legend Snippet: Effect of KCl, NaCl and glycine betaine on the activity of ADP-PFK/GK from Methanosarcinales . Effect of KCl or NaCl on the: (A) MevePFK/GK (halophilic), (B) MmazPFK/GK (non-halophilic), and (C) ancMsPFK/GK (ancestor) activity in the absence and presence of 1 M glycine betaine. (D) Activity of: MevePFK/GK, MmazPFK/GK and ancMsPFK/GK as a function of glycine betaine concentration in the absence of salt. In all cases, activity determinations were made at saturating substrate concentrations. Activity was expressed as the percentage of that obtained in the absence of both salt and glycine betaine.

    Techniques Used: Activity Assay, Concentration Assay

    14) Product Images from "Computational and Experimental Analysis of Redundancy in the Central Metabolism of Geobacter sulfurreducens"

    Article Title: Computational and Experimental Analysis of Redundancy in the Central Metabolism of Geobacter sulfurreducens

    Journal: PLoS Computational Biology

    doi: 10.1371/journal.pcbi.0040036

    Optimal Equivalent Reactions Sets Studied The sets were identified in the metabolism of G. sulfurreducens using the FVA analysis during acetate oxidation with either fumarate or Fe(III) citrate as the acceptor, (A) pyruvate to acetyl-CoA and (B) succinyl-CoA to succinate; and non-optimal central metabolism alternate pathways studied, (C) the redundant pathways for conversion of malate to oxaloacetate and (D) the pathways for synthesis of phosphoenolpyruvate (PEP) from pyruvate. The energetically favorable pathways selected in the model simulations are enclosed in the red box. Ack, Acetate kinase; Adk1, Adenylate kinase; Ato, Acetyl CoA transferase; Fdh, Formate dehydrogenase; Me, Malic enzyme; Mdh, Malate dehydrogenase; Pc, Pyruvate carboxylase; Pdh, Pyruvate dehydrogenase; Pfl, Pyruvate formate lyase; Por, Pyruvate oxidoreductase; Ppa, diphosphatase; Ppck, Phosphoenolpyruvate carboxykinase; Ppdk, pyruvate phosphate dikinase; Ppsa, PEP synthase; Pta, Phosphotransacetylase; Sucoas, Succinyl-CoA synthetase.
    Figure Legend Snippet: Optimal Equivalent Reactions Sets Studied The sets were identified in the metabolism of G. sulfurreducens using the FVA analysis during acetate oxidation with either fumarate or Fe(III) citrate as the acceptor, (A) pyruvate to acetyl-CoA and (B) succinyl-CoA to succinate; and non-optimal central metabolism alternate pathways studied, (C) the redundant pathways for conversion of malate to oxaloacetate and (D) the pathways for synthesis of phosphoenolpyruvate (PEP) from pyruvate. The energetically favorable pathways selected in the model simulations are enclosed in the red box. Ack, Acetate kinase; Adk1, Adenylate kinase; Ato, Acetyl CoA transferase; Fdh, Formate dehydrogenase; Me, Malic enzyme; Mdh, Malate dehydrogenase; Pc, Pyruvate carboxylase; Pdh, Pyruvate dehydrogenase; Pfl, Pyruvate formate lyase; Por, Pyruvate oxidoreductase; Ppa, diphosphatase; Ppck, Phosphoenolpyruvate carboxykinase; Ppdk, pyruvate phosphate dikinase; Ppsa, PEP synthase; Pta, Phosphotransacetylase; Sucoas, Succinyl-CoA synthetase.

    Techniques Used:

    15) Product Images from "Methanol fermentation increases the production of NAD(P)H-dependent chemicals in synthetic methylotrophic Escherichia coli"

    Article Title: Methanol fermentation increases the production of NAD(P)H-dependent chemicals in synthetic methylotrophic Escherichia coli

    Journal: Biotechnology for Biofuels

    doi: 10.1186/s13068-019-1356-4

    Methanol bioconversion for improved lysine synthesis in synthetic methylotrophic E. coli . Enzymes required for the assimilation of methanol into central metabolism are shown in blue: MDH methanol dehydrogenase, HPS 3-hexulose-6-phosphate synthase, and PHI 6-phospho-3-hexuloisomerase. The genes overexpressed in the lysine biosynthetic pathway are shown in green. The cofactor generation pathway was reconstructed by expressing POS5 from S. cerevisiae to convert extra NADH and generate NADPH, which is shown in red
    Figure Legend Snippet: Methanol bioconversion for improved lysine synthesis in synthetic methylotrophic E. coli . Enzymes required for the assimilation of methanol into central metabolism are shown in blue: MDH methanol dehydrogenase, HPS 3-hexulose-6-phosphate synthase, and PHI 6-phospho-3-hexuloisomerase. The genes overexpressed in the lysine biosynthetic pathway are shown in green. The cofactor generation pathway was reconstructed by expressing POS5 from S. cerevisiae to convert extra NADH and generate NADPH, which is shown in red

    Techniques Used: Expressing

    Improved lysine production in synthetic methylotrophic E. coli . a Methylotrophic E. coli was engineered for the bioconversion of methanol to improve lysine production. The extra NADH from methanol consumption was engineered to generate NADPH for lysine production. b Lysine production in the strains BL21/ΔfrmA-ML and BL21/ΔfrmA-ML-POS5 cultivated with and without methanol. c Methanol consumption in the strains BL21/ΔfrmA-ML and BL21/ΔfrmA-ML-POS5. d The intracellular NADH pools in the strains BL21/ΔfrmA-ML and BL21/ΔfrmA-ML-POS5 cultivated with and without methanol. e The intracellular NADPH pools in the strains BL21/ΔfrmA-ML-POS5 cultivated with 55 mM glucose and 50 mM methanol. f Lysine production from 13 C-methanol in the strains BL21/ΔfrmA-Mdh2-Hps-Phi, BL21/ΔfrmA-ML, and BL21/ΔfrmA-ML-POS5. Error bars indicate standard error of the mean ( n = 3)
    Figure Legend Snippet: Improved lysine production in synthetic methylotrophic E. coli . a Methylotrophic E. coli was engineered for the bioconversion of methanol to improve lysine production. The extra NADH from methanol consumption was engineered to generate NADPH for lysine production. b Lysine production in the strains BL21/ΔfrmA-ML and BL21/ΔfrmA-ML-POS5 cultivated with and without methanol. c Methanol consumption in the strains BL21/ΔfrmA-ML and BL21/ΔfrmA-ML-POS5. d The intracellular NADH pools in the strains BL21/ΔfrmA-ML and BL21/ΔfrmA-ML-POS5 cultivated with and without methanol. e The intracellular NADPH pools in the strains BL21/ΔfrmA-ML-POS5 cultivated with 55 mM glucose and 50 mM methanol. f Lysine production from 13 C-methanol in the strains BL21/ΔfrmA-Mdh2-Hps-Phi, BL21/ΔfrmA-ML, and BL21/ΔfrmA-ML-POS5. Error bars indicate standard error of the mean ( n = 3)

    Techniques Used:

    16) Product Images from "3′-NADP and 3′-NAADP, Two Metabolites Formed by the Bacterial Type III Effector AvrRxo1 *"

    Article Title: 3′-NADP and 3′-NAADP, Two Metabolites Formed by the Bacterial Type III Effector AvrRxo1 *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M116.751297

    ADP formation by AvrRxo1 is stimulated by NAD and NAAD but not by the structurally similar dinucleotide UpA. A burst/chase experiment using the PK/LDH assay shows that conversion of ATP to ADP by AvrRxo1 is stimulated by NAD, NAAD, and NADH but not by the dinucleotide UpA. AvrRxo1 was added as indicated to a reaction mixture including PK, LDH, PEP, ATP, and a potential phosphate acceptor substrate or water. The reaction was incubated for 15 min to allow accumulation of pyruvate through ATP regeneration by PK from any newly formed ADP upon consumption of PEP. Subsequently, 200 μ m NADH were titrated into the setup, and the absorbance at 340 nm was measured. Traces of A 340 shown are of reactions in which the following compounds were present: H 2 O ( black ), NAD ( green ), NAAD ( blue ), UpA ( dotted gray ). The reaction setup with ATP, NAD, and no AvrRxo1 ( dotted black ) showed negligible activity. The maximum assay velocity was determined by incubation with 200 μ m ADP ( gray ) for 15 min prior to NADH addition.
    Figure Legend Snippet: ADP formation by AvrRxo1 is stimulated by NAD and NAAD but not by the structurally similar dinucleotide UpA. A burst/chase experiment using the PK/LDH assay shows that conversion of ATP to ADP by AvrRxo1 is stimulated by NAD, NAAD, and NADH but not by the dinucleotide UpA. AvrRxo1 was added as indicated to a reaction mixture including PK, LDH, PEP, ATP, and a potential phosphate acceptor substrate or water. The reaction was incubated for 15 min to allow accumulation of pyruvate through ATP regeneration by PK from any newly formed ADP upon consumption of PEP. Subsequently, 200 μ m NADH were titrated into the setup, and the absorbance at 340 nm was measured. Traces of A 340 shown are of reactions in which the following compounds were present: H 2 O ( black ), NAD ( green ), NAAD ( blue ), UpA ( dotted gray ). The reaction setup with ATP, NAD, and no AvrRxo1 ( dotted black ) showed negligible activity. The maximum assay velocity was determined by incubation with 200 μ m ADP ( gray ) for 15 min prior to NADH addition.

    Techniques Used: Lactate Dehydrogenase Assay, Incubation, Activity Assay

    AvrRxo1 is a highly efficient NAD/NAAD kinase. Steady state Michaelis-Menten kinetics using the PK/LDH assay revealed that the K m values of AvrRxo1 for NAD ( green ) and NAAD ( blue ) are identical at 1.2 ± 0.1 m m at saturating Mg 2+ -ATP concentrations (3 m m ). Turnover by the enzyme is rapid with k cat values of 430 ± 10 s −1 for NAD and 270 ± 10 s −1 for NAAD. A comparable k cat for ATP ( black ) at saturating NAD concentrations (4 m m ) of 460 ± 10 s −1 was determined. The K m for ATP is slightly lower as for NAD with a value of 1.0 ± 0.1 m m . S.E. of triplicates are given as bars for each point measured. The 95% confidence interval for each fit is indicated by dotted lines .
    Figure Legend Snippet: AvrRxo1 is a highly efficient NAD/NAAD kinase. Steady state Michaelis-Menten kinetics using the PK/LDH assay revealed that the K m values of AvrRxo1 for NAD ( green ) and NAAD ( blue ) are identical at 1.2 ± 0.1 m m at saturating Mg 2+ -ATP concentrations (3 m m ). Turnover by the enzyme is rapid with k cat values of 430 ± 10 s −1 for NAD and 270 ± 10 s −1 for NAAD. A comparable k cat for ATP ( black ) at saturating NAD concentrations (4 m m ) of 460 ± 10 s −1 was determined. The K m for ATP is slightly lower as for NAD with a value of 1.0 ± 0.1 m m . S.E. of triplicates are given as bars for each point measured. The 95% confidence interval for each fit is indicated by dotted lines .

    Techniques Used: Lactate Dehydrogenase Assay

    17) Product Images from "Clostridium botulinum C2 Toxin Delays Entry into Mitosis and Activation of p34cdc2 Kinase and cdc25-C Phosphatase in HeLa cells"

    Article Title: Clostridium botulinum C2 Toxin Delays Entry into Mitosis and Activation of p34cdc2 Kinase and cdc25-C Phosphatase in HeLa cells

    Journal: Infection and Immunity

    doi:

    Cytotoxic effect of C. botulinum C2 toxin on synchronous HeLa cells. (A) Time course of C2 toxin-induced actin ADP-ribosylation. At 6 h after release from the amethopterin block, C2 toxin (200 ng of C2II and 100 ng of C2I per ml) was added to synchronized HeLa cells. Cells were incubated at 37°C; immediately and every 30 min after toxin addition, cells were lysed and lysate proteins (100 μg) were subjected to an in vitro ADP-ribosylation assay with C2I. The autoradiogram of [ 32 P]ADP-ribosylated actin is shown. Lane 1, control (without C2 toxin); lanes 2 to 8, incubation for 30 min with C2 toxin, 60 min with C2, 90 min with C2, 120 min with C2, 150 min with C2, 180 min with C2, and 210 min with C2, respectively. (B) C2 toxin-induced morphological changes and F-actin redistribution. Synchronized control cells (8 h after release from the amethopterin block) as well as synchronized cells treated with C2 toxin for 2 h (6 to 8 h after release from the block; 200 ng of C2II and 100 ng of C2I per ml) were fixed, and F-actin was stained with phalloidin-rhodamine.
    Figure Legend Snippet: Cytotoxic effect of C. botulinum C2 toxin on synchronous HeLa cells. (A) Time course of C2 toxin-induced actin ADP-ribosylation. At 6 h after release from the amethopterin block, C2 toxin (200 ng of C2II and 100 ng of C2I per ml) was added to synchronized HeLa cells. Cells were incubated at 37°C; immediately and every 30 min after toxin addition, cells were lysed and lysate proteins (100 μg) were subjected to an in vitro ADP-ribosylation assay with C2I. The autoradiogram of [ 32 P]ADP-ribosylated actin is shown. Lane 1, control (without C2 toxin); lanes 2 to 8, incubation for 30 min with C2 toxin, 60 min with C2, 90 min with C2, 120 min with C2, 150 min with C2, 180 min with C2, and 210 min with C2, respectively. (B) C2 toxin-induced morphological changes and F-actin redistribution. Synchronized control cells (8 h after release from the amethopterin block) as well as synchronized cells treated with C2 toxin for 2 h (6 to 8 h after release from the block; 200 ng of C2II and 100 ng of C2I per ml) were fixed, and F-actin was stained with phalloidin-rhodamine.

    Techniques Used: Blocking Assay, Incubation, In Vitro, Staining

    18) Product Images from "Overexpression of Nmnat3 efficiently increases NAD and NGD levels and ameliorates age‐associated insulin resistance, et al. Overexpression of Nmnat3 efficiently increases NAD and NGD levels and ameliorates age‐associated insulin resistance"

    Article Title: Overexpression of Nmnat3 efficiently increases NAD and NGD levels and ameliorates age‐associated insulin resistance, et al. Overexpression of Nmnat3 efficiently increases NAD and NGD levels and ameliorates age‐associated insulin resistance

    Journal: Aging Cell

    doi: 10.1111/acel.12798

    Overexpression of Nmnat3 significantly increased NGD and NHD levels in vivo. (a) Chemical structures and representative chromatogram of standard NAD , NGD , and NHD . 10 pmol standard solution was injected into the FT ‐ MS ( LTQ Orbitrap XL ). (b–d) Absolute quantification of NAD (b), NGD (c), and NHD (d) levels using skeletal muscle tissue samples prepared from WT and Nmnat3 Tg mice. Data are presented as mean ± SD ( n = 4 for each group)
    Figure Legend Snippet: Overexpression of Nmnat3 significantly increased NGD and NHD levels in vivo. (a) Chemical structures and representative chromatogram of standard NAD , NGD , and NHD . 10 pmol standard solution was injected into the FT ‐ MS ( LTQ Orbitrap XL ). (b–d) Absolute quantification of NAD (b), NGD (c), and NHD (d) levels using skeletal muscle tissue samples prepared from WT and Nmnat3 Tg mice. Data are presented as mean ± SD ( n = 4 for each group)

    Techniques Used: Over Expression, In Vivo, Injection, Mass Spectrometry, Mouse Assay

    19) Product Images from "Methanol fermentation increases the production of NAD(P)H-dependent chemicals in synthetic methylotrophic Escherichia coli"

    Article Title: Methanol fermentation increases the production of NAD(P)H-dependent chemicals in synthetic methylotrophic Escherichia coli

    Journal: Biotechnology for Biofuels

    doi: 10.1186/s13068-019-1356-4

    Improved lysine production in synthetic methylotrophic E. coli . a Methylotrophic E. coli was engineered for the bioconversion of methanol to improve lysine production. The extra NADH from methanol consumption was engineered to generate NADPH for lysine production. b Lysine production in the strains BL21/ΔfrmA-ML and BL21/ΔfrmA-ML-POS5 cultivated with and without methanol. c Methanol consumption in the strains BL21/ΔfrmA-ML and BL21/ΔfrmA-ML-POS5. d The intracellular NADH pools in the strains BL21/ΔfrmA-ML and BL21/ΔfrmA-ML-POS5 cultivated with and without methanol. e The intracellular NADPH pools in the strains BL21/ΔfrmA-ML-POS5 cultivated with 55 mM glucose and 50 mM methanol. f Lysine production from 13 C-methanol in the strains BL21/ΔfrmA-Mdh2-Hps-Phi, BL21/ΔfrmA-ML, and BL21/ΔfrmA-ML-POS5. Error bars indicate standard error of the mean ( n = 3)
    Figure Legend Snippet: Improved lysine production in synthetic methylotrophic E. coli . a Methylotrophic E. coli was engineered for the bioconversion of methanol to improve lysine production. The extra NADH from methanol consumption was engineered to generate NADPH for lysine production. b Lysine production in the strains BL21/ΔfrmA-ML and BL21/ΔfrmA-ML-POS5 cultivated with and without methanol. c Methanol consumption in the strains BL21/ΔfrmA-ML and BL21/ΔfrmA-ML-POS5. d The intracellular NADH pools in the strains BL21/ΔfrmA-ML and BL21/ΔfrmA-ML-POS5 cultivated with and without methanol. e The intracellular NADPH pools in the strains BL21/ΔfrmA-ML-POS5 cultivated with 55 mM glucose and 50 mM methanol. f Lysine production from 13 C-methanol in the strains BL21/ΔfrmA-Mdh2-Hps-Phi, BL21/ΔfrmA-ML, and BL21/ΔfrmA-ML-POS5. Error bars indicate standard error of the mean ( n = 3)

    Techniques Used:

    Methanol bioconversion for improved lysine synthesis in synthetic methylotrophic E. coli . Enzymes required for the assimilation of methanol into central metabolism are shown in blue: MDH methanol dehydrogenase, HPS 3-hexulose-6-phosphate synthase, and PHI 6-phospho-3-hexuloisomerase. The genes overexpressed in the lysine biosynthetic pathway are shown in green. The cofactor generation pathway was reconstructed by expressing POS5 from S. cerevisiae to convert extra NADH and generate NADPH, which is shown in red
    Figure Legend Snippet: Methanol bioconversion for improved lysine synthesis in synthetic methylotrophic E. coli . Enzymes required for the assimilation of methanol into central metabolism are shown in blue: MDH methanol dehydrogenase, HPS 3-hexulose-6-phosphate synthase, and PHI 6-phospho-3-hexuloisomerase. The genes overexpressed in the lysine biosynthetic pathway are shown in green. The cofactor generation pathway was reconstructed by expressing POS5 from S. cerevisiae to convert extra NADH and generate NADPH, which is shown in red

    Techniques Used: Expressing

    20) Product Images from "Methanol fermentation increases the production of NAD(P)H-dependent chemicals in synthetic methylotrophic Escherichia coli"

    Article Title: Methanol fermentation increases the production of NAD(P)H-dependent chemicals in synthetic methylotrophic Escherichia coli

    Journal: Biotechnology for Biofuels

    doi: 10.1186/s13068-019-1356-4

    Improved lysine production in synthetic methylotrophic E. coli . a Methylotrophic E. coli was engineered for the bioconversion of methanol to improve lysine production. The extra NADH from methanol consumption was engineered to generate NADPH for lysine production. b Lysine production in the strains BL21/ΔfrmA-ML and BL21/ΔfrmA-ML-POS5 cultivated with and without methanol. c Methanol consumption in the strains BL21/ΔfrmA-ML and BL21/ΔfrmA-ML-POS5. d The intracellular NADH pools in the strains BL21/ΔfrmA-ML and BL21/ΔfrmA-ML-POS5 cultivated with and without methanol. e The intracellular NADPH pools in the strains BL21/ΔfrmA-ML-POS5 cultivated with 55 mM glucose and 50 mM methanol. f Lysine production from 13 C-methanol in the strains BL21/ΔfrmA-Mdh2-Hps-Phi, BL21/ΔfrmA-ML, and BL21/ΔfrmA-ML-POS5. Error bars indicate standard error of the mean ( n = 3)
    Figure Legend Snippet: Improved lysine production in synthetic methylotrophic E. coli . a Methylotrophic E. coli was engineered for the bioconversion of methanol to improve lysine production. The extra NADH from methanol consumption was engineered to generate NADPH for lysine production. b Lysine production in the strains BL21/ΔfrmA-ML and BL21/ΔfrmA-ML-POS5 cultivated with and without methanol. c Methanol consumption in the strains BL21/ΔfrmA-ML and BL21/ΔfrmA-ML-POS5. d The intracellular NADH pools in the strains BL21/ΔfrmA-ML and BL21/ΔfrmA-ML-POS5 cultivated with and without methanol. e The intracellular NADPH pools in the strains BL21/ΔfrmA-ML-POS5 cultivated with 55 mM glucose and 50 mM methanol. f Lysine production from 13 C-methanol in the strains BL21/ΔfrmA-Mdh2-Hps-Phi, BL21/ΔfrmA-ML, and BL21/ΔfrmA-ML-POS5. Error bars indicate standard error of the mean ( n = 3)

    Techniques Used:

    Methanol bioconversion for improved lysine synthesis in synthetic methylotrophic E. coli . Enzymes required for the assimilation of methanol into central metabolism are shown in blue: MDH methanol dehydrogenase, HPS 3-hexulose-6-phosphate synthase, and PHI 6-phospho-3-hexuloisomerase. The genes overexpressed in the lysine biosynthetic pathway are shown in green. The cofactor generation pathway was reconstructed by expressing POS5 from S. cerevisiae to convert extra NADH and generate NADPH, which is shown in red
    Figure Legend Snippet: Methanol bioconversion for improved lysine synthesis in synthetic methylotrophic E. coli . Enzymes required for the assimilation of methanol into central metabolism are shown in blue: MDH methanol dehydrogenase, HPS 3-hexulose-6-phosphate synthase, and PHI 6-phospho-3-hexuloisomerase. The genes overexpressed in the lysine biosynthetic pathway are shown in green. The cofactor generation pathway was reconstructed by expressing POS5 from S. cerevisiae to convert extra NADH and generate NADPH, which is shown in red

    Techniques Used: Expressing

    21) Product Images from "Phosphorylation of the Amino Terminus of Maize Sucrose Synthase in Relation to Membrane Association and Enzyme Activity 1"

    Article Title: Phosphorylation of the Amino Terminus of Maize Sucrose Synthase in Relation to Membrane Association and Enzyme Activity 1

    Journal: Plant Physiology

    doi: 10.1104/pp.103.036780

    Phosphorylation affects the Suc cleavage activity of SUS. A, Specific Suc cleavage activities ( μ mol UDP-Glc min −1 mg −1 ) at pH 5.5, 6.5, 7.5, and 8.5 of S170A SUS1 recombinants at time zero (black bars) or after a 30-min incubation in vitro in the presence (+, gray bars) or absence (−, white bars) of CDPK II . B, Immunoblot (IB) analyses of CDPK II and S170A SUS1 recombinants at time zero or after a 30-min incubation in vitro in the presence (+) or absence (−) of CDPK II , with the antibodies listed to the right of each panel. The positions of PAGE molecular mass markers are shown in kilodaltons on the left of the figure. C, Specific Suc cleavage activities ( μ mol UDP-Glc min −1 mg −1 ) at pH 5.5, 6.5, 7.5, and 8.5 of wild-type SUS1 recombinants at time zero (black bars) or after a 30-min incubation in vitro in the presence (+, gray bars) or absence (−, white bars) of CDPK II .
    Figure Legend Snippet: Phosphorylation affects the Suc cleavage activity of SUS. A, Specific Suc cleavage activities ( μ mol UDP-Glc min −1 mg −1 ) at pH 5.5, 6.5, 7.5, and 8.5 of S170A SUS1 recombinants at time zero (black bars) or after a 30-min incubation in vitro in the presence (+, gray bars) or absence (−, white bars) of CDPK II . B, Immunoblot (IB) analyses of CDPK II and S170A SUS1 recombinants at time zero or after a 30-min incubation in vitro in the presence (+) or absence (−) of CDPK II , with the antibodies listed to the right of each panel. The positions of PAGE molecular mass markers are shown in kilodaltons on the left of the figure. C, Specific Suc cleavage activities ( μ mol UDP-Glc min −1 mg −1 ) at pH 5.5, 6.5, 7.5, and 8.5 of wild-type SUS1 recombinants at time zero (black bars) or after a 30-min incubation in vitro in the presence (+, gray bars) or absence (−, white bars) of CDPK II .

    Techniques Used: Activity Assay, Gas Chromatography, Incubation, In Vitro, Polyacrylamide Gel Electrophoresis

    Low pH affects the conformation of the amino terminus of SUS without affecting oligomerization state of the native protein. A, Immunoprecipitation (IP) of native s-SUS was performed at pH 7.5 or pH 5.5 with blank Protein-G beads (PG-only) or with the antibodies listed at the top of the figure. Immunoblots (IB) were performed on the IP pellets with the antibody listed to the right of the figure. The positions of PAGE molecular mass markers are shown in kilodaltons on the left of the figure. B, Sucrose cleavage activity ( μ mol UDP-Glc min −1 mL −1 × 10 −3 ) of native s-SUS resolved by size-exclusion chromatography at pH 6.0 (▪, solid line) or pH 7.5 (○, dashed line). The elution positions of molecular mass standards are shown in kilodaltons (kD) under the graph.
    Figure Legend Snippet: Low pH affects the conformation of the amino terminus of SUS without affecting oligomerization state of the native protein. A, Immunoprecipitation (IP) of native s-SUS was performed at pH 7.5 or pH 5.5 with blank Protein-G beads (PG-only) or with the antibodies listed at the top of the figure. Immunoblots (IB) were performed on the IP pellets with the antibody listed to the right of the figure. The positions of PAGE molecular mass markers are shown in kilodaltons on the left of the figure. B, Sucrose cleavage activity ( μ mol UDP-Glc min −1 mL −1 × 10 −3 ) of native s-SUS resolved by size-exclusion chromatography at pH 6.0 (▪, solid line) or pH 7.5 (○, dashed line). The elution positions of molecular mass standards are shown in kilodaltons (kD) under the graph.

    Techniques Used: Immunoprecipitation, Western Blot, Polyacrylamide Gel Electrophoresis, Activity Assay, Gas Chromatography, Size-exclusion Chromatography

    22) Product Images from "A ?38 Deletion Variant of Human Transketolase as a Model of Transketolase-Like Protein 1 Exhibits No Enzymatic Activity"

    Article Title: A ?38 Deletion Variant of Human Transketolase as a Model of Transketolase-Like Protein 1 Exhibits No Enzymatic Activity

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0048321

    Representative SDS-PAGE analysis of purified TKTΔ38 and full-length TKT. Note the smaller molecular weight of the deletion variant compared to the wild-type form.
    Figure Legend Snippet: Representative SDS-PAGE analysis of purified TKTΔ38 and full-length TKT. Note the smaller molecular weight of the deletion variant compared to the wild-type form.

    Techniques Used: SDS Page, Purification, Molecular Weight, Variant Assay

    Steady-state kinetic analysis of enzymatic activity of full-length TKT and variant TKTΔ38. Enzymatic activity for conversion of physiological substrates X5P and R5P into products G3P and S7P was analyzed in a coupled spectrophotometric assay at 340 nm (NADH depletion) and 30°C. Full conditions are detailed in the Experimental Procedures. Note that we were unable to detect any enzymatic activity in case of variant TKTΔ38.
    Figure Legend Snippet: Steady-state kinetic analysis of enzymatic activity of full-length TKT and variant TKTΔ38. Enzymatic activity for conversion of physiological substrates X5P and R5P into products G3P and S7P was analyzed in a coupled spectrophotometric assay at 340 nm (NADH depletion) and 30°C. Full conditions are detailed in the Experimental Procedures. Note that we were unable to detect any enzymatic activity in case of variant TKTΔ38.

    Techniques Used: Activity Assay, Variant Assay, Spectrophotometric Assay

    Far-UV CD spectra and thermal unfolding of TKT and variant TKTΔ38. ( A ) CD spectra of full-length TKT and TKTΔ38 were recorded at a protein concentration of 0.1 mg/mL in 50 mM sodium phosphate buffer, pH 7.6 containing 2.5 mM MgCl 2 and 100 µM ThDP at 20°C. In case of the deletion variant, the buffer additionally contained 500 mM NaCl. ( B ) Thermal unfolding of TKT and TKTΔ38 as detected by the change of the far-UV CD signal at 222 nm. Unfolding was analyzed both in high-salt buffer (50 mM sodium phosphate, pH 7.6, 500 mM NaCl, 2.5 mM MgCl 2 , 100 µM ThDP) and low-salt buffer (same as above but devoid of NaCl). Note that TKTΔ38 is too unstable in low-salt buffer as to allow a spectroscopic analysis under these conditions. Full conditions are detailed in the Experimental Procedures.
    Figure Legend Snippet: Far-UV CD spectra and thermal unfolding of TKT and variant TKTΔ38. ( A ) CD spectra of full-length TKT and TKTΔ38 were recorded at a protein concentration of 0.1 mg/mL in 50 mM sodium phosphate buffer, pH 7.6 containing 2.5 mM MgCl 2 and 100 µM ThDP at 20°C. In case of the deletion variant, the buffer additionally contained 500 mM NaCl. ( B ) Thermal unfolding of TKT and TKTΔ38 as detected by the change of the far-UV CD signal at 222 nm. Unfolding was analyzed both in high-salt buffer (50 mM sodium phosphate, pH 7.6, 500 mM NaCl, 2.5 mM MgCl 2 , 100 µM ThDP) and low-salt buffer (same as above but devoid of NaCl). Note that TKTΔ38 is too unstable in low-salt buffer as to allow a spectroscopic analysis under these conditions. Full conditions are detailed in the Experimental Procedures.

    Techniques Used: Variant Assay, Protein Concentration

    Active site structure of human TKT highlighting the sequence, which has been deleted in TKTΔ38 to generate a viable model of TKTL1. The structure of human TKT (pdb code 3 mos) with bound cofactors ThDP (yellow sticks) and Ca 2+ (red sphere) is shown in cartoon representation. The individual subunits of the functional dimer are shown in green (subunit A) and orange (subunit B), respectively. The deleted sequence comprising residues Gly76-Pro113 of subunit A is colored in magenta. Two His residues of this sequence, which are supposedly critical for cofactor binding and enzymatic activity, are shown in stick representation.
    Figure Legend Snippet: Active site structure of human TKT highlighting the sequence, which has been deleted in TKTΔ38 to generate a viable model of TKTL1. The structure of human TKT (pdb code 3 mos) with bound cofactors ThDP (yellow sticks) and Ca 2+ (red sphere) is shown in cartoon representation. The individual subunits of the functional dimer are shown in green (subunit A) and orange (subunit B), respectively. The deleted sequence comprising residues Gly76-Pro113 of subunit A is colored in magenta. Two His residues of this sequence, which are supposedly critical for cofactor binding and enzymatic activity, are shown in stick representation.

    Techniques Used: Sequencing, Functional Assay, Binding Assay, Activity Assay

    Analysis of cofactor binding in full-length TKT and variant TKTΔ38 by near-UV CD spectroscopy and 1H NMR spectroscopy. (A) Near-UV CD spectra of 2 mg/ml protein in 50 mM glycyl-glycine buffer, pH 7.6 and 500 mM NaCl. Note the absence of the negative CD signal at around 320 nm in case of TKTΔ38, which indicates an impaired binding of the thiamin cofactor in the deletion variant. (B) 1H NMR spectroscopic analysis of supernatant obtained after acid quench treatment of as-isolated TKTΔ38 and full-length TKT. A down-field section of the NMR spectrum (10.0–9.5 ppm) is shown, where the signal of C2-H of the cofactor thiazolium appears. We were unable to detect even traces of the thiamin cofactor in case of TKTΔ38 in contrast to full-length TKT that contains tightly bound ThDP.
    Figure Legend Snippet: Analysis of cofactor binding in full-length TKT and variant TKTΔ38 by near-UV CD spectroscopy and 1H NMR spectroscopy. (A) Near-UV CD spectra of 2 mg/ml protein in 50 mM glycyl-glycine buffer, pH 7.6 and 500 mM NaCl. Note the absence of the negative CD signal at around 320 nm in case of TKTΔ38, which indicates an impaired binding of the thiamin cofactor in the deletion variant. (B) 1H NMR spectroscopic analysis of supernatant obtained after acid quench treatment of as-isolated TKTΔ38 and full-length TKT. A down-field section of the NMR spectrum (10.0–9.5 ppm) is shown, where the signal of C2-H of the cofactor thiazolium appears. We were unable to detect even traces of the thiamin cofactor in case of TKTΔ38 in contrast to full-length TKT that contains tightly bound ThDP.

    Techniques Used: Binding Assay, Variant Assay, Spectroscopy, Nuclear Magnetic Resonance, Isolation

    Analytical gel filtration studies on TKTΔ38 and full-length TKT. ( A ) Individual elution profiles (absorbance at 280 nm) of a protein standard mixture, TKT and TKTΔ38. ( B ) Plot of log MW (molecular weight) versus V E /V 0 (V E , elution volume; V 0 , void volume). Full conditions are detailed in the Experimental Procedures.
    Figure Legend Snippet: Analytical gel filtration studies on TKTΔ38 and full-length TKT. ( A ) Individual elution profiles (absorbance at 280 nm) of a protein standard mixture, TKT and TKTΔ38. ( B ) Plot of log MW (molecular weight) versus V E /V 0 (V E , elution volume; V 0 , void volume). Full conditions are detailed in the Experimental Procedures.

    Techniques Used: Filtration, Molecular Weight

    23) Product Images from "Vanillin formation from ferulic acid in Vanilla planifolia is catalysed by a single enzyme"

    Article Title: Vanillin formation from ferulic acid in Vanilla planifolia is catalysed by a single enzyme

    Journal: Nature Communications

    doi: 10.1038/ncomms5037

    The ability of enzymes synthesized by in vitro transcription/translation to catalyse conversion of ferulic acid glucoside into vanillin glucoside. The experiment shown in ( a – d ) involved incubation of the transcription/translation protein solutions with Vp VAN, Vp ΔSPVAN, Vp Δ137VAN and Vp Δ61VAN with 1 mM of ferulic acid glucoside for 1 h in 1 mM dithiothreitol at 30 °C. ( a ) EIC 337: extracted ion chromatogram m / z vanillin glucoside (M+Na + ) obtained following incubation of Vp VAN with ferulic acid glucoside ( b ) EIC 337: extracted ion chromatogram m / z vanillin glucoside (M+Na + ) obtained following incubation of Vp ΔSPVAN with ferulic acid glucoside ( c ) EIC 337: extracted ion chromatogram m / z vanillin glucoside (M+Na + ) obtained following incubation of Vp Δ137VAN with ferulic acid glucoside. ( d ) EIC 337: extracted ion chromatogram m / z vanillin glucoside (M+ Na + ) obtained following incubation of Vp Δ61VAN with ferulic acid glucoside. ( e ) EIC 337: extracted ion chromatogram m / z vanillin glucoside (M+Na + ) obtained following incubation of a control transcription translation/ protein solution devoid of any protein of interest with ferulic acid glucoside. ( f ) EIC 379: extracted ion chromatogram m / z ferulic acid glucoside (M+Na + ) obtained following incubation of a control transcription translation/ protein solution devoid of any protein of interest with ferulic acid glucoside. Intens., intensity.
    Figure Legend Snippet: The ability of enzymes synthesized by in vitro transcription/translation to catalyse conversion of ferulic acid glucoside into vanillin glucoside. The experiment shown in ( a – d ) involved incubation of the transcription/translation protein solutions with Vp VAN, Vp ΔSPVAN, Vp Δ137VAN and Vp Δ61VAN with 1 mM of ferulic acid glucoside for 1 h in 1 mM dithiothreitol at 30 °C. ( a ) EIC 337: extracted ion chromatogram m / z vanillin glucoside (M+Na + ) obtained following incubation of Vp VAN with ferulic acid glucoside ( b ) EIC 337: extracted ion chromatogram m / z vanillin glucoside (M+Na + ) obtained following incubation of Vp ΔSPVAN with ferulic acid glucoside ( c ) EIC 337: extracted ion chromatogram m / z vanillin glucoside (M+Na + ) obtained following incubation of Vp Δ137VAN with ferulic acid glucoside. ( d ) EIC 337: extracted ion chromatogram m / z vanillin glucoside (M+ Na + ) obtained following incubation of Vp Δ61VAN with ferulic acid glucoside. ( e ) EIC 337: extracted ion chromatogram m / z vanillin glucoside (M+Na + ) obtained following incubation of a control transcription translation/ protein solution devoid of any protein of interest with ferulic acid glucoside. ( f ) EIC 379: extracted ion chromatogram m / z ferulic acid glucoside (M+Na + ) obtained following incubation of a control transcription translation/ protein solution devoid of any protein of interest with ferulic acid glucoside. Intens., intensity.

    Techniques Used: Synthesized, In Vitro, Incubation

    Direct coupled transcription/translation of the PCR-generated DNA for Vp VAN and for Vp VAN devoid of its 21 amino-acid-long ER-targeting signal peptide ( Vp ΔSPVAN). L -[ 35 S]-methionine was included to specifically monitor the formation of de novo synthesized radiolabelled proteins by SDS–PAGE. The ability of Vp VAN synthesized by in vitro transcription/translation to catalyse conversion of ferulic acid into vanillin was monitored by LC–MS using total and selected ion monitoring. ( a ) Proteins present in the in vitro transcription/translation assay visualized by Coomassie brilliant blue staining. ( b ) The [ 35 S]-labelled Vp ΔSPVAN and Vp VAN proteins formed from the two PCR products as visualized by autoradiography. In each transcription/translation experiment, a single radiolabelled protein band of the expected approximate mass was obtained. ( c ) Incubation of the transcription translation protein solution containing Vp VAN with 5 mM of ferulic acid for 1 h in 2.5 mM dithiothreitol at 30 °C, total ion chromatogram (TIC) following LC–MS analysis; ( d ) EIC: 153: extracted ion chromatogram for m / z vanillin (M+H + ) for specific detection of vanillin formation. ( e ) EIC 153: extracted ion chromatogram for m / z vanillin (M+H + ) of a control experiment in which ferulic acid was administered to a transcription translation protein solution not expressing Vp VAN. Intens., intensity.
    Figure Legend Snippet: Direct coupled transcription/translation of the PCR-generated DNA for Vp VAN and for Vp VAN devoid of its 21 amino-acid-long ER-targeting signal peptide ( Vp ΔSPVAN). L -[ 35 S]-methionine was included to specifically monitor the formation of de novo synthesized radiolabelled proteins by SDS–PAGE. The ability of Vp VAN synthesized by in vitro transcription/translation to catalyse conversion of ferulic acid into vanillin was monitored by LC–MS using total and selected ion monitoring. ( a ) Proteins present in the in vitro transcription/translation assay visualized by Coomassie brilliant blue staining. ( b ) The [ 35 S]-labelled Vp ΔSPVAN and Vp VAN proteins formed from the two PCR products as visualized by autoradiography. In each transcription/translation experiment, a single radiolabelled protein band of the expected approximate mass was obtained. ( c ) Incubation of the transcription translation protein solution containing Vp VAN with 5 mM of ferulic acid for 1 h in 2.5 mM dithiothreitol at 30 °C, total ion chromatogram (TIC) following LC–MS analysis; ( d ) EIC: 153: extracted ion chromatogram for m / z vanillin (M+H + ) for specific detection of vanillin formation. ( e ) EIC 153: extracted ion chromatogram for m / z vanillin (M+H + ) of a control experiment in which ferulic acid was administered to a transcription translation protein solution not expressing Vp VAN. Intens., intensity.

    Techniques Used: Polymerase Chain Reaction, Generated, Synthesized, SDS Page, In Vitro, Liquid Chromatography with Mass Spectroscopy, Staining, Autoradiography, Incubation, Expressing

    24) Product Images from "Nitric Oxide Directly Promotes Vascular Endothelial Insulin Transport"

    Article Title: Nitric Oxide Directly Promotes Vascular Endothelial Insulin Transport

    Journal: Diabetes

    doi: 10.2337/db13-0627

    Effects of knockdown of Txnip on insulin uptake. bAECs were transfected with either Txnip siRNA or scrambled control siRNA. Forty-eight hours after the transfection, cells were processed for Western blotting or serum starved for 6 h followed by incubation with or without 50 nmol/L FITC-insulin ± 0.3 μmol/L SNP for 30 min before they were fixed and doubly stained with anti-FITC (red, revealed by Cy3) and anti-Txnip (green, revealed by Cy2) primary antibodies. A : Representative Western blots. Caveolin-1 was used as a control to assess nonspecific off-target effects of siRNA silencing. GAPDH was used as a loading control. B : Mean values for the ratio of Txnip to GAPDH measured by Western blotting. * P
    Figure Legend Snippet: Effects of knockdown of Txnip on insulin uptake. bAECs were transfected with either Txnip siRNA or scrambled control siRNA. Forty-eight hours after the transfection, cells were processed for Western blotting or serum starved for 6 h followed by incubation with or without 50 nmol/L FITC-insulin ± 0.3 μmol/L SNP for 30 min before they were fixed and doubly stained with anti-FITC (red, revealed by Cy3) and anti-Txnip (green, revealed by Cy2) primary antibodies. A : Representative Western blots. Caveolin-1 was used as a control to assess nonspecific off-target effects of siRNA silencing. GAPDH was used as a loading control. B : Mean values for the ratio of Txnip to GAPDH measured by Western blotting. * P

    Techniques Used: Transfection, Western Blot, Incubation, Staining

    25) Product Images from "Paradoxical Inhibition of Glycolysis by Pioglitazone Opposes the Mitochondriopathy Caused by AIF Deficiency"

    Article Title: Paradoxical Inhibition of Glycolysis by Pioglitazone Opposes the Mitochondriopathy Caused by AIF Deficiency

    Journal: EBioMedicine

    doi: 10.1016/j.ebiom.2017.02.013

    PIO interacts with and inhibits purified GAPDH. (A) Western blot analysis of purified GAPDH in the absence (left panel) or presence (right panel) of added dithiothreitol (DTT). Noticeably, no higher aggregation forms were detected in the stacking gel. (B) Quantities of different aggregation forms (tetra-, tri-, di-, and monomeric) in the absence of DTT (open bars: initial GAPDH suspension, GAPDH plus DMSO, GAPDH plus DMSO-solubilized PIO, or in the presence of DTT (green filled bars) added before or after PIO). (C) GAPDH (2.1 mIU/ml) fluorescence in the absence (blue dots) or presence (green dots) of 12.5 μM PIO against UV (280 nm) irradiation. The presence of 1.25‰ DMSO had per se a partial protecting effect against UV (grey dots). No changes in fluorescence were observed when the enzyme was kept away from UV irradiation (black dots). Noticeably initial fluorescence in the presence or absence of PIO, or DMSO was not different ruling out potential interference of these compounds with fluorescence assay conditions (excitation: 280 nm, 10 nm bandpass; emission: 330 nm, 10 nm bandpass). (D) Initial activity of rabbit skeletal muscle GAPDH (0.05 mIU/ml) measured in the forward direction (NADH accumulation) ( Velick, 1955 ) and of increasing amounts of PIO. (E) Activity measured in the backward direction (NADH oxidation) in the absence (blue open bar) or presence (green open bar) of 200 μM PIO, and in the presence of 1.7 mM cysteine and absence (blue filled bar) or presence (green filled bar) of 200 μM PIO. (F) Lineweaver-Burk plots of forward GAPDH activity (0.075 mIU/ml) in the absence (black line) or presence (green line) of 62.5 μM PIO. GAP, d -glyceraldehyde 3-phosphate.
    Figure Legend Snippet: PIO interacts with and inhibits purified GAPDH. (A) Western blot analysis of purified GAPDH in the absence (left panel) or presence (right panel) of added dithiothreitol (DTT). Noticeably, no higher aggregation forms were detected in the stacking gel. (B) Quantities of different aggregation forms (tetra-, tri-, di-, and monomeric) in the absence of DTT (open bars: initial GAPDH suspension, GAPDH plus DMSO, GAPDH plus DMSO-solubilized PIO, or in the presence of DTT (green filled bars) added before or after PIO). (C) GAPDH (2.1 mIU/ml) fluorescence in the absence (blue dots) or presence (green dots) of 12.5 μM PIO against UV (280 nm) irradiation. The presence of 1.25‰ DMSO had per se a partial protecting effect against UV (grey dots). No changes in fluorescence were observed when the enzyme was kept away from UV irradiation (black dots). Noticeably initial fluorescence in the presence or absence of PIO, or DMSO was not different ruling out potential interference of these compounds with fluorescence assay conditions (excitation: 280 nm, 10 nm bandpass; emission: 330 nm, 10 nm bandpass). (D) Initial activity of rabbit skeletal muscle GAPDH (0.05 mIU/ml) measured in the forward direction (NADH accumulation) ( Velick, 1955 ) and of increasing amounts of PIO. (E) Activity measured in the backward direction (NADH oxidation) in the absence (blue open bar) or presence (green open bar) of 200 μM PIO, and in the presence of 1.7 mM cysteine and absence (blue filled bar) or presence (green filled bar) of 200 μM PIO. (F) Lineweaver-Burk plots of forward GAPDH activity (0.075 mIU/ml) in the absence (black line) or presence (green line) of 62.5 μM PIO. GAP, d -glyceraldehyde 3-phosphate.

    Techniques Used: Purification, Western Blot, Fluorescence, Irradiation, Activity Assay

    26) Product Images from "ADP-Dependent Kinases From the Archaeal Order Methanosarcinales Adapt to Salt by a Non-canonical Evolutionarily Conserved Strategy"

    Article Title: ADP-Dependent Kinases From the Archaeal Order Methanosarcinales Adapt to Salt by a Non-canonical Evolutionarily Conserved Strategy

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.01305

    Halophilic traits of homology models constructed for ADP-PFK/GK from Halobacteria, Eukarya , halophilic and non-halophilic Methanosarcinales . (A) Polar and charged surface area expressed as percentage of ASA. (B) Electrostatic potential surface of representative models were generated, being blue for positive and red for negative charge (±3 k B T / e ). (C) Residues of the protein core (inner shell), using as criteria a surface residue exposition less than 5 Å 2 . A representative model of each group is shown; Natronorubrum bangense ( Halobacteria ), Homo sapiens ( Eukarya ), Methanohalobium evestigatum (halophilic Methanosarcinales ), and Methanosarcina mazei (non-halophilic Methanosarcinales ).
    Figure Legend Snippet: Halophilic traits of homology models constructed for ADP-PFK/GK from Halobacteria, Eukarya , halophilic and non-halophilic Methanosarcinales . (A) Polar and charged surface area expressed as percentage of ASA. (B) Electrostatic potential surface of representative models were generated, being blue for positive and red for negative charge (±3 k B T / e ). (C) Residues of the protein core (inner shell), using as criteria a surface residue exposition less than 5 Å 2 . A representative model of each group is shown; Natronorubrum bangense ( Halobacteria ), Homo sapiens ( Eukarya ), Methanohalobium evestigatum (halophilic Methanosarcinales ), and Methanosarcina mazei (non-halophilic Methanosarcinales ).

    Techniques Used: Construct, Generated

    Effect of KCl, NaCl and glycine betaine on the activity of ADP-PFK/GK from Methanosarcinales . Effect of KCl or NaCl on the: (A) MevePFK/GK (halophilic), (B) MmazPFK/GK (non-halophilic), and (C) ancMsPFK/GK (ancestor) activity in the absence and presence of 1 M glycine betaine. (D) Activity of: MevePFK/GK, MmazPFK/GK and ancMsPFK/GK as a function of glycine betaine concentration in the absence of salt. In all cases, activity determinations were made at saturating substrate concentrations. Activity was expressed as the percentage of that obtained in the absence of both salt and glycine betaine.
    Figure Legend Snippet: Effect of KCl, NaCl and glycine betaine on the activity of ADP-PFK/GK from Methanosarcinales . Effect of KCl or NaCl on the: (A) MevePFK/GK (halophilic), (B) MmazPFK/GK (non-halophilic), and (C) ancMsPFK/GK (ancestor) activity in the absence and presence of 1 M glycine betaine. (D) Activity of: MevePFK/GK, MmazPFK/GK and ancMsPFK/GK as a function of glycine betaine concentration in the absence of salt. In all cases, activity determinations were made at saturating substrate concentrations. Activity was expressed as the percentage of that obtained in the absence of both salt and glycine betaine.

    Techniques Used: Activity Assay, Concentration Assay

    Kinetic stability of extant and ancestral ADP-dependent kinases from Methanosarcinales . PFK-ADP residual activity for Methanosarcinales enzymes were determined over time at different KCl concentrations; kinetic stability of MevePFK (A) and MmazPFK (B) was assayed at 40°C while for ancMsPFK (C) 60°C was employed. At the indicated times an aliquot was removed, centrifuged and its activity assayed at 30°C.
    Figure Legend Snippet: Kinetic stability of extant and ancestral ADP-dependent kinases from Methanosarcinales . PFK-ADP residual activity for Methanosarcinales enzymes were determined over time at different KCl concentrations; kinetic stability of MevePFK (A) and MmazPFK (B) was assayed at 40°C while for ancMsPFK (C) 60°C was employed. At the indicated times an aliquot was removed, centrifuged and its activity assayed at 30°C.

    Techniques Used: Activity Assay

    27) Product Images from "Homozygous NMNAT2 mutation in sisters with polyneuropathy and erythromelalgia"

    Article Title: Homozygous NMNAT2 mutation in sisters with polyneuropathy and erythromelalgia

    Journal: bioRxiv

    doi: 10.1101/610907

    Temperature studies of WT and T94M NMNAT2. Data represent mean ± SEM of n experiments as indicated. (A) Apo-enzymes’ stability at different temperatures. Buffered enzyme solutions (40 μg/ml hNMNAT2 WT or 30 μg/ml T94M mutant in 50 mM HEPES/NaOH buffer, pH 7.5, 1 mM TCEP, 20 % glycerol) were pre-incubated for 1 hour at the indicated temperatures, then assayed at 37 °C. Values (n = 6) are referred to the untreated enzyme kept at +4 °C (arbitrary 100%). (B) Apo-enzymes’ stability at 37 °C. Enzyme solutions were incubated and collected at the indicated time intervals, then assayed at 37 °C. Values (n = 4) are referred to that of time zero (arbitrary 100%). T test p values (*) for T94M vs WT: 0.013 at 8 min, 0.010 at 14 min, 0.013 at 20 min, and 0.014 at 28 min. (C) Enzymes’ stability at 37 °C in the presence of substrates. Enzyme solutions supplied with 100 μM both NMN and ATP were treated and assayed as in panel B. (D) Optimal temperature. Enzyme rates were measured after warming the assay mixtures at the indicated temperatures. Values (n = 4) are referred to the relative maximal rate observed for each enzyme (arbitrary 100%). After this assay, the mixtures at 47 °C were rapidly cooled down to 37 °C then new NMNAT2 aliquots were added and rates measured again, demonstrating full recovery of the original activity. This excludes any effect caused by heating on the ancillary enzyme ADH.
    Figure Legend Snippet: Temperature studies of WT and T94M NMNAT2. Data represent mean ± SEM of n experiments as indicated. (A) Apo-enzymes’ stability at different temperatures. Buffered enzyme solutions (40 μg/ml hNMNAT2 WT or 30 μg/ml T94M mutant in 50 mM HEPES/NaOH buffer, pH 7.5, 1 mM TCEP, 20 % glycerol) were pre-incubated for 1 hour at the indicated temperatures, then assayed at 37 °C. Values (n = 6) are referred to the untreated enzyme kept at +4 °C (arbitrary 100%). (B) Apo-enzymes’ stability at 37 °C. Enzyme solutions were incubated and collected at the indicated time intervals, then assayed at 37 °C. Values (n = 4) are referred to that of time zero (arbitrary 100%). T test p values (*) for T94M vs WT: 0.013 at 8 min, 0.010 at 14 min, 0.013 at 20 min, and 0.014 at 28 min. (C) Enzymes’ stability at 37 °C in the presence of substrates. Enzyme solutions supplied with 100 μM both NMN and ATP were treated and assayed as in panel B. (D) Optimal temperature. Enzyme rates were measured after warming the assay mixtures at the indicated temperatures. Values (n = 4) are referred to the relative maximal rate observed for each enzyme (arbitrary 100%). After this assay, the mixtures at 47 °C were rapidly cooled down to 37 °C then new NMNAT2 aliquots were added and rates measured again, demonstrating full recovery of the original activity. This excludes any effect caused by heating on the ancillary enzyme ADH.

    Techniques Used: Mutagenesis, Incubation, Activity Assay

    28) Product Images from "Arginine demethylation is catalysed by a subset of JmjC histone lysine demethylases"

    Article Title: Arginine demethylation is catalysed by a subset of JmjC histone lysine demethylases

    Journal: Nature Communications

    doi: 10.1038/ncomms11974

    The mechanisms of arginine and lysine demethylation are similar. ( a ) The arginine demethylation reaction of KDM6B requires both 2OG and Fe(II) for active demethylation. The degree of arginine demethylation of H3(14–34)K27Rme2a catalysed by KDM6B was quantified by MALDI-TOF mass spectrometry. Data show the mean±s.e.m. ( b ) 1 H NMR analyses of KDM6B-catalysed arginine demethylation. The 1 H spectra are of a reaction mixture containing KDM6B (9 μM), H3(14–34)K27Rme2a peptide (1 mM), 2OG (500 μM), ascorbate (1 mM) and iron(II) (100 μM) after 7 min (blue) and 20 min (red) at 298 K. The resonance corresponding to the methyl group of monomethylated arginine (Rme) is highlighted. ( c ) Graphs showing the degree of succinate production and peptide demethylation of H3(14–34)K27Rme2a catalysed by KDM6B as quantified by 1 H NMR (700 MHz). ( d ) Detection of formaldehyde release during KDM6B-catalysed arginine demethylation. Dimedone reacts with formaldehyde in aqueous solution to form stable adducts that are detectable using 1 H NMR (insert, the formaldehyde-derived carbons in the adducts are highlighted red). Incubation of a reaction mixture containing KDM6B (6.5 μM), H3(18–30)K27Rme2a peptide (1 mM), 2OG (4 mM), ascorbate (1 mM) and iron(II) (100 μM) and dimedone (667 μM) revealed the formation of two dimedone adducts using 1 H NMR (700 MHz). Protons responsible for each peak are shown in red.
    Figure Legend Snippet: The mechanisms of arginine and lysine demethylation are similar. ( a ) The arginine demethylation reaction of KDM6B requires both 2OG and Fe(II) for active demethylation. The degree of arginine demethylation of H3(14–34)K27Rme2a catalysed by KDM6B was quantified by MALDI-TOF mass spectrometry. Data show the mean±s.e.m. ( b ) 1 H NMR analyses of KDM6B-catalysed arginine demethylation. The 1 H spectra are of a reaction mixture containing KDM6B (9 μM), H3(14–34)K27Rme2a peptide (1 mM), 2OG (500 μM), ascorbate (1 mM) and iron(II) (100 μM) after 7 min (blue) and 20 min (red) at 298 K. The resonance corresponding to the methyl group of monomethylated arginine (Rme) is highlighted. ( c ) Graphs showing the degree of succinate production and peptide demethylation of H3(14–34)K27Rme2a catalysed by KDM6B as quantified by 1 H NMR (700 MHz). ( d ) Detection of formaldehyde release during KDM6B-catalysed arginine demethylation. Dimedone reacts with formaldehyde in aqueous solution to form stable adducts that are detectable using 1 H NMR (insert, the formaldehyde-derived carbons in the adducts are highlighted red). Incubation of a reaction mixture containing KDM6B (6.5 μM), H3(18–30)K27Rme2a peptide (1 mM), 2OG (4 mM), ascorbate (1 mM) and iron(II) (100 μM) and dimedone (667 μM) revealed the formation of two dimedone adducts using 1 H NMR (700 MHz). Protons responsible for each peak are shown in red.

    Techniques Used: Mass Spectrometry, Nuclear Magnetic Resonance, Derivative Assay, Incubation

    JmjC KDMs bind methylated arginine peptides in a catalytically-productive binding mode. ( a and b ) Views from an X-ray crystal structure of KDM4A in complex with nickel, NOG (a 2OG mimetic) and an H4R3me2s peptide (residues 1–15). Two orientations of peptide binding were refined; one orientation ( a ) positions a methyl group of symmetric dimethylarginine residue sufficiently close to the metal centre to allow catalysis (within 4.5 Å, for crystallographic reasons, nickel was substituted for iron). The other orientation ( b ) is likely not catalytically productive. Fo-Fc OMIT maps contoured to 3σ around the H4R3me2s residues are shown. ( c ) Overlay of the two binding orientations observed for the H4R3me2s peptide in the KDM4A active site. Only one orientation (blue) positions a methyl group close to the catalytic metal centre. ( d ) Overlay of the H4R3me2s peptide (catalytically-productive orientation only) and an H3K9me3 peptide (residues 7–14) bound in the KDM4A active site. The methylated arginine and lysine residues show similar binding modes.
    Figure Legend Snippet: JmjC KDMs bind methylated arginine peptides in a catalytically-productive binding mode. ( a and b ) Views from an X-ray crystal structure of KDM4A in complex with nickel, NOG (a 2OG mimetic) and an H4R3me2s peptide (residues 1–15). Two orientations of peptide binding were refined; one orientation ( a ) positions a methyl group of symmetric dimethylarginine residue sufficiently close to the metal centre to allow catalysis (within 4.5 Å, for crystallographic reasons, nickel was substituted for iron). The other orientation ( b ) is likely not catalytically productive. Fo-Fc OMIT maps contoured to 3σ around the H4R3me2s residues are shown. ( c ) Overlay of the two binding orientations observed for the H4R3me2s peptide in the KDM4A active site. Only one orientation (blue) positions a methyl group close to the catalytic metal centre. ( d ) Overlay of the H4R3me2s peptide (catalytically-productive orientation only) and an H3K9me3 peptide (residues 7–14) bound in the KDM4A active site. The methylated arginine and lysine residues show similar binding modes.

    Techniques Used: Methylation, Binding Assay

    Mechanism of lysine and arginine demethylation. ( a ) JmjC lysine demethylases (KDM2–7 subfamilies) catalyse oxidative decarboxylation of 2OG to form succinate, carbon dioxide and a reactive iron(IV)-oxo intermediate; this intermediate then facilitates hydroxylation of the lysine N ɛ -methyl group to form an unstable hemiaminal. Fragmentation of the hemiaminal releases formaldehyde and the unmethylated lysine residue. ( b ) Proposed mechanism for JmjC-catalysed arginine demethylation. ( c ) Hydroxylation of the N- methyl, N -isopropyllysine derivative is catalysed by various JmjC KDMs at a position analogous to that required for demethylation of asymmetrically dimethylated arginine residues. Methyl groups are bold red lines, dashed arrows represent proposed reactions and HCHO is formaldehyde. Each JmjC KDM/RDM/oxidation reaction is coupled to the conversion of 2OG and oxygen to carbon dioxide and succinate.
    Figure Legend Snippet: Mechanism of lysine and arginine demethylation. ( a ) JmjC lysine demethylases (KDM2–7 subfamilies) catalyse oxidative decarboxylation of 2OG to form succinate, carbon dioxide and a reactive iron(IV)-oxo intermediate; this intermediate then facilitates hydroxylation of the lysine N ɛ -methyl group to form an unstable hemiaminal. Fragmentation of the hemiaminal releases formaldehyde and the unmethylated lysine residue. ( b ) Proposed mechanism for JmjC-catalysed arginine demethylation. ( c ) Hydroxylation of the N- methyl, N -isopropyllysine derivative is catalysed by various JmjC KDMs at a position analogous to that required for demethylation of asymmetrically dimethylated arginine residues. Methyl groups are bold red lines, dashed arrows represent proposed reactions and HCHO is formaldehyde. Each JmjC KDM/RDM/oxidation reaction is coupled to the conversion of 2OG and oxygen to carbon dioxide and succinate.

    Techniques Used:

    29) Product Images from "Methanol-Essential Growth of Corynebacterium glutamicum: Adaptive Laboratory Evolution Overcomes Limitation due to Methanethiol Assimilation Pathway"

    Article Title: Methanol-Essential Growth of Corynebacterium glutamicum: Adaptive Laboratory Evolution Overcomes Limitation due to Methanethiol Assimilation Pathway

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms21103617

    The ribulose monophosphate pathway (RuMP) implemented in  C. glutamicum . ( A ) Δ rpe  and ( B ) Δ rpi  concepts for methanol-dependent complementation of two metabolic cut-offs of the pentose phosphate pathway in  C. glutamicum , respectively. Substrates in grey boxes: MeOH, methanol; metabolites in black boxes: E4P, erythrose 4-phosphate; F6P, fructose 6-phosphate; FA, formaldehyde; GAP, glyceraldehyde 3-phosphate; Hu6P, hexulose 6-phosphate; R5P, ribose 5-phosphate; Ru5P, ribulose 5-phosphate; S7P, sedoheptulose 7-phosphate; Xul, xylulose; Xu5P, xylulose 5-phosphate; interconnected pathways, violet boxes: PPP, pentose phosphate pathway; native or homologous overexpression of genes in orange circles:  rpe , ribulose 5-phosphate epimerase;  rpi , ribose 5-phosphate isomerase;  tal , transaldolase;  tkt , transketolase;  xylA , xylose isomerase;  xylB , xylulokinase; heterologous overexpression of  xylA  gene (xylose isomerase) from  X. campestris  in green circle; heterologous overexpression of RuMP pathway genes from  B. subtilis  in blue circles:  hxlA , 3-hexulose 6-phosphate synthase;  hxlB , 6-phospho 3-hexulose isomerase; heterologous overexpression of  mdh  gene (methanol dehydrogenase) from  B. methanolicus  in pink circle; red arrows, knocked out reactions; green arrows, complementing reactions.
    Figure Legend Snippet: The ribulose monophosphate pathway (RuMP) implemented in C. glutamicum . ( A ) Δ rpe and ( B ) Δ rpi concepts for methanol-dependent complementation of two metabolic cut-offs of the pentose phosphate pathway in C. glutamicum , respectively. Substrates in grey boxes: MeOH, methanol; metabolites in black boxes: E4P, erythrose 4-phosphate; F6P, fructose 6-phosphate; FA, formaldehyde; GAP, glyceraldehyde 3-phosphate; Hu6P, hexulose 6-phosphate; R5P, ribose 5-phosphate; Ru5P, ribulose 5-phosphate; S7P, sedoheptulose 7-phosphate; Xul, xylulose; Xu5P, xylulose 5-phosphate; interconnected pathways, violet boxes: PPP, pentose phosphate pathway; native or homologous overexpression of genes in orange circles: rpe , ribulose 5-phosphate epimerase; rpi , ribose 5-phosphate isomerase; tal , transaldolase; tkt , transketolase; xylA , xylose isomerase; xylB , xylulokinase; heterologous overexpression of xylA gene (xylose isomerase) from X. campestris in green circle; heterologous overexpression of RuMP pathway genes from B. subtilis in blue circles: hxlA , 3-hexulose 6-phosphate synthase; hxlB , 6-phospho 3-hexulose isomerase; heterologous overexpression of mdh gene (methanol dehydrogenase) from B. methanolicus in pink circle; red arrows, knocked out reactions; green arrows, complementing reactions.

    Techniques Used: Over Expression

    30) Product Images from "Anti-alcohol abuse drug disulfiram inhibits human PHGDH via disruption of its active tetrameric form through a specific cysteine oxidation"

    Article Title: Anti-alcohol abuse drug disulfiram inhibits human PHGDH via disruption of its active tetrameric form through a specific cysteine oxidation

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-41187-0

    Cross-linking experiment of PHGDH with BS3 at various DSF concentrations A . MW marker. B 0 µM. C 1 µM. D 5 µM. E 10 µM. F 50 µM. G 100 µM. H 250 µM. I 500 µM.). PHGDH was incubated with DSF during 30′ before cross-linking. Lane B was used as control without DSF. Lane A (MW marker) was used to deduce the oligomerization state of PHGDH. Original exposure of the uncropped gel is available in the Supplementary Information File (Fig. S4 ).
    Figure Legend Snippet: Cross-linking experiment of PHGDH with BS3 at various DSF concentrations A . MW marker. B 0 µM. C 1 µM. D 5 µM. E 10 µM. F 50 µM. G 100 µM. H 250 µM. I 500 µM.). PHGDH was incubated with DSF during 30′ before cross-linking. Lane B was used as control without DSF. Lane A (MW marker) was used to deduce the oligomerization state of PHGDH. Original exposure of the uncropped gel is available in the Supplementary Information File (Fig. S4 ).

    Techniques Used: Marker, Incubation

    31) Product Images from "Identification of a small molecule inhibitor of 3-phosphoglycerate dehydrogenase to target serine biosynthesis in cancers"

    Article Title: Identification of a small molecule inhibitor of 3-phosphoglycerate dehydrogenase to target serine biosynthesis in cancers

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1521548113

    CBR-5884 does not affect the lactate dehydrogenase (LDH) oligomerization state but inhibits a truncated form of PHGDH. ( A ) LDH was preincubated with CBR-5884 (0, 50, 200, or 400 μM) for 30 min before cross-linking with BS3 (0.25 or 2.5 mM), followed
    Figure Legend Snippet: CBR-5884 does not affect the lactate dehydrogenase (LDH) oligomerization state but inhibits a truncated form of PHGDH. ( A ) LDH was preincubated with CBR-5884 (0, 50, 200, or 400 μM) for 30 min before cross-linking with BS3 (0.25 or 2.5 mM), followed

    Techniques Used:

    32) Product Images from "Novel synthetic chalcone‐coumarin hybrid for Aβ aggregation reduction, antioxidation, and neuroprotection, et al. Novel synthetic chalcone‐coumarin hybrid for Aβ aggregation reduction, antioxidation, and neuroprotection"

    Article Title: Novel synthetic chalcone‐coumarin hybrid for Aβ aggregation reduction, antioxidation, and neuroprotection, et al. Novel synthetic chalcone‐coumarin hybrid for Aβ aggregation reduction, antioxidation, and neuroprotection

    Journal: CNS Neuroscience & Therapeutics

    doi: 10.1111/cns.13058

    Enhanced expression of HSPB1, NRF2, and CREB pathways following test compound administration in Aβ‐GFP SH‐SY5Y cells. On day 2, differentiated SH‐SY5Y cells were pretreated with 1 μmol/L LM‐031, Lico A, or coumarin for 8 hours, and Aβ‐GFP expression was induced for 6 days. Relative A, HSPB1 and soluble Aβ‐GFP, B, NRF2, GCLC, and NQO1, and C, CREB, pCREB, BDNF, BCL2, and BAX protein levels were analyzed through immunoblotting using specific antibodies. Protein levels were normalized to β‐actin or β‐tubulin internal control. Relative protein levels are shown on the right side of the representative Western blot images. To normalize, the expression level in uninduced (−Dox) cells was set at 100%. For Aβ‐GFP, the soluble level in induced (+Dox) cells was set at 100%. P values between induced and uninduced cells ( # P
    Figure Legend Snippet: Enhanced expression of HSPB1, NRF2, and CREB pathways following test compound administration in Aβ‐GFP SH‐SY5Y cells. On day 2, differentiated SH‐SY5Y cells were pretreated with 1 μmol/L LM‐031, Lico A, or coumarin for 8 hours, and Aβ‐GFP expression was induced for 6 days. Relative A, HSPB1 and soluble Aβ‐GFP, B, NRF2, GCLC, and NQO1, and C, CREB, pCREB, BDNF, BCL2, and BAX protein levels were analyzed through immunoblotting using specific antibodies. Protein levels were normalized to β‐actin or β‐tubulin internal control. Relative protein levels are shown on the right side of the representative Western blot images. To normalize, the expression level in uninduced (−Dox) cells was set at 100%. For Aβ‐GFP, the soluble level in induced (+Dox) cells was set at 100%. P values between induced and uninduced cells ( # P

    Techniques Used: Expressing, Western Blot

    33) Product Images from "Phosphorylation of the Amino Terminus of Maize Sucrose Synthase in Relation to Membrane Association and Enzyme Activity 1"

    Article Title: Phosphorylation of the Amino Terminus of Maize Sucrose Synthase in Relation to Membrane Association and Enzyme Activity 1

    Journal: Plant Physiology

    doi: 10.1104/pp.103.036780

    Phosphorylation affects the Suc cleavage activity of SUS. A, Specific Suc cleavage activities ( μ mol UDP-Glc min −1 mg −1 ) at pH 5.5, 6.5, 7.5, and 8.5 of S170A SUS1 recombinants at time zero (black bars) or after a 30-min incubation in vitro in the presence (+, gray bars) or absence (−, white bars) of CDPK II . B, Immunoblot (IB) analyses of CDPK II and S170A SUS1 recombinants at time zero or after a 30-min incubation in vitro in the presence (+) or absence (−) of CDPK II , with the antibodies listed to the right of each panel. The positions of PAGE molecular mass markers are shown in kilodaltons on the left of the figure. C, Specific Suc cleavage activities ( μ mol UDP-Glc min −1 mg −1 ) at pH 5.5, 6.5, 7.5, and 8.5 of wild-type SUS1 recombinants at time zero (black bars) or after a 30-min incubation in vitro in the presence (+, gray bars) or absence (−, white bars) of CDPK II .
    Figure Legend Snippet: Phosphorylation affects the Suc cleavage activity of SUS. A, Specific Suc cleavage activities ( μ mol UDP-Glc min −1 mg −1 ) at pH 5.5, 6.5, 7.5, and 8.5 of S170A SUS1 recombinants at time zero (black bars) or after a 30-min incubation in vitro in the presence (+, gray bars) or absence (−, white bars) of CDPK II . B, Immunoblot (IB) analyses of CDPK II and S170A SUS1 recombinants at time zero or after a 30-min incubation in vitro in the presence (+) or absence (−) of CDPK II , with the antibodies listed to the right of each panel. The positions of PAGE molecular mass markers are shown in kilodaltons on the left of the figure. C, Specific Suc cleavage activities ( μ mol UDP-Glc min −1 mg −1 ) at pH 5.5, 6.5, 7.5, and 8.5 of wild-type SUS1 recombinants at time zero (black bars) or after a 30-min incubation in vitro in the presence (+, gray bars) or absence (−, white bars) of CDPK II .

    Techniques Used: Activity Assay, Gas Chromatography, Incubation, In Vitro, Polyacrylamide Gel Electrophoresis

    Low pH affects the conformation of the amino terminus of SUS without affecting oligomerization state of the native protein. A, Immunoprecipitation (IP) of native s-SUS was performed at pH 7.5 or pH 5.5 with blank Protein-G beads (PG-only) or with the antibodies listed at the top of the figure. Immunoblots (IB) were performed on the IP pellets with the antibody listed to the right of the figure. The positions of PAGE molecular mass markers are shown in kilodaltons on the left of the figure. B, Sucrose cleavage activity ( μ mol UDP-Glc min −1 mL −1 × 10 −3 ) of native s-SUS resolved by size-exclusion chromatography at pH 6.0 (▪, solid line) or pH 7.5 (○, dashed line). The elution positions of molecular mass standards are shown in kilodaltons (kD) under the graph.
    Figure Legend Snippet: Low pH affects the conformation of the amino terminus of SUS without affecting oligomerization state of the native protein. A, Immunoprecipitation (IP) of native s-SUS was performed at pH 7.5 or pH 5.5 with blank Protein-G beads (PG-only) or with the antibodies listed at the top of the figure. Immunoblots (IB) were performed on the IP pellets with the antibody listed to the right of the figure. The positions of PAGE molecular mass markers are shown in kilodaltons on the left of the figure. B, Sucrose cleavage activity ( μ mol UDP-Glc min −1 mL −1 × 10 −3 ) of native s-SUS resolved by size-exclusion chromatography at pH 6.0 (▪, solid line) or pH 7.5 (○, dashed line). The elution positions of molecular mass standards are shown in kilodaltons (kD) under the graph.

    Techniques Used: Immunoprecipitation, Western Blot, Polyacrylamide Gel Electrophoresis, Activity Assay, Gas Chromatography, Size-exclusion Chromatography

    34) Product Images from "Decreased NAA in Gray Matter is Correlated with Decreased Availability of Acetate in White Matter in Postmortem Multiple Sclerosis Cortex"

    Article Title: Decreased NAA in Gray Matter is Correlated with Decreased Availability of Acetate in White Matter in Postmortem Multiple Sclerosis Cortex

    Journal: Neurochemical research

    doi: 10.1007/s11064-013-1151-8

    Treatment with the electron transport chain inhibitor antimycin A reduced NAA levels in SH-SY5Y cells without inducing neuronal cell loss or degeneration of neurites. a Representative HPLC chromatogram showing NAA peak retention time of 5.10 min with
    Figure Legend Snippet: Treatment with the electron transport chain inhibitor antimycin A reduced NAA levels in SH-SY5Y cells without inducing neuronal cell loss or degeneration of neurites. a Representative HPLC chromatogram showing NAA peak retention time of 5.10 min with

    Techniques Used: High Performance Liquid Chromatography

    The effect of antimycin A on respiration and levels of the NAA substrates, l -aspartate and acetyl-CoA, was determined in SH-SY5Y cells. OCR and ECAR were measured simultaneously in SH-SY5Y neuroblastoma cells before and after antimycin A treatment. a
    Figure Legend Snippet: The effect of antimycin A on respiration and levels of the NAA substrates, l -aspartate and acetyl-CoA, was determined in SH-SY5Y cells. OCR and ECAR were measured simultaneously in SH-SY5Y neuroblastoma cells before and after antimycin A treatment. a

    Techniques Used:

    35) Product Images from "Dengue virus nonstructural 3 protein interacts directly with human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and reduces its glycolytic activity"

    Article Title: Dengue virus nonstructural 3 protein interacts directly with human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and reduces its glycolytic activity

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-39157-7

    DENV2 NS3 protein decreases the intracellular GAPDH glycolytic activity in DENV2-infected and NS3-transfected cells. Intracellular GAPDH activity was monitored in a coupled reaction with TPI, in which dihydroxyacetone phosphate was converted into glyceraldehyde-3-phosphate, the GAPDH substrate. Kinetics of NADH were monitored by the increase in absorbance at 340 nm every 1 min for 90 min, using 50 µg of the cell extract of DENV2-infected ( A ) or NS3-transfected BHK-21 cells ( B ) as the source of the enzyme. The conversion of absorbance units into NADH production was determined by molar absorptivity of NADH (6.22 mM −1 cm −1 ). The concentration of NADH was converted into GAPDH activity, considering that one unit of enzyme activity corresponds to the reduction of 1 µM of β-NAD/min ( C ).
    Figure Legend Snippet: DENV2 NS3 protein decreases the intracellular GAPDH glycolytic activity in DENV2-infected and NS3-transfected cells. Intracellular GAPDH activity was monitored in a coupled reaction with TPI, in which dihydroxyacetone phosphate was converted into glyceraldehyde-3-phosphate, the GAPDH substrate. Kinetics of NADH were monitored by the increase in absorbance at 340 nm every 1 min for 90 min, using 50 µg of the cell extract of DENV2-infected ( A ) or NS3-transfected BHK-21 cells ( B ) as the source of the enzyme. The conversion of absorbance units into NADH production was determined by molar absorptivity of NADH (6.22 mM −1 cm −1 ). The concentration of NADH was converted into GAPDH activity, considering that one unit of enzyme activity corresponds to the reduction of 1 µM of β-NAD/min ( C ).

    Techniques Used: Activity Assay, Infection, Transfection, Concentration Assay

    36) Product Images from "Identification of a small molecule inhibitor of 3-phosphoglycerate dehydrogenase to target serine biosynthesis in cancers"

    Article Title: Identification of a small molecule inhibitor of 3-phosphoglycerate dehydrogenase to target serine biosynthesis in cancers

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1521548113

    CBR-5884 does not affect the lactate dehydrogenase (LDH) oligomerization state but inhibits a truncated form of PHGDH. ( A ) LDH was preincubated with CBR-5884 (0, 50, 200, or 400 μM) for 30 min before cross-linking with BS3 (0.25 or 2.5 mM), followed
    Figure Legend Snippet: CBR-5884 does not affect the lactate dehydrogenase (LDH) oligomerization state but inhibits a truncated form of PHGDH. ( A ) LDH was preincubated with CBR-5884 (0, 50, 200, or 400 μM) for 30 min before cross-linking with BS3 (0.25 or 2.5 mM), followed

    Techniques Used:

    37) Product Images from "Paradoxical Inhibition of Glycolysis by Pioglitazone Opposes the Mitochondriopathy Caused by AIF Deficiency"

    Article Title: Paradoxical Inhibition of Glycolysis by Pioglitazone Opposes the Mitochondriopathy Caused by AIF Deficiency

    Journal: EBioMedicine

    doi: 10.1016/j.ebiom.2017.02.013

    PIO interacts with and inhibits purified GAPDH. (A) Western blot analysis of purified GAPDH in the absence (left panel) or presence (right panel) of added dithiothreitol (DTT). Noticeably, no higher aggregation forms were detected in the stacking gel. (B) Quantities of different aggregation forms (tetra-, tri-, di-, and monomeric) in the absence of DTT (open bars: initial GAPDH suspension, GAPDH plus DMSO, GAPDH plus DMSO-solubilized PIO, or in the presence of DTT (green filled bars) added before or after PIO). (C) GAPDH (2.1 mIU/ml) fluorescence in the absence (blue dots) or presence (green dots) of 12.5 μM PIO against UV (280 nm) irradiation. The presence of 1.25‰ DMSO had per se a partial protecting effect against UV (grey dots). No changes in fluorescence were observed when the enzyme was kept away from UV irradiation (black dots). Noticeably initial fluorescence in the presence or absence of PIO, or DMSO was not different ruling out potential interference of these compounds with fluorescence assay conditions (excitation: 280 nm, 10 nm bandpass; emission: 330 nm, 10 nm bandpass). (D) Initial activity of rabbit skeletal muscle GAPDH (0.05 mIU/ml) measured in the forward direction (NADH accumulation) ( Velick, 1955 ) and of increasing amounts of PIO. (E) Activity measured in the backward direction (NADH oxidation) in the absence (blue open bar) or presence (green open bar) of 200 μM PIO, and in the presence of 1.7 mM cysteine and absence (blue filled bar) or presence (green filled bar) of 200 μM PIO. (F) Lineweaver-Burk plots of forward GAPDH activity (0.075 mIU/ml) in the absence (black line) or presence (green line) of 62.5 μM PIO. GAP, d -glyceraldehyde 3-phosphate.
    Figure Legend Snippet: PIO interacts with and inhibits purified GAPDH. (A) Western blot analysis of purified GAPDH in the absence (left panel) or presence (right panel) of added dithiothreitol (DTT). Noticeably, no higher aggregation forms were detected in the stacking gel. (B) Quantities of different aggregation forms (tetra-, tri-, di-, and monomeric) in the absence of DTT (open bars: initial GAPDH suspension, GAPDH plus DMSO, GAPDH plus DMSO-solubilized PIO, or in the presence of DTT (green filled bars) added before or after PIO). (C) GAPDH (2.1 mIU/ml) fluorescence in the absence (blue dots) or presence (green dots) of 12.5 μM PIO against UV (280 nm) irradiation. The presence of 1.25‰ DMSO had per se a partial protecting effect against UV (grey dots). No changes in fluorescence were observed when the enzyme was kept away from UV irradiation (black dots). Noticeably initial fluorescence in the presence or absence of PIO, or DMSO was not different ruling out potential interference of these compounds with fluorescence assay conditions (excitation: 280 nm, 10 nm bandpass; emission: 330 nm, 10 nm bandpass). (D) Initial activity of rabbit skeletal muscle GAPDH (0.05 mIU/ml) measured in the forward direction (NADH accumulation) ( Velick, 1955 ) and of increasing amounts of PIO. (E) Activity measured in the backward direction (NADH oxidation) in the absence (blue open bar) or presence (green open bar) of 200 μM PIO, and in the presence of 1.7 mM cysteine and absence (blue filled bar) or presence (green filled bar) of 200 μM PIO. (F) Lineweaver-Burk plots of forward GAPDH activity (0.075 mIU/ml) in the absence (black line) or presence (green line) of 62.5 μM PIO. GAP, d -glyceraldehyde 3-phosphate.

    Techniques Used: Purification, Western Blot, Fluorescence, Irradiation, Activity Assay

    38) Product Images from "The Plastid Casein Kinase 2 Phosphorylates Rubisco Activase at the Thr-78 Site but Is Not Essential for Regulation of Rubisco Activation State"

    Article Title: The Plastid Casein Kinase 2 Phosphorylates Rubisco Activase at the Thr-78 Site but Is Not Essential for Regulation of Rubisco Activation State

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2016.00404

    The cpck2 mutant is generally similar to wild type Arabidopsis suggesting that phosphorylation of RCA at the Thr-78 site is not playing an essential role in vivo . Induction kinetics of photosynthesis following pretreatment in (A) low light or (B) darkness. (C) Rubisco activation state after exposure of leaves for 60 min to low or high light as in the experiment in (A) . (D) Relative plant growth rate in short days determined based on leaf area expansion. (E) Chloroplast structure showing normal granal development and accumulation of starch granules at the end of the day.
    Figure Legend Snippet: The cpck2 mutant is generally similar to wild type Arabidopsis suggesting that phosphorylation of RCA at the Thr-78 site is not playing an essential role in vivo . Induction kinetics of photosynthesis following pretreatment in (A) low light or (B) darkness. (C) Rubisco activation state after exposure of leaves for 60 min to low or high light as in the experiment in (A) . (D) Relative plant growth rate in short days determined based on leaf area expansion. (E) Chloroplast structure showing normal granal development and accumulation of starch granules at the end of the day.

    Techniques Used: Mutagenesis, In Vivo, Activation Assay

    39) Product Images from "Inhibition of Calcium-Dependent Protein Kinase 1 (CDPK1) In Vitro by Pyrazolopyrimidine Derivatives Does Not Correlate with Sensitivity of Cryptosporidium parvum Growth in Cell Culture"

    Article Title: Inhibition of Calcium-Dependent Protein Kinase 1 (CDPK1) In Vitro by Pyrazolopyrimidine Derivatives Does Not Correlate with Sensitivity of Cryptosporidium parvum Growth in Cell Culture

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.01915-15

    Comparison of the activities of TgCDPK1 and CpCDPK1 enzyme inhibitors and inhibition of C. parvum growth. (A) Comparison of the sensitivities of the CpCDPK1 and TgCDPK1 enzymes to inhibitors in vitro . Linear regression is shown; dashed lines indicate
    Figure Legend Snippet: Comparison of the activities of TgCDPK1 and CpCDPK1 enzyme inhibitors and inhibition of C. parvum growth. (A) Comparison of the sensitivities of the CpCDPK1 and TgCDPK1 enzymes to inhibitors in vitro . Linear regression is shown; dashed lines indicate

    Techniques Used: Inhibition, In Vitro

    PP analogs inhibit CpCDPK1 and TgCDPK1 with similar potencies.
    Figure Legend Snippet: PP analogs inhibit CpCDPK1 and TgCDPK1 with similar potencies.

    Techniques Used:

    40) Product Images from "Role of Hydroxysteroid Dehydrogenase-Like 2 (HSDL2) in Human Ovarian Cancer"

    Article Title: Role of Hydroxysteroid Dehydrogenase-Like 2 (HSDL2) in Human Ovarian Cancer

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    doi: 10.12659/MSM.909418

    HSDL2 knockdown inhibited the migration and invasion of human ovarian cancer OVCAR-3 cells. ( A, C ) Representative Transwell migration ( A ) and invasion ( C ) assay of OVCAR-3 cells transfected with shHSDL2-1 lentivirus. ( B, D ) Quantification of migration ( B ) and invasion ( D ) abilities of OVCAR-3 transfected with shHSDL2-1 lentivirus. (** P
    Figure Legend Snippet: HSDL2 knockdown inhibited the migration and invasion of human ovarian cancer OVCAR-3 cells. ( A, C ) Representative Transwell migration ( A ) and invasion ( C ) assay of OVCAR-3 cells transfected with shHSDL2-1 lentivirus. ( B, D ) Quantification of migration ( B ) and invasion ( D ) abilities of OVCAR-3 transfected with shHSDL2-1 lentivirus. (** P

    Techniques Used: Migration, Transfection

    HSDL2 knockdown repressed the growth of human ovarian cancer OVCAR-3 cells in vivo . ( A ) Tumors grew more slowly after injection of OVCAR-3 cells transfected with shHSDL2-1 lentivirus than those transfected with shCtrl lentivirus. ( A ) The sizes of OVCAR-3 implants at 5 weeks after injection. ( B ) Average weight of the tumors 5 weeks after cell injection, n=10. (* P
    Figure Legend Snippet: HSDL2 knockdown repressed the growth of human ovarian cancer OVCAR-3 cells in vivo . ( A ) Tumors grew more slowly after injection of OVCAR-3 cells transfected with shHSDL2-1 lentivirus than those transfected with shCtrl lentivirus. ( A ) The sizes of OVCAR-3 implants at 5 weeks after injection. ( B ) Average weight of the tumors 5 weeks after cell injection, n=10. (* P

    Techniques Used: In Vivo, Injection, Transfection

    HSDL2 expression in ovarian cancer tissue samples. ( A ) qPCR showed that the levels of HSDL2 mRNA in cancer tissues were significantly different from those in normal tissues ( P
    Figure Legend Snippet: HSDL2 expression in ovarian cancer tissue samples. ( A ) qPCR showed that the levels of HSDL2 mRNA in cancer tissues were significantly different from those in normal tissues ( P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    HSDL2 knockdown augmented cell apoptosis in human ovarian cancer lines. Representative images of cell apoptosis analysis of human ovarian cancer OVCAR-3 ( A ) and SKOV3 ( B ) cells infected with lentivirus shCtrl ( left ), shHSDL2-1, or shHSDL2-2 ( right ). Cell apoptosis in OVCAR-3 ( C, D ) and SKOV3 ( E ) cells was promoted by HSDL2 knockdown. Data shown are the mean ±SD of cell percentage in apoptosis from 3 separate experiments. (** P
    Figure Legend Snippet: HSDL2 knockdown augmented cell apoptosis in human ovarian cancer lines. Representative images of cell apoptosis analysis of human ovarian cancer OVCAR-3 ( A ) and SKOV3 ( B ) cells infected with lentivirus shCtrl ( left ), shHSDL2-1, or shHSDL2-2 ( right ). Cell apoptosis in OVCAR-3 ( C, D ) and SKOV3 ( E ) cells was promoted by HSDL2 knockdown. Data shown are the mean ±SD of cell percentage in apoptosis from 3 separate experiments. (** P

    Techniques Used: Infection

    HSDL2 knockdown inhibited human ovarian cancer cell colony formation. ( A, B ) Photomicrographs of Giemsa-stained OVCAR-3 ( A ) and SKOV3 (B) clones in 6-well plates at 10 days post-infection. ( C–E ) The average number of OVCAR-3 ( C, D ) and SKOV3 ( E ) colonies was suppressed by HSDL2 knockdown. Data shown are the mean ±SD of cell colonies from 3 separate experiments. (* P =0.017; ** P
    Figure Legend Snippet: HSDL2 knockdown inhibited human ovarian cancer cell colony formation. ( A, B ) Photomicrographs of Giemsa-stained OVCAR-3 ( A ) and SKOV3 (B) clones in 6-well plates at 10 days post-infection. ( C–E ) The average number of OVCAR-3 ( C, D ) and SKOV3 ( E ) colonies was suppressed by HSDL2 knockdown. Data shown are the mean ±SD of cell colonies from 3 separate experiments. (* P =0.017; ** P

    Techniques Used: Staining, Infection

    HSDL2 knockdown leads to cell cycle arrest. ( A, B ) Representative image of cell cycle phase distribution following transfection with shHSDL2-1, shHSDL2-2, or shCtrl lentivirus. ( C–E ) Cell cycle phase distribution is expressed as a percentage of total cells. ** P
    Figure Legend Snippet: HSDL2 knockdown leads to cell cycle arrest. ( A, B ) Representative image of cell cycle phase distribution following transfection with shHSDL2-1, shHSDL2-2, or shCtrl lentivirus. ( C–E ) Cell cycle phase distribution is expressed as a percentage of total cells. ** P

    Techniques Used: Transfection

    HSDL2 knockdown inhibited cell proliferation in the human ovarian cancer cell lines as assessed by the Cellomics ArrayScan VTI and MTT assays. ( A, B ) Representative images of OVCAR-3 ( A ) and SKOV3 ( B ) cells infected with shCtrl lentivirus ( top ), shHSDL2-1, or shHSDL2-2 lentivirus ( bottom ) at different time points. ( C, E, G ) Proliferation of OVCAR-3 ( C, E ) and SKOV3 ( G ) was significantly blocked when HSDL2 expression was inhibited. The results are presented as the mean ±SD of 3 separate experiments. (** P
    Figure Legend Snippet: HSDL2 knockdown inhibited cell proliferation in the human ovarian cancer cell lines as assessed by the Cellomics ArrayScan VTI and MTT assays. ( A, B ) Representative images of OVCAR-3 ( A ) and SKOV3 ( B ) cells infected with shCtrl lentivirus ( top ), shHSDL2-1, or shHSDL2-2 lentivirus ( bottom ) at different time points. ( C, E, G ) Proliferation of OVCAR-3 ( C, E ) and SKOV3 ( G ) was significantly blocked when HSDL2 expression was inhibited. The results are presented as the mean ±SD of 3 separate experiments. (** P

    Techniques Used: MTT Assay, Infection, Expressing

    Efficient HSDL2 knockdown at the mRNA and protein levels in the human ovarian cancer OVCAR-3 cells using lentiviral-mediated RNAi. ( A, B ) HSDL2 mRNA ( A ) and protein ( B ) expression in human ovarian cancer cell lines SKOV3, HO8910, and OVCAR-3 and human normal ovarian epithelial cell line IOSE80. ( C, D ) Representative images of GFP expression in OVCAR-3 cells ( C ) and SKOV3 ( D ) after infection of shCtrl, shHSDL2-1, or shHSDL2-2 lentiviruses. ( E–G ) HSDL2 mRNA expression was analyzed by qPCR and was reduced by shHSDL2-1 or shHSDL2-2 lentiviruses compared to the shCtrl group, respectively. GAPDH was used as an internal control and the data represent the mean ±SD of 3 independent experiments. (** P
    Figure Legend Snippet: Efficient HSDL2 knockdown at the mRNA and protein levels in the human ovarian cancer OVCAR-3 cells using lentiviral-mediated RNAi. ( A, B ) HSDL2 mRNA ( A ) and protein ( B ) expression in human ovarian cancer cell lines SKOV3, HO8910, and OVCAR-3 and human normal ovarian epithelial cell line IOSE80. ( C, D ) Representative images of GFP expression in OVCAR-3 cells ( C ) and SKOV3 ( D ) after infection of shCtrl, shHSDL2-1, or shHSDL2-2 lentiviruses. ( E–G ) HSDL2 mRNA expression was analyzed by qPCR and was reduced by shHSDL2-1 or shHSDL2-2 lentiviruses compared to the shCtrl group, respectively. GAPDH was used as an internal control and the data represent the mean ±SD of 3 independent experiments. (** P

    Techniques Used: Expressing, Infection, Real-time Polymerase Chain Reaction

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