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  • 85
    ATCC aspergillus fumigatus
    Culturability of A. <t>fumigatus</t> spores grown under different conidiation temperatures (± standard error, n=3 experiments).
    Aspergillus Fumigatus, supplied by ATCC, used in various techniques. Bioz Stars score: 85/100, based on 319 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Millipore malate dehydrogenase mdh
    Design of the MCG pathway for efficient acetyl-CoA synthesis. a The MCG pathway can convert one C3 sugar to two acetyl-CoA via fixation of one CO 2 equivalent. PEP, phosphoenolpyruvate; Ac-CoA, acetyl-CoA; <t>Ppc,</t> phosphoenolpyruvate carboxylase; <t>Mdh,</t> malate dehydrogenase; Mtk, malate thiokinase; Mcl, malyl-CoA lyase; Gcl, glyoxylate carboligase; Tsr, tartronate semialdehyde reductase; Gk, glycerate kinase; Eno, enolase. b The MCG pathway, coupling with glycolate dehydrogenase, can assimilate glycolate to acetyl-CoA without net carbon loss. Gdh, glycolate dehydrogenase. The net reactions are shown in the yellow boxes
    Malate Dehydrogenase Mdh, supplied by Millipore, used in various techniques. Bioz Stars score: 98/100, based on 104 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Bristol Myers mdh
    Design of the MCG pathway for efficient acetyl-CoA synthesis. a The MCG pathway can convert one C3 sugar to two acetyl-CoA via fixation of one CO 2 equivalent. PEP, phosphoenolpyruvate; Ac-CoA, acetyl-CoA; <t>Ppc,</t> phosphoenolpyruvate carboxylase; <t>Mdh,</t> malate dehydrogenase; Mtk, malate thiokinase; Mcl, malyl-CoA lyase; Gcl, glyoxylate carboligase; Tsr, tartronate semialdehyde reductase; Gk, glycerate kinase; Eno, enolase. b The MCG pathway, coupling with glycolate dehydrogenase, can assimilate glycolate to acetyl-CoA without net carbon loss. Gdh, glycolate dehydrogenase. The net reactions are shown in the yellow boxes
    Mdh, supplied by Bristol Myers, used in various techniques. Bioz Stars score: 93/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    mdh1  (Abcam)
    94
    Abcam mdh1
    Deletion of SLC1A3 Impedes the ETC and Phenocopies Depletion of the Mitochondrial Aspartate Transporters AGC1 and AGC2 under Glutamine Deprivation (A) Schematic representation of the malate-aspartate shuttle (MAS). In brief, the MAS is a system that allows the transfer of electrons from cytosolic NADH to produce mitochondrial NADH where it is oxidized in the ETC. In the cytoplasm <t>MDH1</t> catalyzes the reduction of oxaloacetate (OAA), where it accepts an electron from NADH to produce malate and NAD + . Malate can then enter the mitochondria where it is oxidized by MDH2 to OAA, resulting in the formation of mitochondrial NADH. Mitochondrial OAA is transaminated into aspartate by GOT2 whereby aspartate exits the mitochondria in exchange for cytosolic glutamate through a carrier. OAA is recovered in the cytosol by GOT1. By coupling aspartate-glutamate exchange, the aspartate-glutamate carrier is essential for the shuttle and is thought to represent the rate-limiting step. (B) Respiratory profiles of HCT116 WT cells transiently depleted of SLC1A3 and grown for 24 hr in glutamine-free medium, in the presence of mitochondrial inhibitors (oligomycin, FCCP [carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone] antimycin A/rotenone). Arrows indicate incubation of cells with the indicated inhibitors. Data are presented as mean ± SEM of one representative experiment (n = 6 wells). (C) Oxygen consumption rates (OCR) of HCT116 WT and p53-null clones 2 days after glutamine deprivation as in (B). Data are presented as mean ± SEM of one representative experiment (n = 6 wells). (D) Proliferation of HCT116 WT cells transiently depleted of AGC1 or AGC2 using siRNA and cultured in glutamine-free condition (LHS) or complete medium (RHS). Data are presented as mean ± SEM of one representative experiment (averages of triplicate wells). The downregulation of these two proteins was confirmed by western blot (middle panel). (E) HCT116 WT cells transiently depleted of AGC1 or AGC2 using siRNA were grown for 2 days in glutamine-free medium and pulsed with [U- 13 C]aspartate for the final 24 hr. Extracellular levels of aspartate (m+4), alanine, and serine normalized to cell number were quantified over 24 hr. Data are presented as mean ± SEM of one representative experiment (averages of triplicate wells). (F and G) HCT116 WT cells transiently depleted of AGC1 or AGC2 using siRNA were grown for 2 days in glutamine-free medium and pulsed with [U- 13 C]aspartate for the final 24 hr. Intracellular TCA-cycle intermediates (F) and glutamate and glutamine levels (G) were measured. Data are presented as mean ± SEM of one representative experiment (averages of triplicate wells). .
    Mdh1, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 71 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Medicago cytosolic mdh
    The phylogenetic tree of ( A ) fumarase and ( B ) <t>MDH.</t> Trees were built using putative amino acids sequences from ( A ) fumarase and ( B ) MDH. Subcellular compartments are highlighted as in figure 2 : Mitochondrial isoforms are highlighted by yellow background, peroxissomal isoforms by orange background, and <t>cytosolic</t> isoforms by blue background. Sequences of putative proteins from plants are highlighted by green circle, yeast by red circle, animals by blue circle, algae by dark green circle, bacteria by pink circle, and cyanobacteria by gray circle.
    Cytosolic Mdh, supplied by Medicago, used in various techniques. Bioz Stars score: 86/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Novartis mdh consulting
    The phylogenetic tree of ( A ) fumarase and ( B ) <t>MDH.</t> Trees were built using putative amino acids sequences from ( A ) fumarase and ( B ) MDH. Subcellular compartments are highlighted as in figure 2 : Mitochondrial isoforms are highlighted by yellow background, peroxissomal isoforms by orange background, and <t>cytosolic</t> isoforms by blue background. Sequences of putative proteins from plants are highlighted by green circle, yeast by red circle, animals by blue circle, algae by dark green circle, bacteria by pink circle, and cyanobacteria by gray circle.
    Mdh Consulting, supplied by Novartis, used in various techniques. Bioz Stars score: 88/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Addgene inc mdh1 ires gfp vector
    The phylogenetic tree of ( A ) fumarase and ( B ) <t>MDH.</t> Trees were built using putative amino acids sequences from ( A ) fumarase and ( B ) MDH. Subcellular compartments are highlighted as in figure 2 : Mitochondrial isoforms are highlighted by yellow background, peroxissomal isoforms by orange background, and <t>cytosolic</t> isoforms by blue background. Sequences of putative proteins from plants are highlighted by green circle, yeast by red circle, animals by blue circle, algae by dark green circle, bacteria by pink circle, and cyanobacteria by gray circle.
    Mdh1 Ires Gfp Vector, supplied by Addgene inc, used in various techniques. Bioz Stars score: 88/100, based on 44 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore recombinant e coli malate dehydrogenase
    Detection and quantitation of malate dehydrogenase activity in the Leuginase® preparation. Graph shows the malate dehydrogenase (MDH) activity measured in terms of NADH production after 2:10 min at 37 °C, for different amounts of Leuginase® (5 IU/μL) and Aginasa® (5 IU/μL), and known quantities of recombinant  E. coli  malate dehydrogenase obtained from Sigma-Aldrich. Bars represent mean and SD of technical triplicates. Three different vials of Leuginase® were tested. The malate dehydrogenase activity in 0.1 μL of Leuginase® equals that of 0.025 μg of the recombinant MDH.
    Recombinant E Coli Malate Dehydrogenase, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Addgene inc mdh1 pgk gfp 2 0
    Detection and quantitation of malate dehydrogenase activity in the Leuginase® preparation. Graph shows the malate dehydrogenase (MDH) activity measured in terms of NADH production after 2:10 min at 37 °C, for different amounts of Leuginase® (5 IU/μL) and Aginasa® (5 IU/μL), and known quantities of recombinant  E. coli  malate dehydrogenase obtained from Sigma-Aldrich. Bars represent mean and SD of technical triplicates. Three different vials of Leuginase® were tested. The malate dehydrogenase activity in 0.1 μL of Leuginase® equals that of 0.025 μg of the recombinant MDH.
    Mdh1 Pgk Gfp 2 0, supplied by Addgene inc, used in various techniques. Bioz Stars score: 94/100, based on 34 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Lundbeck mdh consulting
    Detection and quantitation of malate dehydrogenase activity in the Leuginase® preparation. Graph shows the malate dehydrogenase (MDH) activity measured in terms of NADH production after 2:10 min at 37 °C, for different amounts of Leuginase® (5 IU/μL) and Aginasa® (5 IU/μL), and known quantities of recombinant  E. coli  malate dehydrogenase obtained from Sigma-Aldrich. Bars represent mean and SD of technical triplicates. Three different vials of Leuginase® were tested. The malate dehydrogenase activity in 0.1 μL of Leuginase® equals that of 0.025 μg of the recombinant MDH.
    Mdh Consulting, supplied by Lundbeck, used in various techniques. Bioz Stars score: 88/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Merck & Co mdh reports personal fees
    Detection and quantitation of malate dehydrogenase activity in the Leuginase® preparation. Graph shows the malate dehydrogenase (MDH) activity measured in terms of NADH production after 2:10 min at 37 °C, for different amounts of Leuginase® (5 IU/μL) and Aginasa® (5 IU/μL), and known quantities of recombinant  E. coli  malate dehydrogenase obtained from Sigma-Aldrich. Bars represent mean and SD of technical triplicates. Three different vials of Leuginase® were tested. The malate dehydrogenase activity in 0.1 μL of Leuginase® equals that of 0.025 μg of the recombinant MDH.
    Mdh Reports Personal Fees, supplied by Merck & Co, used in various techniques. Bioz Stars score: 92/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Addgene inc mdh1 pgk gfp vector
    Detection and quantitation of malate dehydrogenase activity in the Leuginase® preparation. Graph shows the malate dehydrogenase (MDH) activity measured in terms of NADH production after 2:10 min at 37 °C, for different amounts of Leuginase® (5 IU/μL) and Aginasa® (5 IU/μL), and known quantities of recombinant  E. coli  malate dehydrogenase obtained from Sigma-Aldrich. Bars represent mean and SD of technical triplicates. Three different vials of Leuginase® were tested. The malate dehydrogenase activity in 0.1 μL of Leuginase® equals that of 0.025 μg of the recombinant MDH.
    Mdh1 Pgk Gfp Vector, supplied by Addgene inc, used in various techniques. Bioz Stars score: 85/100, based on 38 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    ICF Macro 2010 mdhs
    Detection and quantitation of malate dehydrogenase activity in the Leuginase® preparation. Graph shows the malate dehydrogenase (MDH) activity measured in terms of NADH production after 2:10 min at 37 °C, for different amounts of Leuginase® (5 IU/μL) and Aginasa® (5 IU/μL), and known quantities of recombinant  E. coli  malate dehydrogenase obtained from Sigma-Aldrich. Bars represent mean and SD of technical triplicates. Three different vials of Leuginase® were tested. The malate dehydrogenase activity in 0.1 μL of Leuginase® equals that of 0.025 μg of the recombinant MDH.
    2010 Mdhs, supplied by ICF Macro, used in various techniques. Bioz Stars score: 88/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Abcam anti mdh1
    Elevated expression of <t>MDH1</t> and MDH2 in primary NSCLCs. (A) Transcript abundance (probe intensity) of the indicated genes in primary NSCLCs from the GSE31210 dataset. (B) Linear regression analysis was performed using GraphPad Prism to determine the relationship between the gene expression levels of MDH1 and MDH2 in the GSE31210 dataset. (C) Heat map representation of the relative mRNA levels of the MDH1 and MDH2 from the GSE33532 cohort. (D) Transcript abundance (probe intensity) of the indicated genes in primary NSCLCs from the GSE33532 dataset. (E) Heat map representation of the relative mRNA levels of the MDH1 and MDH2 genes from GSE33532 dataset. Differential expression was determined using Mann-Whitney U test combined with the false discovery rate (FDR) correction. Microarray gene expression data of two cohorts (GSE31210 and GSE33532) from Gene Expression Omnibus (GEO) database were analyzed. Differential expression was determined using Mann-Whitney U test combined with the false discovery rate (FDR) correction.
    Anti Mdh1, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Abcam coli his tagged mdh
    Elevated expression of <t>MDH1</t> and MDH2 in primary NSCLCs. (A) Transcript abundance (probe intensity) of the indicated genes in primary NSCLCs from the GSE31210 dataset. (B) Linear regression analysis was performed using GraphPad Prism to determine the relationship between the gene expression levels of MDH1 and MDH2 in the GSE31210 dataset. (C) Heat map representation of the relative mRNA levels of the MDH1 and MDH2 from the GSE33532 cohort. (D) Transcript abundance (probe intensity) of the indicated genes in primary NSCLCs from the GSE33532 dataset. (E) Heat map representation of the relative mRNA levels of the MDH1 and MDH2 genes from GSE33532 dataset. Differential expression was determined using Mann-Whitney U test combined with the false discovery rate (FDR) correction. Microarray gene expression data of two cohorts (GSE31210 and GSE33532) from Gene Expression Omnibus (GEO) database were analyzed. Differential expression was determined using Mann-Whitney U test combined with the false discovery rate (FDR) correction.
    Coli His Tagged Mdh, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore malic dehydrogenase from porcine heart
    Elevated expression of <t>MDH1</t> and MDH2 in primary NSCLCs. (A) Transcript abundance (probe intensity) of the indicated genes in primary NSCLCs from the GSE31210 dataset. (B) Linear regression analysis was performed using GraphPad Prism to determine the relationship between the gene expression levels of MDH1 and MDH2 in the GSE31210 dataset. (C) Heat map representation of the relative mRNA levels of the MDH1 and MDH2 from the GSE33532 cohort. (D) Transcript abundance (probe intensity) of the indicated genes in primary NSCLCs from the GSE33532 dataset. (E) Heat map representation of the relative mRNA levels of the MDH1 and MDH2 genes from GSE33532 dataset. Differential expression was determined using Mann-Whitney U test combined with the false discovery rate (FDR) correction. Microarray gene expression data of two cohorts (GSE31210 and GSE33532) from Gene Expression Omnibus (GEO) database were analyzed. Differential expression was determined using Mann-Whitney U test combined with the false discovery rate (FDR) correction.
    Malic Dehydrogenase From Porcine Heart, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore strain e coli bl21 δfrma nudf mdh hps phi
    The optimization of methanol metabolism in the synthetic E. coli <t>BL21/ΔfrmA-NudF-Mdh2-Hps-Phi.</t> a The optimization of a nitrogen source. b The optimization of methanol concentration. Error bars indicate standard error of the mean ( n = 3)
    Strain E Coli Bl21 δfrma Nudf Mdh Hps Phi, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Culturability of A. fumigatus spores grown under different conidiation temperatures (± standard error, n=3 experiments).

    Journal: Fungal biology

    Article Title: The Allergenicity of Aspergillus fumigatus conidia is influenced by growth temperature

    doi: 10.1016/j.funbio.2011.03.006

    Figure Lengend Snippet: Culturability of A. fumigatus spores grown under different conidiation temperatures (± standard error, n=3 experiments).

    Article Snippet: The cultivation of Aspergillus fumigatus (ATCC #34506) took place under three controlled temperatures (17°C, 25°C, and 32°C).

    Techniques:

    Allergenicity per 10 7 spores of Aspergillus fumigatus conidia cultivated under different sporulation temperatures. Error bars represent standard error values over three independent experimental replicates. ( Inset ) Protein content per 10 7 spores in conidia

    Journal: Fungal biology

    Article Title: The Allergenicity of Aspergillus fumigatus conidia is influenced by growth temperature

    doi: 10.1016/j.funbio.2011.03.006

    Figure Lengend Snippet: Allergenicity per 10 7 spores of Aspergillus fumigatus conidia cultivated under different sporulation temperatures. Error bars represent standard error values over three independent experimental replicates. ( Inset ) Protein content per 10 7 spores in conidia

    Article Snippet: The cultivation of Aspergillus fumigatus (ATCC #34506) took place under three controlled temperatures (17°C, 25°C, and 32°C).

    Techniques:

    Design of the MCG pathway for efficient acetyl-CoA synthesis. a The MCG pathway can convert one C3 sugar to two acetyl-CoA via fixation of one CO 2 equivalent. PEP, phosphoenolpyruvate; Ac-CoA, acetyl-CoA; Ppc, phosphoenolpyruvate carboxylase; Mdh, malate dehydrogenase; Mtk, malate thiokinase; Mcl, malyl-CoA lyase; Gcl, glyoxylate carboligase; Tsr, tartronate semialdehyde reductase; Gk, glycerate kinase; Eno, enolase. b The MCG pathway, coupling with glycolate dehydrogenase, can assimilate glycolate to acetyl-CoA without net carbon loss. Gdh, glycolate dehydrogenase. The net reactions are shown in the yellow boxes

    Journal: Nature Communications

    Article Title: Augmenting the Calvin–Benson–Bassham cycle by a synthetic malyl-CoA-glycerate carbon fixation pathway

    doi: 10.1038/s41467-018-04417-z

    Figure Lengend Snippet: Design of the MCG pathway for efficient acetyl-CoA synthesis. a The MCG pathway can convert one C3 sugar to two acetyl-CoA via fixation of one CO 2 equivalent. PEP, phosphoenolpyruvate; Ac-CoA, acetyl-CoA; Ppc, phosphoenolpyruvate carboxylase; Mdh, malate dehydrogenase; Mtk, malate thiokinase; Mcl, malyl-CoA lyase; Gcl, glyoxylate carboligase; Tsr, tartronate semialdehyde reductase; Gk, glycerate kinase; Eno, enolase. b The MCG pathway, coupling with glycolate dehydrogenase, can assimilate glycolate to acetyl-CoA without net carbon loss. Gdh, glycolate dehydrogenase. The net reactions are shown in the yellow boxes

    Article Snippet: Protein synthesis and purification Ppc, Eno, and Mdh were purchased from Sigma-Aldrich.

    Techniques:

    Specific enzymatic activity of 100 n m Eu‐MDH supplemented with 20 μ m EuCl 3 with SEM ( n =3). Conditions: 20 m m PIPES pH 7.2, 1 m m PES and KCN, 80 μ m DCPIP, 45 °C.

    Journal: Chembiochem

    Article Title: Similar but Not the Same: First Kinetic and Structural Analyses of a Methanol Dehydrogenase Containing a Europium Ion in the Active Site

    doi: 10.1002/cbic.201800130

    Figure Lengend Snippet: Specific enzymatic activity of 100 n m Eu‐MDH supplemented with 20 μ m EuCl 3 with SEM ( n =3). Conditions: 20 m m PIPES pH 7.2, 1 m m PES and KCN, 80 μ m DCPIP, 45 °C.

    Article Snippet: Each well contained assay mix [180 μL, PIPES (pH 7.2, 20 mm ), DCPIP (100 μm ), PES (1 mm ), KCN (1 mm )], Eu‐MDH (200 nm ), and—variously—LaCl3 , PrCl3 , EuCl3 , or YbCl3 (0.4 mm , 10 μL), or Millipore water (10 μL).

    Techniques: Activity Assay

    Titration of different REEs (20 μ m ) against the Eu‐MDH (200 n m ). Conditions: 20 m m PIPES pH 7.2, 1 m m PES, 1 m m KCN, 100 μ m DCPIP, 45 °C. Normalized specific enzymatic activity with standard error of the mean (SEM, n =4) is shown.

    Journal: Chembiochem

    Article Title: Similar but Not the Same: First Kinetic and Structural Analyses of a Methanol Dehydrogenase Containing a Europium Ion in the Active Site

    doi: 10.1002/cbic.201800130

    Figure Lengend Snippet: Titration of different REEs (20 μ m ) against the Eu‐MDH (200 n m ). Conditions: 20 m m PIPES pH 7.2, 1 m m PES, 1 m m KCN, 100 μ m DCPIP, 45 °C. Normalized specific enzymatic activity with standard error of the mean (SEM, n =4) is shown.

    Article Snippet: Each well contained assay mix [180 μL, PIPES (pH 7.2, 20 mm ), DCPIP (100 μm ), PES (1 mm ), KCN (1 mm )], Eu‐MDH (200 nm ), and—variously—LaCl3 , PrCl3 , EuCl3 , or YbCl3 (0.4 mm , 10 μL), or Millipore water (10 μL).

    Techniques: Titration, Activity Assay

    Deletion of SLC1A3 Impedes the ETC and Phenocopies Depletion of the Mitochondrial Aspartate Transporters AGC1 and AGC2 under Glutamine Deprivation (A) Schematic representation of the malate-aspartate shuttle (MAS). In brief, the MAS is a system that allows the transfer of electrons from cytosolic NADH to produce mitochondrial NADH where it is oxidized in the ETC. In the cytoplasm MDH1 catalyzes the reduction of oxaloacetate (OAA), where it accepts an electron from NADH to produce malate and NAD + . Malate can then enter the mitochondria where it is oxidized by MDH2 to OAA, resulting in the formation of mitochondrial NADH. Mitochondrial OAA is transaminated into aspartate by GOT2 whereby aspartate exits the mitochondria in exchange for cytosolic glutamate through a carrier. OAA is recovered in the cytosol by GOT1. By coupling aspartate-glutamate exchange, the aspartate-glutamate carrier is essential for the shuttle and is thought to represent the rate-limiting step. (B) Respiratory profiles of HCT116 WT cells transiently depleted of SLC1A3 and grown for 24 hr in glutamine-free medium, in the presence of mitochondrial inhibitors (oligomycin, FCCP [carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone] antimycin A/rotenone). Arrows indicate incubation of cells with the indicated inhibitors. Data are presented as mean ± SEM of one representative experiment (n = 6 wells). (C) Oxygen consumption rates (OCR) of HCT116 WT and p53-null clones 2 days after glutamine deprivation as in (B). Data are presented as mean ± SEM of one representative experiment (n = 6 wells). (D) Proliferation of HCT116 WT cells transiently depleted of AGC1 or AGC2 using siRNA and cultured in glutamine-free condition (LHS) or complete medium (RHS). Data are presented as mean ± SEM of one representative experiment (averages of triplicate wells). The downregulation of these two proteins was confirmed by western blot (middle panel). (E) HCT116 WT cells transiently depleted of AGC1 or AGC2 using siRNA were grown for 2 days in glutamine-free medium and pulsed with [U- 13 C]aspartate for the final 24 hr. Extracellular levels of aspartate (m+4), alanine, and serine normalized to cell number were quantified over 24 hr. Data are presented as mean ± SEM of one representative experiment (averages of triplicate wells). (F and G) HCT116 WT cells transiently depleted of AGC1 or AGC2 using siRNA were grown for 2 days in glutamine-free medium and pulsed with [U- 13 C]aspartate for the final 24 hr. Intracellular TCA-cycle intermediates (F) and glutamate and glutamine levels (G) were measured. Data are presented as mean ± SEM of one representative experiment (averages of triplicate wells). .

    Journal: Cell Metabolism

    Article Title: A Role for p53 in the Adaptation to Glutamine Starvation through the Expression of SLC1A3

    doi: 10.1016/j.cmet.2018.07.005

    Figure Lengend Snippet: Deletion of SLC1A3 Impedes the ETC and Phenocopies Depletion of the Mitochondrial Aspartate Transporters AGC1 and AGC2 under Glutamine Deprivation (A) Schematic representation of the malate-aspartate shuttle (MAS). In brief, the MAS is a system that allows the transfer of electrons from cytosolic NADH to produce mitochondrial NADH where it is oxidized in the ETC. In the cytoplasm MDH1 catalyzes the reduction of oxaloacetate (OAA), where it accepts an electron from NADH to produce malate and NAD + . Malate can then enter the mitochondria where it is oxidized by MDH2 to OAA, resulting in the formation of mitochondrial NADH. Mitochondrial OAA is transaminated into aspartate by GOT2 whereby aspartate exits the mitochondria in exchange for cytosolic glutamate through a carrier. OAA is recovered in the cytosol by GOT1. By coupling aspartate-glutamate exchange, the aspartate-glutamate carrier is essential for the shuttle and is thought to represent the rate-limiting step. (B) Respiratory profiles of HCT116 WT cells transiently depleted of SLC1A3 and grown for 24 hr in glutamine-free medium, in the presence of mitochondrial inhibitors (oligomycin, FCCP [carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone] antimycin A/rotenone). Arrows indicate incubation of cells with the indicated inhibitors. Data are presented as mean ± SEM of one representative experiment (n = 6 wells). (C) Oxygen consumption rates (OCR) of HCT116 WT and p53-null clones 2 days after glutamine deprivation as in (B). Data are presented as mean ± SEM of one representative experiment (n = 6 wells). (D) Proliferation of HCT116 WT cells transiently depleted of AGC1 or AGC2 using siRNA and cultured in glutamine-free condition (LHS) or complete medium (RHS). Data are presented as mean ± SEM of one representative experiment (averages of triplicate wells). The downregulation of these two proteins was confirmed by western blot (middle panel). (E) HCT116 WT cells transiently depleted of AGC1 or AGC2 using siRNA were grown for 2 days in glutamine-free medium and pulsed with [U- 13 C]aspartate for the final 24 hr. Extracellular levels of aspartate (m+4), alanine, and serine normalized to cell number were quantified over 24 hr. Data are presented as mean ± SEM of one representative experiment (averages of triplicate wells). (F and G) HCT116 WT cells transiently depleted of AGC1 or AGC2 using siRNA were grown for 2 days in glutamine-free medium and pulsed with [U- 13 C]aspartate for the final 24 hr. Intracellular TCA-cycle intermediates (F) and glutamate and glutamine levels (G) were measured. Data are presented as mean ± SEM of one representative experiment (averages of triplicate wells). .

    Article Snippet: Primary antibodies used were as follows: SLC1A3 (5685), AGC1 (64169), MDH2 (11908), phospho-p53 (S15) (9284) from Cell Signaling Technology; p53 (DO-1, sc-126), p21 (sc-397), ACTIN (sc-1616), AGC2 (sc-393303), MDM2 (SMP14, sc-965) from Santa Cruz Biotechnology; GOT1 (ab170950), GOT2 (ab90562), MDH1 (ab180152) from Abcam; glutamine synthetase (610517) from BD Transduction Laboratories.

    Techniques: Incubation, Clone Assay, Cell Culture, Western Blot

    Reductive Carboxylation Supports Glycolytic Flux via NADH Channeling between MDH1 and GAPDH (A) Percentage of contribution to ATP production for the indicated enzymes, as predicted by metabolic modeling in mT80 and mT7 models. (B) Total levels of NAD + /NADH in shIDH1 and shMDH1 mT7 and mT80 cells, compared to shNTC controls, in basal conditions measured by enzymatic assay. (C) Schematic representation of labeling pattern originating from 4- 2 H 1 -glucose. Deuterium atoms are represented as green filled circles. (D and E) Proportion of total pool of malate m+1 originating from 4- 2 H 1 -glucose in mT7, mT45, and mT80 cells (D) and shMDH1 mT80 cells (E). (F) Levels of secreted lactate m+3 upon incubation of shMDH1 mT7 and mT80 cells with (U)- 13 C-glucose. Data are normalized to shNTC controls. (G) Proportion of total pool for citrate m+1 and malate m+1 originating from 1- 13 C-glutamine in mT7, mT45, and mT80 cells upon treatment with 0.5 μM of the GAPDH inhibitor heptelidic acid. (H) GAPDH IP on lysates of mT7, mT45, and mT80 cells. The interaction between GAPDH and MDH1 is shown by coIP. Immunoglobulin G (IgG) is used in negative isotype controls. Representative images from two independent experiments. (I) Immunofluorescence images of mT7, mT45, and mT80 cells stained with DAPI (blue), GAPDH (green), and MDH1 (red). (B and D–G) Data are mean ± SEM from at least three independent cultures. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, two-sided t test (B and E–G). ∗ p ≤ 0.05, one-way ANOVA (D). GAA, glutamate aspartate antiporter.

    Journal: Molecular Cell

    Article Title: NADH Shuttling Couples Cytosolic Reductive Carboxylation of Glutamine with Glycolysis in Cells with Mitochondrial Dysfunction

    doi: 10.1016/j.molcel.2018.01.034

    Figure Lengend Snippet: Reductive Carboxylation Supports Glycolytic Flux via NADH Channeling between MDH1 and GAPDH (A) Percentage of contribution to ATP production for the indicated enzymes, as predicted by metabolic modeling in mT80 and mT7 models. (B) Total levels of NAD + /NADH in shIDH1 and shMDH1 mT7 and mT80 cells, compared to shNTC controls, in basal conditions measured by enzymatic assay. (C) Schematic representation of labeling pattern originating from 4- 2 H 1 -glucose. Deuterium atoms are represented as green filled circles. (D and E) Proportion of total pool of malate m+1 originating from 4- 2 H 1 -glucose in mT7, mT45, and mT80 cells (D) and shMDH1 mT80 cells (E). (F) Levels of secreted lactate m+3 upon incubation of shMDH1 mT7 and mT80 cells with (U)- 13 C-glucose. Data are normalized to shNTC controls. (G) Proportion of total pool for citrate m+1 and malate m+1 originating from 1- 13 C-glutamine in mT7, mT45, and mT80 cells upon treatment with 0.5 μM of the GAPDH inhibitor heptelidic acid. (H) GAPDH IP on lysates of mT7, mT45, and mT80 cells. The interaction between GAPDH and MDH1 is shown by coIP. Immunoglobulin G (IgG) is used in negative isotype controls. Representative images from two independent experiments. (I) Immunofluorescence images of mT7, mT45, and mT80 cells stained with DAPI (blue), GAPDH (green), and MDH1 (red). (B and D–G) Data are mean ± SEM from at least three independent cultures. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, two-sided t test (B and E–G). ∗ p ≤ 0.05, one-way ANOVA (D). GAA, glutamate aspartate antiporter.

    Article Snippet: Cells were washed three times with TBS and incubated overnight at 4°C with a solution containing mouse anti-human GAPDH (Abcam cat. no. ab8245) and rabbit anti-human MDH1 (Abcam cat. no. ab180152) at a 1:100 dilution.

    Techniques: Enzymatic Assay, Labeling, Incubation, Co-Immunoprecipitation Assay, Immunofluorescence, Staining

    Aspartate Transamination Supports Flux through MDH1 and Generation of Malate (A) Schematic representation of MAS and labeling patterns originating from (U)- 13 C-aspartate. (B and C) Proportion of total pool of malate m+4 (B) and fumarate m+4 (C) in mT7, mT45, and mT80 cells grown in the presence of U- 13 C-aspartate upon treatment with vehicle control or 0.5 μM rotenone. (D) Malate m+4 levels originating from (U)- 13 C-aspartate in mT80 cells upon silencing of GOT1. Data are normalized to intracellular levels of aspartate m+4 and are mean ± SD from one independent experiment. (E and F) Cell growth of mT7, mT45, and mT80 cells grown in 25 mM galactose and supplemented with 5 mM aspartate (E) upon treatment with 2 mM of the transaminase inhibitor aminooxyacetate (F). Data are normalized on cell growth of vehicle control (E) or on cell growth in the presence of aspartate only (F). (G) Cell growth of mT80 cells grown in 25 mM galactose and supplemented with 5 mM aspartate upon silencing of GOT1. Data are normalized to the cell growth rate of vehicle control. (H and I) Total levels of NAD + /NADH in mT7, mT45, and mT80 cells (H) or shMDH1 mT80 cells (I) upon supplementation with 5 mM aspartate. Data are normalized on vehicle control. (J and K) Secretion of lactate of mT7, mT45, and mT80 cells (J) or shMDH1 mT80 cells (K) upon supplementation with 5 mM aspartate. Data are normalized on vehicle control. (B, C, and E–K) Data are mean ± SEM from at least three independent cultures. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, two-sided t test; n.s., not significant (B, C, E, and G–K). ∗∗∗ p ≤ 0.001, one-way ANOVA (F).

    Journal: Molecular Cell

    Article Title: NADH Shuttling Couples Cytosolic Reductive Carboxylation of Glutamine with Glycolysis in Cells with Mitochondrial Dysfunction

    doi: 10.1016/j.molcel.2018.01.034

    Figure Lengend Snippet: Aspartate Transamination Supports Flux through MDH1 and Generation of Malate (A) Schematic representation of MAS and labeling patterns originating from (U)- 13 C-aspartate. (B and C) Proportion of total pool of malate m+4 (B) and fumarate m+4 (C) in mT7, mT45, and mT80 cells grown in the presence of U- 13 C-aspartate upon treatment with vehicle control or 0.5 μM rotenone. (D) Malate m+4 levels originating from (U)- 13 C-aspartate in mT80 cells upon silencing of GOT1. Data are normalized to intracellular levels of aspartate m+4 and are mean ± SD from one independent experiment. (E and F) Cell growth of mT7, mT45, and mT80 cells grown in 25 mM galactose and supplemented with 5 mM aspartate (E) upon treatment with 2 mM of the transaminase inhibitor aminooxyacetate (F). Data are normalized on cell growth of vehicle control (E) or on cell growth in the presence of aspartate only (F). (G) Cell growth of mT80 cells grown in 25 mM galactose and supplemented with 5 mM aspartate upon silencing of GOT1. Data are normalized to the cell growth rate of vehicle control. (H and I) Total levels of NAD + /NADH in mT7, mT45, and mT80 cells (H) or shMDH1 mT80 cells (I) upon supplementation with 5 mM aspartate. Data are normalized on vehicle control. (J and K) Secretion of lactate of mT7, mT45, and mT80 cells (J) or shMDH1 mT80 cells (K) upon supplementation with 5 mM aspartate. Data are normalized on vehicle control. (B, C, and E–K) Data are mean ± SEM from at least three independent cultures. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, two-sided t test; n.s., not significant (B, C, E, and G–K). ∗∗∗ p ≤ 0.001, one-way ANOVA (F).

    Article Snippet: Cells were washed three times with TBS and incubated overnight at 4°C with a solution containing mouse anti-human GAPDH (Abcam cat. no. ab8245) and rabbit anti-human MDH1 (Abcam cat. no. ab180152) at a 1:100 dilution.

    Techniques: Labeling

    Reductive Glutamine Carboxylation Regulates NAD Redox Balance and Supports Glycolysis in Response to Mitochondrial Dysfunction Reduced turnover of NADH by mitochondria leads to impairment of the MAS and increase of cytosolic NADH. This in turn induces reductive carboxylation of glutamine, providing carbons for NADH-coupled MDH1, thus regulating NAD redox state and enhancing GAPDH activity. Increased glycolytic turnover supports ATP production in the cytosol, and this is associated with cell migration.

    Journal: Molecular Cell

    Article Title: NADH Shuttling Couples Cytosolic Reductive Carboxylation of Glutamine with Glycolysis in Cells with Mitochondrial Dysfunction

    doi: 10.1016/j.molcel.2018.01.034

    Figure Lengend Snippet: Reductive Glutamine Carboxylation Regulates NAD Redox Balance and Supports Glycolysis in Response to Mitochondrial Dysfunction Reduced turnover of NADH by mitochondria leads to impairment of the MAS and increase of cytosolic NADH. This in turn induces reductive carboxylation of glutamine, providing carbons for NADH-coupled MDH1, thus regulating NAD redox state and enhancing GAPDH activity. Increased glycolytic turnover supports ATP production in the cytosol, and this is associated with cell migration.

    Article Snippet: Cells were washed three times with TBS and incubated overnight at 4°C with a solution containing mouse anti-human GAPDH (Abcam cat. no. ab8245) and rabbit anti-human MDH1 (Abcam cat. no. ab180152) at a 1:100 dilution.

    Techniques: Activity Assay, Migration

    Mitochondrial Dysfunction Is Linked with Cell Migration (A) Enrichment p values (−log 10 ) of gene ontology (GO) biological processes involved in cell migration and cytoskeleton remodeling as obtained with measurements of protein abundance by proteomics. Red dashed line indicates false discovery rate (FDR) = 0.05. (B and D) Migration speed of mT7, mT45, and mT80 cells (B) or shMDH1 mT80 cells (D) measured by wound healing assay. (C and E) Values of J ATP consumption due to cytoskeleton remodeling based on calculations from OCR and ECAR data upon treatment with 1 μM nocodazole in mT7, mT45, and mT80 cells (C) or mT80 shMDH1 cells (E). (F) Immunofluorescence images of mT7, mT45, and mT80 cells stained with DAPI (blue), phalloidin (green), or antibody against MDH1 (red). White arrows indicate areas of co-localization between MDH1 and actin in mT80 cells. (G) Quantification of co-localization between MDH1 and phalloidin (actin). Data were obtained from 20–30 ROIs per condition. (B–E) Data are mean ± SEM from three to four independent cultures and were normalized on mean values of each experiment. ∗ p ≤ 0.05 and ∗∗∗ p ≤ 0.001, ANOVA (B, C, and G) or Dunnett’s test (D).

    Journal: Molecular Cell

    Article Title: NADH Shuttling Couples Cytosolic Reductive Carboxylation of Glutamine with Glycolysis in Cells with Mitochondrial Dysfunction

    doi: 10.1016/j.molcel.2018.01.034

    Figure Lengend Snippet: Mitochondrial Dysfunction Is Linked with Cell Migration (A) Enrichment p values (−log 10 ) of gene ontology (GO) biological processes involved in cell migration and cytoskeleton remodeling as obtained with measurements of protein abundance by proteomics. Red dashed line indicates false discovery rate (FDR) = 0.05. (B and D) Migration speed of mT7, mT45, and mT80 cells (B) or shMDH1 mT80 cells (D) measured by wound healing assay. (C and E) Values of J ATP consumption due to cytoskeleton remodeling based on calculations from OCR and ECAR data upon treatment with 1 μM nocodazole in mT7, mT45, and mT80 cells (C) or mT80 shMDH1 cells (E). (F) Immunofluorescence images of mT7, mT45, and mT80 cells stained with DAPI (blue), phalloidin (green), or antibody against MDH1 (red). White arrows indicate areas of co-localization between MDH1 and actin in mT80 cells. (G) Quantification of co-localization between MDH1 and phalloidin (actin). Data were obtained from 20–30 ROIs per condition. (B–E) Data are mean ± SEM from three to four independent cultures and were normalized on mean values of each experiment. ∗ p ≤ 0.05 and ∗∗∗ p ≤ 0.001, ANOVA (B, C, and G) or Dunnett’s test (D).

    Article Snippet: Cells were washed three times with TBS and incubated overnight at 4°C with a solution containing mouse anti-human GAPDH (Abcam cat. no. ab8245) and rabbit anti-human MDH1 (Abcam cat. no. ab180152) at a 1:100 dilution.

    Techniques: Migration, Wound Healing Assay, Immunofluorescence, Staining

    The phylogenetic tree of ( A ) fumarase and ( B ) MDH. Trees were built using putative amino acids sequences from ( A ) fumarase and ( B ) MDH. Subcellular compartments are highlighted as in figure 2 : Mitochondrial isoforms are highlighted by yellow background, peroxissomal isoforms by orange background, and cytosolic isoforms by blue background. Sequences of putative proteins from plants are highlighted by green circle, yeast by red circle, animals by blue circle, algae by dark green circle, bacteria by pink circle, and cyanobacteria by gray circle.

    Journal: Genome Biology and Evolution

    Article Title: Evolution and Functional Implications of the Tricarboxylic Acid Cycle as Revealed by Phylogenetic Analysis

    doi: 10.1093/gbe/evu221

    Figure Lengend Snippet: The phylogenetic tree of ( A ) fumarase and ( B ) MDH. Trees were built using putative amino acids sequences from ( A ) fumarase and ( B ) MDH. Subcellular compartments are highlighted as in figure 2 : Mitochondrial isoforms are highlighted by yellow background, peroxissomal isoforms by orange background, and cytosolic isoforms by blue background. Sequences of putative proteins from plants are highlighted by green circle, yeast by red circle, animals by blue circle, algae by dark green circle, bacteria by pink circle, and cyanobacteria by gray circle.

    Article Snippet: In addition, overexpression of cytosolic MDH in alfalfa (Medicago sativa ) increased aluminum tolerance through metal chelation in the soil ( ).

    Techniques:

    Detection and quantitation of malate dehydrogenase activity in the Leuginase® preparation. Graph shows the malate dehydrogenase (MDH) activity measured in terms of NADH production after 2:10 min at 37 °C, for different amounts of Leuginase® (5 IU/μL) and Aginasa® (5 IU/μL), and known quantities of recombinant  E. coli  malate dehydrogenase obtained from Sigma-Aldrich. Bars represent mean and SD of technical triplicates. Three different vials of Leuginase® were tested. The malate dehydrogenase activity in 0.1 μL of Leuginase® equals that of 0.025 μg of the recombinant MDH.

    Journal: EBioMedicine

    Article Title: Low Bioavailability and High Immunogenicity of a New Brand of E. colil-Asparaginase with Active Host Contaminating Proteins

    doi: 10.1016/j.ebiom.2018.03.005

    Figure Lengend Snippet: Detection and quantitation of malate dehydrogenase activity in the Leuginase® preparation. Graph shows the malate dehydrogenase (MDH) activity measured in terms of NADH production after 2:10 min at 37 °C, for different amounts of Leuginase® (5 IU/μL) and Aginasa® (5 IU/μL), and known quantities of recombinant E. coli malate dehydrogenase obtained from Sigma-Aldrich. Bars represent mean and SD of technical triplicates. Three different vials of Leuginase® were tested. The malate dehydrogenase activity in 0.1 μL of Leuginase® equals that of 0.025 μg of the recombinant MDH.

    Article Snippet: Mass inference was obtained by comparison to a standard curve made with recombinant E. coli malate dehydrogenase (Sigma-Aldrich, cat# SRP6105).

    Techniques: Quantitation Assay, Activity Assay, Recombinant

    Elevated expression of MDH1 and MDH2 in primary NSCLCs. (A) Transcript abundance (probe intensity) of the indicated genes in primary NSCLCs from the GSE31210 dataset. (B) Linear regression analysis was performed using GraphPad Prism to determine the relationship between the gene expression levels of MDH1 and MDH2 in the GSE31210 dataset. (C) Heat map representation of the relative mRNA levels of the MDH1 and MDH2 from the GSE33532 cohort. (D) Transcript abundance (probe intensity) of the indicated genes in primary NSCLCs from the GSE33532 dataset. (E) Heat map representation of the relative mRNA levels of the MDH1 and MDH2 genes from GSE33532 dataset. Differential expression was determined using Mann-Whitney U test combined with the false discovery rate (FDR) correction. Microarray gene expression data of two cohorts (GSE31210 and GSE33532) from Gene Expression Omnibus (GEO) database were analyzed. Differential expression was determined using Mann-Whitney U test combined with the false discovery rate (FDR) correction.

    Journal: Journal of Cancer

    Article Title: Characterization of the Role of the Malate Dehydrogenases to Lung Tumor Cell Survival

    doi: 10.7150/jca.19373

    Figure Lengend Snippet: Elevated expression of MDH1 and MDH2 in primary NSCLCs. (A) Transcript abundance (probe intensity) of the indicated genes in primary NSCLCs from the GSE31210 dataset. (B) Linear regression analysis was performed using GraphPad Prism to determine the relationship between the gene expression levels of MDH1 and MDH2 in the GSE31210 dataset. (C) Heat map representation of the relative mRNA levels of the MDH1 and MDH2 from the GSE33532 cohort. (D) Transcript abundance (probe intensity) of the indicated genes in primary NSCLCs from the GSE33532 dataset. (E) Heat map representation of the relative mRNA levels of the MDH1 and MDH2 genes from GSE33532 dataset. Differential expression was determined using Mann-Whitney U test combined with the false discovery rate (FDR) correction. Microarray gene expression data of two cohorts (GSE31210 and GSE33532) from Gene Expression Omnibus (GEO) database were analyzed. Differential expression was determined using Mann-Whitney U test combined with the false discovery rate (FDR) correction.

    Article Snippet: Protein lysates prepared using Complete Lysis-M buffer (Roche Diagnostics, Risch-Rotkreutz, Switzerland) supplemented with protease inhibitors (Roche Diagnostics, Risch-Rotkreutz, Switzerland) were mixed with Laemmeli's loading buffer, boiled and separated on 15% SDS-PAGE at 40 mA followed by transfer to nitrocellulose membranes for 90 min at 120 V. Membranes were blocked for 1h with 5% non‐fat milk in TBS at room temperature and subsequently probed with the following antibodies: anti-MDH1 (ab76616, Abcam), anti-MDH2 (clone # 11908, Cell Signaling, Danvers, MA, USA) and anti-Actin (Sigma Aldrich, St Louis, MO, USA).

    Techniques: Expressing, MANN-WHITNEY, Microarray

    MDH1 has significantly higher activity than MDH2 in tumor cells. (A) Enzyme kinetic analysis of MDH1 and MDH2 activities measured in 8 tumor cell lines for 30 min. (B) Cumulative from the tumor cell line panel (black bar) and cumulative data from the 3 normal cells (gray bar). Activity was normalized per mg protein. Error bars, ±SEM. ***p

    Journal: Journal of Cancer

    Article Title: Characterization of the Role of the Malate Dehydrogenases to Lung Tumor Cell Survival

    doi: 10.7150/jca.19373

    Figure Lengend Snippet: MDH1 has significantly higher activity than MDH2 in tumor cells. (A) Enzyme kinetic analysis of MDH1 and MDH2 activities measured in 8 tumor cell lines for 30 min. (B) Cumulative from the tumor cell line panel (black bar) and cumulative data from the 3 normal cells (gray bar). Activity was normalized per mg protein. Error bars, ±SEM. ***p

    Article Snippet: Protein lysates prepared using Complete Lysis-M buffer (Roche Diagnostics, Risch-Rotkreutz, Switzerland) supplemented with protease inhibitors (Roche Diagnostics, Risch-Rotkreutz, Switzerland) were mixed with Laemmeli's loading buffer, boiled and separated on 15% SDS-PAGE at 40 mA followed by transfer to nitrocellulose membranes for 90 min at 120 V. Membranes were blocked for 1h with 5% non‐fat milk in TBS at room temperature and subsequently probed with the following antibodies: anti-MDH1 (ab76616, Abcam), anti-MDH2 (clone # 11908, Cell Signaling, Danvers, MA, USA) and anti-Actin (Sigma Aldrich, St Louis, MO, USA).

    Techniques: Activity Assay

    MDH1 has functional and prognostic value in NSCLCs. (A) Total cellular malate dehydrogenase activity measured in 11 cell lines. The blue bar is cumulative data from the 3 normal cells and the red bar is cumulative from 8 tumor cell lines. Activity was normalized per mg protein. (B) Kaplan-Meier Analysis of MDH1 expression and overall survival in 226 NSCLC patients. (C) Tissue-Microarray analysis (TMA) of the MDH1 and MDH2 protein expression level in 33 NSCLCs and 33 normal adjacent lung tissues. Representative images are shown. (D) Expression level of the malate dehydrogenases in a cell line panel consisting of NSCLCs and normal lung cells. (E) Effect of genetic depletion on cell viability in the cell line panel, by two independent siRNAs, targeting MDH1 and MDH2, respectively. Cell death is presented as fold over control siRNA. Error bars, ±SEM. *p

    Journal: Journal of Cancer

    Article Title: Characterization of the Role of the Malate Dehydrogenases to Lung Tumor Cell Survival

    doi: 10.7150/jca.19373

    Figure Lengend Snippet: MDH1 has functional and prognostic value in NSCLCs. (A) Total cellular malate dehydrogenase activity measured in 11 cell lines. The blue bar is cumulative data from the 3 normal cells and the red bar is cumulative from 8 tumor cell lines. Activity was normalized per mg protein. (B) Kaplan-Meier Analysis of MDH1 expression and overall survival in 226 NSCLC patients. (C) Tissue-Microarray analysis (TMA) of the MDH1 and MDH2 protein expression level in 33 NSCLCs and 33 normal adjacent lung tissues. Representative images are shown. (D) Expression level of the malate dehydrogenases in a cell line panel consisting of NSCLCs and normal lung cells. (E) Effect of genetic depletion on cell viability in the cell line panel, by two independent siRNAs, targeting MDH1 and MDH2, respectively. Cell death is presented as fold over control siRNA. Error bars, ±SEM. *p

    Article Snippet: Protein lysates prepared using Complete Lysis-M buffer (Roche Diagnostics, Risch-Rotkreutz, Switzerland) supplemented with protease inhibitors (Roche Diagnostics, Risch-Rotkreutz, Switzerland) were mixed with Laemmeli's loading buffer, boiled and separated on 15% SDS-PAGE at 40 mA followed by transfer to nitrocellulose membranes for 90 min at 120 V. Membranes were blocked for 1h with 5% non‐fat milk in TBS at room temperature and subsequently probed with the following antibodies: anti-MDH1 (ab76616, Abcam), anti-MDH2 (clone # 11908, Cell Signaling, Danvers, MA, USA) and anti-Actin (Sigma Aldrich, St Louis, MO, USA).

    Techniques: Functional Assay, Activity Assay, Expressing, Microarray

    Reductive Carboxylation Supports Glycolytic Flux via NADH Channeling between MDH1 and GAPDH (A) Percentage of contribution to ATP production for the indicated enzymes, as predicted by metabolic modeling in mT80 and mT7 models. (B) Total levels of NAD + /NADH in shIDH1 and shMDH1 mT7 and mT80 cells, compared to shNTC controls, in basal conditions measured by enzymatic assay. (C) Schematic representation of labeling pattern originating from 4- 2 H 1 -glucose. Deuterium atoms are represented as green filled circles. (D and E) Proportion of total pool of malate m+1 originating from 4- 2 H 1 -glucose in mT7, mT45, and mT80 cells (D) and shMDH1 mT80 cells (E). (F) Levels of secreted lactate m+3 upon incubation of shMDH1 mT7 and mT80 cells with (U)- 13 C-glucose. Data are normalized to shNTC controls. (G) Proportion of total pool for citrate m+1 and malate m+1 originating from 1- 13 C-glutamine in mT7, mT45, and mT80 cells upon treatment with 0.5 μM of the GAPDH inhibitor heptelidic acid. (H) GAPDH IP on lysates of mT7, mT45, and mT80 cells. The interaction between GAPDH and MDH1 is shown by coIP. Immunoglobulin G (IgG) is used in negative isotype controls. Representative images from two independent experiments. (I) Immunofluorescence images of mT7, mT45, and mT80 cells stained with DAPI (blue), GAPDH (green), and MDH1 (red). (B and D–G) Data are mean ± SEM from at least three independent cultures. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, two-sided t test (B and E–G). ∗ p ≤ 0.05, one-way ANOVA (D). GAA, glutamate aspartate antiporter.

    Journal: Molecular Cell

    Article Title: NADH Shuttling Couples Cytosolic Reductive Carboxylation of Glutamine with Glycolysis in Cells with Mitochondrial Dysfunction

    doi: 10.1016/j.molcel.2018.01.034

    Figure Lengend Snippet: Reductive Carboxylation Supports Glycolytic Flux via NADH Channeling between MDH1 and GAPDH (A) Percentage of contribution to ATP production for the indicated enzymes, as predicted by metabolic modeling in mT80 and mT7 models. (B) Total levels of NAD + /NADH in shIDH1 and shMDH1 mT7 and mT80 cells, compared to shNTC controls, in basal conditions measured by enzymatic assay. (C) Schematic representation of labeling pattern originating from 4- 2 H 1 -glucose. Deuterium atoms are represented as green filled circles. (D and E) Proportion of total pool of malate m+1 originating from 4- 2 H 1 -glucose in mT7, mT45, and mT80 cells (D) and shMDH1 mT80 cells (E). (F) Levels of secreted lactate m+3 upon incubation of shMDH1 mT7 and mT80 cells with (U)- 13 C-glucose. Data are normalized to shNTC controls. (G) Proportion of total pool for citrate m+1 and malate m+1 originating from 1- 13 C-glutamine in mT7, mT45, and mT80 cells upon treatment with 0.5 μM of the GAPDH inhibitor heptelidic acid. (H) GAPDH IP on lysates of mT7, mT45, and mT80 cells. The interaction between GAPDH and MDH1 is shown by coIP. Immunoglobulin G (IgG) is used in negative isotype controls. Representative images from two independent experiments. (I) Immunofluorescence images of mT7, mT45, and mT80 cells stained with DAPI (blue), GAPDH (green), and MDH1 (red). (B and D–G) Data are mean ± SEM from at least three independent cultures. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, two-sided t test (B and E–G). ∗ p ≤ 0.05, one-way ANOVA (D). GAA, glutamate aspartate antiporter.

    Article Snippet: For each immunoprecipitation reaction, 35 μg of mouse anti-human GAPDH (Abcam cat. no. ab8245) or mouse anti-human MDH1 (Abcam cat. no. ab76616) were coupled to 1.5 mg Dynabeads M-270 Epoxy beads (Life Technologies, cat. no. 14311D) following manufacturer’s instructions.

    Techniques: Enzymatic Assay, Labeling, Incubation, Co-Immunoprecipitation Assay, Immunofluorescence, Staining

    Aspartate Transamination Supports Flux through MDH1 and Generation of Malate (A) Schematic representation of MAS and labeling patterns originating from (U)- 13 C-aspartate. (B and C) Proportion of total pool of malate m+4 (B) and fumarate m+4 (C) in mT7, mT45, and mT80 cells grown in the presence of U- 13 C-aspartate upon treatment with vehicle control or 0.5 μM rotenone. (D) Malate m+4 levels originating from (U)- 13 C-aspartate in mT80 cells upon silencing of GOT1. Data are normalized to intracellular levels of aspartate m+4 and are mean ± SD from one independent experiment. (E and F) Cell growth of mT7, mT45, and mT80 cells grown in 25 mM galactose and supplemented with 5 mM aspartate (E) upon treatment with 2 mM of the transaminase inhibitor aminooxyacetate (F). Data are normalized on cell growth of vehicle control (E) or on cell growth in the presence of aspartate only (F). (G) Cell growth of mT80 cells grown in 25 mM galactose and supplemented with 5 mM aspartate upon silencing of GOT1. Data are normalized to the cell growth rate of vehicle control. (H and I) Total levels of NAD + /NADH in mT7, mT45, and mT80 cells (H) or shMDH1 mT80 cells (I) upon supplementation with 5 mM aspartate. Data are normalized on vehicle control. (J and K) Secretion of lactate of mT7, mT45, and mT80 cells (J) or shMDH1 mT80 cells (K) upon supplementation with 5 mM aspartate. Data are normalized on vehicle control. (B, C, and E–K) Data are mean ± SEM from at least three independent cultures. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, two-sided t test; n.s., not significant (B, C, E, and G–K). ∗∗∗ p ≤ 0.001, one-way ANOVA (F).

    Journal: Molecular Cell

    Article Title: NADH Shuttling Couples Cytosolic Reductive Carboxylation of Glutamine with Glycolysis in Cells with Mitochondrial Dysfunction

    doi: 10.1016/j.molcel.2018.01.034

    Figure Lengend Snippet: Aspartate Transamination Supports Flux through MDH1 and Generation of Malate (A) Schematic representation of MAS and labeling patterns originating from (U)- 13 C-aspartate. (B and C) Proportion of total pool of malate m+4 (B) and fumarate m+4 (C) in mT7, mT45, and mT80 cells grown in the presence of U- 13 C-aspartate upon treatment with vehicle control or 0.5 μM rotenone. (D) Malate m+4 levels originating from (U)- 13 C-aspartate in mT80 cells upon silencing of GOT1. Data are normalized to intracellular levels of aspartate m+4 and are mean ± SD from one independent experiment. (E and F) Cell growth of mT7, mT45, and mT80 cells grown in 25 mM galactose and supplemented with 5 mM aspartate (E) upon treatment with 2 mM of the transaminase inhibitor aminooxyacetate (F). Data are normalized on cell growth of vehicle control (E) or on cell growth in the presence of aspartate only (F). (G) Cell growth of mT80 cells grown in 25 mM galactose and supplemented with 5 mM aspartate upon silencing of GOT1. Data are normalized to the cell growth rate of vehicle control. (H and I) Total levels of NAD + /NADH in mT7, mT45, and mT80 cells (H) or shMDH1 mT80 cells (I) upon supplementation with 5 mM aspartate. Data are normalized on vehicle control. (J and K) Secretion of lactate of mT7, mT45, and mT80 cells (J) or shMDH1 mT80 cells (K) upon supplementation with 5 mM aspartate. Data are normalized on vehicle control. (B, C, and E–K) Data are mean ± SEM from at least three independent cultures. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, two-sided t test; n.s., not significant (B, C, E, and G–K). ∗∗∗ p ≤ 0.001, one-way ANOVA (F).

    Article Snippet: For each immunoprecipitation reaction, 35 μg of mouse anti-human GAPDH (Abcam cat. no. ab8245) or mouse anti-human MDH1 (Abcam cat. no. ab76616) were coupled to 1.5 mg Dynabeads M-270 Epoxy beads (Life Technologies, cat. no. 14311D) following manufacturer’s instructions.

    Techniques: Labeling

    Reductive Glutamine Carboxylation Regulates NAD Redox Balance and Supports Glycolysis in Response to Mitochondrial Dysfunction Reduced turnover of NADH by mitochondria leads to impairment of the MAS and increase of cytosolic NADH. This in turn induces reductive carboxylation of glutamine, providing carbons for NADH-coupled MDH1, thus regulating NAD redox state and enhancing GAPDH activity. Increased glycolytic turnover supports ATP production in the cytosol, and this is associated with cell migration.

    Journal: Molecular Cell

    Article Title: NADH Shuttling Couples Cytosolic Reductive Carboxylation of Glutamine with Glycolysis in Cells with Mitochondrial Dysfunction

    doi: 10.1016/j.molcel.2018.01.034

    Figure Lengend Snippet: Reductive Glutamine Carboxylation Regulates NAD Redox Balance and Supports Glycolysis in Response to Mitochondrial Dysfunction Reduced turnover of NADH by mitochondria leads to impairment of the MAS and increase of cytosolic NADH. This in turn induces reductive carboxylation of glutamine, providing carbons for NADH-coupled MDH1, thus regulating NAD redox state and enhancing GAPDH activity. Increased glycolytic turnover supports ATP production in the cytosol, and this is associated with cell migration.

    Article Snippet: For each immunoprecipitation reaction, 35 μg of mouse anti-human GAPDH (Abcam cat. no. ab8245) or mouse anti-human MDH1 (Abcam cat. no. ab76616) were coupled to 1.5 mg Dynabeads M-270 Epoxy beads (Life Technologies, cat. no. 14311D) following manufacturer’s instructions.

    Techniques: Activity Assay, Migration

    Mitochondrial Dysfunction Is Linked with Cell Migration (A) Enrichment p values (−log 10 ) of gene ontology (GO) biological processes involved in cell migration and cytoskeleton remodeling as obtained with measurements of protein abundance by proteomics. Red dashed line indicates false discovery rate (FDR) = 0.05. (B and D) Migration speed of mT7, mT45, and mT80 cells (B) or shMDH1 mT80 cells (D) measured by wound healing assay. (C and E) Values of J ATP consumption due to cytoskeleton remodeling based on calculations from OCR and ECAR data upon treatment with 1 μM nocodazole in mT7, mT45, and mT80 cells (C) or mT80 shMDH1 cells (E). (F) Immunofluorescence images of mT7, mT45, and mT80 cells stained with DAPI (blue), phalloidin (green), or antibody against MDH1 (red). White arrows indicate areas of co-localization between MDH1 and actin in mT80 cells. (G) Quantification of co-localization between MDH1 and phalloidin (actin). Data were obtained from 20–30 ROIs per condition. (B–E) Data are mean ± SEM from three to four independent cultures and were normalized on mean values of each experiment. ∗ p ≤ 0.05 and ∗∗∗ p ≤ 0.001, ANOVA (B, C, and G) or Dunnett’s test (D).

    Journal: Molecular Cell

    Article Title: NADH Shuttling Couples Cytosolic Reductive Carboxylation of Glutamine with Glycolysis in Cells with Mitochondrial Dysfunction

    doi: 10.1016/j.molcel.2018.01.034

    Figure Lengend Snippet: Mitochondrial Dysfunction Is Linked with Cell Migration (A) Enrichment p values (−log 10 ) of gene ontology (GO) biological processes involved in cell migration and cytoskeleton remodeling as obtained with measurements of protein abundance by proteomics. Red dashed line indicates false discovery rate (FDR) = 0.05. (B and D) Migration speed of mT7, mT45, and mT80 cells (B) or shMDH1 mT80 cells (D) measured by wound healing assay. (C and E) Values of J ATP consumption due to cytoskeleton remodeling based on calculations from OCR and ECAR data upon treatment with 1 μM nocodazole in mT7, mT45, and mT80 cells (C) or mT80 shMDH1 cells (E). (F) Immunofluorescence images of mT7, mT45, and mT80 cells stained with DAPI (blue), phalloidin (green), or antibody against MDH1 (red). White arrows indicate areas of co-localization between MDH1 and actin in mT80 cells. (G) Quantification of co-localization between MDH1 and phalloidin (actin). Data were obtained from 20–30 ROIs per condition. (B–E) Data are mean ± SEM from three to four independent cultures and were normalized on mean values of each experiment. ∗ p ≤ 0.05 and ∗∗∗ p ≤ 0.001, ANOVA (B, C, and G) or Dunnett’s test (D).

    Article Snippet: For each immunoprecipitation reaction, 35 μg of mouse anti-human GAPDH (Abcam cat. no. ab8245) or mouse anti-human MDH1 (Abcam cat. no. ab76616) were coupled to 1.5 mg Dynabeads M-270 Epoxy beads (Life Technologies, cat. no. 14311D) following manufacturer’s instructions.

    Techniques: Migration, Wound Healing Assay, Immunofluorescence, Staining

    The optimization of methanol metabolism in the synthetic E. coli BL21/ΔfrmA-NudF-Mdh2-Hps-Phi. a The optimization of a nitrogen source. b The optimization of methanol concentration. Error bars indicate standard error of the mean ( n = 3)

    Journal: Biotechnology for Biofuels

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

    doi: 10.1186/s13068-019-1356-4

    Figure Lengend Snippet: The optimization of methanol metabolism in the synthetic E. coli BL21/ΔfrmA-NudF-Mdh2-Hps-Phi. a The optimization of a nitrogen source. b The optimization of methanol concentration. Error bars indicate standard error of the mean ( n = 3)

    Article Snippet: The strain E. coli BL21/ΔfrmA-NudF-Mdh-Hps-Phi was cultivated in LB medium supplemented with 100 µg/mL Amp and 0.5 mM IPTG for 10–12 h. Cells were then transferred to M9 medium with 50 mM 13 C-methanol (Sigma-Aldrich).

    Techniques: Concentration Assay