scholarly journals Flux Analysis Uncovers Key Role of Functional Redundancy in Formaldehyde Metabolism

PLoS Biology ◽  
2005 ◽  
Vol 3 (2) ◽  
pp. e16 ◽  
Author(s):  
Christopher J Marx ◽  
Stephen J Van Dien ◽  
Mary E Lidstrom
Keyword(s):  
Functional Redundancy ◽  
Flux Analysis ◽  
Formaldehyde Metabolism

scholarly journals Selectins mediate eosinophil recruitment in vivo: a comparison with their role in neutrophil influx

Blood ◽  
1996 ◽  
Vol 87 (12) ◽  
pp. 5297-5304 ◽  
Author(s):  
GM Henriques ◽  
JM Miotla ◽  
SB Cordeiro ◽  
BA Wolitzky ◽  
ST Woolley ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Adhesion Molecule ◽  
Functional Redundancy ◽  
Neutrophil Recruitment ◽  
Eosinophil Recruitment ◽  
Neutrophil Influx ◽  
Eosinophil Accumulation

The role of selectins in mediating eosinophil recruitment in vivo was assessed in a model of lipopolysaccharide (LPS)-induced mouse pleurisy. LPS administration resulted in significant eosinophil influx at 24 hours, whereas neutrophil recruitment to the cavity peaked at 4 hours and persisted for 24 hours. The anti-L-selectin monoclonal antibody (MoAb) MEL-14 effectively inhibited (by 97%) eosinophil influx at 24 hours and also inhibited neutrophil recruitment at both times (75% to 95%). Eosinophil recruitment was partially reduced (54%) by the anti-P- selectin MoAb 5H1 but, in contrast, was unaffected by the anti-E- selectin MoAb 10E6. Neutrophil influx at 4 or 24 hours was not affected by the anti-P- or anti-E-selectin MoAbs. However, coadministration of anti-P-selectin and anti-E-selectin was very effective at inhibiting eosinophil influx at 24 hours (86%) and neutrophil influx at 4 (93%) and 24 hours (92%). These results show that all three selectins play a role in LPS-induced eosinophil and neutrophil recruitment in vivo, although P- and E-selectin show a degree of functional redundancy. The demonstration that P-selectin mediates eosinophil but not neutrophil influx suggests that suppressing the function of this adhesion molecule may be beneficial in blocking eosinophil accumulation in pleural inflammation.

scholarly journals YtsJ Has the Major Physiological Role of the Four Paralogous Malic Enzyme Isoforms in Bacillus subtilis

2006 ◽  
Vol 188 (13) ◽  
pp. 4727-4736 ◽  
Author(s):  
Guillaume Lerondel ◽  
Thierry Doan ◽  
Nicola Zamboni ◽  
Uwe Sauer ◽  
Stéphane Aymerich
Keyword(s):  
Enzyme Activity ◽  
Bacillus Subtilis ◽  
Malic Enzyme ◽  
Metabolic Flux ◽  
Physiological Role ◽  
Extreme Case ◽  
Growth Defect ◽  
Flux Analysis ◽  
Malic Enzyme Activity

ABSTRACT The Bacillus subtilis genome contains several sets of paralogs. An extreme case is the four putative malic enzyme genes maeA, malS, ytsJ, and mleA. maeA was demonstrated to encode malic enzyme activity, to be inducible by malate, but also to be dispensable for growth on malate. We report systematic experiments to test whether these four genes ensure backup or cover different functions. Analysis of single- and multiple-mutant strains demonstrated that ytsJ has a major physiological role in malate utilization for which none of the other three genes could compensate. In contrast, maeA, malS, and mleA had distinct roles in malate utilization for which they could compensate one another. The four proteins exhibited malic enzyme activity; MalS, MleA, and MaeA exhibited 4- to 90-fold higher activities with NAD+ than with NADP+. YtsJ activity, in contrast, was 70-fold higher with NADP+ than with NAD+, with Km values of 0.055 and 2.8 mM, respectively. lacZ fusions revealed strong transcription of ytsJ, twofold higher in malate than in glucose medium, but weak transcription of malS and mleA. In contrast, mleA was strongly transcribed in complex medium. Metabolic flux analysis confirmed the major role of YtsJ in malate-to-pyruvate interconversion. While overexpression of the NADP-dependent Escherichia coli malic enzyme MaeB did not suppress the growth defect of a ytsJ mutant on malate, overexpression of the transhydrogenase UdhA from E. coli partially suppressed it. These results suggest an additional physiological role of YtsJ beyond that of malate-to-pyruvate conversion.

scholarly journals The Role of Pool Size Measurements in Improving Flux Estimations in Non-Stationary Metabolic Flux Analysis

2019 ◽  
Vol 116 (3) ◽  
pp. 132a
Author(s):  
Anna Sher ◽  
Daniel Fridman ◽  
Jamey Young ◽  
Cynthia J. Musante
Keyword(s):  
Metabolic Flux Analysis ◽  
Metabolic Flux ◽  
Pool Size ◽  
Flux Analysis

scholarly journals Metabolomic and molecular insights into sickle cell disease and innovative therapies

2019 ◽  
Vol 3 (8) ◽  
pp. 1347-1355 ◽  
Author(s):  
Morayo G. Adebiyi ◽  
Jeanne M. Manalo ◽  
Yang Xia
Keyword(s):  
Sickle Cell Disease ◽  
Disease Progression ◽  
Sickle Cell ◽  
Intracellular Signaling ◽  
Metabolic Reprogramming ◽  
Flux Analysis ◽  
High Morbidity ◽  
Cell Disease ◽  
Sickle Hemoglobin

Abstract Sickle cell disease (SCD) is an autosomal-recessive hemolytic disorder with high morbidity and mortality. The pathophysiology of SCD is characterized by the polymerization of deoxygenated intracellular sickle hemoglobin, which causes the sickling of erythrocytes. The recent development of metabolomics, the newest member of the “omics” family, has provided a powerful new research strategy to accurately measure functional phenotypes that are the net result of genomic, transcriptomic, and proteomic changes. Metabolomics changes respond faster to external stimuli than any other “ome” and are especially appropriate for surveilling the metabolic profile of erythrocytes. In this review, we summarize recent pioneering research that exploited cutting-edge metabolomics and state-of-the-art isotopically labeled nutrient flux analysis to monitor and trace intracellular metabolism in SCD mice and humans. Genetic, structural, biochemical, and molecular studies in mice and humans demonstrate unrecognized intracellular signaling pathways, including purinergic and sphingolipid signaling networks that promote hypoxic metabolic reprogramming by channeling glucose metabolism to glycolysis via the pentose phosphate pathway. In turn, this hypoxic metabolic reprogramming induces 2,3-bisphosphoglycerate production, deoxygenation of sickle hemoglobin, polymerization, and sickling. Additionally, we review the detrimental role of an impaired Lands’ cycle, which contributes to sickling, inflammation, and disease progression. Thus, metabolomic profiling allows us to identify the pathological role of adenosine signaling and S1P-mediated erythrocyte hypoxic metabolic reprogramming and hypoxia-induced impaired Lands' cycle in SCD. These findings further reveal that the inhibition of adenosine and S1P signaling cascade and the restoration of an imbalanced Lands' cycle have potent preclinical efficacy in counteracting sickling, inflammation, and disease progression.

Metabolic Endocrine Regulators of Hematopoietic Stem Cell Formation and Function.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1496-1496
Author(s):  
James Harris ◽  
Claire C Cutting ◽  
Michael Dovey ◽  
Wolfram Goessling ◽  
Trista North
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Energy Production ◽  
Functional Redundancy ◽  
Hematopoietic Stem ◽  
Glucose Levels ◽  
Obesity And Diabetes ◽  
Elevated Glucose ◽  
Glucose Treatment

Abstract Abstract 1496 Poster Board I-519 Obesity and subsequent diabetes have emerged as major health problems in the U.S. While the consequences of elevated blood glucose levels on the cardiovascular system and other organs are well known, the direct effects on the hematopoietic system are more elusive. Similarly, the impact of gestational diabetes on embryonic hematopoiesis has not been examined in detail. The zebrafish has emerged as an important model system to study conserved regulators of organ development and homeostasis. In order to evaluate the role of elevated glucose levels on hematopoietic stem cell (HSC) production, zebrafish embryos were exposed to increasing doses of D-glucose from 5 somites to 36 hours post fertilization (hpf); HSC number, as indicated by in situ hybridization for the conserved markers runx1 and cmyb in the Aorta-Gonad-Mesonephros (AGM) region, was increased at 0.5, 1% and 2% glucose; results were confirmed by in analysis of CD41 expression. Quantification using FACS analysis of fluorescent HSC reporter embryos and qPCR revealed a 2-3-fold enhancement following 1% glucose treatment. Other mono, di-, and trisaccharide sugars had similar effects, causing increased numbers of HSCs, however, L-glucose had no impact. BrdU incorporation in the AGM region was elevated after 1% glucose treatment, while acridine orange staining revealed an inhibitory effect on apoptosis. To evaluate potential mediators of these glucose-responsive effects, embryos were injected with antisense morpholino oligonucleotides (MO) against both the insulin (insr), and insulin-like growth factor receptors (igfr); insr and igfr receptors can each bind insulin, released following elevations in blood sugar levels. MO knockdown of insra or igfrb, but not igfra, influenceded runx1+ HSCs substantially, indicating an important role of these endocrine regulatory signaling pathways in HSC formation. However, D-glucose completely reversed these effects, implying either functional redundancy, or a multi-step, multi-effector process of HSC regulation by endocrine factors. To further clarify when insr- and/or igfr-mediated activity was influencing HSC formation and to correlate that effect with elevated glucose exposure, embryos were treated for defined periods with either 1% glucose, insulin, or IGF; exposure from 10 somites to 24 hpf influences the formation and arterial/venous specification of dorsal aorta, the conserved site of initial definitive HSC production, while exposure from 24 to 36 hpf regulates HSC induction. IGF exerted a positive effect on HSCs only after the establishment of the hematopoietic niche (>24hpf). Glucose treatment, however, positively influenced HSC formation at all time points examined, suggesting it works not only in the HSC niche to induce HSCs, but also prior to HSC formation. MO knockdown of the glucose transporter glut1 resulted in diminished HSC production, confirming a direct role of glucose in this process. To determine whether the effect of glucose elevation was mediated by changes in cellular energy production, embryos were exposed to chemical inhibitors of oxidative phosphorylation. Cyanide and oxaloacetate reversed the beneficial effects of D-glucose, indicating that energy production can modulate HSC formation. Investigation into the functional redundancy and cross-regulation of insulin and IGF on HSC self-renewal and the evolutionary conservation of the effects of energy metabolism on HSC production are ongoing; further studies will be needed to determine if glucose maintains an influential role on HSC homeostasis or bone marrow recovery following injury. These results could have an impact on methods for HSC modulation for therapeutic purposes, and may further unveil specific risks of obesity and diabetes for hematopoiesis and HSC homeostasis during gestation and in the adult. Disclosures: Goessling: Fate Therapeutics: Consultancy, Patents & Royalties. North: Fate Therapeutics: Consultancy, Patents & Royalties.

scholarly journals Defining pathways of spindle checkpoint silencing: functional redundancy between Cdc20 ubiquitination and p31comet

2011 ◽  
Vol 22 (22) ◽  
pp. 4227-4235 ◽  
Author(s):  
Luying Jia ◽  
Bing Li ◽  
Ross T. Warrington ◽  
Xing Hao ◽  
Shixuan Wang ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Spindle Checkpoint ◽  
Functional Redundancy ◽  
Mitotic Checkpoint ◽  
Human Cells ◽  
Unresolved Issue ◽  
Anaphase Promoting Complex ◽  
Anaphase Onset ◽  
Mitotic Checkpoint Complex

The spindle checkpoint senses unattached or improperly attached kinetochores during mitosis, inhibits the anaphase-promoting complex or cyclosome (APC/C), and delays anaphase onset to prevent aneuploidy. The mitotic checkpoint complex (MCC) consisting of BubR1, Bub3, Mad2, and Cdc20 is a critical APC/C-inhibitory checkpoint complex in human cells. At the metaphase–anaphase transition, the spindle checkpoint turns off, and MCC disassembles to allow anaphase onset. The molecular mechanisms of checkpoint inactivation are poorly understood. A major unresolved issue is the role of Cdc20 autoubiquitination in this process. Although Cdc20 autoubiquitination can promote Mad2 dissociation from Cdc20, a nonubiquitinatable Cdc20 mutant still dissociates from Mad2 during checkpoint inactivation. Here, we show that depletion of p31comet delays Mad2 dissociation from Cdc20 mutants that cannot undergo autoubiquitination. Thus both p31comet and ubiquitination of Cdc20 are critical mechanisms of checkpoint inactivation. They act redundantly to promote Mad2 dissociation from Cdc20.

Co-overexpression of AtSHMT1 and AtFDH induces sugar synthesis and enhances the role of original pathways during formaldehyde metabolism in tobacco

Plant Science ◽  
2021 ◽  
Vol 305 ◽  
pp. 110829
Author(s):  
Xing Zhao ◽  
Zhidong Zeng ◽  
Wenjia Cao ◽  
Dawood Khan ◽  
Muhammad Ikram ◽  
...  
Keyword(s):  
Formaldehyde Metabolism

scholarly journals Metabolic flux analysis of wild-type Escherichia coli and mutants deficient in pyruvate-dissimilating enzymes during the fermentative metabolism of glucuronate

Microbiology ◽  
2010 ◽  
Vol 156 (6) ◽  
pp. 1860-1872 ◽  
Author(s):  
Abhishek Murarka ◽  
James M. Clomburg ◽  
Ramon Gonzalez
Keyword(s):  
Escherichia Coli ◽  
Metabolic Flux Analysis ◽  
Metabolic Flux ◽  
Reducing Power ◽  
Building Blocks ◽  
Exponential Phase ◽  
Flux Analysis ◽  
Wild Type ◽  
Fermentative Metabolism

The fermentative metabolism of d-glucuronic acid (glucuronate) in Escherichia coli was investigated with emphasis on the dissimilation of pyruvate via pyruvate formate-lyase (PFL) and pyruvate dehydrogenase (PDH). In silico and in vivo metabolic flux analysis (MFA) revealed that PFL and PDH share the dissimilation of pyruvate in wild-type MG1655. Surprisingly, it was found that PDH supports fermentative growth on glucuronate in the absence of PFL. The PDH-deficient strain (Pdh−) exhibited a slower transition into the exponential phase and a decrease in specific rates of growth and glucuronate utilization. Moreover, a significant redistribution of metabolic fluxes was found in PDH- and PFL-deficient strains. Since no role had been proposed for PDH in the fermentative metabolism of E. coli, the metabolic differences between MG1655 and Pdh− were further investigated. An increase in the oxidative pentose phosphate pathway (ox-PPP) flux was observed in response to PDH deficiency. A comparison of the ox-PPP and PDH pathways led to the hypothesis that the role of PDH is the supply of reducing equivalents. The finding that a PDH deficiency lowers the NADH : NAD+ ratio supported the proposed role of PDH. Moreover, the NADH : NAD+ ratio in a strain deficient in both PDH and the ox-PPP (Pdh−Zwf−) was even lower than that observed for Pdh−. Strain Pdh−Zwf− also exhibited a slower transition into the exponential phase and a lower growth rate than Pdh−. Finally, a transhydrogenase-deficient strain grew more slowly than wild-type but did not show the slower transition into the exponential phase characteristic of Pdh− mutants. It is proposed that PDH fulfils two metabolic functions. First, by creating the appropriate internal redox state (i.e. appropriate NADH : NAD+ ratio), PDH ensures the functioning of the glucuronate utilization pathway. Secondly, the NADH generated by PDH can be converted to NADPH by the action of transhydrogenases, thus serving as biosynthetic reducing power in the synthesis of building blocks and macromolecules.

scholarly journals Bifunctional Malic/Malolactic Enzyme Provides a Novel Mechanism for NADPH-Balancing in Bacillus subtilis

mBio ◽  
2021 ◽  
Vol 12 (2) ◽  
Author(s):  
Manuel Hörl ◽  
Tobias Fuhrer ◽  
Nicola Zamboni
Keyword(s):  
Bacillus Subtilis ◽  
Metabolic Flux ◽  
Central Carbon Metabolism ◽  
Flux Analysis ◽  
Content Type ◽  
Malolactic Enzyme ◽  
First Time

ABSTRACT The redox cofactor NADPH is required as a reducing equivalent in about 100 anabolic reactions throughout metabolism. To ensure fitness under all conditions, the demand is fulfilled by a few dehydrogenases in central carbon metabolism that reduce NADP+ with electrons derived from the catabolism of nutrients. In the case of Bacillus subtilis growing on glucose, quantitative flux analyses indicate that NADPH production largely exceeds biosynthetic needs, suggesting a hitherto unknown mechanism for NADPH balancing. We investigated the role of the four malic enzymes present in B. subtilis that could bring about a metabolic cycle for transhydrogenation of NADPH into NADH. Using quantitative 13C metabolic flux analysis, we found that isoform YtsJ alone contributes to NADPH balancing in vivo and demonstrated relevant NADPH-oxidizing activity by YtsJ in vitro. To our surprise, we discovered that depending on NADPH, YtsJ switches activity from a pyruvate-producing malic enzyme to a lactate-generating malolactic enzyme. This switch in activity allows YtsJ to adaptively compensate for cellular NADPH over- and underproduction upon demand. Finally, NADPH-dependent bifunctional activity was also detected in the YtsJ homolog in Escherichia coli MaeB. Overall, our study extends the known redox cofactor balancing mechanisms by providing first-time evidence that the type of catalyzed reaction by an enzyme depends on metabolite abundance. IMPORTANCE A new mechanism for NADPH balancing was discovered in Bacillus subtilis. It pivots on the bifunctional enzyme YtsJ, which is known to catalyze NADP-dependent malate decarboxylation. We found that in the presence of excessive NADPH, the same enzyme switches to malolactic activity and creates a transhydrogenation cycle that ultimately converts NADPH to NADH. This provides a regulated mechanism to immediately adjust NADPH/NADP+ in response to instantaneous needs.

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