scholarly journals Amino Acids Rather than Glucose Account for the Majority of Cell Mass in Proliferating Mammalian Cells

2016 ◽  
Vol 36 (5) ◽  
pp. 540-549 ◽  
Author(s):  
Aaron M. Hosios ◽  
Vivian C. Hecht ◽  
Laura V. Danai ◽  
Marc O. Johnson ◽  
Jeffrey C. Rathmell ◽  
...  
Keyword(s):  
Amino Acids ◽  
Mammalian Cells ◽  
Cell Mass

Abstract A36: Amino acids rather than glucose accounts for the majority of cell mass in rapidly proliferating mammalian cells

2016 ◽  
Author(s):  
Aaron M. Hosios ◽  
Vivian C. Hecht ◽  
Marc Johnson ◽  
Jeffrey C. Rathmell ◽  
Scott R. Manalis ◽  
...  
Keyword(s):  
Amino Acids ◽  
Mammalian Cells ◽  
Cell Mass

scholarly journals Autophagy Deficiency by Atg4B Loss Leads to Metabolomic Alterations in Mice

Metabolites ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 481
Author(s):  
Gemma G. Martínez-García ◽  
Raúl F. Pérez ◽  
Álvaro F. Fernández ◽  
Sylvere Durand ◽  
Guido Kroemer ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Amino Acids ◽  
Mammalian Cells ◽  
Tissue Homeostasis ◽  
In Vivo Studies ◽  
Protective Mechanism ◽  
Cardiac Damage ◽  
Wide Range ◽  
First Time

Autophagy is an essential protective mechanism that allows mammalian cells to cope with a variety of stressors and contributes to maintaining cellular and tissue homeostasis. Due to these crucial roles and also to the fact that autophagy malfunction has been described in a wide range of pathologies, an increasing number of in vivo studies involving animal models targeting autophagy genes have been developed. In mammals, total autophagy inactivation is lethal, and constitutive knockout models lacking effectors of this route are not viable, which has hindered so far the analysis of the consequences of a systemic autophagy decline. Here, we take advantage of atg4b−/− mice, an autophagy-deficient model with only partial disruption of the process, to assess the effects of systemic reduction of autophagy on the metabolome. We describe for the first time the metabolic footprint of systemic autophagy decline, showing that impaired autophagy results in highly tissue-dependent alterations that are more accentuated in the skeletal muscle and plasma. These changes, which include changes in the levels of amino-acids, lipids, or nucleosides, sometimes resemble those that are frequently described in conditions like aging, obesity, or cardiac damage. We also discuss different hypotheses on how impaired autophagy may affect the metabolism of several tissues in mammals.

scholarly journals Rheology of rounded mammalian cells over continuous high-frequencies

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Gotthold Fläschner ◽  
Cosmin I. Roman ◽  
Nico Strohmeyer ◽  
David Martinez-Martin ◽  
Daniel J. Müller
Keyword(s):  
High Frequency ◽  
Mammalian Cells ◽  
Mechanical Response ◽  
Cell Mass ◽  
Low Frequency ◽  
Mass Measurements ◽  
High Frequencies ◽  
Polymer Relaxation ◽  
Continuous Frequency ◽  
Rheological Experiments

AbstractUnderstanding the viscoelastic properties of living cells and their relation to cell state and morphology remains challenging. Low-frequency mechanical perturbations have contributed considerably to the understanding, yet higher frequencies promise to elucidate the link between cellular and molecular properties, such as polymer relaxation and monomer reaction kinetics. Here, we introduce an assay, that uses an actuated microcantilever to confine a single, rounded cell on a second microcantilever, which measures the cell mechanical response across a continuous frequency range ≈ 1–40 kHz. Cell mass measurements and optical microscopy are co-implemented. The fast, high-frequency measurements are applied to rheologically monitor cellular stiffening. We find that the rheology of rounded HeLa cells obeys a cytoskeleton-dependent power-law, similar to spread cells. Cell size and viscoelasticity are uncorrelated, which contrasts an assumption based on the Laplace law. Together with the presented theory of mechanical de-embedding, our assay is generally applicable to other rheological experiments.

scholarly journals Finding the Binding Site of Peloruside A and its Secondary Effects in Saccharomyces Cerevisiae using a  Chemical Genetics Approach

2021 ◽  
Author(s):  
◽  
Reem Hanna
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Amino Acids ◽  
Flow Cytometry ◽  
Binding Site ◽  
Mammalian Cells ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Peloruside A ◽  
Microtubule Function

<p>Peloruside A, a natural product isolated from the marine sponge Mycale hentscheli, is a microtubule-stabilising agent that has a similar mechanism of action to the anticancer drug paclitaxel and is cytotoxic to cultured mammalian cells. Peloruside appears to bind to a distinct site on mammalian tubulin that is different from that of the taxoid-site drugs. Because of the high sequence homology between yeast and mammalian tubulin, Saccharomyces cerevisiae (S. cerevisiae) was used as a model organism to characterise the peloruside-binding site with the aim of advancing our understanding about this site on mammalian tubulin. Wild type S. cerevisiae (BY4741) was sensitive to peloruside at uM concentrations; however, a strain that lacks the mad2 (Mitotic Arrest Deficient 2) gene showed increased sensitivity to the drug at much lower uM concentrations. This gene is a component of the spindle-assembly checkpoint complex that delays the onset of anaphase in cells with defects in mitotic spindle assembly. The main aims of this project were to define the binding site of peloruside A using yeast tubulin to see if microtubule function and/or morphology is altered in yeast by peloruside, and to identify any secondary drug targets "friends of the target" through chemical genetic interactions profiling (Homozygous deletion profiling microarray). Site-directed mutagenesis was used to mutate two conserved amino acids (A296T; R306H) known to confer resistance to peloruside in mammalian cells. Based on a published computer model of the peloruside binding site on mammalian tubulin, we also mutated three other amino acids, two that were predicted to affect peloruside binding (Q291M and N337L), and one that was predicted to affect laulimalide binding but have little affect on peloruside binding (V333W). We also included a negative control that was predicted to have no effect on peloruside binding (R282Q) and would affect epothilone binding. We found that of the six point mutations, only Q291M failed to confer resistance in yeast and instead it increased the inhibition to the drug. Using a bud index assay, confocal microscopy, and flow cytometry, 40-50 uM peloruside was shown to block cells in G2/M of the cell cycle, confirming a direct action of the drug on microtubule function. Homozygous profiling (HOP) microarray analysis of a deletion mutant set of yeast genes was also carried out to identify gene products that interact with peloruside in order to link the drug to specific networks or biochemical pathways in the cells. From site-directed mutagenesis, we concluded that peloruside binds to yeast B-tubulin in the region predicted by the published model of the binding site, and therefore mapping the site on yeast tubulin could provide useful information about the mammalian binding site for peloruside. The bud index, flow cytometry, and confocal microscopy experiments provided further evidence that peloruside interacts with yeast tubulin. From HOP we found that peloruside has roles in the cell cycle, as expected, and has effects on protein transport, secretion, cell wall synthesis, and steroid biosynthesis pathways.</p>

scholarly journals A Genetically Encoded Picolyl Azide for Improved Live Cell Copper Click Labeling

2021 ◽  
Vol 9 ◽  
Author(s):  
Birthe Meineke ◽  
Johannes Heimgärtner ◽  
Alexander J. Craig ◽  
Michael Landreh ◽  
Lindon W. K. Moodie ◽  
...  
Keyword(s):  
Amino Acids ◽  
Mammalian Cells ◽  
Stop Codon ◽  
Bioorthogonal Chemistry ◽  
Bioorthogonal Reaction ◽  
Noncanonical Amino Acids ◽  
Selective Reactivity ◽  
Direct Incorporation ◽  
Chelating Groups ◽  
Amber Suppression

Bioorthogonal chemistry allows rapid and highly selective reactivity in biological environments. The copper-catalyzed azide–alkyne cycloaddition (CuAAC) is a classic bioorthogonal reaction routinely used to modify azides or alkynes that have been introduced into biomolecules. Amber suppression is an efficient method for incorporating such chemical handles into proteins on the ribosome, in which noncanonical amino acids (ncAAs) are site specifically introduced into the polypeptide in response to an amber (UAG) stop codon. A variety of ncAA structures containing azides or alkynes have been proven useful for performing CuAAC chemistry on proteins. To improve CuAAC efficiency, biologically incorporated alkyne groups can be reacted with azide substrates that contain copper-chelating groups. However, the direct incorporation of copper-chelating azides into proteins has not been explored. To remedy this, we prepared the ncAA paz-lysine (PazK), which contains a picolyl azide motif. We show that PazK is efficiently incorporated into proteins by amber suppression in mammalian cells. Furthermore, PazK-labeled proteins show improved reactivity with alkyne reagents in CuAAC.

scholarly journals Homeostasis of branched-chain amino acids is critical for the activity of TOR signaling in Arabidopsis

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Pengfei Cao ◽  
Sang-Jin Kim ◽  
Anqi Xing ◽  
Craig A Schenck ◽  
Lu Liu ◽  
...  
Keyword(s):  
Amino Acids ◽  
Mammalian Cells ◽  
Branched Chain Amino Acids ◽  
Nutrient Sensing ◽  
Tor Signaling ◽  
Functional Connection ◽  
Primary Input ◽  
Tor Kinase ◽  
Arabidopsis Mutant ◽  
Branched Chain

The target of rapamycin (TOR) kinase is an evolutionarily conserved hub of nutrient sensing and metabolic signaling. In plants, a functional connection of TOR activation with glucose availability was demonstrated, while it is yet unclear whether branched-chain amino acids (BCAAs) are a primary input of TOR signaling as they are in yeast and mammalian cells. Here, we report on the characterization of an Arabidopsis mutant over-accumulating BCAAs. Through chemical interventions targeting TOR and by examining mutants of BCAA biosynthesis and TOR signaling, we found that BCAA over-accumulation leads to up-regulation of TOR activity, which causes reorganization of the actin cytoskeleton and actin-associated endomembranes. Finally, we show that activation of TOR is concomitant with alteration of cell expansion, proliferation and specialized metabolism, leading to pleiotropic effects on plant growth and development. These results demonstrate that BCAAs contribute to plant TOR activation and reveal previously uncharted downstream subcellular processes of TOR signaling.

scholarly journals Metabolic characterization of the bovine blastocyst, inner cell mass, trophectoderm and blastocoel fluid

Reproduction ◽  
2003 ◽  
pp. 299-308 ◽  
Author(s):  
N Gopichandran ◽  
HJ Leese
Keyword(s):  
Amino Acids ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Lactate Production ◽  
Cell Populations ◽  
Lower Glucose ◽  
Amino Acid Consumption ◽  
Production And Consumption ◽  
Acid Consumption

The formation of a viable blastocyst is dependent upon the establishment of a correct inner cell mass (ICM):trophectoderm cell ratio but little is known about the metabolism of the two cell populations or about the composition of blastocoel fluid. In this study, the metabolism of intact bovine blastocysts, isolated ICM and trophectoderm was examined in terms of glucose and pyruvate uptake, lactate production, and amino acid consumption or production. The concentration of these nutrients in blastocoel fluid was also determined. The metabolism of glucose, pyruvate and lactate differed significantly between the isolated ICM and trophectoderm. Isolated trophectoderm had a higher pyruvate (P<0.001) and lower glucose (P<0.05) consumption, and higher lactate production (P<0.05) than did ICM. The consumption or production of amino acids by ICM and trophectoderm also differed, with the trophectoderm displaying a higher turnover (the sum of production and consumption). The ICM and trophectoderm both depleted arginine, aspartate and leucine, whereas the production of alanine was consistent. Isolated ICM depleted a further six amino acids, which appeared during trophectoderm culture; the reverse trend was observed for the remaining amino acids. The concentration of lactate in blastocoel fluid was significantly higher than in synthetic oviductal fluid supplemented with amino acids and BSA (SOFaaBSA; P<0.05). However, glucose (P<0.05) and pyruvate (P<0.001) concentrations were both lower. Aspartate, glutamate, glycine, alanine and tryptophan were present at significantly higher concentrations in blastocoel fluid than in SOFaaBSA, whereas threonine and asparagine concentrations were significantly lower. The metabolism of composite blastocysts, obtained by summing the consumption and production profiles of the ICM and trophectoderm, and taking into account their respective number of cells, was higher than that of intact blastocysts, indicating that upon isolation of the two cell populations there may be disruption to paracrine interactions or the onset of culture-induced cellular stress or both.

Construction of an inducible stable cell line for efficient incorporation of unnatural amino acids in mammalian cells

2017 ◽  
Vol 489 (4) ◽  
pp. 490-496 ◽  
Author(s):  
Ziwei Zhang ◽  
Huan Xu ◽  
Longlong Si ◽  
Yi Chen ◽  
Bo Zhang ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Line ◽  
Unnatural Amino Acids ◽  
Mammalian Cells ◽  
Stable Cell Line
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