scholarly journals Visualizing Cancer Cell Metabolic Dynamics Regulated With Aromatic Amino Acids Using DO-SRS and 2PEF Microscopy

2021 ◽  
Vol 8 ◽  
Author(s):  
Pegah Bagheri ◽  
Khang Hoang ◽  
Anthony A. Fung ◽  
Sahran Hussain ◽  
Lingyan Shi
Keyword(s):  
Oxidative Stress ◽  
Amino Acids ◽  
Protein Synthesis ◽  
Spatial Distribution ◽  
Lipid Droplets ◽  
De Novo ◽  
Culture Media ◽  
Aromatic Amino Acids ◽  
Unsaturated Lipids ◽  
Metabolic Activities

Oxidative imbalance plays an essential role in the progression of many diseases that include cancer and neurodegenerative diseases. Aromatic amino acids (AAA) such as phenylalanine and tryptophan have the capability of escalating oxidative stress because of their involvement in the production of Reactive Oxygen Species (ROS). Here, we use D2O (heavy water) probed stimulated Raman scattering microscopy (DO-SRS) and two Photon Excitation Fluorescence (2PEF) microscopy as a multimodal imaging approach to visualize metabolic changes in HeLa cells under excess AAA such as phenylalanine or trytophan in culture media. The cellular spatial distribution of de novo lipogenesis, new protein synthesis, NADH, Flavin, unsaturated lipids, and saturated lipids were all imaged and quantified in this experiment. Our studies reveal ∼10% increase in de novo lipogenesis and the ratio of NADH to flavin, and ∼50% increase of the ratio of unsaturated lipids to saturated lipid in cells treated with excess phenylalanine or trytophan. In contrast, these cells exhibited a decrease in the protein synthesis rate by ∼10% under these AAA treatments. The cellular metabolic activities of these biomolecules are indicators of elevated oxidative stress and mitochondrial dysfunction. Furthermore, 3D reconstruction images of lipid droplets were acquired and quantified to observe their spatial distribution around cells’ nuceli under different AAA culture media. We observed a higher number of lipid droplets in excess AAA conditions. Our study showcases that DO-SRS imaging can be used to quantitatively study how excess AAA regulates metabolic activities of cells with subcellular resolution in situ.

scholarly journals Action of growth hormone in vitro on the net uptake and incorporation into protein of amino acids in muscle from intact rabbits given protein-deficient diets

1976 ◽  
Vol 35 (1) ◽  
pp. 1-10 ◽  
Author(s):  
M. R. Turner ◽  
P. J. Reeds ◽  
K. A. Munday
Keyword(s):  
Amino Acids ◽  
Growth Hormone ◽  
Protein Synthesis ◽  
Amino Acid ◽  
De Novo ◽  
Amino Acid Uptake ◽  
Acid Uptake ◽  
Amino Acid Incorporation ◽  
Acid Incorporation

1. Net amino acid uptake, and incorporation into protein have been measured in vitro in the presence and absence of porcine growth hormone (GH) in muscle from intact rabbits fed for 5 d on low-protein (LP), protein-free (PF) or control diets.2. In muscle from control and LP animals GH had no effect on the net amino acid uptake but stimulated amino acid incorporation into protein, although this response was less in LP animals than in control animals.3. In muscle from PF animals, GH stimulated both amino acid incorporation into protein and the net amino acid uptake, a type of response which also occurs in hypophysectomized animals. The magnitude of the effect of GH on the incorporation of amino acids into protein was reduced in muscle from PF animals.4. The effect of GH on the net amino acid uptake in PF animals was completely blocked by cycloheximide; the uptake effect of GH in these animals was dependent therefore on de novo protein synthesis.5. It is proposed that in the adult the role of growth hormone in protein metabolism is to sustain cellular protein synthesis when there is a decrease in the level of substrate amino acids, similar to that which occurs during a short-term fast or when the dietary protein intake is inadequate.

Influence of ammonia concentration on [15N]ammonia uptake andde novosynthesis of different amino acids by mixed rumen microorganisms from the sheep rumenin vitro

1999 ◽  
Vol 1999 ◽  
pp. 212-212 ◽  
Author(s):  
C. Atasoglu ◽  
C.J. Newbold ◽  
R.J. Wallace
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
De Novo ◽  
Ammonia Concentration ◽  
Dependent Manner ◽  
Microbial Protein ◽  
Concentration Dependent Manner ◽  
Rumen Microorganisms ◽  
Ammonia Uptake

Ammonia is thought to be the main source of nitrogen for protein synthesis by the rumen microorganisms, but peptides and amino acids derived from protein degradation are also incorporated into microbial protein. Recent experiments carried out by Atasogluet al.(1998) demonstrated that preformed amino acids decrease the uptake of ammonia into microbial protein and microbial amino acids in a concentration-dependent manner. However, little is known about how rumen ammonia concentrations affect ammonia uptake into microbial protein. The present study was undertaken to determine the influence of rumen ammonia concentrations on ammonia incorporation andde novosynthesis of individual amino acids by the mixed rumen microorganismsin vitro.

scholarly journals Pinocytosis and intracellular degradation of exogenous protein: modulation by amino acids.

1983 ◽  
Vol 96 (6) ◽  
pp. 1586-1591 ◽  
Author(s):  
J M Besterman ◽  
J A Airhart ◽  
R B Low ◽  
D E Rannels
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Free Amino Acids ◽  
Free Amino ◽  
De Novo ◽  
Serum Proteins ◽  
Fluid Phase ◽  
Cellular Protein ◽  
Intracellular Degradation ◽  
Exogenous Protein

Intracellular degradation of exogenous (serum) proteins provides a source of amino acids for cellular protein synthesis. Pinocytosis serves as the mechanism for delivering exogenous protein to the lysosomes, the major site of intracellular degradation of exogenous protein. To determine whether the availability of extracellular free amino acids altered pinocytic function, we incubated monolayers of pulmonary alveolar macrophages with the fluid-phase marker, [14C]sucrose, and we dissected the pinocytic process by kinetic analysis. Additionally, intracellular degradation of endogenous and exogenous protein was monitored by measuring phenylalanine released from the cell monolayers in the presence of cycloheximide. Results revealed that in response to a subphysiological level of essential amino acids or to amino acid deprivation, (a) the rate of fluid-phase pinocytosis increased in such a manner as to preferentially increase both delivery to and size of an intracellular compartment believed to be the lysosomes, (b) the degradation of exogenously supplied albumin increased, and (c) the fraction of phenylalanine derived from degradation of exogenous albumin and reutilized for de novo protein synthesis increased. Thus, modulation of the pinosome-lysosome pathway may represent a homeostatic mechanism sensitive to the availability of extracellular free amino acids.

Effects of branched-chain-enriched amino acids and insulin on forearm leucine kinetics

1999 ◽  
Vol 97 (4) ◽  
pp. 437-448 ◽  
Author(s):  
Michela ZANETTI ◽  
Rocco BARAZZONI ◽  
Edward KIWANUKA ◽  
Paolo TESSARI
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Muscle Protein ◽  
Aromatic Amino Acids ◽  
Leucine Kinetics ◽  
Before And After ◽  
Amino Acid Mixtures ◽  
Leucine Transport ◽  
Branched Chain ◽  
Plasma Leucine

Although amino acid mixtures enriched in branched-chain amino acids (BCAA) and deficient in aromatic amino acids (AAA) are often used together with insulin and glucose in clinical nutrition, their physiological effects on muscle protein anabolism are not known. To this aim, we studied forearm leucine kinetics in post-absorptive volunteers, before and after the systemic infusion of BCAA-enriched, AAA-deficient amino acids along with insulin and the euglycaemic clamp. The results were compared with the effects of insulin infusion alone. A compartmental leucine forearm model was employed at steady state. Hyperaminoacidaemia with hyperinsulinaemia (to ≈ 80–100 μ-units/ml) increased the leucine plasma concentration (+70%; P< 0.001), inflow into the forearm cell (+150%; P< 0.01), disposal into protein synthesis (+100%; P< 0.01), net intracellular retention (P< 0.01), net forearm balance (by ≈ 6-fold; P< 0.01) and net deamination to α-ketoisocaproate (4-methyl-2-oxopentanoate) (+9%; P< 0.05). Leucine release from forearm proteolysis and outflow from the forearm cell were unchanged. In contrast, hyperinsulinaemia alone decreased plasma leucine concentrations (-35%; P< 0.001) and leucine inflow (-20%; P< 0.05) and outflow (-30%; P< 0.01) into and out of forearm cell(s), it increased net intracellular leucine retention (P< 0.03), and it did not change leucine release from forearm proteolysis (-20%; P = 0.138), net leucine deamination to α-ketoisocaproate, leucine disposal into protein synthesis or net forearm protein balance. By considering all data together, leucine disposal into protein synthesis was directly correlated with leucine inflow into the cell (r = 0.71; P< 0.0001). These data indicate that the infusion of BCAA-enriched, AAA-deficient amino acids along with insulin is capable of stimulating forearm (i.e. muscle) protein anabolism in normal volunteers by enhancing intracellular leucine transport and protein synthesis. These effects are probably due to hyperaminoacidaemia and/or its interaction with hyperinsulinaemia, since they were not observed under conditions of hyperinsulinaemia alone.

Transmembrane transport and intracellular kinetics of amino acids in human skeletal muscle

1995 ◽  
Vol 268 (1) ◽  
pp. E75-E84 ◽  
Author(s):  
G. Biolo ◽  
R. Y. Fleming ◽  
S. P. Maggi ◽  
R. R. Wolfe
Keyword(s):  
Skeletal Muscle ◽  
Amino Acids ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Human Skeletal Muscle ◽  
De Novo ◽  
Transmembrane Transport ◽  
Acid Transport ◽  
De Novo Synthesis ◽  
Intracellular Amino Acid

We have used stable isotopic tracers of amino acids to measure in vivo transmembrane transport of phenylalanine, leucine, lysine, alanine, and glutamine as well as the rates of intracellular amino acid appearance from proteolysis, de novo synthesis, and disappearance to protein synthesis in human skeletal muscle. Calculations were based on data obtained by the arteriovenous catheterization of the femoral vessels and muscle biopsy. We found that the fractional contribution of transport from the bloodstream to the total intracellular amino acid appearance depends on the individual amino acid, varying between 0.63 +/- 0.02 for phenylalanine and 0.22 +/- 0.02 for alanine. Rates of alanine and glutamine de novo synthesis were approximately eight and five times their rate of appearance from protein breakdown, respectively. The model-derived rate of protein synthesis was highly correlated with the same value calculated by means of the tracer incorporation technique. Furthermore, amino acid transport rates were in the range expected from literature values. Consequently, we conclude that our new model provides a valid means of quantifying the important aspects of protein synthesis, breakdown, and amino acid transport in human subjects.

scholarly journals A Yarrowia lipolytica Strain Engineered for Pyomelanin Production

2021 ◽  
Vol 9 (4) ◽  
pp. 838
Author(s):  
Macarena Larroude ◽  
Djamila Onésime ◽  
Olivier Rué ◽  
Jean-Marc Nicaud ◽  
Tristan Rossignol
Keyword(s):  
Amino Acids ◽  
Yarrowia Lipolytica ◽  
De Novo ◽  
Aromatic Amino Acids ◽  
Acid Synthesis ◽  
Homogentisic Acid ◽  
Lipolytica Strain ◽  
Acid Pathway ◽  
Extracellular Environment ◽  
Yeast Yarrowia Lipolytica

The yeast Yarrowia lipolytica naturally produces pyomelanin. This pigment accumulates in the extracellular environment following the autoxidation and polymerization of homogentisic acid, a metabolite derived from aromatic amino acids. In this study, we used a chassis strain optimized to produce aromatic amino acids for the de novo overproduction of pyomelanin. The gene 4HPPD, which encodes an enzyme involved in homogentisic acid synthesis (4-hydroxyphenylpyruvic acid dioxygenase), was characterized and overexpressed in the chassis strain with up to three copies, leading to pyomelanin yields of 4.5 g/L. Homogentisic acid is derived from tyrosine. When engineered strains were grown in a phenylalanine-supplemented medium, pyomelanin production increased, revealing that the yeast could convert phenylalanine to tyrosine, or that the homogentisic acid pathway is strongly induced by phenylalanine.

Steps in amino-acid incorporation into mammary tissue

1958 ◽  
Vol 149 (936) ◽  
pp. 392-400 ◽  
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Mammary Gland ◽  
Amino Acid ◽  
De Novo ◽  
Protein Biosynthesis ◽  
Mammary Tissue ◽  
Mammary Cells ◽  
Intact Cells ◽  
Ideal System

The mammary gland in full lactation had for long been recognized as an ideal system for the study of the biosynthesis of protein. The discoveries during the last 5 years of the incorporation of labelled amino acids into the microsomes of cell homogenates and of other reactions of amino acids which might be on the pathway to protein synthesis, encouraged us to study the fate of amino acids in various systems prepared from mammary cells. De novo protein synthesis had not yet been proved in any system which contained no intact cells. So far no net increase in any defined protein fraction during incubations has been found or indeed looked for in our experiments. Naturally one hopes that such studies of the fate of labelled amino acids in cell-free preparations will reveal the detail of enzymic reactions which will prove to be part of the mechanisms of protein biosynthesis. Three types of reactions of amino acids in cell-free preparations from homogenates of many tissues have been studied most extensively. (1) The acyl activation of amino acids to form amino acid-acid adenylates in the presence of ATP and ‘activating enzymes’. (2) The formation of compounds of cell sap-ribonucleic acid ( SRN A ) with amino acids in the presence of ATP and ‘activating enzymes’. (3) The incorporation of amino acids into intracellular particles either from free amino acid or by transfer from amino-acid- SRN A compounds in the presence of ATP , guanosine triphosphate ( GTP ) and ‘activating enzymes’. In this paper we are giving a survey of the results of studies on these three types of reactions in systems prepared from mammary tissue and we are relating these to results obtained with other systems elsewhere. Some comparative studies of the incorporation of labelled amino acids into protein fractions of intact mammary cells (minced tissue) are also presented. All the original results given here were obtained from experiments with guinea-pig mammary gland preparations from animals 2 to 6 days after parturition. Experimental detail will be reported elsewhere.

In Vitro Uptake of Labelled Amino Acids by the Skin of Children with Generalized Congenital Analgesia

1969 ◽  
Vol 08 (01) ◽  
pp. 83-88
Author(s):  
J. Merzel ◽  
G. Blumen ◽  
B. J. Schmidt ◽  
J. C. Maia ◽  
I. Raw ◽  
...  
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Aromatic Amino Acids ◽  
Relationship Of ◽  
In Vitro Uptake

SummaryThe “in vitro” uptake of labelled tyrosine, tryptophan and leucine by the skin of two children with GCA (Generalized Congenital Analgesia) was compared with that in normal (white and negro) individuals. Through radioactivity counts and radioautographic procedures it was shown that the uptake of the two aromatic amino acids was reduced in the GCA individuals the protein synthesis seemed to be normal.The possible relationship of these results to an abnormal metabolite isolated from the urine of these patients is discussed.

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