Effects of amino acids and ethanolamine on choline uptake and phosphatidylcholine biosynthesis in baby hamster kidney-21 cells

1992 ◽  
Vol 70 (12) ◽  
pp. 1319-1324 ◽  
Author(s):  
Xiliang Zha ◽  
Francis T. Jay ◽  
Patrick C. Choy
Keyword(s):  
Amino Acids ◽  
Culture Media ◽  
Specific Radioactivity ◽  
Kinetic Studies ◽  
Choline Uptake ◽  
Dependent Manner ◽  
Baby Hamster Kidney ◽  
Concentration Dependent Manner ◽  
Cell Culture Media ◽  
Phosphatidylcholine Biosynthesis

The effects of amino acids and ethanolamine on choline uptake and phosphatidylcholine biosynthesis in baby hamster kidney (BHK-21) cells were investigated. The cells were incubated with labelled choline in the presence of an amino acid or ethanolamine. The uptake of labelled choline was noncompetitively inhibited by amino acids. Glycine, L-alanine, L-serine, L-leucine, L-aspartate, and L-arginine were effective inhibitors and a maximum of 22% inhibition of choline uptake was obtained with 5 mM glycine. Analyses of the labellings in the choline-containing metabolites revealed that the conversion of choline to CDP-choline and subsequently phosphatidylcholine was not affected by the presence of amino acids. The uptake of choline was also inhibited by ethanolamine in a concentration-dependent manner. Kinetic studies on the uptake of choline indicated that the inhibition by ethanolamine was competitive in nature. Although ethanolamine is a potent inhibitor of choline kinase, analyses of the labellings in the choline-containing metabolites indicated that the conversion of choline to phosphocholine was not affected in the cells incubated with ethanolamine. Ethanolamine did not change the pool sizes of phosphocholine and CDP-choline. Based on the specific radioactivity of CDP-choline and the labelling of phosphatidylcholine, the rates of phosphatidylcholine biosynthesis were not significantly different between the control and the ethanolamine-treated cells. In view of the concentrations of amino acids (millimolar) and ethanolamine (micromolar) in most cell culture media, it appeared that only amino acids were important metabolites for the regulation of choline uptake in BHK-21 cells. We conclude that both amino acids and ethanolamine have no direct effect on the biosynthesis of phosphatidylcholine.Key words: choline uptake, phosphatidylcholine biosynthesis, amino acids, ethanolamine, BHK-21 cells.

scholarly journals Development of novel MOG analogues with increased stability to explore MCT2 and α-ketoglutarate biology in vivo

2021 ◽  
Author(s):  
Louise Fets ◽  
Patrícia M. Nunes ◽  
Sebastien Campos ◽  
Mariana Silva dos Santos ◽  
Natalie Bevan ◽  
...  
Keyword(s):  
Culture Media ◽  
Pharmacokinetic Profile ◽  
Monocarboxylate Transporter ◽  
Metabolic Effects ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Cell Culture Media ◽  
Pharmacokinetic Properties

ABSTRACTα-ketoglutarate (αKG) is a central metabolic node with far-reaching influence on cellular physiology. The αKG analogue N-oxalylglycine (NOG) and its membrane-permeable pro-drug derivative dimethyloxalylglycine (DMOG) have been broadly used as tool compounds both in vitro and in vivo to study αKG-dependent processes. In cell culture media, DMOG is rapidly converted to MOG, a substrate of the monocarboxylate transporter MCT2. The expression level of MCT2 determines the intracellular concentration of NOG, and, as such, influences the molecular targets NOG engages with. Here we show that DMOG and MOG are highly unstable also in mouse blood. We therefore designed and characterised a series of MOG analogues with two aims: to improve pharmacokinetic properties, and to explore the pharmacophore of MCT2, a relatively understudied member of the SLC16 family. We report MOG analogues that maintain MCT2-dependent uptake, including NOG-generating compounds that replicate the metabolic effects of MOG in a concentration-dependent manner. One such analogue, IPOG, shows significantly increased blood stability, and an improved overall pharmacokinetic profile, leading to increased NOG accumulation in MCT2-expressing tumours versus isogenic controls.

scholarly journals The Amino Terminus of Varicella-Zoster Virus (VZV) Glycoprotein E Is Required for Binding to Insulin-Degrading Enzyme, a VZV Receptor

2007 ◽  
Vol 81 (16) ◽  
pp. 8525-8532 ◽  
Author(s):  
Qingxue Li ◽  
Tammy Krogmann ◽  
Mir A. Ali ◽  
Wei-Jen Tang ◽  
Jeffrey I. Cohen
Keyword(s):  
Amino Acids ◽  
Varicella Zoster Virus ◽  
Catalytic Domain ◽  
Amino Terminus ◽  
Dependent Manner ◽  
Insulin Degrading Enzyme ◽  
Concentration Dependent Manner ◽  
Glycoprotein E ◽  
Degrading Enzyme ◽  
Varicella Zoster

ABSTRACT Varicella-zoster virus (VZV) glycoprotein E (gE) is required for VZV infection. Although gE is well conserved among alphaherpesviruses, the amino terminus of VZV gE is unique. Previously, we showed that gE interacts with insulin-degrading enzyme (IDE) and facilitates VZV infection and cell-to-cell spread of the virus. Here we define the region of VZV gE required to bind IDE. Deletion of amino acids 32 to 71 of gE, located immediately after the predicted signal peptide, resulted in loss of the ability of gE to bind IDE. A synthetic peptide corresponding to amino acids 24 to 50 of gE blocked its interaction with IDE in a concentration-dependent manner. However, a chimeric gE in which amino acids 1 to 71 of VZV gE were fused to amino acids 30 to 545 of herpes simplex virus type 2 gE did not show an increased level of binding to IDE compared with that of full-length HSV gE. Thus, amino acids 24 to 71 of gE are required for IDE binding, and the secondary structure of gE is critical for the interaction. VZV gE also forms a heterodimer with glycoprotein gI. Deletion of amino acids 163 to 208 of gE severely reduced its ability to form a complex with gI. The amino portion of IDE, as well an IDE mutant in the catalytic domain of the protein, bound to gE. Therefore, distinct motifs of VZV gE are important for binding to IDE or to gI.

scholarly journals The effect of calcium antagonists on the growth of Entamoeba histolytica in vitro

2010 ◽  
Vol 4 (1) ◽  
pp. 5-10
Author(s):  
Zahra’a Abdul-Raheem Ahmed ◽  
Ali H. Ad’hiah ◽  
Amna N. Jasim
Keyword(s):  
Entamoeba Histolytica ◽  
Calcium Antagonists ◽  
Culture Media ◽  
Agar Medium ◽  
Reproduction Rate ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Infusion Agar ◽  
Ethylene Diaminetetraacetic Acid

he E. histolytica parasite was maintained in vitro using Locke-egg medium (LEM) and Liver infusion agar medium (LIAM). The effect of two calcium antagonists (Nifedipine and Ethylene-diaminetetraacetic acid EDTA) on the growth and activity of the parasite in the two culture media was investigated. The calcium antagonists Nifedipine and EDTA inhibited the reproduction rate of E. histolytica in a concentration-dependent manner. For Nifedipine, a concentration of 41.6 mg/ml inhibited the reproduction rate to 99.7% in both media. The EDTA had an approximate effect (98.2 and 95.8)% at a concentration of 0.83 mg/ml in LEM and LIAM media, respectively. Additionally, some cases of a parasite encystment were observed in LEM medium that was treated with Nifedipine.

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.

Effect of amino acids on choline uptake and phosphatidylcholine biosynthesis in the isolated hamster heart

1988 ◽  
Vol 66 (5) ◽  
pp. 418-424 ◽  
Author(s):  
Grant M. Hatch ◽  
Willem K. Stevens ◽  
Patrick C. Choy
Keyword(s):  
Amino Acids ◽  
Direct Interaction ◽  
Choline Uptake ◽  
Choline Transport ◽  
Neutral Amino Acids ◽  
Acidic Amino Acids ◽  
Phosphatidylcholine Biosynthesis ◽  
Concentration Threshold ◽  
Hamster Heart ◽  
Dose Dependent

Choline uptake by the hamster heart has been shown to be enhanced by exogenous glycine. In this study, the effect of neutral, basic, and acidic amino acids on choline uptake was assessed. Hamster hearts were perfused with labelled choline, and in the presence of L-alanine, L-serine, or L-phenylalanine (≥0.1 mM), choline uptake was enhanced 20–38%. L-Arginine, L-lysine, L-aspartate, and L-glutamate did not influence choline uptake. The rate of phosphatidylcholine biosynthesis was unaffected by all amino acids tested. Enhancement of choline uptake by neutral amino acids was not additive or dose dependent but required a concentration threshold. The enhancement of choline uptake by neutral amino acids was not influenced by preperfusion with the same amino acid. Exogenous choline had no effect on the uptake of amino acids. We postulate that choline and the neutral amino acids are not cotransported and modulation of choline uptake is facilitated by direct interaction of the neutral amino acids with the choline transport system.

scholarly journals A big cheese in biotherapeutics: Lactoyl leucine and isoleucine are bioavailable alternatives for canonical amino acids in cell culture media

2021 ◽  
Author(s):  
Corinna Schmidt ◽  
Maria Wehsling ◽  
Maxime Le Mignon ◽  
Gregor Wille ◽  
Yannick Rey ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Culture ◽  
Cho Cells ◽  
Culture Media ◽  
Chinese Hamster ◽  
Batch Processes ◽  
Next Generation ◽  
Cell Culture Media ◽  
Derivatives Of

Increasing demands for protein-based therapeutics such as monoclonal antibodies, fusion proteins, bispecific molecules and antibody fragments require researchers to constantly find innovative solutions. To increase yields and decrease costs of next generation bioprocesses, highly concentrated cell culture media formulations are developed but often limited by the low solubility of amino acids such as tyrosine, cystine, leucine and isoleucine, in particular at physiological pH. This work sought to investigate highly soluble and bioavailable derivatives of leucine and isoleucine that are applicable for fed-batch processes. N-lactoyl-leucine and N-lactoyl-isoleucine sodium salts were tested in cell culture media and proved to be beneficial to increase the overall solubility of cell culture media formulations. These modified amino acids proved to be bioavailable for various Chinese hamster ovary (CHO) cells and were suitable for replacement of canonical amino acids in cell culture feeds. The quality of the final recombinant protein was studied in bioprocesses using the derivatives, and the mechanism of cleavage was investigated in CHO cells. Altogether, both N-lactoyl amino acids represent an advantageous alternative to canonical amino acids to develop highly concentrated cell culture media formulations to support next generation bioprocesses.

Anionic amino acid uptake by microvillous membrane vesicles from human placenta

1989 ◽  
Vol 257 (5) ◽  
pp. C1005-C1011 ◽  
Author(s):  
A. J. Moe ◽  
C. H. Smith
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Glutamate Uptake ◽  
Membrane Vesicles ◽  
Amino Acid Uptake ◽  
Maternal Blood ◽  
Acid Uptake ◽  
Dependent Manner ◽  
Concentration Dependent Manner

The transport mechanisms for anionic amino acids in trophoblast microvillous (maternal facing) membrane were investigated by characterization of L-[3H]aspartate and L-[3H]glutamate uptake in membrane vesicles. Uptake of the anionic amino acids was by a single high-affinity Na+-dependent K+-stimulated cotransporter that is pH sensitive and electrogenic. A second Na+-dependent transporter could not be discriminated, and there was no observable Na+-independent uptake. An outwardly directed K+ gradient (100 mM KCl inside) resulted in a 5- to 10-fold stimulation in glutamate uptake in the presence of Na+. Intravesicular KCl had no effect on transporter affinity but increased transporter velocity in a concentration-dependent manner. Inhibition of Na+-K+-dependent uptake of L-aspartate and L-glutamate (20 mM, 30 s) by 2 mM unlabeled amino acids demonstrated stereoselectivity for L-glutamate but not for L-aspartate. The neutral amino acids (L-alanine, L-threonine, L-serine, L-cysteine, L-phenylalanine) were not effective inhibitors. These data are consistent with an anionic amino acid transporter in the microvillous membrane of the trophoblast, which has characteristics qualitatively similar to the X-AG system found in other epithelia. This system may mediate the concentrative placental uptake of anionic amino acids from maternal blood in utero.

Effect of protein precipitating agents on the recovery of plasma free amino acids

1991 ◽  
Vol 71 (3) ◽  
pp. 953-957 ◽  
Author(s):  
G. W. Sedgwick ◽  
T. W. Fenton ◽  
J. R. Thompson
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Free Amino Acid ◽  
Free Amino ◽  
Trichloroacetic Acid ◽  
Protein Concentration ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Amino Acid Concentrations ◽  
Plasma Free Amino Acids

The effect of the protein precipitants acetone, acetonitrile, perchloric acid and trichloroacetic acid on free amino acid concentrations in supernatants from ovine plasma and bovine serum albumin solutions was determined. The organic precipitants decreased (P < 0.05) free amino acid concentrations in a protein concentration dependent manner while the acid precipitants had no effect. Key words: Amino acids, protein precipitating agents

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