Neither glutamate nor alanine but arginine sensitizes BY-2 cells to arsenate

2021 ◽  
Author(s):  
Nur-E-Nazmun Nahar ◽  
Md Yeasin Prodhan ◽  
Yoshiharu Mimata ◽  
Anna Yonezawa ◽  
Toshiyuki Nakamura ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Growth ◽  
Stressed Condition ◽  
Enhancement Mechanism ◽  
Arsenate Stress

Abstract Arsenic is toxic for plants. Our previous results showed that the application of proline enhanced the sensitivity of tobacco BY-2 cells to arsenate. In order to clarify the enhancement mechanism, we investigated the effects of other amino acids on the arsenate-stressed BY-2 cells. Glutamate at up to 10 mM did not affect the cell growth in the absence or presence of arsenate. Arginine at up to 10 mM did not affect the growth in the absence of arsenate but arginine at 10 mM enhanced the inhibition of the cell growth by arsenate. Alanine at up to 10 mM did not affect the cell growth under non-stressed condition but alanine at 10 mM significantly improved the cell growth under arsenate stress. These results suggest that alanine mitigates arsenate stress in BY-2 cells and that arginine like proline enhances the sensitivity of BY-2 cells to arsenate.

The uptake of amino acids by mouse cells (strain LS) during growth in batch culture and chemostat culture: the influence of cell growth rate

1967 ◽  
Vol 168 (1013) ◽  
pp. 421-438 ◽  
Keyword(s):  
Amino Acids ◽  
Growth Rate ◽  
Amino Acid ◽  
Cell Growth ◽  
Glutamic Acid ◽  
Batch Culture ◽  
Amino Acid Uptake ◽  
Essential Amino Acids ◽  
Growth Yields ◽  
Acid Uptake

The uptake of thirteen essential amino acids by mouse LS cells in suspension culture was determined by bacteriological assay methods. Chemostat continuous-flow cultures were used to determine the effect of different cell growth rates on the quantitative amino acid requirements for growth. The growth yields of the cells ( Y = g cell dry weight produced/g amino acid utilized) were calculated for each of the essential amino acids. A mixture of the non-essential amino acids, serine, alanine and glycine increased the cell yield from the essential amino acids. The growth yields from nearly all the essential amino acids in batch culture were increased when glutamic acid was substituted for the glutamine in the medium. The growth yields from the amino acids in batch culture were much less at the beginning than at the end of the culture. The highest efficiencies of conversion of amino acids to cell material were obtained by chemostat culture. When glutamic acid largely replaced the glutamine in the medium the conversion of amino acid nitrogen to cell nitrogen was 100 % efficient (that is, the theoretical yield was obtained) at the optimum growth rate (cell doubling time, 43 h). The maximum population density a given amino acid mixture will support can be calculated from the data. It is concluded that in several routinely used tissue culture media the cell growth is limited by the amino acid supply. In batch culture glutamine was wasted by (1) its spontaneous decomposition to pyrrolidone carboxylic acid and ammonia, and (2) its enzymic breakdown to glutamic acid and ammonia, but also glutamine was used less efficiently than glutamic acid. Study of the influence of cell growth rate on amino acid uptake rates per unit mass of cells indicated that a marked change in amino acid metabolism occurred at a specific growth rate of 0.4 day -1 (cell doubling time, 43 h). With decrease in specific growth rate below 0.4 day -1 there was a marked stimulation of amino acid uptake rate per cell and essential amino acids were consumed increasingly for functions other than synthesis of cell material.

scholarly journals Functional Domains of c-myc Promoter Binding Protein 1 Involved in Transcriptional Repression and Cell Growth Regulation

1999 ◽  
Vol 19 (4) ◽  
pp. 2880-2886 ◽  
Author(s):  
Asish K. Ghosh ◽  
Robert Steele ◽  
Ratna B. Ray
Keyword(s):  
Amino Acids ◽  
Cell Growth ◽  
Dna Binding ◽  
Transcriptional Repression ◽  
Growth Regulation ◽  
Transcriptional Repressor ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Repressor Activity

ABSTRACT We initially identified c-myc promoter binding protein 1 (MBP-1), which negatively regulates c-myc promoter activity, from a human cervical carcinoma cell expression library. Subsequent studies on the biological role of MBP-1 demonstrated induction of cell death in fibroblasts and loss of anchorage-independent growth, reduced invasive ability, and tumorigenicity of human breast carcinoma cells. To investigate the potential role of MBP-1 as a transcriptional regulator, a chimeric protein containing MBP-1 fused to the DNA binding domain of the yeast transactivator factor GAL4 was constructed. This fusion protein exhibited repressor activity on the herpes simplex virus thymidine kinase promoter via upstream GAL4 DNA binding sites. Structure-function analysis of mutant MBP-1 in the context of the GAL4 DNA binding domain revealed that MBP-1 transcriptional repressor domains are located in the N terminus (amino acids 1 to 47) and C terminus (amino acids 232 to 338), whereas the activation domain lies in the middle (amino acids 140 to 244). The N-terminal domain exhibited stronger transcriptional repressor activity than the C-terminal region. When the N-terminal repressor domain was transferred to a potent activator, transcription was strongly inhibited. Both of the repressor domains contained hydrophobic regions and had an LXVXL motif in common. Site-directed mutagenesis in the repressor domains indicated that the leucine residues in the LXVXL motif are required for transcriptional repression. Mutation of the leucine residues in the common motif of MBP-1 also abrogated the repressor activity on the c-mycpromoter. In addition, the leucine mutant forms of MBP-1 failed to suppress cell growth in fibroblasts like wild-type MBP-1. Taken together, our results indicate that MBP-1 is a complex cellular factor containing multiple transcriptional regulatory domains that play an important role in cell growth regulation.

Temperature-sensitive mutants of MscL mechanosensitive channel

2019 ◽  
Vol 166 (3) ◽  
pp. 281-288 ◽  
Author(s):  
Naoto Owada ◽  
Megumi Yoshida ◽  
Kohei Morita ◽  
Kenjiro Yoshimura
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Structural Change ◽  
Cell Growth ◽  
Thermodynamic Stability ◽  
Transmembrane Helix ◽  
Mechanosensitive Channel ◽  
Temperature Sensitive ◽  
Temperature Sensitive Mutants ◽  
Membrane Stretching

Abstract MscL is a mechanosensitive channel that undergoes a global conformational change upon application of membrane stretching. To elucidate how the structural stability and flexibility occur, we isolated temperature-sensitive (Ts) mutants of Escherichia coli MscL that allowed cell growth at 32°C but not at 42°C. Two Ts mutants, L86P and D127V, were identified. The L86P mutation occurred in the second transmembrane helix, TM2. Substitution of residues neighbouring L86 with proline also led to a Ts mutation, but the substitution of L86 with other amino acids did not result in a Ts phenotype, indicating that the Ts phenotype was due to a structural change of TM2 helix by the introduction of a proline residue. The D127V mutation was localized in the electrostatic belt of the bundle of cytoplasmic helices, indicating that stability of the pentameric bundle of the cytoplasmic helix affects MscL structure. Together, this study described a novel class of MscL mutations that were correlated with the thermodynamic stability of the MscL structure.

scholarly journals Decoupling Protein Production from Cell Growth Enhances the Site-Specific Incorporation of Noncanonical Amino Acids in E. coli

2020 ◽  
Vol 9 (11) ◽  
pp. 3052-3066
Author(s):  
Meritxell Galindo Casas ◽  
Patrick Stargardt ◽  
Juergen Mairhofer ◽  
Birgit Wiltschi
Keyword(s):  
Amino Acids ◽  
Cell Growth ◽  
Protein Production ◽  
Noncanonical Amino Acids ◽  
Site Specific ◽  
E Coli

scholarly journals Nutrient-responsive mTOR signalling grows on Sterile ground

2007 ◽  
Vol 403 (1) ◽  
Author(s):  
Simon J. Cook ◽  
Simon J. Morley
Keyword(s):  
Amino Acids ◽  
Growth Factors ◽  
Growth Factor ◽  
Protein Kinase ◽  
Cell Growth ◽  
Cell Size ◽  
Protein Translation ◽  
Small Gtpase ◽  
Mtor Activity ◽  
Mtor Signalling

The control of cell growth, that is cell size, is largely controlled by mTOR (the mammalian target of rapamycin), a large serine/threonine protein kinase that regulates ribosome biogenesis and protein translation. mTOR activity is regulated both by the availability of growth factors, such as insulin/IGF-1 (insulin-like growth factor 1), and by nutrients, notably the supply of certain key amino acids. The last few years have seen a remarkable increase in our understanding of the canonical, growth factor-regulated pathway for mTOR activation, which is mediated by the class I PI3Ks (phosphoinositide 3-kinases), PKB (protein kinase B), TSC1/2 (the tuberous sclerosis complex) and the small GTPase, Rheb. However, the nutrient-responsive input into mTOR is important in its own right and is also required for maximal activation of mTOR signalling by growth factors. Despite this, the details of the nutrient-responsive signalling pathway(s) controlling mTOR have remained elusive, although recent studies have suggested a role for the class III PI3K hVps34. In this issue of the Biochemical Journal, Findlay et al. demonstrate that the protein kinase MAP4K3 [mitogen-activated protein kinase kinase kinase kinase-3, a Ste20 family protein kinase also known as GLK (germinal centre-like kinase)] is a new component of the nutrient-responsive pathway. MAP4K3 activity is stimulated by administration of amino acids, but not growth factors, and this is insensitive to rapamycin, most likely placing MAP4K3 upstream of mTOR. Indeed, MAP4K3 is required for phosphorylation of known mTOR targets such as S6K1 (S6 kinase 1), and overexpression of MAP4K3 promotes the rapamycin-sensitive phosphorylation of these same targets. Finally, knockdown of MAP4K3 levels causes a decrease in cell size. The results suggest that MAP4K3 is a new component in the nutrient-responsive pathway for mTOR activation and reveal a completely new function for MAP4K3 in promoting cell growth. Given that mTOR activity is frequently deregulated in cancer, there is much interest in new strategies for inhibition of this pathway. In this context, MAP4K3 looks like an attractive drug target since inhibitors of this enzyme should switch off mTOR, thereby inhibiting cell growth and proliferation, and promoting apoptosis.

Extracellular Metabolite Biomarkers of Bortezomib Resistance in Multiple Myeloma Indicate Involvement of Unexpected Metabolic Pathways.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1839-1839
Author(s):  
Aalim M Weljie ◽  
Paola Neri ◽  
Farzana Sayani ◽  
Nizar J Bahlis
Keyword(s):  
Amino Acids ◽  
Multiple Myeloma ◽  
Cell Growth ◽  
Cell Lines ◽  
Single Sample ◽  
Metabolic Phenotype ◽  
Growth Media ◽  
Resonance Spectroscopy ◽  
Waste Products ◽  
Extracellular Metabolites

Abstract Abstract 1839 Poster Board I-865 Introduction and Objectives: Bortezomib (BZ) is a chemotherapeutic agent approved for the treatment of multiple myeloma (MM). BZ acts through proteasome inhibition, inducing significant ER stress and ideally resulting in cell death. Unfortunately, nearly 20% of MM patients are primarily resistant to BZ treatment and responses to BZ are difficult to predict based on the currently available clinical, cytogenetic and genomic biomarkers. Our function hypothesis is that extracellular metabolites have a greater potential to be found in circulating biofluids as biomarkers. As a result we used a metabolite ‘footprinting’ approach in cell growth media to examine the metabolic consequences of BZ treatment using eight human MM cell lines, three of which have been determined to be less sensitive to BZ treatment than the others with a 10 fold difference in their IC50 at 24 hours (5 nM vs 50 nM). Our aims were 1) to establish whether analysis of growth media was suitable for monitoring metabolic changes and 2) to determine specific biopatterns of BZ resistance. Methods: Eight MM cell lines (MM1S, MM1R, INA6, U266, RPMI8266, OPM2, KMS11 and PCL1) were cultured under standard conditions without (control group, 10% FBS) or with bortezomib added (10nM). Media samples were taken for metabolic analysis at 6 and 24 hours for a total of 32 media profiles. Metabolite profiling was accomplished using gas chromatography mass spectrometry (GC-MS) and nuclear magnetic resonance spectroscopy (NMR). GC-MS data was analysed using AMDIS (NIST), and NMR data using Chenomx NMR Suite. Significant metabolites were identified using multivariate regression analysis by supervised projection methods (two-way orthogonal partial least squares discriminant analysis, O2PLS-DA) using SIMCA-P (Umetrics). Results: An average of 756 chemical or metabolite components per sample were profiled, which was reduced to a subset of 116 unique features that were shared in at least 75% of samples. An initial O2PLS-DA model was successfully built from the GC-MS feature set using both growth time (p=0.03) and BZ status (p=6.9e-13) in the Y-matrix. Figure 1 shows a scores plot, where each point represents a single sample, and the position is calculated as a combination of the underlying metabolite concentrations. Changes in cell growth were consistent with the known uptake of carbohydrate substrates and elimination of various amino acids and waste products such as lactate. The remarkable metabolic difference between BZ-treated and untreated cells resulted from reduced energy-related metabolites such as citric acid cycle intermediates and sugars, with a concomitant increase in selected amino acids. Intriguingly, the BZ-insensitive cell cultures exhibit overall metabolic phenotypes much more similar to the BZ-sensitive cultures than to the untreated group, with the exception of a single sample after 6 hours (denoted with an asterisk in Figure 1) which showed an averaged profile. To further probe the phenomenon of BZ resistance, the treated group was analyzed independently, with the 37 most influential components providing discriminating ability between the BZ-insensitive and BZ-sensitive cells (p= 0.04) in an OPLS-DA model. Conclusions: We conclude that metabolite footprinting is a reliable and robust method for monitoring metabolic events for both cell growth and BZ treatment. Furthermore, BZ-insensitivity is accompanied by a notable shift from carbohydrate metabolism to fatty acid metabolism, while the overall metabolic phenotype remains very similar in both BZ-sensitive and insensitive strains in the presence of the drug. This result suggests that BZ function remains largely intact in both sensitive and insensitive cell lines, and resistance is conferred through alternate mechanisms with measureable metabolic endpoints. Success in measuring extracellular metabolites also supports the notion of serum-accessible biomarkers or biopatterns of BZ resistance. The unique genetic instability underlying each cell line may provide a further avenue for characterizing resistance mechanisms along with analysis of various intracellular components. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Cold-stress responses in the Antarctic basidiomycetous yeast Mrakia blollopis

2016 ◽  
Vol 3 (7) ◽  
pp. 160106 ◽  
Author(s):  
Masaharu Tsuji
Keyword(s):  
Amino Acids ◽  
Cell Growth ◽  
Cold Stress ◽  
Stress Responses ◽  
Aromatic Amino Acids ◽  
Tca Cycle ◽  
Basidiomycetous Yeast ◽  
Subzero Temperature ◽  
Subzero Temperatures ◽  
The Antarctic

Microbes growing at subzero temperatures encounter numerous growth constraints. However, fungi that inhabit cold environments can grow and decompose organic compounds under subzero temperatures. Thus, understanding the cold-adaptation strategies of fungi under extreme environments is critical for elucidating polar-region ecosystems. Here, I report that two strains of the Antarctic basidiomycetous yeast Mrakia blollopis exhibited distinct growth characteristics under subzero conditions: SK-4 grew efficiently, whereas TKG1-2 did not. I analysed the metabolite responses elicited by cold stress in these two M. blollopis strains by using capillary electrophoresis–time-of-flight mass spectrometry. M. blollopis SK-4, which grew well under subzero temperatures, accumulated high levels of TCA-cycle metabolites, lactic acid, aromatic amino acids and polyamines in response to cold shock. Polyamines are recognized to function in cell-growth and developmental processes, and aromatic amino acids are also known to improve cell growth at low temperatures. By contrast, in TKG1-2, which did not grow efficiently, cold stress strongly induced the metabolites of the TCA cycle, but other metabolites were not highly accumulated in the cell. Thus, these differences in metabolite responses could contribute to the distinct abilities of SK-4 and TKG1-2 cells to grow under subzero temperature conditions.

The GATOR–Rag GTPase pathway inhibits mTORC1 activation by lysosome-derived amino acids

Science ◽  
2020 ◽  
Vol 370 (6514) ◽  
pp. 351-356
Author(s):  
Geoffrey G. Hesketh ◽  
Fotini Papazotos ◽  
Judy Pawling ◽  
Dushyandi Rajendran ◽  
James D. R. Knight ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Growth ◽  
Target Of Rapamycin ◽  
Lysosomal Degradation ◽  
Oncogenic Ras ◽  
Mechanistic Target Of Rapamycin ◽  
Independent Mechanism ◽  
Guanosine Triphosphatase ◽  
Rag Gtpase ◽  
Mtorc1 Activation

The mechanistic target of rapamycin complex 1 (mTORC1) couples nutrient sufficiency to cell growth. mTORC1 is activated by exogenously acquired amino acids sensed through the GATOR–Rag guanosine triphosphatase (GTPase) pathway, or by amino acids derived through lysosomal degradation of protein by a poorly defined mechanism. Here, we revealed that amino acids derived from the degradation of protein (acquired through oncogenic Ras-driven macropinocytosis) activate mTORC1 by a Rag GTPase–independent mechanism. mTORC1 stimulation through this pathway required the HOPS complex and was negatively regulated by activation of the GATOR-Rag GTPase pathway. Therefore, distinct but functionally coordinated pathways control mTORC1 activity on late endocytic organelles in response to distinct sources of amino acids.

Imbalance of non-essential amino acids and cell growth

1963 ◽  
Vol 31 (1) ◽  
pp. 13-18 ◽  
Author(s):  
Norma H. Best ◽  
S. Mills ◽  
Kay K. Leach ◽  
N.N. Durham ◽  
F.R. Leach
Keyword(s):  
Amino Acids ◽  
Cell Growth ◽  
Essential Amino Acids
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