scholarly journals Spontaneous hydrolysis and spurious metabolic properties of α-ketoglutarate esters

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Seth J. Parker ◽  
Joel Encarnación-Rosado ◽  
Kate E. R. Hollinshead ◽  
David M. Hollinshead ◽  
Leonard J. Ash ◽  
...  
Keyword(s):  
Cell Lines ◽  
Aqueous Media ◽  
Cellular Metabolism ◽  
Inhibitory Effects ◽  
Extracellular Acidification ◽  
Spontaneous Hydrolysis ◽  
Metabolic Properties ◽  
Independent Effects ◽  
Rapid Hydrolysis ◽  
Pleiotropic Functions

Abstractα-ketoglutarate (KG), also referred to as 2-oxoglutarate, is a key intermediate of cellular metabolism with pleiotropic functions. Cell-permeable esterified analogs are widely used to study how KG fuels bioenergetic and amino acid metabolism and DNA, RNA, and protein hydroxylation reactions, as cellular membranes are thought to be impermeable to KG. Here we show that esterified KG analogs rapidly hydrolyze in aqueous media, yielding KG that, in contrast to prevailing assumptions, imports into many cell lines. Esterified KG analogs exhibit spurious KG-independent effects on cellular metabolism, including extracellular acidification, arising from rapid hydrolysis and de-protonation of α-ketoesters, and significant analog-specific inhibitory effects on glycolysis or mitochondrial respiration. We observe that imported KG decarboxylates to succinate in the cytosol and contributes minimally to mitochondrial metabolism in many cell lines cultured in normal conditions. These findings demonstrate that nuclear and cytosolic KG-dependent reactions may derive KG from functionally distinct subcellular pools and sources.

scholarly journals Inhibitory effects of methanol extracts of selected plants on the proliferation of two human melanoma cell lines

2018 ◽  
Vol 17 (6) ◽  
pp. 1081 ◽  
Author(s):  
Alaa Fraihat ◽  
Luma Alatrash ◽  
Reem Abbasi ◽  
Bashaer Abu-Irmaileh ◽  
Saja Hamed ◽  
...  
Keyword(s):  
Cell Lines ◽  
Melanoma Cell ◽  
Human Melanoma ◽  
Human Melanoma Cell ◽  
Inhibitory Effects ◽  
Methanol Extracts ◽  
Melanoma Cell Lines

Immunofluorescent localization of the ubiquitin-activating enzyme, E1, to the nucleus and cytoskeleton

1993 ◽  
Vol 264 (1) ◽  
pp. C93-C102 ◽  
Author(s):  
J. S. Trausch ◽  
S. J. Grenfell ◽  
P. M. Handley-Gearhart ◽  
A. Ciechanover ◽  
A. L. Schwartz
Keyword(s):  
Cell Lines ◽  
Chinese Hamster ◽  
Eukaryotic Cell ◽  
Nuclear Staining ◽  
Immunofluorescent Localization ◽  
Acid Protein ◽  
Ubiquitin Conjugation ◽  
Double Label ◽  
Pleiotropic Functions ◽  
Amino Acid Protein

Ubiquitin, a 76-amino acid protein, is covalently attached to abnormal and short-lived proteins, thus marking them for ATP-dependent proteolysis in eukaryotic cells. Ubiquitin is found within the cytoplasm, nucleus, microvilli, autophagic vacuoles, and lysosomes. The ubiquitin-activating enzyme, E1, catalyzes the first step in ubiquitin conjugation. To date, very little is known about the subcellular distribution of this enzyme. We have utilized immunofluorescence and immunoblotting to examine the cellular distribution of E1 in several eukaryotic cell lines, including HeLa, smooth muscle A7r5, choriocarcinoma BeWo, Pt K1, and Chinese hamster ovary (CHO) E36. E1 was identified in both cytoplasmic and nuclear compartments in all cell lines examined. However, the relative abundance within these compartments differed markedly between the cell lines. Even within a single cell line, nuclear distribution was not uniform, and certain cells demonstrated an absence of nuclear staining. E1 resides predominantly within the nucleus in BeWo. In contrast, its distribution in CHO and Pt K1 cells is mainly cytoplasmic. Within the cytoplasm, three pools of E1 were identified by double-label immunofluorescence. The first of these colocalized with phalloidin, indicating association of E1 with actin filaments. A second cytoplasmic pool colocalized with tubulin and was predominantly perinuclear in its distribution. The third pool associated with intermediate filaments. This suggests that E1 is associated with all three components of the cytoskeleton. The distribution of E1 was unaltered in a mutant line of CHO E36 designated ts20, in which the E1 can be thermally inactivated. The variable distribution of E1 among cell lines, including its apparent cytoskeletal association, suggests pleiotropic functions of this enzyme and the ubiquitin-conjugating system.

scholarly journals Draft Genome Sequence of the Symbiotically Competent Cyanobacterium Nostoc sp. Strain KVJ20

2019 ◽  
Vol 8 (45) ◽  
Author(s):  
Marie-Josée H. Halsør ◽  
Anton Liaimer ◽  
Seila Pandur ◽  
Inger L. U. Ræder ◽  
Arne O. Smalås ◽  
...  
Keyword(s):  
Secondary Metabolites ◽  
Cell Lines ◽  
Cancer Cell ◽  
Genome Sequence ◽  
Draft Genome ◽  
Cancer Cell Lines ◽  
Draft Genome Sequence ◽  
Inhibitory Effects ◽  
Content Type

Nostoc sp. strain KVJ20 was isolated from the symbiotic organs of the liverwort Blasia pusilla. This cyanobacterium has been shown to have broad symbiotic competence, and bacterial extracts have inhibitory effects on cancer cell lines and microbes. An array of genes for the production of secondary metabolites is present.

scholarly journals Cytotoxicity and Mitochondrial Effects of Phenolic and Quinone-Based Mitochondria-Targeted and Untargeted Antioxidants on Human Neuronal and Hepatic Cell Lines: A Comparative Analysis

Biomolecules ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 1605
Author(s):  
Carlos Fernandes ◽  
Afonso J. C. Videira ◽  
Caroline D. Veloso ◽  
Sofia Benfeito ◽  
Pedro Soares ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Lines ◽  
Mitochondrial Membrane ◽  
Hepatic Cell ◽  
Therapeutic Application ◽  
Extracellular Acidification ◽  
Mitochondrial Oxidative Stress ◽  
Drug Candidates ◽  
Cellular Oxygen ◽  
Hepatic Cell Lines

Mitochondriotropic antioxidants (MC3, MC6.2, MC4 and MC7.2) based on dietary antioxidants and analogs (caffeic, hydrocaffeic, trihydroxyphenylpropanoic and trihydroxycinnamic acids) were developed. In this study, we evaluate and compare the cytotoxicity profile of novel mitochondria-targeted molecules (generally known as MitoCINs) on human HepG2 and differentiated SH-SY5Y cells with the quinone-based mitochondria-targeted antioxidants MitoQ and SkQ1 and with two non-targeted antioxidants, resveratrol and coenzyme Q10 (CoQ10). We further evaluate their effects on mitochondrial membrane potential, cellular oxygen consumption and extracellular acidification rates. Overall, MitoCINs derivatives reduced cell viability at concentrations about six times higher than those observed with MitoQ and SkQ1. A toxicity ranking for both cell lines was produced: MC4 < MC7.2 < MC3 < MC6.2. These results suggest that C-6 carbon linker and the presence of a pyrogallol group result in lower cytotoxicity. MC3 and MC6.2 affected the mitochondrial function more significantly relative to MitoQ, SkQ1, resveratrol and CoQ10, while MC4 and MC7.2 displayed around 100–1000× less cytotoxicity than SkQ1 and MitoQ. Based on the mitochondrial and cytotoxicity cellular data, MC4 and MC7.2 are proposed as leads that can be optimized to develop safe drug candidates with therapeutic application in mitochondrial oxidative stress-related diseases.

scholarly journals Metabolic drug survey highlights cancer cell dependencies and vulnerabilities

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Tea Pemovska ◽  
Johannes W. Bigenzahn ◽  
Ismet Srndic ◽  
Alexander Lercher ◽  
Andreas Bergthaler ◽  
...  
Keyword(s):  
Cell Lines ◽  
High Throughput Screening ◽  
Drug Response ◽  
Fatty Acid Synthase ◽  
Leukemia Cell ◽  
Cellular Metabolism ◽  
Acid Synthesis ◽  
Metabolic Drug ◽  
Response Profiles ◽  
Synthase Inhibitor

AbstractInterrogation of cellular metabolism with high-throughput screening approaches can unravel contextual biology and identify cancer-specific metabolic vulnerabilities. To systematically study the consequences of distinct metabolic perturbations, we assemble a comprehensive metabolic drug library (CeMM Library of Metabolic Drugs; CLIMET) covering 243 compounds. We, next, characterize it phenotypically in a diverse panel of myeloid leukemia cell lines and primary patient cells. Analysis of the drug response profiles reveals that 77 drugs affect cell viability, with the top effective compounds targeting nucleic acid synthesis, oxidative stress, and the PI3K/mTOR pathway. Clustering of individual drug response profiles stratifies the cell lines into five functional groups, which link to specific molecular and metabolic features. Mechanistic characterization of selective responses to the PI3K inhibitor pictilisib, the fatty acid synthase inhibitor GSK2194069, and the SLC16A1 inhibitor AZD3965, bring forth biomarkers of drug response. Phenotypic screening using CLIMET represents a valuable tool to probe cellular metabolism and identify metabolic dependencies at large.

Recruitment of purinergically stimulated Cl- channels from granule membrane to plasma membrane

1996 ◽  
Vol 271 (2) ◽  
pp. C612-C619 ◽  
Author(s):  
D. Merlin ◽  
X. Guo ◽  
K. Martin ◽  
C. Laboisse ◽  
D. Landis ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Lines ◽  
Inhibitory Effects ◽  
Fungal Metabolite ◽  
Cl Secretion ◽  
Fusion Inhibitors ◽  
Granule Membrane ◽  
Cl Channels ◽  
Ht 29 ◽  
Cl Conductance

HT29-Cl.16E and HT29-Cl.19A are two different subcloned cell lines derived from the human adenocarcinoma cell line HT-29. They are similar in their ability to grow and differentiate to polarized epithelial cells but differ in that HT29-Cl.16E is goblet cell-like with many mucin granules, whereas HT29-Cl.19A lacks mucin granules. Extracellular ATP stimulates Cl- secretion in both cell lines through luminal purinergic P20 receptors and, in HT29-Cl.16E, also mucin secretion release. To evaluate whether fusion of mucin granules is associated with an increase in Cl- conductance of the plasma membrane, the effects of two fusion inhibitors on luminal Cl- conductance were measured. Blockage of actin depolymerization with phalloidin (1 microM) inhibited purinergically stimulated but not adenosine 3',5'-cyclic monophosphate (cAMP)-stimulated luminal Cl- efflux by 50% in HT29-Cl.16E. The same treatment was without effect in HT29-Cl.19A. The fungal metabolite wortmannin, which is an inhibitor of regulated exocytosis in leukocytes, at 100 nM inhibited Cl- secretion by 70% in HT29-Cl.16E. This inhibition was not a direct effect on purinergically stimulated Cl- channels because wortmannin concentrations of up to 1 microM did not affect the secretory response in HT29-Cl.19A. The wortmannin inhibition of Cl- secretion is associated with an inhibition of granule fusion as judged by electron microscopy. The differential inhibition of Cl- secretion in the related HT-29 clones that differ with respect to the presence of mucin granules indicates that 1) the granule fusion inhibitors, phalloidin and wortmannin, have no direct inhibitory effects on purinergically and cAMP-activated Cl- channels, 2) a major portion of purinergically but not cAMP-activated Cl- channels is associated with granule fusion in HT29-Cl.16E, and 3) the signaling pathways for Cl- secretion and granule fusion are not completely identical.

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