scholarly journals Degradation of Extracellular NAD+ Intermediates in Cultures of Human HEK293 Cells

Metabolites ◽  
2019 ◽  
Vol 9 (12) ◽  
pp. 293 ◽  
Author(s):  
Veronika Kulikova ◽  
Konstantin Shabalin ◽  
Kirill Nerinovski ◽  
Alexander Yakimov ◽  
Maria Svetlova ◽  
...  
Keyword(s):  
Cell Culture ◽  
Nicotinic Acid ◽  
Culture Medium ◽  
Cultured Cells ◽  
Culture Conditions ◽  
Hek293 Cells ◽  
Vitamin B3 ◽  
Serum Free ◽  
Nicotinamide Riboside ◽  
Nicotinamide Mononucleotide

Nicotinamide adenine dinucleotide (NAD) is an essential redox carrier, whereas its degradation is a key element of important signaling pathways. Human cells replenish their NAD contents through NAD biosynthesis from extracellular precursors. These precursors encompass bases nicotinamide (Nam) and nicotinic acid and their corresponding nucleosides nicotinamide riboside (NR) and nicotinic acid riboside (NAR), now collectively referred to as vitamin B3. In addition, extracellular NAD+ and nicotinamide mononucleotide (NMN), and potentially their deamidated counterparts, nicotinic acid adenine dinucleotide (NAAD) and nicotinic acid mononucleotide (NAMN), may serve as precursors of intracellular NAD. However, it is still debated whether nucleotides enter cells directly or whether they are converted to nucleosides and bases prior to uptake into cells. Here, we studied the metabolism of extracellular NAD+ and its derivatives in human HEK293 cells using normal and serum-free culture medium. Using medium containing 10% fetal bovine serum (FBS), mono- and dinucleotides were degraded to the corresponding nucleosides. In turn, the nucleosides were cleaved to their corresponding bases. Degradation was also observed in culture medium alone, in the absence of cells, indicating that FBS contains enzymatic activities which degrade NAD+ intermediates. Surprisingly, NR was also rather efficiently hydrolyzed to Nam in the absence of FBS. When cultivated in serum-free medium, HEK293 cells efficiently cleaved NAD+ and NAAD to NMN and NAMN. NMN exhibited rather high stability in cell culture, but was partially metabolized to NR. Using pharmacological inhibitors of plasma membrane transporters, we also showed that extracellular cleavage of NAD+ and NMN to NR is a prerequisite for using these nucleotides to maintain intracellular NAD contents. We also present evidence that, besides spontaneous hydrolysis, NR is intensively metabolized in cell culture by intracellular conversion to Nam. Our results demonstrate that both the cultured cells and the culture medium mediate a rather active conversion of NAD+ intermediates. Consequently, in studies of precursor supplementation and uptake, the culture conditions need to be carefully defined.

scholarly journals Equilibrative Nucleoside Transporters Mediate the Import of Nicotinamide Riboside and Nicotinic Acid Riboside into Human Cells

2021 ◽  
Vol 22 (3) ◽  
pp. 1391
Author(s):  
Andrey Kropotov ◽  
Veronika Kulikova ◽  
Kirill Nerinovski ◽  
Alexander Yakimov ◽  
Maria Svetlova ◽  
...  
Keyword(s):  
Nicotinic Acid ◽  
Mammalian Cells ◽  
Experimental Models ◽  
The Body ◽  
Nucleoside Transporters ◽  
Hek293 Cells ◽  
Nucleoside Transporter ◽  
Vitamin B3 ◽  
Animal Cells ◽  
Nicotinamide Riboside

Nicotinamide riboside (NR), a new form of vitamin B3, is an effective precursor of nicotinamide adenine dinucleotide (NAD+) in human and animal cells. The introduction of NR into the body effectively increases the level of intracellular NAD+ and thereby restores physiological functions that are weakened or lost in experimental models of aging and various pathologies. Despite the active use of NR in applied biomedicine, the mechanism of its transport into mammalian cells is currently not understood. In this study, we used overexpression of proteins in HEK293 cells, and metabolite detection by NMR, to show that extracellular NR can be imported into cells by members of the equilibrative nucleoside transporter (ENT) family ENT1, ENT2, and ENT4. After being imported into cells, NR is readily metabolized resulting in Nam generation. Moreover, the same ENT-dependent mechanism can be used to import the deamidated form of NR, nicotinic acid riboside (NAR). However, NAR uptake into HEK293 cells required the stimulation of its active utilization in the cytosol such as phosphorylation by NR kinase. On the other hand, we did not detect any NR uptake mediated by the concentrative nucleoside transporters (CNT) CNT1, CNT2, or CNT3, while overexpression of CNT3, but not CNT1 or CNT2, moderately stimulated NAR utilization by HEK293 cells.

Development of a novel ELISA for human insulin using monoclonal antibodies produced in serum-free cell culture medium

2007 ◽  
Vol 40 (1-2) ◽  
pp. 98-103 ◽  
Author(s):  
Megha S. Even ◽  
Chad B. Sandusky ◽  
Neal D. Barnard ◽  
Jehangir Mistry ◽  
Madhur K. Sinha
Keyword(s):  
Cell Culture ◽  
Monoclonal Antibodies ◽  
Human Insulin ◽  
Culture Medium ◽  
Free Cell ◽  
Cell Culture Medium ◽  
Serum Free

scholarly journals Silk fibroin induces chondrogenic differentiation of canine adipose–derived multipotent mesenchymal stromal cells/mesenchymal stem cells

2019 ◽  
Vol 10 ◽  
pp. 204173141983505 ◽  
Author(s):  
Metka Voga ◽  
Natasa Drnovsek ◽  
Sasa Novak ◽  
Gregor Majdic
Keyword(s):  
Stem Cells ◽  
Cell Culture ◽  
Stromal Cells ◽  
Chondrogenic Differentiation ◽  
Culture Medium ◽  
Collagen Type ◽  
Multipotent Mesenchymal Stromal Cells ◽  
Culture Conditions ◽  
Standard Cell ◽  
Cell Culture Medium

Under appropriate culture conditions, mesenchymal stem cells (MSC), also called more properly multipotent mesenchymal stromal cells (MMSC), can be induced toward differentiation into different cell lineages. In order to guide stem cell fate within an environment resembling the stem cell niche, different biomaterials are being developed. In the present study, we used silk fibroin (SF) as a biomaterial supporting the growth of MMSC and studied its effect on chondrogenesis of canine adipose–derived MMSC (cADMMSC). Adipose tissue was collected from nine privately owned dogs. MMSC were cultured on SF films and SF scaffolds in a standard cell culture medium. Cell morphology was evaluated by scanning electron microscopy (SEM). Chondrogenic differentiation was evaluated by alcian blue staining and mRNA expression of collagen type 1, collagen type 2, Sox9, and Aggrecan genes. cADMMSC cultured on SF films and SF scaffolds stained positive using alcian blue. SEM images revealed nodule-like structures with matrix vesicles and fibers resembling chondrogenic nodules. Gene expression of chondrogenic markers Sox9 and Aggrecan were statistically significantly upregulated in cADMMSC cultured on SF films in comparison to negative control cADMMSC. This result suggests that chondrogenesis of cADMMSC could occur when cells were grown on SF films in a standard cell culture medium without specific culture conditions, which were previously considered necessary for induction of chondrogenic differentiation.

scholarly journals Non-Mulberry and Mulberry Silk Protein Sericins as Potential Media Supplement for Animal Cell Culture

2016 ◽  
Vol 2016 ◽  
pp. 1-13 ◽  
Author(s):  
Neety Sahu ◽  
Shilpa Pal ◽  
Sunaina Sapru ◽  
Joydip Kundu ◽  
Sarmistha Talukdar ◽  
...  
Keyword(s):  
Cell Culture ◽  
Culture Medium ◽  
Culture Media ◽  
Animal Cell ◽  
Animal Cell Culture ◽  
Silk Protein ◽  
Antheraea Mylitta ◽  
Serum Free ◽  
Growth Supplement ◽  
Fibrosarcoma Cells

Silk protein sericins, in the recent years, find application in cosmetics and pharmaceuticals and as biomaterials. We investigate the potential of sericin, extracted from both mulberryBombyx moriand different non-mulberry sources, namely, tropical tasar,Antheraea mylitta; muga,Antheraea assama; and eri,Samia ricini,as growth supplement in serum-free culture medium. Sericin supplemented media containing different concentrations of sericins from the different species are examined for attachment, growth, proliferation, and morphology of fibrosarcoma cells. The optimum sericin supplementation seems to vary with the source of sericins. The results indicate that all the sericins promote the growth of L929 cells in serum-free culture media; however,S. ricinisericin seems to promote better growth of cells amongst other non-mulberry sericins.

Preliminary clinical trials of serum free cell culture medium as a treatment for chronic atrophic ulcers

1997 ◽  
Vol 20 (3) ◽  
pp. 115-120 ◽  
Author(s):  
E. Lindenbaum ◽  
Y. Har-Shai ◽  
Y. Ullmann ◽  
A. L. Feitelberg ◽  
M. Tendler ◽  
...  
Keyword(s):  
Cell Culture ◽  
Clinical Trials ◽  
Culture Medium ◽  
Free Cell ◽  
Cell Culture Medium ◽  
Serum Free

scholarly journals Nicotinamide and acute kidney injury

2021 ◽  
Author(s):  
Miguel Fontecha-Barriuso ◽  
Ana Lopez-Diaz ◽  
Sol Carriazo ◽  
Alberto Ortiz ◽  
Ana Belen Sanz
Keyword(s):  
Acute Kidney Injury ◽  
Glomerular Filtration ◽  
Kidney Injury ◽  
Kynurenine Pathway ◽  
Vitamin B3 ◽  
Nicotinamide Riboside ◽  
Nicotinamide Mononucleotide ◽  
Nad Synthesis ◽  
The Impact ◽  
Reduced Activity

ABSTRACT In a recent issue of ckj, Piedrafita et al reported that urine tryptophan and kynurenine are reduced in cardiac bypass surgery patients that develop acute kidney injury (AKI), suggesting reduced activity of the kynurenine pathway of nicotinamide adenine dinucleotide (NAD+) synthesis from tryptophan. However, nicotinamide (NAM) supplementation aiming at repleting NAD+ did not replete kidney NAD+ and did not improve glomerular filtration or reduce histological injury in ischemic-reperfusion kidney injury in mice. The lack of improvement of kidney injury is partially at odds with prior reports that did not study kidney NAD+, glomerular filtration or histology in NAM-treated wild-type mice with AKI. We now present an overview of research on therapy with vitamin B3 vitamers and derivate molecules [niacin, NAM, nicotinamide riboside (NR), NRH and nicotinamide mononucleotide (NMN)] in kidney injury, including an overview of ongoing clinical trials, and discuss the potential explanations for diverging reports on the impact of these therapeutic approaches on preclinical acute and chronic kidney disease.

Endothelial cell cystine uptake and glutathione increase with N,N-bis(2-chloroethyl)-N-nitrosourea exposure

1992 ◽  
Vol 262 (3) ◽  
pp. L301-L304 ◽  
Author(s):  
S. M. Deneke ◽  
R. A. Lawrence ◽  
S. G. Jenkinson
Keyword(s):  
Cell Culture ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Culture Medium ◽  
Cultured Cells ◽  
Potent Inhibitor ◽  
Cell Types ◽  
Cell Culture Medium ◽  
Intracellular Accumulation ◽  
Pulmonary Endothelial Cells

Glutathione (gamma-glutamylcysteinylglycine, GSH) is an important cellular antioxidant. In typical cultured cell preparations GSH synthesis is limited by the availability of intracellular cysteine. Because extracellular cystine is the chief source of intracellular cysteine in cultured cells, increasing cystine transport can result in increased intracellular GSH. Depletion of GSH or exposure to oxidants has been shown to stimulate cystine transport in bovine pulmonary endothelial cells and other cell types. BCNU [N,N-bis(2-chloroethyl)-N-nitrosourea] is a potent inhibitor of glutathione reductase (GSSG-Red). We examined the effects of BCNU on cystine uptake by bovine pulmonary artery endothelial cells (BPAEC). We hypothesized that blocking GSSG-Red could result in increased cellular uptake of cystine to replenish decreases in GSH caused by oxidation. Levels of BCNU between 0.005 and 0.05 mM added to the cell culture medium inhibited GSSG-Red at 2, 4, and 24 h after addition. BCNU treatment resulted in concentration-dependent increases in both cystine uptake and GSH levels after 24 h of exposure. The increases in uptake were specific for cystine and glutamate and were sodium independent, suggesting induction of a xc(-)-like transport system. No intracellular accumulation of GSSG was measured nor was any significant depletion of GSH noted at any time of BCNU exposure.

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