Mycoplasma contamination of animal cell cultures: A simple, rapid detection method

Em, Levine

doi:10.1016/0014-4827(72)90484-3

Cited by 178 publications

(30 citation statements)

References 49 publications

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“…Cell lines were monitored for mycoplasma by standard culture techniques (3) as well as conversion of uridine to uracil (6). The presence of mycoplasmas did not have any measurable effect upon mutase activity in the extract experiments.…”

Section: Methodsmentioning

confidence: 99%

Studies of Methylmalonyl Coenzyme A Carbonylmutase Activity in Methylmalonic Acidemia. I. Correlation of Clinical, Hepatic, and Fibroblast Data

et al. 1975

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Extract (MM-emia). With the exception of one patient (4) all MM-emiaMethylmalonyl-CoA carbonylmutase (mutasel activity was measured in fibroblast extracts from 15 patients with methylmalonic acidemia and in extracts of postmortem tissues from 6 of these children. Propionate oxidation and synthesis of 5'-deoxyadenosylcobalamin (AdoCbl, the vitamin B I Z coenzyme that is part of the mutase holoenzyme) were measured in intact fibroblasts. Mutase activity was low in the absence of added AdoCbl in fibroblast extracts from both control subjects and patients. When the assay included supplemental AdoCbl, mutase activity increased in the control subjects ( t o 24.0 pmol succinatelmg proteinlmin) and in extracts from eight of the patients (20.8 pmol/mg proteinlmin), but showed almost no change in extracts from the other seven patients (0.16 pmol/mg proteinlmin). We have defined the eight fibroblast lines that showed normal mutase activity in the presence of AdoCbl as "responsive lines" and the other seven lines as "nonresponsive." In the liver or kidney extracts of postmortem tissues. mutase activity responded to AdoCbl supplementation if fibroblast mutase activity from that patient had responded, and failed to respond if fibroblast activity failed to respond. Mean propionate oxidation in intact fibroblasts was much higher in control lines than in either responsive or nonresponsive lines (0.728 vs 0.097 vs 0.080 nmol C0,/106 cells/hr, respectively). AdoCbl synthesis was normal (0.27 pg AdoChl/mg cells wet weight) in nonresponsive fibroblasts but was undetectable ( <0.005 pg/mg cells) in the responsive lines. Thus, the deficiency of mutase activity in responsive fibroblast lines is due to the failure to synthesize significant amounts of AdoCbl, whereas the deficiency in nonresponsive lines is due to some other abnormality, presumably a defect in the mutase apoenzyme. SpeculationFibroblasts can be used to define whether patients with methylmalonic acidemia have an error of vitamin B,, metabolism if both mutase activity and AdoCbl synthesis are measured. The response of fibroblast mutase activity to the addition of AdoCbl reflects accurately the responsiveness of the enzyme in other tissues of the body. If an error of vitamin B,, metabolism is diagnosed with cultured fibroblasts, a prolonged trial of vitamin B,, therapv is warranted in the patient to seek evidence of in virzo responsibeness and clinical benefit from vitamin therapy.individuals exhibit an enzymatic block a t the terminal, vitamin BIZ-dependent methylnialonyl-CoA carbonylmutase (mutase, EC 5.4.99.2) step, either as the result of apoenzyme failure or defective coenzyme synthesis. In the former situation, excretion of methylmalonic acid ( M M A ) does not decrease with massive doses of vitamin B,,. whereas in the latter, significant drops in urinary M M A are noted. Mahoney and coworkers (9) have demonstrated in tissue culture that fibroblasts from one patient who responds to vitamin B,, therapy did not synthesize 5'-deoxyadenosylcobalamin coenzyme adequately. C...

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Section: Methodsmentioning

confidence: 99%

Studies of Methylmalonyl Coenzyme A Carbonylmutase Activity in Methylmalonic Acidemia. I. Correlation of Clinical, Hepatic, and Fibroblast Data

et al. 1975

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“…All experiments were completed within 2 months after reactivation of the cells from liquid nitrogen. No mycoplasma contamination was detected in cultures of these cells when they were examined by fluorescent staining (1) or by uridine phosphorylase assay (19). Cell number was measured with a Coulter Counter.…”

mentioning

confidence: 99%

Characterization of a ts mutant of BALB/3T3 cells and correction of the defect by in vitro addition of extracts from wild-type cells.

Zeng¹,

Donegan²,

Ozer³

et al. 1984

Mol. Cell. Biol.

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“…Ltd, according to [28]). The cells were tested for mycoplasma contamination as described by Levine [31]. In the labeling experiments we used medium A which contained per liter : 474 ml distilled water, 100 ml dialysed newborn calf serum, 300 ml Tc-199 medium, 42 ml 0.068 M Na2H PO4, 18 ml 0.076 M KH2P04 and 60ml of a Hanks solution (10 times concentrated) with a lowered NaCl content (149.3 g x 1-') to maintain a physiological osmolarity.…”

mentioning

confidence: 99%

Source of Amino Acids Used for Protein Synthesis in HeLa Cells

Venrooij

Moonen

Loon-Klaassen

1974

European Journal of Biochemistry

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HeLa cells, prelabeled with [3H]leucine for several generations, were pulse-labeled with [14C]-leucine. The 14C/3H ratio of the leucine in the medium, in the total amino acid pool and bound to transfer RNA was determined.It was found that the 14C/3H ratio of the leucyl-tRNA was identical with that of the extracellular medium while that of the amino acid pool was much lower. These results confirm previous reports that selection of amino acids for protein synthesis takes place before the amino acids are released in tracellularly .Further, it was concluded that amino acids resulting from intracellular proteolysis are not reutilized directly under the conditions of our experiments.A possible model of intracellular amino acid pathways is discussed.It is still unsettled whether or not amino acids on their way to incorporation into protein pass through some intracellular pool. Initially, it was generally accepted that the intracellular amino acid pool served as precursor pool for protein synthesis [1,2]. This view had changed somewhat as a result of the experiments of Kipnis et al.[3]. They found a constant rate of incorporation of radioactive amino acids into protein almost from the moment that radioactive amino acids were offered to the cells, unaffected by the slow equilibrium between the medium and the intracellular pool of amino acids. Therefore it must be concluded that the amino acids used for protein synthesis are selected from a pool of constant specific activity. Kipnis and coworkers [3] proposed a functional heterogeneity of the intracellular amino acid pool and suggested that amino acids to be used for protein synthesis were compartimentalized in a specific precursor pool. This compartimentalization could be achieved either chemically or structurally within the cell. Although the results of Kipnis and coworkers were confirmed and discussed by several authors [4-9,15,17,22,23], the concept of an intracellular precursor pool for protein synthesis is still alive [lo-12,30,32,34]. Berg [13], working with sea urchin embryos, found that valine to be used for protein synthesis was selected directly from the extracellular fluid when the supply of valine in the medium was sufficient. The valine of the intracellular pool was used only when insufficient amounts of valine were present extracellularly. Although his results were contradicted by those of Fry and Gross [14], they suggested that a sufficient supply of amino acids and consequently a maximal rate of protein synthesis were essential for correct interpretation of the results.Further evidence for the direct incorporation into protein of the extracellular amino acids came from chase experiments in which the intracellular pool of amino acids was prelabeled [15-181. Thus it was found that the radioactive amino acids from the intracellular pool were not incorporated into protein.Despite increasing evidence that under certain conditions extracellular amino acids are preferentially used for protein synthesis it is still not known whether or not all amino acids for pro...

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Mycoplasma contamination of animal cell cultures: A simple, rapid detection method

Cited by 178 publications

References 49 publications

Studies of Methylmalonyl Coenzyme A Carbonylmutase Activity in Methylmalonic Acidemia. I. Correlation of Clinical, Hepatic, and Fibroblast Data

Studies of Methylmalonyl Coenzyme A Carbonylmutase Activity in Methylmalonic Acidemia. I. Correlation of Clinical, Hepatic, and Fibroblast Data

Characterization of a ts mutant of BALB/3T3 cells and correction of the defect by in vitro addition of extracts from wild-type cells.

Source of Amino Acids Used for Protein Synthesis in HeLa Cells

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