Determination of polyamines in urine

Dreyfuss, Gideon; Dvir, R; Harell, A; Chayen, R.

doi:10.1016/0009-8981(73)90344-6

Cited by 67 publications

(21 citation statements)

References 13 publications

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“…These observations were confirmed in our laboratory using a modification of Spock's original assay system (8), and by Bowman and coworkers (3) using oyster gill preparations in place of the rabbit tracheal explants. This C D F has also been demonstrated to be present in the cell-free medium obtained from phytohemagglutinin-stimulated leukocytes of C F patients and their parents, as well as in the cell-free medium of long term lymphoid lines derived from CF-affected and carrier subjects (7). In addition, the cell-free medium from cultured skin fibroblasts from homozygotes and heterozygotes for CF demonstrates C D F in both the oyster gill system (10) and the rabbit tracheal bioassay system, the latter in the presence of human immunoglobulin (IgC) (2).…”

Section: Extractmentioning

confidence: 84%

“…Polyamine preparations were dansylated (2) and the dansyl derivatives were extracted into benzene, dried in an there were some inevitable losses in the n-butanol extration procedure (7,9) and the overall recovery was between 50% and 70% in different experiments. This variation in recovery did not affect the accuracy of the tissue estimations, since in all experiments C F and control tissues were paired and there was considerably less variation within batches than that between batches.…”

Section: Methodsmentioning

confidence: 99%

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Polyamines in Cystic Fibrosis

McEVOY

Hartley

1975

Pediatr Res

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POLYAMINES IN CYSTIC FIBROSIS 72 1 gainscores (before or after treatment), however, there was a significantly higher variance in the pyritinol group in four parameters. The latter findings suggest medication effects for a not as yet specifiable subpopulation. The problem of how to.define objective indications for a psychoactive drug was therefore considered to be the most difficult task for such a study.

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Section: Extractmentioning

confidence: 84%

Section: Methodsmentioning

confidence: 99%

Polyamines in Cystic Fibrosis

McEVOY

Hartley

1975

Pediatr Res

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“…The ainines were quantified by the dansylation technique [18] using alkaline ether extraction of the dansyl derivatives [19] with minor modifications [14].…”

Section: Atzlz1ysiy Ofl)oljutninesmentioning

confidence: 99%

Polyamine depletion results in impairment of polyribosome formation and protein synthesis before onset of DNA synthesis in mitogen-activated human lymphocytes

Hölttä

Hovi

1985

Eur J Biochem

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Lymphocytes, exposed to mitogens in culture, show enhanced protein and RNA synthesis before the onset of DNA synthesis. Inhibition by DL-a-difluoromethylornithine of polyamine synthesis in phytohaemagglutininactivated human lymphocytes resulted in a suppression of protein synthesis, which was evident before the initiation of DNA synthesis. The mitogen-induced increase in the incorporation of [3H]thymidine into DNA was subsequently inhibited in parallel with the activity of thymidine kinase in the polyamine-depleted cells. Ultraviolet absorbance measurement of the ribosomes after sucrose gradient centrifugation revealed a suppression of polyribosome formation that coincided with the decrease in the rate of protein synthesis. The disturbance in the polysomal profiles did not appear to be due to a shortage of mRNA, since the synthesis of poly(A)-rich mRNA was reduced less than that of rRNA after inhibiting polyamine synthesis. Entry of both the pre-existing and newly synthesized ribosomal subunits into polysomal structures was found to be impaired. These results thus suggest an important role for polyamines in the initiation step of protein synthesis.The natural polyamines, putrescine, spermidine and spermine, are organic cellular cations, which have been shown to play an important role in cell proliferation. Studies with the polyamine-deficient mutants isolated from bacteria [l , 21 fungi and yeast [3 ~ 51 have clearly shown that polyamines are needed for their optimal growth. Mammalian cell lines unable to synthesize polyamines [6-81 may even be more strictly dependent on exogenous polyamines for survival than prokaryotes, like Escherichia coli [2].

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“…The activity of ornithine decarboxylase was measured as described by Janne and Williams-Ashman (11) with minor modifications. The assay mixture (0.25 ml) consisted of 50 mM Tris-hydrochloride buffer (pH 7.4), 3 ,uCi of DL-[1-_4C]ornithine, 0.05 mM L-ornithine, 0.1 mM pyridoxal phosphate, 4 mM dithiothreitol, 4 mM EDTA, and a 100,000 x g supernatant fraction of the cells. S-Adenosylmethionine decarboxylase activity was determined in the presence of 2.5 mM putrescine with 0.2 mM S-adenosyl-L-[1-14C]methionine (specific activity, 10 mCi/mmol) as the substrate (12).…”

Section: Methodsmentioning

confidence: 99%

Mutant strain of Chinese hamster ovary cells with no detectable ornithine decarboxylase activity.

Pohjanpelto¹,

Hölttä²,

Jänne³

1985

Mol. Cell. Biol.

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We previously described an arginase-deficient, polyamine-dependent Chinese hamster ovary cell line which grows in serum-free medium. From this strain we isolated a new mutant strain that has no detectable catalytic ornithine decarboxylase activity. The mutant cells contain, however, immunoreactive ornithine decarboxylaselike protein roughly in the same quantity as the parent strain. The mutant and the parent cell line strains also contain similar amounts of ornithine decarboxylase-mRNA hybridizable to a specific cDNA. If polyamines are omitted from the medium, proliferation of the mutant cells is considerably retarded and ceases in 6 to 10 days. Addition of ornithine or a-difluoromethylornithine, a specific inhibitor of ornithine decarboxylase, has no effect on these cells. Putrescine and spermidine decreased in the mutant cells to undetectable levels during polyamine starvation, whereas spermine was reduced to 1/5th of that found in the control cultures. Polyamines appear to be indispensable for the mutant strain, but this was obvious only after the amount of polyamines, found as impurities in bovine serum albumin used in the medium, was reduced by dialysis to 10-12 M. Because sera contain polyamines, the ability of the mutant strain to grow in serum-free medium is a great advantage in elucidation of the mechanisms of polyamine function.The polyamines putrescine, spermidine, and spermine are found in all cells, and they appear to be required for optimal growth of both bacteria and eucaryotes. Bacterial mutants that are completely devoid of polyamines have been isolated. These mutants have a considerably retarded rate of growth, but they can replicate indefinitely in the absence of polyamines (4). However, some yeast cell mutants unable to synthesize polyamines will gradually stop replicating (28, 31).We previously isolated a strain of Chinese hamster ovary cells (CHO), designated A7, which grows under serum-free conditions and has deficient arginase activity. Hence, it is unable to synthesize ornithine, the precursor of polyamines. If ornithine and polyamines are omitted from the growth medium, the cells are depleted of polyamines, and their growth is gradually retarded (8,21). At the same time, the cells lose most of their actin filaments and microtubules (21), and they also develop major chromosome aberrations (14,19,20). Thus, polyamine starvation of the A7 cells affects structures characteristic of eucaryotic cells. Steglich and Scheffler later isolated an ornithine decarboxylase-deficient mutant of CHO cells, designated C54, with 3% of the ornithine decarboxylase activity of the wild type (26). These cells, however, showed only a slight retardation of growth when polyamines were omitted from the growth medium based on the otherwise rich nutrient mixture F10 (25).In this communication we report isolation from A7 cells of a mutant strain designated P22 that lacks catalytically active ornithine decarboxylase enzyme and thus cannot synthesize putrescine. When external sources of polyamines were excluded, the P...

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Determination of polyamines in urine

Cited by 67 publications

References 13 publications

Polyamines in Cystic Fibrosis

Polyamines in Cystic Fibrosis

Polyamine depletion results in impairment of polyribosome formation and protein synthesis before onset of DNA synthesis in mitogen-activated human lymphocytes

Mutant strain of Chinese hamster ovary cells with no detectable ornithine decarboxylase activity.

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