On the Synthesis of RNA in Lymphocytes Stimulated by Phytohemagglutinin. 1. Induction of Uridine-Kinase and the Conversion of Uridine to UTP

Hausen, Peter; Stein, Hans

doi:10.1111/j.1432-1033.1968.tb00226.x

Cited by 71 publications

(21 citation statements)

References 17 publications

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“…Alternatively, assuming an unlimited permeation through the membrane, an increased conversion of the uridine into substances which are not released from the cell, may lead to an increased accumulation of label within the cell. An increased activity of uridine kinase has in fact been reported to occur in phytohemagglutinin treated human lymphocytes [7,8]. This was also found in bovine lymphocytes as shown in Fig.…”

Section: Resultssupporting

confidence: 57%

“…Furthermore, RNA labelling seems to depend strictly on the activity of the membrane transport system. It has been shown previously that the uptake of uridine into lymphocytes as a function of uridine concentration follows a typical saturation curve [8]. I n the following experiments such saturation curves were monitored for phytohemagglutinin treated and untreated control cells, in the absence and in the presence of limiting amounts of dipyridamole.…”

Section: Resultsmentioning

confidence: 87%

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Effect of Phytohemagglutinin on Lymphocyte Membrane Transport

Hausen

1971

European Journal of Biochemistry

156

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Uptake of labelled uridine into lymphocytes is restricted. Several lines of evidence, including experiments with dipyridamole, an inhibitor for membrane transport processes, and kinetic experiments indicate that transport of uridine through the cell membrane is the rate-limiting step in uridine uptake. Addition of phytohemagglutinin results in an immediate increase of uridine uptake. Saturation experiments have shown that this change is due to an increase in V , whereas the apparent K , value remains constant. The hypothesis is advanced, that phytohemagglutinin induces an increase in the number of functional carrier sites of the membrane transport system.Resting lymphocytes respond to the addition of phytohemagglutinin with a number of biochemical changes which ultimately lead to the transformation of small lymphocytes to large pyroninophilic cells.The latter actively synthesize RNA and DNA and undergo mitotic division. The mechanism of this differentiative process is still obscure although numerous alterations of the cell metabolism have been recorded.

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Section: Resultssupporting

confidence: 57%

Section: Resultsmentioning

confidence: 87%

Effect of Phytohemagglutinin on Lymphocyte Membrane Transport

Hausen

1971

European Journal of Biochemistry

156

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“…The rate of labeling is determined by the specific activity of the immediate precursor pools and by the activities of the enzymes involved in synthesis. Thus, the increased specific activity of the uridine triphosphate (UTP) pool is probably responsible for part of the enhanced labeling of RNA in regenerating liver immediately after partial hepatectomy (44) and for the labeling of RNA in the kidney of the castrate mouse (45); in phytohemagglutinin-stimulated lymphocytes (46,47) and in isoproterenol-stimulated mouse salivary glands (48) newly induced uridine-kinase activity selectively increases the specific activities of the uridine nucleotide pools. The specific activities of UTP pools were not measured in the experiments reported in this paper; if, however, aldosterone had influenced the pool, like cortisone (49) would seem more likely to have increased the specific activity of the pool and hence to have produced an effect contrary to the results reported, rather than to have caused them.…”

Section: Xf)mentioning

confidence: 99%

Kinetics of RNA synthesis in toad bladder epithelium: action of aldosterone during the latent period

Vancura¹,

Sharp²,

Malt³

1971

J. Clin. Invest.

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A B S T R A C T Processing of RNA in the toad bladder was analyzed by polyacrylamide-gel electrophoresis to determine whether aldosterone causes any changes in the 1 hr before it potentiates transport of sodium ion. No change was found in the quantity or in the specific activity of bulk RNA labeled with uridine-5-3H. In vivo and in vitro with either uridine-5-'H or with methionine-(methyl)-'H as precursors, processing of RNA was extremely slow. Heterodisperse RNA was obvious after 30 min of continuous labeling, but labeling of the 40S precursor of ribosomal RNA was not apparent for 60 min. Labeling of mature 28S and 18S RNA first became apparent after 8 hr. -S RNA was the principal fastmigrating species labeled at 30 min, and 4S RNA was not heavily labeled until 1 hr. Aldosterone (5 x 10-mole/liter) produced no changes. If care were not taken to inhibit metabolism of native bacteria colonizing the bladder, bacterial RNA of high specific activity predominated. We conclude that RNA metabolism in the toad bladder is extraordinarily slow, that a major acceleration of de novo synthesis in response to physiologic doses of aldosterone was not demonstrable, and that some reports to the contrary may have been influenced by artifacts from bacterial RNA metabolism. Earlier evidence for obligatory alterations in RNA metabolism during the latent period is not strong.

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“…A drastic increase in the incorporation of radioactive uridine into RNA soon after the addition of the stimulating agent phytoheniagglutinin indicated a rapid induction of RNA synthesis. The interpretation of these labelling experiments is difficult since the uptake of the labeled precursor into the intracellular nucleotide pool is increased considerably in lymphocytes treated with phytohemagglutinin [9,10]. Therefore it was decided to investigate the RNA synthesizing capacity of cell free systems derived from transforming lymphocytes.…”

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On the Synthesis of RNA in Lymphocytes Stimulated by Phytohemagglutinin

Hausen

Stein

Peters

1969

European Journal of Biochemistry

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The experimental conditions to study lymphocyte transformation in cultures derived from bovine lymph nodes are described. Both deoxyribonucleoprotein-bound and soluble RNA polymerase can conveniently be tested in cell lysates. During the course of transformation the activities of the two enzymes begin to rise a t the same time when the cells begin to accumulate stable RNA. At 30 h after addition of phytohemagglutinin both enzyme activities are enhanced by a factor of two over the controls.The transformation of small lymphocytes to large pyroninophilic cells under tho stimulus of phytohemagglutinin provides an excellent model system to study the conversion of resting cells to actively growing cells. The assumption that the synthesis of RNA may play a key role in this process has focused attention on the RNA metabolism in transforming lymphocytes [ 1 -81. A drastic increase in the incorporation of radioactive uridine into RNA soon after the addition of the stimulating agent phytoheniagglutinin indicated a rapid induction of RNA synthesis. The interpretation of these labelling experiments is difficult since the uptake of the labeled precursor into the intracellular nucleotide pool is increased considerably in lymphocytes treated with phytohemagglutinin [9,10]. Therefore it was decided to investigate the RNA synthesizing capacity of cell free systems derived from transforming lymphocytes. This approach avoids the discussion of "pool quesstions" and might give information as to what extent the RNA synthesizing apparatus of the cell is changed after stimulation.Different methods for studying RNA synthesis in cell free systems derived from animal cells have been described. Using bacterial RNA polymerase isolated chromatin has a template activity which changes with the physiological state of the cell [ll]. At low salt concentration isolated nuclei show RNA synthesizing activity which possibly reflects the activity of ribosomal operons within the nucleolus [12-151. The activity of cellular RNA polymerase bound to the chromatin can be tested at high salt concentration [16,17]. Finally, a soluble enzyme which uses exogeneous DNA as template for RNA synthesis can be extracted from the cells [is--211. The activities observed in these different assay systems probably reflect different aspects of tbe i n vivo conditions. Studies on the activity of chromatin-bound and of the soluble RNA polymerase will be described in this communication.I n order to perform such experiments large amounts of lymphocytes are needed. The limited supply of human blood used earlier [lo] prompted the investigation of other sources for lymphocytes. Bovine lymph node cells were found to respond to phytohemagglutinin in a similar way as human blood lymphocytes. These cells have been used throughout the studies. Culture conditions and the changes in leucine and uridine labelling as well as the net synthesis of DNA, RNA and protein after phytohemagglutinin stimulation will be described in the first section of this communication.Lymphocytes are particular...

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On the Synthesis of RNA in Lymphocytes Stimulated by Phytohemagglutinin. 1. Induction of Uridine-Kinase and the Conversion of Uridine to UTP

Cited by 71 publications

References 17 publications

Effect of Phytohemagglutinin on Lymphocyte Membrane Transport

Effect of Phytohemagglutinin on Lymphocyte Membrane Transport

Kinetics of RNA synthesis in toad bladder epithelium: action of aldosterone during the latent period

On the Synthesis of RNA in Lymphocytes Stimulated by Phytohemagglutinin

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