RNA Metabolism in Lymphocytes Stimulated by Phytohemagglutinin: Initial Responses to Phytohemagglutinin

Cooper, Herbert L.; Rubin, Arnold D.

doi:10.1182/blood.v25.6.1014.1014

Cited by 203 publications

(33 citation statements)

References 25 publications

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“…Obviously turnover does take place, since non-stimulated lymphocytes do show labelling with radioactive uridine but no accumulation of RNA. A degradation of preexisting RNA in cells treated with phytohemagglutinin has been described by Rubin and Cooper [3]. Our experiments do not give any indication of such a degradation.…”

Section: Discussionsupporting

confidence: 72%

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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Section: Discussionsupporting

confidence: 72%

On the Synthesis of RNA in Lymphocytes Stimulated by Phytohemagglutinin

Hausen

Stein

Peters

1969

European Journal of Biochemistry

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“…These morphological manifestations are considered characteristic for cell metabolism activation; the resulting stages of lymphocyte activation have been described in general [2,4,10,25,32,39]. Moreover, it has been reported in the literature that RNA synthesis is enhanced in lymphocytes as early as the first hour of PHA stimulation [24], and continues by growing exponentially for at least 24 hr [6,23,41]. Although the PHA stimulation of lymphocytes is not synchronous in all ceils [1,26], it is possible to assess the course of nucleolar activation according to the sequence of morphological changes taking place.…”

Section: Discussionmentioning

confidence: 99%

“…Changes in the structure of nucleoli after 2 and 4 hr of stimulation showed similar features: the nucleolus became slightly enlarged to 1.3-2.0/~m, and the nucleoli area no longer contained such extensive blocks of condensed chromatin as was the case with nonstimulated cells (Figs. 2,4,6). The inner structure of the nucleolus began to undergo substantial changes in two respects: the shape of the fibrillar centers and the quantity of fibrillar centers.…”

Section: And 4 Hours Of Pha Lymphocyte Stimulationmentioning

confidence: 99%

Three-dimensional reconstructions of nucleolus-organizing regions in PHA-stimulated human lymphocytes

HOZAK¹

1989

Biology of the Cell

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Ultrastructure and three-dimensional distribution of nucleolus-organizing regions have been studied on ultrathin serial sections of PHA-stimulated human lymphocytes. During the 48 hr of activation the size of fibrillar centers (FCs) decreased from 0.6-0.9 microns to 0.2-0.3 microns and the number of FCs increased rapidly from one to 75-107 per cell. The number of fibrillar complexes (i.e. associations of a different number of FCs connected by the dense fibrillar component) also increased but did not reach the maximum number of nucleolar organizers presented here. Three-dimensional computer reconstructions of fibrillar complexes showed that lymphocyte activation was accompanied by early (2-4 hr) changes in the shape of the primary fibrillar center. Invagination of the dense fibrillar component on its surface occurred and division into two or more smaller FCs followed. Gradually, the typical structure of the nucleolus with several fibrillar complexes and many FCs was formed. These results confirm the hypothesis of fibrillar complex-nucleolar organizer correlation published recently.

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“…[3H]dT (25 pl; 0.15 nmol) was added and the mixtures incubated at 37°C for 3 h. Thecells were then washed three times with cold HBSS (2 ml) and lysed by suspension in cold glacial acetic acid: ethyl alcohol (I : 2,2 ml) for 10 min. The mixtures were centrifuged and DNA extracted from the precipitates with perchloric acid (Feinendegen et al, 1961;Cooper & Rubin, 1965).…”

Section: Methodsmentioning

confidence: 99%

The Anti‐Folate Effect of Methionine on Bone Marrow of Normal and Vitamin B₁₂ Deficient Rats

1975

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The effects of dietary vitamin B12 and methionine deficiency, and the in vitro addition of methionine, homocysteine, or folic acid on the methylation of dUMP to dTMP were studied in rat bone marrow culture. Vitamin B12 or methionine deficiency had no effect on the methylation reaction or on bone marrow folate levels although the vitamin B12 content in bone marrow was reduced in vitamin B12 deficiency. In vitro addition of vitamin B12 or folic acid also had no effect on the methylation of dUMP. In vitro addition of methionine reduced the methylation of dUMP and increased the proportion of 5-methyltetrahydrofolate at the expense of other folate coenzymes. The reason for this 'anti-folate' effect of methionine, which is the opposite to that found in liver, was not clear. The presence of 5,10-methylenetetrahydrofolate reductase and 5-methyltetrahydrofolate-homocysteine methyltransferase were confirmed in rat bone marrow and they were inhibited by S-adenosylmethionine and methionine, respectively, in a similar fashion to that found with the liver enzymes. Homocysteine had no effect on the proportions of the various folate coenzymes in bone marrow but did inhibit the incorporation of deoxyuridine and deoxythymidine into DNA. It appeared that homocysteine exerted at a non-folate dependent step beyond the formation of dTMP.

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RNA Metabolism in Lymphocytes Stimulated by Phytohemagglutinin: Initial Responses to Phytohemagglutinin

Cited by 203 publications

References 25 publications

On the Synthesis of RNA in Lymphocytes Stimulated by Phytohemagglutinin

On the Synthesis of RNA in Lymphocytes Stimulated by Phytohemagglutinin

Three-dimensional reconstructions of nucleolus-organizing regions in PHA-stimulated human lymphocytes

The Anti‐Folate Effect of Methionine on Bone Marrow of Normal and Vitamin B₁₂ Deficient Rats

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RNA Metabolism in Lymphocytes Stimulated by Phytohemagglutinin: Initial Responses to Phytohemagglutinin

Cited by 203 publications

References 25 publications

On the Synthesis of RNA in Lymphocytes Stimulated by Phytohemagglutinin

On the Synthesis of RNA in Lymphocytes Stimulated by Phytohemagglutinin

Three-dimensional reconstructions of nucleolus-organizing regions in PHA-stimulated human lymphocytes

The Anti‐Folate Effect of Methionine on Bone Marrow of Normal and Vitamin B12 Deficient Rats

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The Anti‐Folate Effect of Methionine on Bone Marrow of Normal and Vitamin B₁₂ Deficient Rats