Insulin effects on glucose utilization by human lymphocytes

In recently isolated human lymphocytes, no effect of insulin on (U-14C) glucose incorporation into CO2, triglycerides and glycogen, was found over a wide range of insulin concentration. The rates of glucose oxidation, synthesis of triglycerides and synthesis of glycogen are much lower than those observed in chicken and rat adipocytes. The authors discard the possibility of using the human lymphocyte as an instrument in the study of the insulin action on normal and altered human physiology.     

Effect of phytohaemagglutinin on the nuclear RNA polymerase activity of human lymphocytes

The DNA-dependent RNA polymerase activity of isolated nuclei from human peripheral blood has been shown to increase following stimulation with phytohaemagglutinin (PHA). Using the toxin α-amanitin it has been possible to demonstrate that within 4 h of the addition of PHA there is a two-fold increase in the amanitin-resistant polymerase activity (polymerase A) with little increase in the sensitive polymerase activity (polymerase B). 24 h following PHA stimulation the amanitin-resistant activity is stimulated 4–5 fold and the amanitin-sensitive activity less than two-fold. The susceptibility of this increased amanitin-resistant activity to low doses of actinomycin D both in vivo and in vitro indicates that the amanitin-resistant enzyme is mainly engaged in ribosomal RNA precursor synthesis. These changes in DNA-dependent RNA polymerase activity closely correspond to the observed changes in ribosomal and non-ribosomal RNA synthesis following lymphocyte stimulation.The increased polymerase A activity is diminished by a 1 h incubation of the cells with cycloheximide added 24 h after PHA whereas polymerase B activity remains unaffected. This indicates that the polymerase A activity observed after transformation is dependent on continuing protein synthesis.In our incubation conditions the polymerase activity observed in isolated nuclei appeared to be almost wholly attributable to elongation of nascent RNA molecules attached to the endogenous DNA template.     

The effects of busulphan on the chromosomes of normal human lymphocytes

In vitro exposure of human lymphocytes to busulphan (BUS) produced an increase in chromosome aberrations and in sister-chromatid exchange (SCE) frequency. The distribution of chromosome breaks throughout the karyotype was non-random and they occurred mainly in the G-negative bands. Certain bands had a marked susceptibility to BUS and comparisons with the human chromosome-break distributions reported for a number of drugs revealed that some of these bands were equally susceptible to other alkylating agents. Both the number of chromosome gaps and breaks and the SCE frequency increased with BUS concentration, but only the SCE dose--response was a clearly defined linear relationship. Therefore a standard SCE dose--response curve was constructed for future comparison with the results of similar investigations of patients on BUS therapy.     

The effects of busulphan on the chromosomes of normal human lymphocytes

In vitro exposure of human lymphocytes to busulphan (BUS) produced an increase in chromosome aberrations and in sister-chromatid exchange (SCE) frequency. The distribution of chromosome breaks throughout the karyotype was non-random and they occurred mainly in the G-negative bands. Certain bands had a marked susceptibility to BUS and comparisons with the human chromosome-break distributions reported for a number of drugs revealed that some of these bands were equally susceptible to other alkylating agents. Both the number of chromosome gaps and breaks and the SCE frequency increased with BUS concentration, but only the SCE dose-response was a clearly defined linear relationship. Therefore a standard SCE dose-response curve was constructed for future comparison with the results of similar investigations of patients on BUS therapy.     

Behaviour of lymphocytes from cancer patients and normal individuals cultured with phytohaemagglutinin.

Blood lymphocytes from cancer patients with solid tumours without any previous immunosuppressive treatment and from normal individuals, were cultured in vitro with a wide range of phytohaemagglutinin (PHA). Sixty two per cent of all the cancer patients studied show a minimal of no response to PHA in comparison with the normal population. The rest (38%), show a quantitative identical response than normals. However, the maximal response in these patients occur in the high PHA doses, while the normal individuals show their maximal activity with low PHA doses. The low or no PHA response showed by the 62% of patients, may indicate they have impaired cellular immunity. The high response showed by the other 38%, may indicate that the patients of this group have high cellular immunity capacity. This immunity, however, higher PHA doses are required to reach the maximal response compared with the seems to be different from that of normal individuals, since higher PHA doses are required in cancer patients to reach maximal response. These results also suggest that a large range of PHA doses may be important to detect the degree of cellular immunity in cancer patients compared with the normal population. One or two random PHA doses, may not show a distinction.     

A comparison of the effects of phytohaemagglutinin and of calcium ionophore A23187 on the metabolism of glycerolipids in small lymphocytes.

1. The effects of phytohaemagglutinin and of a Ca2+ ionophore (A23187) on glycerolipid metabolism in lymphocytes from pig lymph nodes were compared (a) by studying the incorporation of [32P]Pi and [3H]glycerol, and (b) by following the redistribution of [3H]glycerol among the lipids caused by these agents in pulse-chase experiments. 2. Phytohaemagglutinin only stimulated 32P incorporation into phosphatidylinositol and, to a slight extent, phosphatidate. Removal of most of the extracellular Ca2+ somewhat decreased this response. 3. Ionophore A23187 stimulated the labelling of phosphatidate and phosphatidylinositol with 32P to a much greater extent than did phytohaemagglutinin: the increase in phosphatidate labelling, but not that of phosphatidylinositol, was almost abolished by the removal of extracellular Ca2+. 4. The combined effects of phytohaemagglutinin and ionophore appeared to be additive, rather than synergistic. 5. Treatment with ionophore A23187 somewhat decreased the total incorporation of [3H]glycerol into glycerolipids, possibly because it lowered cell ATP content. In these experiments di- and tri-acylglycerol behaved anomalously, triacylglycerol labelling being suppressed completely, whereas that of diacylglycerol was enhanced. The pulse-chase results revealed that triacylglycerol was converted into diacylglycerol in the ionophore-treated cells, and the availability of this diacylglycerol probably led to the enhanced labelling of phosphatidate and phosphatidylinositol in the these cells. 6. Thus an increase in intracellular Ca2+ concentration appeared to have three effects on glycerolipid metabolism: (a) slight inhibition of some metabolic step preceding phosphatidate synthesis, (b) inhibition of diacylglycerol acyltransferase and (c) activation of a triacylglycerol lipase. 7. In contrast, it seems likely that the only effect of phytohaemagglutinin is to stimulate phosphatidylinositol breakdown. 8. Pig polymorphonuclear leucocytes treated with ionophore A23187 showed metabolic changes that were similar to those demonstrated with lymphocytes. 9. A possible similarity is suggested between Ca2+-stimulated triacylglycerol lipase in lymphocytes and polymorphonuclear leucocytes and previous observations of enhanced triacylglycerol metabolism in stimulated cells whose metabolic functions involve membrane fusion.     

Effect of orotic acid on the mitosis of phytohaemagglutinin stimulated human lymphocytes in culture

Summary The addition of excess orotic acid to cultures of normal human lymphocytes stimulated with phytohaemagglutinin causes a reduction in the mitotic rate. It is suggested that this is caused by competition for the available 5-phosphoribosyl-1-pyrophosphate in the cell leading to a reduced purine synthesis. The significance of these results with regard to orotic aciduria is discussed.

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Zusammenfassung Zugabe von Orotsäure im überschuß zu Kulturen normaler menschlicher Lymphocyten, die mit Phytohämagglutinin stimuliert wurden, bewirkt eine Reduktion der Mitoserate. Es wird vorgeschlagen, daß dies die Folge einer Kompetition für das vorhandene 5-Phosphoribosyl-1-pyrophosphat ist, welche zu einer reduzierten Purinsynthese fü hrt

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I wish to thank the Smith Kline and French Foundation for financial support and Miss B. Wilson for technical assistance.     

Myelin Basic Protein inhibits the calcium response to phytohaemagglutinin in human lymphocytes

Myelin Basic Protein, one of the major membrane protein component of the central nervous system, was used to probe the molecular mechanism of cellular activation by phytohaemagglutinin.Pre-treatment of human lymphocytes with myelin basic protein results in a lower rising of cytosolic concentration of free calcium after stimulation with phytohaemagglutinin.This effect is dependent on myelin basic protein concentration and on the preincubation time of the protein with the cells. It is not due to a interaction between myelin basic protein and phytohaemagglutinin, but appears to be a consequence of the binding of the protein to the cell surface.The reduction of the rise of cytosolic calcium induced by phytohaemagglutinin is specific for the myelin basic protein because other proteins like albumin and protamine have no effect.