A.D. Perris

The role of calcium in the mitotic stimulation of rat thymocytes by detergents, agmatine and poly-L-lysine.

Abstract Low concentrations of the detergents cetyltrimethyl ammonium bromide, dodecyl sodium sulphate, and Triton X-100 stimulate mitotic activity in rat thymocyte populations maintained in vitro. These compounds have no mitotic effect on cells maintained in calcium-free medium. The maximum mitotic activity in the presence of a detergent such as dodecyl sodium sulphate occurs when the CaCl2 concentration is 0.04 mM while the same activity in detergent-free medium occurs only when the CaCl2 concentration is 2.5 mM. Similarly, the organic cations agmatine and poly- l -lysine also stimulated mitosis in calcium-containing medium, but not in calcium-free medium. It is concluded that all these compounds stimulate mitosis only indirectly by sensitising the mitotically competent segment of a thymocyte population to the action of calcium.

Stimulation by calcium of the entry of thymic lymphocytes into the deoxyribonucleic acid-synthetic (S) phase of the cell cycle☆

Abstract Calcium increases mitotic activity in rat thymocyte populations by stimulating the entry of cells into the deoxyribonucleic acid-synthetic (S) phase of their growth-division cycle. The ion does not affect the passage of cells through the premitotic G2 phase of the growth-division cycle.

Dissolution of the condensed chromatin structures of isolated thymocyte nuclei and the disruption of deoxyribonucleoprotein by inorganic phosphate and a phosphoprotein

Phosvitin, a phosphoprotein, is able to severely disrupt deoxyribonucleoprotein (DNP) and thereby cause a large release of saline-soluble DNA from the saline-insoluble DNP isolated from normal rat thymocytes. This ability of phosvitin to disrupt DNP was reduced by calcium and magnesium ions. Equivalent amounts of inorganic phosphate (in the form of Na2HPO4) were unable to cause a similar large release of saline-soluble DNA from nucleoprotein. Both inorganic phosphate and the phosphoprotein caused the complete disappearance of the highly condensed deoxyribonucleoprotein (chromatin) structures in nuclei isolated from normal thymocytes. Condensed structures reappeared within these isolated nuclei when they were exposed to lysinerich histones. The support which these observations lend to current hypotheses on the roles of phosphate and phosphoproteins in the determination of the degree of condensation of nuclear chromatin structures in the whole cell under normal and pathological conditions is discussed.

The relation between nuclear structural changes and the appearance of free deoxyribonucleic acid in irradiated rat thymocytes

Abstract Concurrent with the microscopically visible loss of nuclear structure in irradiated (1000 r of γ-radiation) thymocytes, there is an increase in the amount of acid-insoluble, but saline-soluble DNA, and a parallel decrease in the amount of deoxyribonucleoprotein. The magnitude of these changes can be increased by raising the concentration of inorganic phosphate in the medium. Some compounds and conditions which are known to decrease phosphate uptake, such as 2,4 dinitrophenol, high concentrations of nicotinamide, low temperature or anaerobiosis, inhibit both the nucleoprotein changes and the loss of nuclear structure. It is suggested that inorganic phosphate displaces histone from DNA in the nuclear chromatin granules and that this disruption of the nucleoprotein complex can explain the loss of nuclear structure and the acquisition of saline-solubility by a part of the DNA. In addition, a possible mechanism by which these changes can cause the death of the cell is discussed.

Early postirradiation changes leading to the loss of nuclear structure in rat thymocytes

Abstract The loss of thymocyte nuclear structure begins 45 to 60 min after irradiation. During this cytological latent period there are two distinct phases of response to radiation which combine sequentially to destroy the normal nuclear structure. The damage which develops during the first phase can become fully and irreversibly established in the absence of extracellular phosphate, but further development of damage in the second phase initially requires the presence of an adequate supply of extracellular phosphate. Between 30 and 45 min after irradiation the continued progress of events becomes independent of the extracellular phosphate supply and 10 min thereafter it can no longer be blocked by dinitrophenol. The most probable mechanisms underlying these phases are discussed.

The effects of anoxia on the nuclear morphology of thymus cells in normal and irradiated rats

Incubation of thymocytes in the anoxic thymus of the dead rat for periods longer than 30 min damages them. This damage causes no visible change in nuclear structure if the cells remain in the anoxic thymus, but when they are released therefrom and suspended in vitro their nuclei become structurally homogeneous (“pycnotic”). The fraction of cells which subsequently lose their nuclear structure in vitro is proportional to the concentration of inorganic phosphate in the medium. The amount of damage inflicted upon the cells during incubation in the anoxic thymus can be reduced by introducing tris(hydroxymethyl)aminomethane (Tris) buffer into the animal before its death. The establishment of primary radiation damage in thymocytes can be considerably reduced by incubation in the anoxic thymus of a Tris-treated, dead animal during the first 2 h after irradiation. The development of radiation damage cannot be reduced if the 2-h sojourn in the anoxic thymus is delayed until 30 min after irradiation. The beneficial effect of postirradiation oxygen deprivation is at least equal to that produced by the classical “oxygen effect”.