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Preincubation of cytolytic T lymphocytes (CTLs) generated in secondary C57BL/6 anti-DBA/2 mixed leukocyte cultures with an inhibitor of cellular cholesterol synthesis (25-OH-cholesterol) for 24 hr strongly depressed the cytolytic activity as determined in a 3-hr 51Cr assay. The effect of the inhibitor was reversed by the simultaneous addition of cholesterol or of mevalonic acid during the preincubation period (mevalonate is the product of the regulatory enzyme in the sterol synthesis pathway, 3-hydroxy-3-methylglutaryl-CoA reductase (NADP) [mevalonate:NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.34]). Because, under the same culture conditions, inhibition of DNA synthesis had no effect on CTL activity, the experiments suggest that the effect of 25-OH-cholesterol is related to its inhibitory effect on sterol synthesis, resulting in decreased levels of membrane-bound cholesterol, rather than to inhibition of cellular proliferation.

Incubation of peripheral blood or isolated lymphocytes of C57L/J mice with phytohemagglutinin stimulated the incorporation of thymidine into DNA of lymphocytes as they transformed into large lymphoblasts. DNA synthesis began after about 24 hr of incubation and reached a peak at 48 hours. The de-novo synthesis of sterols from acetate was stimulated much earlier, at 4 hr of incubation, and the rate reached a maximum at 24 hr, approximately at the time DNA synthesis began. Rates of incorporation of radioactivity from [14-C]acetate into fatty acids and into CO2 by phytohemagglutinin-treated blood were not significantly different from control values. Phytohemagglutinin stimulation of sterol synthesis could be abolished by the addition of certain oxygenated derivatives of cholesterol (e.g., 25-hydroxycholesterol and 20alpha-hydroxycholesterol) which specifically depress the activity of the regulatory enzyme in the sterol synthesis pathway, 3-hydroxy-3-methylglutaryl CoA reductase [mevalonate:NADP-nOXIDOREDUCTASE (CoA acylating); EC 1.1.1.34]. This treatment also abolished DNA synthesis and blastogenesis which otherwise followed the peak of sterol synthesis. Furthermore, DNA synthesis was repressed only if the inhibitor was added early enough to prevent sterol synthesis from reaching its usual maximum. When the compound was added after the rate of sterol synthesis had reached its maximum, DNA synthesis was not affected. These findings suggest that the synthesis of cholesterol is an essential prerequisite for successful initiation and completion of the cell cycle in lymphocytes after phytohemagglutinin activation.

The kinetics of sterol synthesis and DNA synthesis in polyclonally activated, concanavalin A-stimulated spleen cell cultures were analyzed. Inhibition of DNA synthesis by 1-beta-D-arabinofuranosylcytosine (Ara-C) did not abrogate the formation of cytotoxic effector cells. However, inhibition of sterol synthesis by 25-hydroxycholesterol inhibited formation of cytotoxic effector cells as well as cellular proliferation. The inhibition of cytotoxicity correlated well with the dose of 25-hydroxycholesterol administered and was dependent on the time of administration. The agent had to be present when sterol synthesis occurred normally during the time lapse before DNA synthesis began. Compactin had the same effect as 25-hydroxycholesterol. The effects of inhibition of sterol biosynthesis on cytotoxicity could be counteracted by addition of cholesterol-containing liposomes. Based on these experiments, the links between proliferation and differentiation in lymphocytes are discussed.

The influence of age, sex and strain on the serum concentration of transcortin (corticosteroid-binding globulin) and vitamin D-binding protein (DBP) in mice was investigated. The effect of age was studied in two strains, C57BL/6JPfd and BALB/cmHeAPfd. The concentration of transcortin and DBP increased with age. In young animals the concentration of each protein showed a significant strain difference, which disappeared in older mice for DBP, but not for transcortin. In 7-day-old animals, no sex difference was observed for either protein, but in older animals a clear sex difference was found for transcortin. Adult males tended to have somewhat higher levels of DBP than adult females, but this difference was significant only on day 70.The variation in transcortin and DBP levels was further investigated in a large number of mouse strains. The DBP concentration did not markedly vary among strains (5·98–9·65 μmol/l in males and 5·08–8·85 μmol/l in females). Transcortin, however, showed marked strain variations, ranging from 0·72 to 2·06 μmol/l in males and from 1·02 to 4·55 μmol/l in females and there was a significant correlation (r= 0·66, n= 26, P<0·001) between the mean transcortin levels in males and females of different strains. Interstrain variation was much higher than intrastrain variation or variation among related strains, suggesting that the transcortin concentration is largely controlled by genetically determined factors.There was a significant correlation (r= 0·82, n= 9, P<0·01) between the mean corticosterone and transcortin concentrations (measured at 21.00 h). Consequently, differences in the free corticosterone levels in the serum of various mouse strains were smaller than the differences in the total corticosterone concentrations. The affinity of transcortin for corticosterone was similar in all but one strain; however, transcortin of the RIIIS/J strain showed a lower affinity for corticosterone.J. Endocr.(1986) 109,141–147

Thymidine reutilization was studied in single cells of the rat bone marrow. Using 3H-TdR in parallel with 125I-UdR in conjunction with the autoradiographic technique, cells of the erythrocytic series, the megakaryocytic group, and the lymphoid cells were analyzed. Reutilization of thymidine was observed only in those cells known to synthesize DNA. An estimate of the amounts of the thymidine reutilized by the salvage pathway indicated that approximately 40-60 per cent of the thymidine in the blast cells is supplied from DNA of dead cells. This value is similar to that reported previously for whole bone marrow cell populations, suggesting the presence of a common thymidine pool within the bone marrow.

Ultrastructural differences between small lymphocytes of the thymic cortex and those located in the cortical zone of lymph nodes of mice were examined by electron microscopy and cytometric methods. The particle size analyzer TGZ 3 (Zeiss) was used to measure the size-frequency distribution of nucleolar cross-section diameters, and the resulting data were computed mathematically and statistically to determine the corresponding mean volumina. It could be shown that thymic, as compared to lymph node cortical small lymphocytes, (1) have a smaller mean nucleolar volume (0.11µ3 versus 0.46µ3); (2) represent a more uniform population with regard to the frequency distribution of the nucleolar cross section diameters; and (3) are of smaller size due to less cytoplasm, while the mean nuclear volume does not differ significantly from that of small lymphocytes located in the cortex of lymph nodes. These findings are discussed in relation to qualitative ultrastructural peculiarities and probable differences in the magnitude of RNA turnover as estimated by the use of tritiated cytidine. The possible significance of cytometrically defined populations of small lymphocytes with regard to sensitized and nonsensitized cells is considered.

Parallel studies of 3H-TdR and 125I-UdR incorporation into regenerating liver following repeated injection into female Swiss albino mice yielded the following results: (i) both 3H-TdR and 125I-UdR are reutilized, although 3H-TdR to a considerably greater extent; (ii) in addition to local reutilization, the data are explained by labeled precursors entering the regenerating liver from the intestine via the portal circulation. Precursor supply from immigrated leukocytes also occurs; and (iii) since a bypass of the thymidine kinase salvage pathway is unlikely, the incorporation of tracer into the regenerating liver must occur from the nucleoside level.

Zusammenfassung Leukozytenkulturen von Normalpersonen, Patienten mit chronischer Hepatitis und solchen mit Zirrhose wurden 3 Tage nach Stimulation mit Phytohämagglutinin mit den spezifischen DNS-Vorläufern125J-Desoxyuridin oder3H-Thymidin markiert. Dabei ist der Gebrauch von Joddesoxyuridin technisch einfacher und liefert schnelle und reproduzierbare Resultate.