Use of the recursion formula of the Gompertz function for the quantitation of PCR-amplified templates.

One common drawback of the currently used procedures to quantitate the polymerase chain reaction (PCR) is that the statistical evaluation of the experimental data depends on many, not just trivial, model assumptions. In the present study we report on an improvement in this crucial step of the quantitative PCR. The experimental design underlying the introduced method is exactly the same as in the case of the so-called PCR. However, by applying growth curve analysis based on the recursion formula of the Gompertz function the kinetics of the accumulation of the amplicon are estimated conjointly from data spanning both the and phases of the reaction. We demonstrate the method by determining the relative number of templates (a 206 bp segment spanning the exon 3 of the X-chromosomal murine Hprt-gene) contained in known orders of dilutions of DNA isolated from the spleen of the C57BL/6J-mouse. [32P]-dATP incorporation was used in duplicate experiments to quantify the amplicons as a function of amplification cycles. Our results: i) indicate that the accumulation of the PCR product as a function of PCR cycles follows a sigmoidal pattern compatible with the Gompertz growth model (P<0.0000001); ii) directly support the thesis that the kinetical pattern of accumulation of amplicons of a given DNA fragment does not depend on the number of corresponding DNA templates provided to the reaction; iii) permit a simple direct evaluation of the parallelity in the course of the accumulation of amplicons from different template numbers as a function of amplification cycles, which is a silent preposition in the evaluation of the so-called PCR; iv) allow an easy quantitation of the relative number of provided templates.

Age dependent selection against HPRT deficient T lymphocytes in the HPRT+/- heterozygous mouse.

The fraction of HPRT deficient T lymphocytes was measured in the HPRT +/- female mouse between birth and an age of about 2 years. The animals were the F1 offspring of the HPRT deficient strain 129MF1 and HPRT competent C57BL/6J-mice. T lymphocytes from spleen were cloned in vitro and HPRT deficient clones were detected by double-labeling with [3H]thymidine and [14C]hypoxanthine. During the first weeks of life the fraction of deficient lymphocytes sharply decreases from about 50% at birth to 10-30% at an age of 10 weeks. In adult animals the fraction of HPRT deficient T cells smoothly further decreases to values about 10% at 80-90 weeks. The equation gamma(t)=[0.547 x exp(-0.405 x t)] + [0.453 x exp(-0.0116 x t)] was found to be a good approximation of the time course of HPRT deficient cells in spleen; gamma(t) is the fraction of deficient cells per competent cell and t is the age of animals in weeks. It is postulated that the selection against HPRT deficient T lymphocytes is the consequence of the reduced proliferative capacity of HPRT deficient cells (=selection factor). The time course of the ratio of deficient cells can be described as a function of the proliferation rate of the HPRT competent T cells and this selection factor. The sharp initial decrease is explained by a high selection pressure against HPRT deficient cells in young animals when the proliferation rate of the expanding T cell population is high and when T cells proliferate in the bone marrow. In adult animals the selection pressure against HPRT deficient cells is reduced, since T cells arise in general in peripheral lymphatic organs, where the salvage pathway is of lesser importance compared to the de novo purine synthesis. Implications of the selection against HPRT deficient lymphocytes for the widely used HPRT mutation assay are discussed.

Studies on the effect of aurintricarboxylic acid (ATA) on cell proliferation of renewable epithelial tissues in the mouse.

The effect of a three-week treatment regimen with 2, 4 or 6 mg aurintricarboxylic acid (ATA) per kg body weight on the cell proliferation of jejunal crypts and dorsal epidermis of the nude mouse was studied using standard autoradiographic methods after in vivo pulse labelling with 3H-thymidine. ATA was slightly toxic to the animals in a dose-dependent manner (p < 0.01), as measured by the decrease of the rate of weight gain of the treated animals. In the small intestine, ATA led to a shrinkage of crypt length by a factor of 10-15% (p < 0.05), mainly by inhibiting cell proliferation in a dose-dependent manner (p < 0.05) and only secondarily and at higher doses by additionally increasing the cell loss. On the contrary, both the 3H-thymidine labelling index and the mitotic index of the basal cell layer of the epidermis are found to be increased under treatment with ATA. Thus, the present findings indicate that ATA may differentially affect the tissue homeostasis of different renewable epithelia in vivo.

Studies on the effect of suramin on cell-proliferation of epithelial tissue renewal in the mouse.

The effect of a three-week treatment regimen with suramin (30, 60 or 90 mg/kg body weight) on the cell proliferation of small intestinal crypts and dorsal epidermis of the nude mouse was studied using standard auto-radiographic methods after in vivo pulse labeling with H-3-thymidine. Suramin was slightly toxic to the animals in a dose-dependent manner (p<0.01) as measured by the decrease of the rate of weight gain of the treated animals. In the small intestine suramin treatment led to crypt shrinkage (factor: about 70-80%; p<0.05) mainly by increasing the rate of cell loss out of the crypt epithelium (p<0.05) and to a lesser degree by inhibiting the proliferation of crypt cells. However, this suramin treatment scheme did not significantly affect the H-3-thymidine labeling index and the mitotic index of the basal cell layer of the epidermis. The present findings indicate that suramin can be used to approach the problem of differential homeostatic reactions of epithelial tissue renewal in vivo.

Dose and dose-rate dependence of the frequency of HPRT deficient T lymphocytes in the spleen of the 137Cs gamma-irradiated mouse.

The frequency of hypoxanthine phosphoribosyl transferase (HPRT) deficient splenic T lymphocytes was measured in the 137Cs gamma-irradiated mouse by the T cell cloning method. Doses from 0.3 to 6 Gy were applied at the dose-rates 0.5 Gy/min, 1 Gy/day and 1 Gy/week. Mutants were determined 8-10 and 30-40 weeks after the end of exposure. Radiation-induced mutant frequency (MFi) was calculated by subtracting the age corrected spontaneous mutant frequency (MFsp) from total mutant frequency (MF) found in irradiated animals. Data were fitted to linear and linear-quadratic dose-response models. MFi depended markedly on dose, dose-rate and time after exposure. When mutants were determined 8-10 weeks after acute irradiation (0.5 Gy/min) the dose-effect curve fitted the linear-quadratic equation MFi = 6.9 x 10(-6) Gy + 1.2 x 10(-6) Gy2, whereas in low dose-rate experiments (1 Gy/day, 1 Gy/week) the dose-effect curves were linear. The slope of the linear regression was about 3 x 10(-6). When low dose-rate-irradiated animals were killed 30-40 weeks after irradiation, MFi was about one-third of that observed after 8 weeks. The dose dose-rate effectiveness factor (DDREF) for radiation mutagenicity was calculated in animals that had been exposed 8-10 weeks previously. For doses < 2 Gy the reduction in effectiveness was about 1.5 when the irradiation dose-rate was < or = 1 Gy/day. For higher doses DDREF was 3-5.

Normal and reverse dose-rate effect for the induction of mutants in somatic cells by ionizing radiation.

In radiobiology the reduction of the dose-rate in general diminishes the degree of the biological effect per unit dose. This phenomenon is characterized by the dose-rate effectiveness factor (DREF). DREF is the factor by which a risk per unit dose obtained from data at high dose and high dose-rate overestimates the risk at low doses and/or low dose-rates. In general, DREF is in the range of 2 to 10. In the first part of this review, a short survey of the modern microdosimetric approach for a better understanding of radiation load on the cellular level and the significance of dose-rate is given. Experiments on the influence of dose-rate on the mutagenicity of ionizing radiation in cultured cells are reviewed. In contrast to other biological effects, in most experiments the reduction of the dose-rate had no or even a reverse dose-rate effect (DREF < or = 1). In the second part results on the influence of dose-rates on the induction of HGPRT-deficient T-lymphocytes in mice irradiated in vivo are given. Mutagenicity decreases with dose-rate and DREF values between 3-10 were measured. Possible reasons for the discrepancy between in vitro and in vivo experiments are discussed.