Pancreatic cell proliferation in normal rats studied by in vivo autoradiography with 3H-thymidine

In vivo 3H-Thymidine autoradiographic investigations of DNA synthesis in acinar, islet and duct cells in the pancreas of normal rats showed that activity was dependent on age. The proliferation of acinar and islet cells, which was high in young animals, decreased exponentially with age; proliferation of the ductal cells on the other hand, increased until the animals became mature. These findings suggest that the physiological regeneration of acinar and islet cells, as well as their replacement after injury in adult animals commences from pancreatic ducts.     

A method of DNA histogram analysis by computer and its evaluation by simultaneous combination with 3H-thymidine autoradiography

A method for determining the fractions of cells in the G1, S, and G2 + M phases of the cell life cycle, by quantifying DNA histograms derived from static fluorescence cytophotometry, was evaluated by simultaneous combination with 3H-thymidine autoradiography. DNA histograms were obtained by cytofluorometry on the Feulgen-stained autoradiographs of HeLa cells, and mouse and rat hepatocytes, after DNA labelling with 3H-thymidine. The synthetic histogram determined by "sum of discrete normal curves" technique was fitted to the experimental data according to a weighted least-squares method by a desk-top computer (HP 85F). The mean relative percent deviations of estimated cell cycle phase fractions from the actual phase fractions determined directly on an autoradiograph was 6.6 +/- 3.3%.     

DNA cytofluorometry on large and small cell nuclei stained with pararosaniline Feulgen

Summary To examine reliability of DNA cytofluorometry with conventional Feulgen staining, measurement was carried out on smears of small and large neurons of human cerebellum, using a Digital-Microfluorometer with incident green light excitation. Nuclear fluorescence of the inner granule neuron and the Purkinje cell came to good agreement with each other when the staining was reduced so that the maximal extinction of the excitation beam nowhere exceeded the value of 0.1. The reduction of the feulgen staining is realized with unimpaired proportionality by treatment with 0.05% Schiff's reagent (pH 2.3) at 7–10° C for 10 min. It is concluded that this staining condition assures accurate determination of DNA content irrespective of size of the nucleus and of pattern of the chromatin distribution.     

Hormone-dependent changes of blood vessels in DMBA-induced rat mammary carcinoma and its regression studied by 3H-thymidine autoradiography

Using DMBA-induced rat breast cancer, the changes in the histology and proliferative activity underlying the phenomenon of tumor regression by hormone therapy were studied by 3H-thymidine autoradiography. The control tumor was found to essentially consist of two histologically different areas, medullary (A area) and tubular or cystic (B area). The cancer cells in the A area were homogeneously proliferating with a cell cycle time of 51h, and among those in the B area, 65% were proliferating with a cell cycle time of 81h while 35% were non-proliferating. Among the various-kinds of hormone therapies, ovariectomy plus male sex hormone administration was most effective in inducing tumor regression. In the regressed tumor, the A area was greatly diminished due to central necrosis and replaced by cystic B area. In the remaining A area, the cell cycle time was lengthened to 97h, and that for the proliferating cells in the B area was as long as 118h. The most striking histological change after ovariectomy plus male sex hormone administration was the diffuse necrosis of the capillary endothelial cell within 24h, followed by hemorrhage, central necrosis in the A area (1W), and final stage of fibrosis (2W). The tumor administered with female sex hormone after ovariectomy showed a rebound growth from the regression, due to the initial reactivation of the endothelial cell proliferation and following stimulation of cancer cell mitotic activity. From these observations, it is concluded that the capillary endothelial cells in DMBA-induced rat breast cancer are estrogen dependent, and that the tumor regression induced by decreased estrogen-level is attributable to the massive necrosis from capillary insufficiency and anoxia.     

Effect of vinblastine on the cell cycle and migration of ameloblasts of mouse incisors as shown by autoradiography using 3H-thymidine

The effects of vinblastine on the cell cycle and the migration of ameloblasts were studied in the lower incisors of mice by labelling the cells with 3H-thymidine ([3H]TdR) and radioautography. A group of mice received 2 micrograms/g of body weight vinblastine intraperitoneally and 6 hr after these animals and those of a control group were injected with 1 microCi/g body weight of [3H]TdR, and sacrificed at time intervals from 0.75 hr to 15 days. The generation time of ameloblasts in the progenitor compartment was 14.8 hr in animals treated with vinblastine and 17 hr in the controls, using the FLM curve method; with the grain dilution method the duration was respectively 29.25 hr and 25.96 hr. The thymidine labelling index of the treated animals was 50% higher than the controls. The velocity of ameloblast migration, determined either by the displacement of the most incisally labelled cell or by the grain dilution method, was lower in the experimental group (2.48 cell positions/hr and 9.18 microns/hr respectively) as compared with the control (3.21 cell positions/hr and 18.88 microns/hr respectively). The results on the ameloblast production rate are contradictory but the slowing down in the velocity of cell migration is compatible with a decrease of the rate of cell production in the progenitor compartment as a vinblastine effect.     

Localization of99mTc in bone by means of autoradiography

The uptake of two different preparations of^99mTechnetium-methylene diphosphonate in fetal rat calvaria is compared. The localization of^99mTc after administration of^99mTc(Sn)-MDP and^99mTc-MDP showed equal distribution in autoradiography.     

Localisation of [3H] clonidine binding in rat neurohypophysis by means of electron-microscopic autoradiography

Summary Electron-microscopic autoradiography of rat neurohypophyses treated with [³H] clonidine, an ₂-agonist, showed that binding apparently occurred preferentially at the neurosecretory endings and blood vessels rather than on the pituicytes. Since it is known that clonidine has a high affinity for plasma proteins, the distribution over the neurosecretory nerve endings would suggest the existence of presynaptic ₂-binding sites on neurosecretory neurones, which could indicate a regulatory function for catecholamines in neurohypophysial hormone release.     

Neuronal uptake of 3H-thymidine

A pronounced incorporation of ³H nucleotides into mitochondrial DNA in isolated suspensions of mitochondria has been demonstrated recently for a number of cell types. Although it is generally assumed that the multiplication of mitochondria is accompanied by replication of their DNA, pronounced incorporation of ³H-thymidine into the mitochondria of higher organisms in vivo has yet to be shown. In the present study, uptake has been measured of ³H-thymidine into neurons of mouse spinal ganglia, cockroach nerve cord ganglia and goldfish retina.The frequency of ³H-thymidinc incorporation into neuronal mitochondria precludes in vivo fractionation of a labelled population. Less than 1% labelling of axonal mitochondria is observed. Although neuronal mitochondria have a high half life and several hundred pass a fixed point along an axon in one day in a unidirectional flow, failure to observe a significant mitochondrial uptake leaves open the problem of the nuclcotide pool serving mitochondrial DNA.     

Bacterial secondary production in freshwater measured by3H-thymidine incorporation method

Bacterial secondary production in lake water was measured by³H-thymidine incorporation into DNA. The application of the method to freshwater systems studied required (1) thymidine concentration > 10 nM (10–25 nM) evaluated from isotope dilution by varying the specific activity of labeled thymidine, (2) short incubation periods less than 1 hour, and (3) partial purification of the DNA fraction for measuring³H-thymidine incorporation. During 2 diel studies, bacterial productivity was compared to phytoplankton primary production and extracellular release of organic carbon. Diel changes in bacterial growth suggested substantial activity during the morning and evening. Possible mechanisms of control of bacterial growth, such as extracellular release of organic carbon, are discussed.     

Analysis of the effect of fluorescence intensity on distributions obtained by flow cytofluorometry

It is shown that one of the major resolution limiting factors in the rapid measurement of fluorescence from individual cells with “fast flow cytofluorometers” is the small number of photons which are counted in each light pulse. A method is described for evaluating this factor for individual systems and for specific cells and stains. Once evaluated, this contribution to the broadening of the distribution can be stripped from the observed distribution to give a closer estimate of the actual distribution of dye in the cell poulation.     

Ultrastructure and 3H-thymidine incorporation by chromosome vesicles in sea urchin embryos

The ultrastructural features of chromosome vesicle formation in early sea urchin embryos and chromosome vesicle uptake of tritiated thymidine is described. Envelopes which resemble typical nuclear envelopes form around the condensed anaphase chromosomes. In late anaphase or early telophase, the chromosomes swell and decondense and it is at this time when tritiated thymidine is incorporated. This study shows that DNA synthesis in the rapidly dividing cells of early sea urchin embryos occurs in chromosome vesicles which form during anaphase.     

3H-thymidine incorporation by interstitial myocardial cells in the rat with a single administration of the label

The hearts were taken 1, 2, 3, 4, 5, 6, 7, 14 and 21 days after a single injection of 3H-thymidine into 20 young male Wistar rats. Label incorporation was determined on half-thin slices derived through the whole left ventricular wall. First labeled nuclei are seen on the day 1 at the level of 0.4 +/- 0.08%, their quantity reaches maximum (1.66 +/- 0.1%) on day 4 and declines to 0.51 +/- 0.1% at day 7. On day 21 0.1 +/- 0.14% cells contained radioactivity. Maximal label concentration was found in middle intramural layer (2.44 +/- 0.24% on day 4), and reached only 1.36 +/- 0.36% in subendocardial and 1.16 +/- 0.24% in subepicardial layer. These data allow us to suppose the existence of linkage between interstitial cells repopulation and functional activity of different myocardial layers, and also the existence of short living and long living fibroblast subpopulations.     

Genome Size Determination in Peronosporales (Oomycota) by Feulgen Image Analysis

Genome size was determined, by nuclear Feulgen staining and image analysis, in 46 accessions of 31 species of Peronosporales (Oomycota), including important plant pathogens such asBremia lactucae, Plasmopara viticola, Pseudoperonospora cubensis,andPseudoperonospora humuli.The 1C DNA contents ranged from 0.046 (45.6 Mb) to 0.163 pg (159.9 Mb). This is 0.041- to 0.144-fold that ofGlycine max(soybean, 1C = 1.134 pg), which was used as an internal standard for genome size determination. The linearity of Feulgen absorbance photometry method over this range was demonstrated by calibration ofAspergillusspecies (1C = 31–38 Mb) againstGlycine,which revealed differences of less than 6% compared to the published CHEF data. The low coefficients of variation (usually between 5 and 10%), repeatability of the results, and compatibility with CHEF data prove the resolution power of Feulgen image analysis. The applicability and limitations of Feulgen photometry are discussed in relation to other methods of genome size determination (CHEF gel electrophoresis, reassociation kinetics, genomic reconstruction) that have been previously applied to Oomycota.     

High-speed autoradiography of 3H-thymidine-labelled nuclei

Summary High-speed autoradiography with stripping film of ³H-thymidine-labelled cells was tested. The tests involved: (a) various times of immersion of emulsion-covered cell preparations in the mixture of dioxane-PPO-POPOP, at 20°C, (b) exposure of cell preparations and blanks for various times at either –70°C or +20°C, with different humidity levels. Autoradiographs of good quality could be produced by 2-min immersion in the scintillator, exposure time 1 h at either temperature and relative humidity 20–30%. A linear relationship between autoradiographic efficiency and exposure time of 1–7 h was found at either temperature, although the efficiency of autoradiographs exposed at –70°C was by approximately 30% higher than that of autoradiographs exposed at +20°C. Background values of autoradiographs dried with a fan and exposed for 1/4–7h at either –70°C or +20°C were 0.6–0.8 grain/100 m². Theoretical calculations and experimental data showed that high-speed autoradiographs are 30–50 times more efficient as compared with conventional stripping film autoradiographs, thus allowing a shortening of the respective exposure time. Theoretical aspects of efficiency and resolution of high-speed autoradiography are considered.This investigation was supported in part by MR II.1 grant. The technical assistance of Mrs. S. Bie is gratefully acknowledged