A two emulsion autoradiographic technique and the discrimination of the three different types of labelling after double labelling with 3H- and 14C-thymidine.

The first part of the paper deals with a two emulsion autoradiographic technique for double labelling experiments with 3H- and 14C-thymidine which permits a clear discrimination of the different types of labelling. In the second part the application of this technique to cell kinetic studies is discussed. Accurate discrimination between the different types of labelling, namely purely 3H-, purely 14C- and double (3H + 14C) labelling, is only possible if the activity ratio of 3H- to 14C-thymidine is sufficiently high. This condition is necessary for a reliable distinction between those grains in the first emulsion which are due to true 3H-labelling and spurious grains which are simultaneously produced in the same emulsion by 14C-beta-particles. Experiments are described to determine the required activity ratio of 3H- to 14C-thymidine.     

Circadian variations in the DNA synthesis of the rat corneal epithelium. A study using double labelling with tritiated thymidine

A prominent circadian rhythm was found in the labelling indices (LI) of the peripheral rat corneal epithelium and of the adjacent conjunctival epithelium, while almost no diurnal variation was found in the central area. Application of a double labelling technique indicated that there are rhythmic pulses of high and low influx of cells into the S phase and similar pulses of efflux of cells from the S phase. Results of the study indicate that there are different cohorts of cycling cells all over the rat corneal epithelium. Cells belonging to a rapidly proliferating cohort are observed in the peripheral cornea. There is a gradual reduction in the fraction of labelled DNA-synthesizing cells towards the centre. The considerably lower fraction of cells taking up tritiated thymidine (3H)TdR in the central cornea may be due to a higher fraction of basal cells having reached higher levels of differentiation. This may result in a shift from the salvage to the de novo pathway. The slowly proliferating cohort seems to have a prolonged S phase duration and displays practically no diurnal variation in the LI. The DNA-synthesizing cells belonging to this latter cohort probably use the salvage pathway for DNA synthesis resulting in uptake of (3H)TdR all over the cornea. The LI is thus not a reliable indicator of cell proliferation in the corneal epithelium, due both to the heterogeneity of the cell proliferation, and in particular due to the lack of labelling of the centrally located DNA-synthesizing cells. To what extent these properties may also be present in other proliferating tissues with different levels of differentiations, may be questioned.     

Re-examination of the effect of beta-adrenergic blocking agents on the proliferation of rat jejunal crypt cells using the stathmokinetic method

Reports of the effects of beta-adrenergic receptor blocking agents on the proliferative activity of rat jejunal crypt cells are contradictory. According to Tutton and Helme (1974) a single injection of propranolol or practolol (10 mg/kg) increased the mitotic index twofold and shortened the duration of the cell cycle of the crypt cells. However, upon repeating the experiments with double the dose of propranolol, Maurer-Schultze et al. (1986) observed no such effects using cell kinetic methods with 3H-thymidine instead of the stathmokinetic method applied by Tutton and Helme. Since the discrepancy in the results may have been due to methodological differences the same stathmokinetic method used by Tutton and Helme has been applied in the present work. However, the results obtained with this method indicate no influence by propranolol on the proliferation of jejunal crypt cells even with a dose of 20 mg/kg. Consequently we were unable to confirm the stimulant effect of propranolol on crypt cell proliferation. The possible causes of the discrepancy between the present results and those of Tutton and Helme are discussed.     

Influence of the beta-adrenergic antagonists propranolol, practolol and oxprenolol on the proliferation of rat jejunal crypt cells

Beta-adrenergic blockade by quite large doses of propranolol, practolol and oxprenolol, once or continuously applied, does not influence jejunal crypt-cell proliferation in the rat. After a single i.p. injection of 20 mg/kg propranolol or practolol and even of 100 mg/kg practolol, the mitotic index, the labelling index and the duration of the S phase do not differ between treated and untreated control animals nor between animals treated with the different drugs. Continuous application of 30 mg/kg/d propranolol, practolol or oxprenolol for 7 or 14 days does not affect the mitotic and labelling indices either, nor does it change the duration of the cycle of the jejunal crypt cells and its phases as determined by the percent labelled mitoses method. These results are in contrast to those reported previously by Tutton & Helme (1974).     

Pattern of labelling of the rat brain stem after intraventricular administration of 3H-leucine; low and high resolution autoradiographic study

The pattern of labelling of proteins of the periventricular grey matter was studied two hours after intraventricular administration of 3H-leucine by low- and high-resolution autoradiography. The pattern was investigated by computer-controlled densitometry. The deposition of radioactive, proteins in the periventricular grey surrounding the mesencephalic part of the aquaeductus Sylvii was compared with that surrounding the fourth ventricle. In the former case, the distribution, of grains was in a circular area 500-600 micrometer in diameter; the densitometric tracing revealed a homogeneous distribution of the label; in the latter case, the distribution was nonhomogeneous and was limited by the tissue components forming the wall of the fourth ventricle. A comparison of the intensity of labelling (performed by a combination of low- and high-resolution autoradiography indicated: a) relatively substantial labelling of proteins of ependymal cells, b) very sparce labelling of subependymal layers, c) very high labelling ot neurones, adjacent to the subependymal layers. The significance of these findings for the interpretation of studies using the intraventricular administration of labelled amino acids for investigating brain macromolecular metabolism is discussed.     

Cell kinetic studies of chick brain cells in culture: an autoradiographic study with [3H]- and [14C]-thymidine

Labelling index, S-phase duration and cell-cycle time of proliferating brain cells from 6-day-old chick embryos in culture were investigated autoradiographically after labelling with [3H]- and/or [14C]-thymidine. The dissociated cells were cultured in the absence or in the presence of brain extract from 8-day-old chick embryos. Cultures contained essentially two cell types, which could be easily distinguished by the size of their nuclei: small nuclei identified as belonging to precursor cells of neurons and large nuclei corresponding to astroglial cells. The labelling index of astroglial cells (16.4%) was about 2 times higher than that of the neuronal cells (9.9%). Under the influence of brain extract the labelling index of neuroblasts was nearly doubled while that of the astroglial cells remained nearly unchanged. From double-labelling experiments with [3H]- and [14C]-thymidine, the same S-phase duration of about 7 hr was found for both cell types cultured with or without brain extract. A cell-cycle duration of 39 hr for neuronal and of 29 hr for astroglial cells was found. The cycle times remained constant under the influence of brain extract. From the measured data mentioned above, a growth fraction of 50% (neuroblasts) and 68% (astroglial cells) was calculated in control cultures without brain extract. After addition of brain extract, the growth fraction increased for both cell types (neuroblasts: 92%; astroglial cells: 80%). The results demonstrate that more cells proliferate in the presence of brain extract, but the durations of the S-phase and the cell cycle remain unchanged.     

Position and growth of upper and lower tooth primordia in prenatal mouse--3D study

The secondary palate formation in mouse has been associated with the period of fast growth of the mandible from embryonic days (ED) 13.0 to 16.0. During that time, the incisors and first molars develop from the bud to the bell stage. We investigated the position and growth of the tooth during prenatal elongation of the lower and upper jaws, and searched for the developmental stage when alignment of opposing teeth was achieved. Computer-aided 3D representations allowed us to represent the position of incisors and molars in the embryonic head from ED 13.5 to 18.0 on the basis of data obtained from histological sections. The atlas-hypophysis connection exhibited minimum change in length and orientation during the prenatal period, and thus was used as a reference line. The length of the teeth was calculated from 3D data. The upper first and second molars were longer than the lower ones. When viewed from the upper side, the upper and lower molar primordia were parallel from ED 13.5 to 15.0. During this period, the upper molars had a more lateral position than the lower ones. This situation was maintained in the anterior extremity of the first molars at later stages, while the posterior part of the upper and lower molar epithelia reached opposition in the medio-lateral direction from ED 16.0. The lower incisors exhibited an apparently backward position when compared to the upper incisors at earlier stages. However, the distance between the prospective anterior tips of the opposing incisors gradually decreased. The part of Meckel's cartilage associated with the lower dental quadrant elongated more than 3-fold from ED 13.5 to 18.0, and the lower jaw grew faster than the upper one. This difference resulted from the fast growth of the lower diastema from ED 14.0 to 18.0. The different growth speeds of the upper and lower jaws did not change the relative antero-posterior adjustment of the upper and lower molars, but contributed to achieving the opposition of the gnawing ends of the incisors.     

Modulation of [3H]-glutamate binding by serotonin in the rat hippocampus: an autoradiographic study.

Serotonin (5-HT) added in vitro (10 microM) increased [3H]-glutamate specific binding in the rat hippocampus, reaching statistical significance in layers rich in N-Methyl-D-Aspartate sensitive glutamate receptors. This effect was explained by a significant increase in the apparent affinity of [3H]-glutamate when 5-HT is added in vitro. Two days after lesion of serotonergic afferents to the hippocampus with 5,7-Dihydroxytryptamine [3H]-glutamate binding was significantly decreased in the CA3 region and stratum lacunosum moleculare of the hippocampus, this reduction being reversed by in vitro addition of 10 microM 5-HT. The decrease observed is due to a significant reduction of quisqualate-insensitive (radiatum CA3) and kainate receptors (strata oriens, radiatum, pyramidal of CA3). Five days after lesion [3H]-glutamate binding increased significantly in the CA3 region of the hippocampus but was not different from sham animals in the other hippocampal layers. Two weeks after lesion [3H]-glutamate binding to quisqualate-insensitive receptors was increased in all the hippocampal layers, while kainate and quisqualate-sensitive receptors were not affected. These data are consistent with the possibility that 5-HT is a direct positive modulator of glutamate receptor subtypes.     

Pool and synthesis of phosphoribosylpyrophosphate in rat embryo cells infected with X14 or H-1 parvovirus

In rat embryo cell cultures infected with X14 or H-1 parvovirus the PRPP pool and the PRPP synthetase activity have been assayed. A radiometric method, prepared by Authors, based on the conversion of [6-14C) orotate to [6-14C) UMP by the mixed enzyme orotate phosphoribosyltransferase and orotidylate decarboxylase and on the separation of UMP by ascending chromatography, has been utilized. The PRPP pool and te PRPP synthetase activity appeared nearly unmodified in the cells infected with X14 or H-1 parvovirus compared to the mock-infected cells. Therefore, the lowered pyrimidine nucleotide synthesis in infected cells, shown in previous studies, may depend, rather than on the diminished PRPP pool, on the lower PRPP utilization; in fact, some inhibition by metabolites, that may be removed by added PRPP, might occur in the infected cells.     

Transit times through the cycle phases of jejunal crypt cells of the mouse. Analysis in terms of the mean values and the variances.

Mean transit times as well as variances of the transit times through the individual phases of the cell cycle have been determined for the crypt epithelial cells of the jejunum of the mouse. To achieve this the fraction of labelled mitoses (FLM) technique has been modified by double labelling with [3H] and [14C]thymidine. Mice were given a first injection of [3H]thymidine, and 2 hr later a second injection of [14C]thymidine. This produces a narrow subpopulation of purely 3H-labelled cells at the beginning of G2-phase and a corresponding subpopulation of purely 14C-labelled cells at the beginning of the S-phase. When these two subpopulations progress through the cell cycle, one obtains FLM waves of purely 3H- and purely 14C-labelled mitoses. These waves have considerably better resolution than the conventional FLM-curves. From the temporal positions of the observed maxima the mean transit times of the cells through the individual phases of the cycle can be determined. Moreover one obtains from the width of the individual waves the variances of the transit times through the individual phases. It has been found, that the variances of the transit times through successive phases are additive. This indicates that the transit times of cells through successive phases are independently distributed. This statistical independence is an implicit assumption in most of the models applied to the analysis of FLM curves, however there had previously been no experimental support of this assumption. A further result is, that the variance of the transit time through any phase of the cycle is proportional to the mean transit time. This implies that the progress of the crypt epithelial cells is subject to an equal degree of randomness in the various phases of the cycle.     

Method of binary classification and risk assessment of individuals with familial polyposis based on [3H]TdR labelling of epithelial cells in colonic crypts

An analysis has been developed to improve the quantitation of abnormal patterns of tritiated thymidine [(3H]TdR) labelling of colonic epithelial cells, in biopsy specimens removed from human subjects at varying degrees of risk for colon cancer. After pulse incubation of specimens of colonic mucosa with [3H]TdR, each subject's microautoradiographic epithelial cell labelling distribution was segregated into eleven compartments over entire colonic crypts. The findings of each subject were then analysed to determine their relative degree of similarity to the findings for two reference populations of interest, i.e. a high-risk and a low-risk population; the individual was then classified as being closer to one or the other of the reference populations. The analysis developed is based upon a comparison of multinomial probabilities for the distributions of the labelled cells within the crypts, and permits the routine categorization of uneven distributions of labelled cells. For each subject, certain linear scores, a prognostic index based on them, and a related presumptive risk, were calculated. The sensitivity with which individuals known to be symptomatic for polyposis, and the specificity with which individuals known to be at lower risk were determined, were 73 and 93% respectively. The results suggest that this method of distinguishing among integer distributions of [3H]TdR- labelled cells in biopsies of colonic mucosa, may provide a useful basis for identifying individuals with familial polyposis, by separating their labelling patterns from those of low-risk subjects.     

Keratin expression in rat intestinal crypt and villus cells. Analysis with a panel of monoclonal antibodies.

Seven monoclonal antibodies were prepared against cytoskeletal components of rat intestinal brush borders. In the following paper (Chandler, J. S., Calnek, D., and Quaroni, A., J. Biol. Chem. 266, 11932-11938), three of them were shown to be specific for, respectively, keratin 8 (RK4), keratin 19 (RK7), and a newly identified type I keratin (keratin 21) (RK5). With these antibodies we have investigated the changes in keratin gene expression accompanying intestinal cell differentiation. Keratin 21 was detected exclusively in differentiated villus cells and in goblet, enteroendocrine, and Paneth cells in the crypts; in the proliferative crypt cells keratin 19 was predominant. Analysis of keratins expressed by cultured rat crypt cells (IEC cells) confirmed the absence of keratin 21 in undifferentiated intestinal cells. Changes in keratin's expression similar to those observed with cell differentiation in the adult intestinal mucosa were also demonstrated during early fetal intestinal development: the stratified epithelium present at 15-16 days of gestation contained predominantly keratin 19 with only a small amount of keratin 8; keratin 21 was first detected at 18-19 days of gestation, concomitant with the appearance of a well formed brush border and an apical cytoplasmic terminal web. These results suggest that keratin tonofilaments may play a role in the morphological and structural alterations accompanying intestinal cell differentiation in vivo.     

Immunocytochemical and autoradiographic studies of the endocrine cells interacting with GABA in the rat stomach

There are now increasing evidences suggesting that GABA is able of direct interaction with certain endocrine cells. In the present study, highly specific anti-GABA-glutaraldehyde antibodies and 3H-GABA uptake were used at the light and electron microscope levels to investigate the occurrence of cells containing endogenous GABA or taking up exogenous GABA in the mucosal antrum and corpus of the rat stomach. Only certain endocrine cell types of both regions were immunostained or grain-labelled. However, the morphology of their secretory granules did not allow to identify the nature of their hormone with certainty but suggested that somatostatin-like cells could interact with GABA. The combination of gastrin and somatostatin immunodetection with 3H-GABA uptake autoradiography at the light microscope level, revealed that a subpopulation of somatostatin-like cells and other still unidentified endocrine cells are able to take up GABA, while the gastrin-like cells are not. These results reinforce the hypothesis that certain endocrine cell types of the diffuse endocrine system of the digestive tract are able to directly interact with GABA.