Discrimination of two fibroblast progenitor populations in early explant cultures of hamster gingiva

Summary Numerous metabolic studies have demonstrated heterogeneity of fibroblast populations in culture, yet little is known about the structure of fibroblast populations in adult tissues in vivo. To determine if populations of both cycling and non-cycling cells are present in gingiva, hamsters were labelled with [³H]-thymidine to label cycling cells in vivo, and explanted biopsies were subsequently incubated with bromodeoxyuridine to label cycling cells in vitro. Cycling cells were identified by combined immunohistochemistry and radioautography. Fibroblasts were recognized by the presence of vimentin and the absence of keratin as determined by immuno-fluorescence. The largest proportion of cells were double-labelled with [³H]-thymidine and bromodeoxyuridine (43.8%) indicating the presence of actively cycling populations that maintained their proliferative status upon explantation. Cultures also exhibited a second population of cells labelled only with bromodeoxyuridine (38.7%) that did not cycle in vivo, but retained the capacity for proliferation in vitro. However, limiting dilution analysis of single-cell suspensions revealed only a single class of progenitors capable of forming large colonies in vitro. Approximately 1 in 190 plated cells was capable of colony-formation, indicating that, upon explantation, a subset of the cycling cells in vitro exhibits extensive proliferative capacity. There was also a small population of cells unlabeled with either [³H]-thymidine or bromodeoxyuridine (9.4%) that appeared to be terminally differentiated. Different substrates, including glass and thin films of gelatin and collagen, did not significantly alter the fraction of cells labelled with [³H]-thymidine. These data demonstrate the existence of 2 separate progenitor-cell populations with different capacities for proliferation in vivo and in vitro.     

The interaction of carbon-14-labelled alkyl carbamates, labelled in the alkyl and carbonyl positions, with DNA in vivo

The binding of ethyl carbamate labelled with carbon-14 in the alkyl or carbonyl group, and of methyl, n-butyl and n-propyl carbamates labelled in the alkyl group, to the DNA of mouse liver, lung and kidney has been studied in male Crackenbush mice. Only ethyl carbamate bound to liver and kidney DNA to any significant extent.The binding of ethyl carbamate labelled with carbon-14 in the C₁, C₂ or the carbonyl position was examined and compared. The levels of binding of [1-¹⁴C]- and [2-¹⁴C]ethyl carbamate to liver DNA were not significantly different (328 ± 34 and 267 ± 24 dpm/mg DNA, respectively), but there was very little binding of the [carbonyl-¹⁴C]ethyl carbamate (26 ± 3 dpm/mg DNA). Furthermore, only 18% of the radioactivity was removed from the DNA labelled with the alkyl-labelled carbamates, whereas 65% of the radioactivity was removed from the DNA labelled with carbonyl-labelled ethyl carbamate on continuous ether extraction. It was concluded that the bound molecule does not contain the carbonyl carbon and is probably an ethyl group.     

The production of lipids alternately labelled with carbon-13

Chlorella cells were shown to have similar fatty acid profiles when grown photoautotrophically or if grown photoheterotrophically with ethanoate (acetate) as carbon source. When supplied with ethanoate labelled with carbon-13 in the methyl group, the alga incorporated it into fatty acids with retention of the sequence of labelling on alternate carbon atoms, thus providing a convenient method for synthesising lipids in a form useful for nuclear magnetic resonance (NMR) studies of lipids in situ in membranes. Marine algae used in fish farming may have higher levels of very highly unsaturated fatty acids; proposals for producing these compounds labelled with carbon-13 are, therefore, presented, based on using centrally labelled glycerol. The scope for producing other substances labelled in a form suitable for NMR studies, such as carotenoids, is discussed.     

Autoradiographic study of cellular structure of the epithelium of the duct of testicular epididymis of rats]

Mature rats were given intraperitoneal injections of H-3-thymidine (1 mkk/g 1-32 hours before being killed. Labelled and non-labelled mitoses and interphase cells of different types were counted in each zone of the epididymis autographs. The diurnal fluctuatiof the mitoticindex (Im) was found: form 0,19% in the day-time to 0.33% in the night at and morning hours (psmaller than 0.05). The average diurnal Im was equal to .23%-0.03. The fist wave of labelled mitoses of the epithelial cells was observed during 32 hours, tg-2 (3-5 hours) and ts(13-14 hours) were graphically calculated. The time tg-2-tm-ts was equal to 19-20 hours. Therprietal (0.87%), basal (1.87%) and oreolar (2.20%) cells of the epidermis duct labelled 1 hour after ijection of H-3-thymidine. The apical cells (3.%) were labelled 8 hours later, while the light ones were not labelled during the whole period of observation. On these grounds, the parietal, basal and oreolar cells are considered to be proliferative cells, while the light and apical ones-to be their derivatives in the epidemis epithelium. Besides, the oreolar cells may be regareded as a foreign element in the epidermis according to their morphological features and ability to migrate throughout the total depth of the epithelial layer.     

A rapid and simple procedure for the preparation of the two bacterial cell wall peptidoglycan nucleotide precursors labeled in their amino sugars

The two bacterial cell wall peptidoglycan precursors UDP-MurNAc-l-Ala-d-iso Glu-l-Lys-d-Ala-d-Ala and UDP-GlcNAc labeled in their amino sugars with either tritium or carbon-14 accumulated in cells ofMicrococcus luteus that were incubated for short periods of time in a minimal medium to which [¹⁴C]glucose or [³H]glucose together with Vancomycin were added. The radioactive nucleotides were extracted from the cells with cold trichloroacetic acid, and their purification was achieved by paper electrophoresis followed by paper chromatography.     

Changes in the DNA lesions induced by alkylating agents during the post-treatment washing and redrying of barley seeds

During the 24 hours’ washing of¹⁴C MNU and¹⁴C MMS-treated seeds the amount of DNA 7-methylguanine as well as the total alkylation of DNA, RNA and proteins in cells of the embryo was enhanced. The drying of shortly washed¹⁴C MNU-treated seeds to 30% and 15% water content led to, a similar increase of alkylation. In accordance with the changes in the biological damage, the amount of single strand breaks and/or alkali labile sites in DNA dropped down during a 19 h washing period of MNU-treated seeds but changed only slightly in MMS-treated and 19 hours washed seeds. The drying of 0·5 or 18 h resp. 19 h washed seeds treated either with MNU or MMS to 15 per cent water content increased the amount of single strand breaks and/or alkali labile sites in DNA.     

The role of myonuclei in muscle regeneration: An in vitro study

It is well established that during muscle regeneration, the satellite cells which are in a state of mitotic arrest, can initiate cell division to produce myoblasts which subsequently fuse to form myotubes. However, whether myonuclei, contained within damaged myotubes, or “freed” as a result of the trauma, play any role in muscle regeneration remains unresolved. In myogenic cultures, it is possible to obtain renewed myogenesis when initial cultures are sub-cultured. The aim of this study, was to obtain evidence of the participation by myonuclei of primary cultures in myogenesis which occurs subsequently in secondary cultures. In culture, myonuclei can be labelled with H³-thymidine and their ultimate fate, either as “free” myonuclei or myonuclei associated with disrupted myotubes can be followed unequivocally. Three types of experiments are performed: (i) Primary myogenic cultures containing only myotubes are subcultured. (ii) Primary myogenic cultures containing myotubes with labelled myonuclei are disrupted and subcultured. (iii) Primary myogenic cultures containing myotubes with unlabelled myonuclei are mixed with labelled mononucleated myogenic cells and sub-cultured. In all instances no evidence of myogenesis from myonuclei is obtained. It is concluded that myonuclei, which were rendered postmitotic during myogenesis, remain so when muscle is disrupted and cannot re-enter the mitotic cycle.     

An automatic carbon-14 gas analyzer for organic compounds and biological samples

The present paper concerns automatic measurement of the radioactivity of substances labeled with carbon-14 isotope. The organic compounds or biological samples are combusted in an oxygen chamber. The oxygen excess is converted by hydrogen into water vapor in a reactor filled with copper and copper oxide. The water is retained by condensation and the carbon dioxide is swept by means of a counting gas, preferably propane or butane or a mixture of such gases, into an anticoincidence counter. The carbon-14 gas analyzer allows the measurement of carbon-14 isotope with high accuracy and sensitivity in a fully automated way, entirely free of manual intervention.     

Labelling patterns of carbon-14 in net plankton during a winter-spring bloom

Weekly surface samples were collected in lower Narragansett Bay, Rhode Island, during the 1975 winter-spring bloom and fractionated by nets to nannoplankton (<20 μm) and total (< 158 μm) size fractions. Each size fraction was assayed for paniculate carbon, nitrogen, carbohydrate, protein, chlorophyll a, and cell counts. The <20 μm values were subtracted from the <158 μm values to estimate the composition of the 20 μm to 158 μm fraction (termed net plankton). As nutrients (primarily nitrogen) decreased to undetectable levels with the culmination of the diatom bloom, the ratios of protein/carbohydrate, carbohydrate/carbon, and carbon/chlorophyll a in the net plankton indicated the diatom population was increasingly nutrient-limited. Each size fraction was also incubated at a saturating light intensity with carbon-14; following filtration, the cells were extracted with solvents to obtain labelled polysaccharide and protein. The daily rates of polysaccharide and protein synthesis in the net plankton declined as the bloom entered the stationary phase. When the diatom population was at its maximum density the majority of the carbon-14 was found in the ethanol-soluble fraction; this may be due to high light intensities or nutrient effects.     

The effects of carbon-13 incorporation into preimplantation mouse embryos on development before and after implantation

Preimplantation mouse embryos were cultured in vitro for 48 hours from the 8-cell to the blastocyst stage in media containing uniformly labelled ¹³C-glucose. The ¹³C content of the blastocysts was 20 atom % according to incorporation studies with ¹⁴C-glucose. No embryotoxic effects of carbon-13 incorporation could be detected on the basis of these criteria of normal development: the percentage of embryos reaching the blastocyst stage during the culture period; the number of cells in these blastocysts; and the development after transplantation to pseudopregnant foster mothers.     

Markierungsmuster nach H3-Thymidineinbau in Kernen der Hypokotylzellen von Sinapis alba L.

DNA-synthesis in the hypocotyls of Sinapis alba L. was studied with H³-thymidine labelling. Cells from hypocotyl segments were stained by the Feulgen-method and squash preparations were made. The following labelling patterns were observed: 1. Labelling of the chromocentres only. 2. Nuclear area evenly labelled. 3. No radioactivity in the chromocentres. This pattern was rarely seen. — The frequency of the first two types in different tissue segments is not equal. In segments with more differentiated cells there was an increase in the percentage of nuclei with radioactivity only in the chromocentres. This could be due to a prolongation of the phase of synthesis in the chromocentres in this tissue. — The total number of labelled nuclei decreases basipetally as well as with the age of the hypocotyl. In hypocotyls of seedlings older than 52 hrs radioactivity appeared only sporadically in the nuclei. The decrease in the number of labelled nuclei is faster than the decline of the corresponding measurable total DNA synthesis in the hypocotyl. This can either be due to extra nuclear DNA synthesis or depend on an increase in DNA synthesis in the later replicating heterochromatic region of the nucleus.     

Labelling of murine epidermal Langerhans cells with H3-thymidine.

Epidermal Langerhans cells may be identified by light microscopy by their strongly positive reaction following incubation for ATPase activity. Intact sheets of epidermis from mice killed at various time intervals following a single pulse label of H3-thymidine were incubated to demonstrate ATPase activity and subsequently processed for autoradiography. In specimens taken one hour after labelling, many basal keratinocytes were labelled but very few ATPase-positive dendritic cells. At subsequent time periods a few pairs of labelled ATPase-positive cells were found but individually labelled cells were not observed. The findings suggest that epidermal Langerhans cells form a very stable (labelling index less than 0.01%) self-replicating population which divides to maintain cell spacing during growth. No evidence was found for migration and interchange of Langerhans cells with the connective tissue, or for an origin of Langerhans cells by transformation of another cell type.     

Changes in cell cycle and chromatin distribution in C6 glioma cells treated by dibutyryl cyclic AMP

C6 glioma cells in culture were treated with 1 mM dibutyryl cyclic AMP (Db-cAMP) for 5, 8, 24 and 72 h. The cells were labelled with [3H]-thymidine before either the end, or the beginning, of the Db-cAMP treatment. The cell cycle passage was monitored by the simultaneous determination of DNA content and DNA synthesis in propidium iodide stained autoradiograms. The data revealed an early (t less than or equal to 3-8 h) and moderate inhibitory effect of Db-cAMP on all phases of the cell cycle except mitosis; some cells (2%) were completely blocked in the S phase. Later (8 less than t less than 24-72 h), the cycling of a substantial part of the population became inhibited in G1 phase. Microdensitometric texture analysis of Feulgen-stained nuclei, performed 24 h after administration of Db-cAMP, showed a higher inhomogeneity of the DNA distribution in cell nuclei, caused by the condensation of a part of the chromatin. This may reflect either changes in genome expression taking part in the process of cAMP induced differentiation or transit of some cells into quiescent G0 or S0 phases.     

Localization of ornithine decarboxylase and changes in polyamine content in root meristems of Zea mays

Polyamine content and the activity of arginine decarboxylase (EC 4.1.1.19) and ornithine decarboxylase (EC 4.1.1.17) were studied with respect to meristematic activity in primary roots and in developing lateral roots of Zea mays L. (cv. Neve Ya'ar 170) seedlings. Comparative localization of active ornithine decarboxylase and of meristematic activity were determined by labelling roots either with α-[5-¹⁴C]-difluoromethyl ornithine or with [³H]-thymidine, respectively.
Lateral roots were formed during the 72 h post-decapitation period, accompanied by an initial decline in putrescine content and by a significant increase in spennidine con-tent at 48–72 h. High levels of spermidine and lower levels of putrescine were found in the primary root apex as well. A marked increase in ornithine and arginine decarboxylase activity, as measured by ¹⁴CO₂ release, was found during the 72 h post-decapitation period of lateral root development. This increase in ornithine decarboxylase activity was confirmed also by a parallel rise in the incorporation of α-[5-¹⁴C]-difluoromethyl ornithine into trichloroacetic acid-insoluble fractions. Microautoradiographs of longitudinal and cross sections of roots, labelled with α-[5-¹⁴C]-difluoromethyl ornithine, showed that ornithine decarboxylase is localized mainly in the meristematic zones, as evidenced by [³H]-thymidine incorporation. A close correlation between meristematic activity and polyamines was demonstrated in situ , suggesting that polyamine content and biosynthesis may have a role in meristematic activity in corn roots.     

The production of ryegrass labelled with carbon-14

Summary The construction and operation of a growth chamber for producing plant material labelled with carbon-14 to a specified degree of uniformity is described.The specific activity of the plant material is measured by a method based on scintillation counting.     

Identification and differentiation of lens cells in tissue culture]

The cells of S-phase labelled prior to cultivation with H3-thymidine and other neighbouring cambial cells of the lens of the pig, cattle and sheep were found to form morphologically underdifferentiated zones of growth. The zones of growth were formed in the culture from differentiating in vivo cells of the lens. The cells of these zones occasionally resembled abortively differentiated lens fibres in vivo. The growth zones of the lens cells in vitro are comparable by its growings in trauma or cataract in vivo. In lens cultures under routine conditions of cultivation there occurs disturbance of normal embryonic histogenesis and abortive differentiation of the already differentiated in vivo cells.     

Quantitative 125-I-autoradiography of individual cells (author's transl)]

Iodine 125, an emitter of beta-radiation with an energy lying between that of tritium and carbon-14, is investigated for its applicability in quantitative autoradiography. Absorption and geometric factors of radiation are elucidated. From this, appropriate measuring conditions are derived. The simultaneous exposure of radioactive standard sources permits the evaluation of absolute amounts of radioactivity. Standard cells with labelled membranes are a suitable source of reference taking into account the physical properties of the isotope. Sheep red blood cells are examined for their suitability as standard cells after enzymatic radioiodination. The absolute number of antigenic substances on the surface of single cells is obtained by determining the specific activity of the labelled antibody molecules, and by measuring the silver grain densities of the cells under investigation and of the standard cells. The radioactivity per standard cell can be assessed by conventional procedures. The new method is applied to the quantification of membrane-bound immunoglobulin molecules of the IgG-type on single human lymphocytes. The determination of an immunologic saturation of the labelled antibody is essential for this purpose. On the lymphocytes of a normal person and of a patient with chronic lymphatic leukaemia quite different amounts of immunoglobulins have been evaluated.     

The position of chromosomes at metaphase in human fibroblasts and their DNA synthesis behaviour

The peripheral location of H³-thymidine labelled chromosomes in spreads of colcemid blocked metaphases from human fibroblasts has been investigated in several karyotypes. Chromosomes show non-random distribution in relation to the periphery of the plate. Longer chromosomes are significantly more peripheral than smaller chromosomes. Those chromosomes which contain large amounts of late-synthesising DNA (Y, 18 and 13) are significantly more peripheral than early synthesising chromosomes of similar length (21–22, 19–20 and 14–15). These locations are compared with those from peripheral leucocytes obtained by other workers. When location and grain number of homologues of nos. 1, 2, 3, 16 and late-X are compared it was found that in late-S labelled cells, the more peripheral of each pair was significantly more heavily labelled; in early-S labelled cells the reverse was the case. Location and patterns of labelling between late-X homologues of XXXXY cells showed that peripheral location was related to more delay in DNA synthesis. These results are discussed in terms of peripheral and central interphase distribution of condensed chromatin. It is concluded that the apparent asynchrony of replication of homologues is principally the result of different degrees of condensation of the chromatin in the late-S phase at the time of synthesis, and these differences are related to the proximity to the nuclear membrane.     

Mercapturic acid biosynthesis: the separate identities of glutathione-S-aryl chloride transferase and ligandin

To examine if, as has been suggested, a peculiar proteolytic activity of thymus cell lysates might explain failures to detect immunoglobulin (Ig) biosynthesis by thymus cells, lysates of ¹⁴C leucine labelled mouse myeloma cells were incubated with a 10³ excess of unlabelled mouse thymus or spleen cell lysates, and then submitted to immune precipitation to isolate labelled Ig chains. Analysis of the immune precipitates by SDS polyacrylamide gel electrophoresis followed by radioautography failed to provide evidence for the purported proteolytic activity of the thymus cell lysates. Furthermore, thymus cell suspensions uncontaminated by plasma cells were biosynthetically labelled, then lysed in the presence or absence of Trasylol, an inhibitor of trypsin-like protesses. No labelled Ig chains could be detected under either condition of cell lysis. Evidence is presented that the detection of Ig chains synthesized by thymus cell suspensions might result from the contamination of these suspensions by plasma cells.     

Kinetics of the calcium induced stratification of human keratinocytes in vitro

In a low concentration of calcium (0.1 mM), keratinocytes form a monolayer with about 30% of cells synthesizing involucrin. After addition of calcium to the culture medium to a concentration of 1.2 mM, the monolayer stratifies within 24 h, with a preferential migration of involucrin positive keratinocytes. In the present study, we tried to determine if keratinocytes control the decision to migrate at a distinct cell cycle point. A percentage labelled mitosis (PLM) curve was constructed for keratinocytes grown in low calcium medium and values for the length of the cell cycle (47 h), S phase duration (11 h) and G2+M period (6 h), were obtained. Monolayer cultures at 80% confluence were switched to high calcium concentration at various times (from 0 to 48 h), after pulse labelling with [3H]-thymidine. Based on the PLM data, the behaviour of cells known to be in S, G1 and G2 at the time of the migration stimulus were followed. No significant difference in the percentage of labelled suprabasal cells was found for any point of the cell cycle. For cells submitting to stratification, in S phase involucrin staining showed that about 60% of the [3H]-thymidine labelled cells were also involucrin negative. These results indicate that upward migration of keratinocytes in cultured epithelium can be triggered at all points in the cell cycle with equal probability and is not restricted to those cells that already contained involucrin.