Effect of Vinblastine On the Cell Cycle and Migration of Ameloblasts of Mouse Incisors As Shown By Autoradiography Using 3H-Thymidine

Abstract. 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 μg/g of body weight vinblastine intraperitoneally and 6 hr after these animals and those of a control group were injected with 1μCi/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 μ/hr respectively) as compared with the control (3.21 cell positions/hr and 18.88 μm/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.     

Hormone-Induced Alterations in Nonhistone Protein

Methylation and phosphorylation of chromosomal nonhistone protein (NHP) has been demonstrated in the salivary gland cells of diptera [5, 7] and implicated in the control of gene expression [35, 36]. Furthermore, hormones can stimulate methyl and phosphoryl side chain metabolism and thus enhance template activity. Salivary glands from late fourth instar female larvae of Sciara coprophila (cortisone-supplemented and normal diet) were incubated in ³H-uridine (10 μCi/ml), ³H-thymidine (10 μCi/ml), ³H-methyl-methionine (20 μCi/ml), ³⁵S-methionine (10 μCi/ml) and ³²P-orthophosphate (1 mc/ml), for varying time periods, to measure RNA synthesis, DNA synthesis, methylation, protein synthesis and phosphorylation, respectively. Following selective extraction of lipid, histone and nucleic acids, glands were prepared for light microscope autoradiography. A more specific labelling pattern, as well as increased grain number on particular bands, interbands and bulbs, was noted on chromosomes from cortisone-fed larvae incubated in ³H-methyl-methionine for 1 min when compared with larvae on the standard diet. Cortisone also increased RNA synthesis and nucleoprotein phosphorylation, but not DNA or protein synthesis. In summary, cortisone enhances the specificity and degree of NHP methylation and phosphorylation at discrete chromosomal loci, i.e. alterations in side chain metabolism which may be responsible for increased RNA synthesis.     

Double labelling of tissue combining tritiated thymidine autoradiography with immunodetection of bromodeoxyuridine: the autoradiographic significance of inhibition of thymidine incorporation into DNA by bromodeoxyuridine given simultaneously

Abstract We describe a reproducible method for combining tritiated thymidine ([³H]TdR) autoradiography with immunoperoxidase detection of bromodeoxyuridine (BrdU) in paraffin-embedded tissues. The technique has been used to examine, in mouse tongue epithelium, the inhibition of incorporation into DNA of [³H]TdR by a simultaneous injection of BrdU in the doses that both compounds are likely to be used in cell proliferation studies. The significance that this inhibition has on prolongation of autoradiograph exposure times, to ensure that all cells that incorporate [³H]TdR are scored as positive, in particular the most lightly labelled cells, has been quantified.
The inhibition of uptake into DNA of [³H]TdR from 0.23 to 1.85 MBq (6.25 to 50 μCi) per animal, produced by a simultaneous injection of 2.5 mg BrdU shows a linear, dose-dependent relationship. Provided the injected dose (in μCi per animal) multiplied by the autoradiographic exposure time (in days) is greater than a value of 700, then all cells that are labelled after incorporation of [³H]TdR alone are also labelled after simultaneous double labelling, despite the latter producing a lower average grain count.     

The Effect of Hydrocortisone on the Epidermal Proliferation in an Organ Culture of Chick Embryonic Skin

Hydrocortisone is regarded as an initiator of keratinization in embryonic skin. The present investigation dealt with the effect of hydrocortisone on the proliferation of epidermal cells during early development: Cell kinetic analyses using ³H-thymidine autoradiography were applied to a skin organ culture prepared from a 13-day chick embryo.
Hydrocortisone at a concentration between 0.01 and 1.0 μg/ml was effective in initiating a morphological change leading to the epidermal keratinization in vitro and caused a marked decrease in the mitotic and labeling indices of epidermal basal cells, the decrease being maximum at 2 days of culture previous to the morphological change.
During continuous labeling with ³H-thymidine, the number of labeled basal cells reached 100% within 2 days in the control and 4 days in the culture treated with hydrocortisone. This confirmed that the growth fraction of epidermal basal cells was 1.0 even after the administration of hydrocortisone.
The duration of each cell cycle phase at 2 days of culture was determined by percent labeled mitoses and double-labeling analyses. It was concluded that hydrocortisone extended the generation time of epidermal basal cells at this time point about three fold over the control. This extension was mainly due to the elongation of the G ₁ phase.     

Radioiodination of Enterotoxin from Clostridium perfringens Type A using Chloramine T

Procedures were examined for labelling enterotoxin isolated from Clostridium perfringens type A. with ¹²⁵I using chloramine T as the oxidizing agent. The iodination method was evaluated critically to establish the optimal conditions for the preparation of iodinated enterotoxin with a high specific radioactivity and without impairing the immunospecificity and biological activity. The use of 250 μg/ml of chloramine T in the reaction mixture. 500–1000 μCi of Na¹²⁵I/10 μg of enterotoxin and a reaction time of 40 s at pH 7–0 produced ¹²⁵I-enterotoxin of both high specific radioactivity and immunospecificity which retained its biological activity. No damage or aggregate formation due to the iodination process was observed. Enterotoxin labelled with high specific activity (135 μCi μg) showed extensive dissociation of ¹²⁵I when stored at 4°C and—20°C. In contrast, toxin labelled with low specific activity (7 μgCi/μg) was stable for as long as two months. The immunoreactivity of all labelled preparations was essentially unchanged after storage for one month.     

The Synthesis and Release of [3H-Tyrosine1]Methionine5-Enkephalin from Guinea Pig Brain Slices

Abstract: Stores of methionine-enkephalin were labelled on the N -terminal by incubation of whole brain slices with [³H]tyrosine (10 °Ci/ml). The ³H radioactivity corresponding to the position of authentic Met-enkephalin after extraction on Amberlite XAD₂ and separation by thin-layer chromatography was taken as an index of synthesis. Maximal incorporation of the labelled tyrosine into Met-enkephalin was attained after 4 h of incubation at 37°C and was inhibited in the presence of 10 μ M cycloheximide. Isolated nerve terminals failed to incorporate any [³H]tyrosine. The labelled compound had opiatelike activity and consisted of the same five amino acids as an authentic standard. Incubations with leucine aminopeptidase indicated that the labelled tyrosine was on the N -terminus and removal of this tyrosine resulted in loss of opiate-like activity. The incorporation of [¹⁴C]glycine, selected as an alternative precursor, was consistent with de novo synthesis and not N -terminal exchange. A radioimmunoassay was also used to quantify the amount of labelled Met-enkephalin. KCl (50 m M ) elicited a Ca²⁺-dependent release of the synthesised [³H]Met-enkephalin from whole brain slices and also from isolated nerve terminals. The release of Met-enkephalin radioimmunoactivity paralleled that of [³H]met-enkephalin. Preliminary investigations have suggested that carbamyl choline inhibited this release and its effect was partially reversed by atropine.     

Metabolic utilization of body stores during the early life of whitefish, Coregonus lavaretus L.

Patterns of oxygen consumption, ammonia and urea excretion were monitored during late embryogenesis, i.e. 5 days before mass hatching and 12 days during the free-swimming stage of whitefish larvae, Coregonus lavaretus. Oxygen consumption increased from 1.31 to 2.53 mgO₂ h⁻¹× 10³ eggs⁻¹ at hatching. Fasted, free-swimming larvae showed increasing oxygen consumption to the tenth day after hatching when it reached 5.52 mgO₂h⁻¹× 10³ larvae⁻¹. Ammonia and urea excretion increased during pre-hatching period from 52.1 to 163.2 and 26.8 to 51.4 μgh⁻¹× 10³ eggs⁻¹, respectively. The nitrogen excretion rate increased between the sixth and tenth day of fasting, i.e. for ammonia from 117.7 to 160.9 and for urea from 35.8 to 52.5 μg h⁻¹× 10³ larvae⁻¹. Cumulative data on nitrogen and energy metabolism indicated that during late embryogenesis, and up to the fifth day after hatching, protein dominated in the energy expenditure. During the free swimming stage, the ratio of fat to protein in energy expenditure rose from 0.86 to 1.99. Combined data for several fish species indicated high dependance of oxygen uptake during the hatching period on egg size and temperature.     

THE PROLIFERATION OF LYMPHOID CELLS IN GUINEA-PIG BONE MARROW

A double isotope DNA labelling method has been used to determine the duration of DNA synthesis (S) in bone marrow lymphoid cells classified by their nuclear diameters in smears. Incorporation of ³H-thymidine was confined almost entirely to marrow lymphoid cells of 8·0-15·0 μm nuclear diameter (large lymphoid cells). After exposure to ³H-thymidine in vivo and ¹⁴C-thymidine 40-104 min later in vitro , the proportion of cells labelled with ³H alone to those labelled with ¹⁴C(±³H) in radioautographic smears, plotted against time indicated the efflux from S per hour. Collectively, 28·3 ± 1·1% of all large lymphoid cells were in S and the efflux from S was 15·1% per hour. With decreasing cell size (nuclear diameter) the efflux fell progressively from 28·3% per hour (11·0 μm) to 9·2% per hour (8·0-8·9 μm) and the proportion of cells in S declined from 54·9 ± 2·3% to 14·8 ± 1·6%. Influx into S, measured in vitro by reversing the sequence of isotopes, closely resembled the corresponding efflux values in vivo relative to cell size. Most DNA synthesizing marrow large lymphoid cells belonged to a subgroup with deeply basophilic cytoplasm. The results demonstrate that basophilic large lymphoid cells in the marrow are actively proliferating and have a mean S phase duration of 6·6 hr. The largest marrow lymphoid cells (11·0 μm) proliferate most rapidly (S phase, 3·5 hr; maximum cell cycle time, 6·4 hr) while S duration is prolonged progressively to 10·9 hr for the smaller cells (8·0-8·9 μm).     

Amino acid incorporation by a natural population of Oscillatoria rubescens. A microautoradiographic study

Abstract The incorporation of a very low concentration (0.015 μM) of an [³H]amino acid mixture was measured for a natural population of Oscillatoria rubescens DC in samples from a eutrophic lake, Lake Nantua (France).
The kinetics of amino acid incorporation in the size fraction ⩾12 μ m showed that uptake was fast and that the maximum was reached after 4 h.
Microautoradiography demonstrated that Oscillatoria rubescens is able to utilize for protein synthesis an external pool of amino acids whose [³H] label becomes distributed generally throughout the cell.     

RNA Synthesis and Processing during Cytodifferentiation in Fetal Rat Pancreas

In fetal rat pancreas cytodifferentiation occurs between day 14 and day 20 of gestation and is accompanied by an exponential increase in the cellular accumulation of tissue specific proteins and an elaboration of the cellular organelles associated with their synthesis and secretion. Evaluation of RNA synthesis by [³H] uridine incorporation into trichloroacetic acid precipitable material showed that during this period the apparent rate of RNA synthesis increased 7.5 fold from 2 × 10³ dpm/μg DNA/h on day 15 to 1.5 × 10⁴ dpm/μg DNA/h on day 19; [³H] leucine uptake showed that the rate of protein synthesis increased about the same extent with the major difference being that the maximum rate of protein synthesis occurred on day 19, one day after the maximum rate of RNA synthesis. The soluble pyrimidine nucleotide pools decreased from 122 pmol/μg DNA on day 14 to 15 pmol/μg DNA on day 16 followed by an increase to 104 pmol/μg DNA on day 19; the purine nucleotide pools decreased from 367 pmol/μg DNA on day 14 to 286 pmol/μg DNA on day 16 and then increased to 635 pmol/μg DNA on day 19. These values roughly paralleled the transitions observed in the rates of RNA and protein synthesis. Agarose-acrylamide slab gel electrophoresis showed an increase in RNA synthesis and an increase in ribosomal RNA synthesis and processing with cytodifferentiation.     

Estimating bacterial mortality by the disappearance of 3H-labeled intracellular DNA

Abstract A method proposed for estimating the rate of bacterial mortality in aquatic environments consists in following the disappearance of radioactive tracer from the macromolecular fraction of ³H-thymidine labeled natural assemblages of bacteria. The data presented in this paper offer a further technical validation of this procedure. Application of the method to North and Mediterranean Sea, estuaries, rivers and lakes yields first order mortality constant in the range 0.008–0.06 h⁻¹. Mortality due to grazing by protozoans retained by 2 μm filtration range from 20 to 90% of the mortality detected by the method.     

Mechanism of Action of the Gonadal Steroids Producing Diminution of Growth of Staphylococcus Aureus

S ummary . Studies were undertaken to characterize the mechanism of action of the gonadal steroids responsible for decreasing growth of Staphylococcus aureus in vitro. Progesterone or testosterone at 20 μg/ml significantly increased the leakage of ¹⁴C activity from staphylococci pre-loaded with ¹⁴C-glucose. This enhancement of leakage was not detected with Gram negative micro-organisms. Hormonal diminution of total uptake of alanine was relatively independent of temperature and of the phase of culture. Anaerobiosis increased the steroidal diminution of alanine uptake c. 2-fold. Fraction-ation of staphylococci following exposure to various ¹⁴C-substrates in the presence of progesterone at 40 μg/ml did not reveal any distinctive influences on macromolecular syntheses. Entry of the labels into cellular pools, however, was altered for 8 of the 10 substrates tested. Exchange experiments detailed the effects of steroids on the efflux of internal alanine and lysine. With progesterone at 40 μg/ml, alanine effluxed from the internal pool 3 times as fast as from the corresponding controls. The opposite effect occurred with lysine and progesterone depressed its exit rate. The stepwise removal of cellular constituents indicated a preferential binding of hormones to cell wall components. Using ¹⁴C-progesterone or ¹⁴C-diethylstilbestrol, 24% and 29%, respectively, of the added hormone was firmly bound to mucopeptide preparations, compared to 1–5% bound to whole cells or isolated cell walls. We suggest that the hormones interfere with the integrated functioning of membrane-associated processes.     

Effects of Monensin on Assembly of Po Protein into Peripheral Nerve Myelin

Abstract: The ionophore monensin has been used in a variety of systems to block secretion of glycoproteins or assembly of glycoproteins into membranes. We examined the effects of monensin on assembly of the Po glycoprotein into PNS myelin, and compared this agent with the glycosylation inhibitor tunicamycin in our system. Sciatic nerves from 9-day-old rat pups were sliced and incubated in vitro . Electron microscopy of the Schwann cells in slices incubated with monensin revealed extensive swelling of the Golgi complex. Incubation with 10⁻⁷ M monensin inhibited total protein synthesis by about 20% and fucose incorporation into protein about 35%. Following isolation of myelin, proteins were separated by sodium dodecyl sulfate gel electrophoresis. Monensin inhibited the appearance of Po in myelin, while causing its accumulation in a denser membrane fraction. In addition, a slightly faster-migrating species of P_o labeled with both [³H]fucose and [¹⁴C]glycine was observed in all fractions. Assembly of basic proteins into myelin was not affected. Preincubation with 10 μg/ml tunicamycin for 30 min prior to incubation with [³H]fucose and [¹⁴C]glycine for 2 h resulted in a 65% decrease in [³H]fucose incorporation into P_o, and the appearance of a new [¹⁴C]glycine-labeled peak that migrated in the region of the 23K protein reported by Smith and Sternberger. [³H]Fucose incorporation was inhibited earlier, and to a greater extent, than protein synthesis. Our results show that processing of the P_o glycoprotein is sensitive to both monensin and tunicamycin, and that monensin partially blocks assembly of P_o into myelin.     

An investigation of the factors influencing mean cell volume in populations of the ciliate Colpidium campylum

Within the temperature range 10°C-20°C, temperature had no effect on the mean cell size of C. campylum. Population density also exerted no noticeable effect on mean cell volume. The quantity of energy consumed, however, had a marked effect. In experiments where less than 8000 μJ were consumed/individual/24 h, the mean cell volume decreased. Above this level of energy consumption mean cell volume maintained a constant level.
The maximum values obtained for cell sizes were 160–190 × 10³μm³ and the minimum values 40–100 × 10³μm³. A response to decreased food concentration and hence decreased energy consumption was obtained within the 24 h experimental period, indicating a rapid response to changed environment by the ciliates.     

Pathogenicity of live bacteria and extracellular products of motile Aeromonas isolated from eels

The pathogenic activities in vitro and in vivo of live bacteria and extracellular products (ECP) of 24 motile Aeromonas strains were investigated. Most Aer. hydrophila and Aer. jandaei isolates were pathogenic for eels (LD₅₀ 10^5·4-10^7·6 cfu fish^-1) but no Aer. sobria , Aer. caviae and Aer. allosaccharophila caused mortality in eels at doses of > 10^8·4 cfu fish^-1. Of these Aeromonas strains, Aer. hydrophila and Aer. jandaei in particular produced elastases and haemolysins against fish erythrocytes. ECP from Aer. hydrophila and Aer. jandaei caused degenerative changes in fish cell lines and were strongly toxic for eels (LD⁵⁰ 1·0–3·2 μg (g fish)^-1) reproducing the symptoms associated with natural disease. ECP from non-pathogenic species were inactive on fish cell lines as well as being poorly lethal for eels (LD⁵⁰ > 9·2 μg (g fish)^-1). All these biological activities of Aeromonas ECP were lost after heat treatment. These findings indicate differences between pathogenic and non-pathogenic Aeromonas species with respect to the expression of virulence factors, and show that elastases, haemolysins and exotoxins play a leading role in the pathogenicity of motile Aeromonas for eels.     

Bacterial production measured by leucine and thymidine incorporation rates in French lakes

1. Production of heterotrophic bacterioplankton was estimated monthly by the tritiated thymidine and leucine incorporation methods during the draining and filling of the mesotrophic Lake Pareloup (over a 2.5-years sampling program).
2. Rates of ³H-leucine (leu) and ³H-thymidine (thy _DNA) incorporation generally paralleled each other but the ratio of leu/thy _DNA incorporation rates was higher for the draining period (34.5 mean) than during and after filling (11.5 mean).
3. After draining, the highest ratios were observed during periods of low temperature and low bacterial specific activity, while DNA labeling by ³H-thymidine was reduced. However, bacterial production estimates obtained by ³H-leucine (BPL) and ³H-thymidine (BPT) incorporation methods were generally well correlated and the average BPL/BPT ratio was equal to 0.78.
4. In addition, both methods were applied during a diel cycle in three lakes of different trophic status. An increase of leu/thy _DNA incorporation rates was noted from the oligotrophic to the eutrophic system. In the absence of Cyanobacteria, BPL and BPT values were quite concordant on average.
5. In situations of unbalanced growth, BPL and BPT values can diverge but when considered over a sufficient period of time they were found to be in agreement.     

Effect of sulfolipid antibodies on the macromolecular synthesis of Mycobacterium smegmatis ATCC607

Abstract The biosynthesis of DNA, RNA, proteins and lipids in the presence of antiserum to sulfolipids was investigated by studying the incorporation of radiolabelled precursors like ³H-thymidine ¹⁴C-uracil, ¹⁴C-leucine and ¹⁴C-Acetate into their respective macromolecules. Antiserum to sulfolipids had a inhibitory effect on the biosynthesis of all these components. Antiserum also exhibited a growth inhibitory effects as compared to normal serum.     

Advances in Rabbit Embryo Culture during Organogenesis

Modified rodent embryo culture techniques were used to maintain 10 1/2 day rabbit embryos in vitro for up to 24 hr. In homologous, immediately centrifuged, heat inactivated serum containing 2 μCi/ml ³H-thymidine, observed status was consistently high at 12 hr but fell thereafter. Liquid scintillation counting and autoradiography revealed rapid uptake of radioactivity during the first 12 hr. The splanchnopleure of the inverted yolk-sac is only partially vascularised in the rabbit and is thus inadequate as a medium of metabolic exchange in extended culture. Nevertheless, this technique will enable a study of the morphology and biochemistry of teratology in vitro in a sensitive species.     

[3H]thymidine labels less than half of the DNA-synthesizing cells in the mouse tumour, carcinoma NT

Abstract. We have studied carcinoma NT, a transplantable mouse adenocarcinoma of spontaneous origin. Cells labelled with [³H]thymidine ([³H]TdR) were restricted to a narrow zone around the periphery of this tumour and were also found in rings up to 50 μ m wide, around isolated blood vessels in the central necrotic area. Labelling with [³H]deoxyuridine ([³H]UdR), another DNA synthesis precursor, produced a very different pattern. The labelled zone around the periphery was much wider than with [³H]TdR, and [³H]UdR labelled cells were found up to 110 μ m from isolated vessels. [³H]iododeoxyuridine ([³H]IUdR) gave the same pattern of labelling as [³H]UdR. In the heavily labelled zone, within 1 mm of the tumour periphery, the labelling index (LI) was 51% after [³H]UdR or [³H]IUdR injection, and only 36% with [³H]TdR.
The data show that at least half of the DNA-synthesizing cells in this tumour did not incorporate [³H]TdR. Previous workers reported cell loss factors for carcinoma NT of 60% calculated from [³H]TdR labelling data and 30% from the rate of loss of [¹²⁵I]UdR. The present work suggests that calculations based on [¹²⁵I]UdR data are more likely to be accurate for carcinoma NT than those using [³H]TdR data.     

Evidence for Chromosome Endoreduplication in Eudorina californica, a Colonial Alga

Several nuclear events seen during the cleavage period in Eudorina suggest that chromosome endoreduplication, proportional to the number of cells to be produced, may occur in the gonidia prior to cleavage. Presumably the DNA concentration is reduced to the haploid level during rapid, successive divisions of the cleavage period.
To test this hypothesis, I determined DNA content of gonidia as they grew from 4 μm to 38 μm in diameter between cleavage periods. During growth from 4 μm to 8 μm in diameter, the DNA concentration remained at the haploid level of 0.17 pg/cell. As gonidia in 64 cell colonies continued to grow from 8 μm to 33 μm in diameter, their DNA concentrations increased 60-fold.
Analysis of the Eudorina DNA by equilibrium centrifugation in CsCl showed only 2 bands with buoyant densities of 1.721 g/cm³ and 1.699 g/cm³, presumed to be nuclear and chloroplast, respectively, on the basis of labelling with ³H-thymidine and ³H-adenine. The 8: 2 ratio of the two bands did not change with increase in cell size and no other bands were detected, suggesting that both nuclear and chloroplast DNAs were synthesised proportionately prior to the cleavage period.