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1.
Abstract. About twice as much tritiated thymidine ([3 H]TdR) is taken up by cells at the bottom of the crypt of the small intestine as by the rapidly cycling mid-crypt cells. However, the uptake of tritiated deoxyuridine ([3 H]UdR) is even throughout the crypt.
Exogenous thymidine is incorporated about four times and eight times more efficiently than deoxyuridine by the cells in the mid-crypt and cells at the bottom of the crypt, respectively. However all S phase cells in the crypt appear to be capable of using either precursors, i.e. either the de novo or salvage pathway.
Since methotrexate (1 or 5 mg/kg) inhibits (at 5 mg/kg completely) the uptake of [3 H]UdR, but has no effect on [3 H]TdR uptake, the de novo and salvage pathways appear to be independent. Within the precision of the methods used in the experiments the 3 hr inhibition of the de novo pathway of deoxythymidylic acid (dTMP) synthesis by methotrexate does not produce any increase in utilization of the salvage pathway measured by incorporation of [3 H]TdR into DNA. the increased efficiency of thymidine utilization by crypt base cells is not attributable to (i) differences in accessibility of thymidine; (ii) differences in the rate of DNA synthesis or (iii) the size of the nuclei. 相似文献
Exogenous thymidine is incorporated about four times and eight times more efficiently than deoxyuridine by the cells in the mid-crypt and cells at the bottom of the crypt, respectively. However all S phase cells in the crypt appear to be capable of using either precursors, i.e. either the de novo or salvage pathway.
Since methotrexate (1 or 5 mg/kg) inhibits (at 5 mg/kg completely) the uptake of [
2.
Abstract The effect of cortisol on the proliferation of the rainbow trout fibroblast cell line, RTG-2, was examined in synchronous and asynchronous cultures. When the transition from G1 to S was synchronized by restoring serum to serum-deprived cultures, the addition of cortisol at the time of serum restoration delayed the entry of cells into S phase. However, if cortisol was added 24 h after serum restoration, at the G1 /S transition point, the subsequent peak of DNA synthesis was unaffected. In asynchronous cultures cortisol inhibited [3 H]-thymidine and [3 H]-uridine but not [3 H]-leucine incorporation into acid-insoluble material. If the exogenous nucleoside concentration was raised, [3 H]-thymidine but not [3 H]-uridine incorporation continued to be inhibited by cortisol. This suggested that cortisol's effect on [3 H]-thymidine incorporation reflected a change in entry into S phase and not just on thymidine uptake and metabolism. Cortisol inhibited the proliferation of RTG-2 in asynchronous cultures. At 1000 ng/ml of cortisol a reduction in cell number became apparent before the RTG-2 cultures were confluent, whereas at 100 ng/ml the reduction only became evident in confluent cultures. The synthetic antiglucocorticoid, RU 486, which acts at the level of the corticosteroid receptor, blocked the growth inhibition by cortisol. These results suggest that cortisol regulates rainbow trout fibroblast proliferation via the corticosteroid receptor and that the G1 /S transition is one point at which this regulation occurs. 相似文献
3.
Abstract. Cellular uptake of [3 H]thymidine ([3 H]TdR) and incorporation into DNA of Ehrlich ascites tumour cells were studied in relation to the cell cycle by measuring the activity in the acid-soluble and insoluble parts of the cell material. Cells were synchronized at various stages of the cell cycle using centrifugal elutriation. The degree of synchrony of the various cell fractions was measured by flow-cytofluorometric DNA analysis. From the cellular uptake, the TdR triphosphate (dTTP) concentration of a mean cell in an unseparated cell population was calculated to be 20 × 10-18 mol/cell. The pool activity of G1 cells was unmeasurable but rose to maximum values at the border of the G1 -S phase. It decreased again during G2 . The [3 H]TdR incorporation into DNA was low during early S phase, reached a maximum value at two-thirds of the S phase and decreased again during late S phase. These changes in DNA synthesis were not due to changes in the dTTP pool being a limiting factor. During maximum DNA synthesis, 10%× min-1 of the dTTP pool was utilized, at which time the pool size also decreased by about 30%. Changes in pool size during the cell cycle have to be taken into account when the results of incorporation of radioactive TdR into DNA are discussed. 相似文献
4.
Abstract. DNA synthesis in rat hepatocytes, from livers regenerating after 70% hepatectomy, was assessed by flow cytometric determination of nuclear DNA content and by incorporation of [3 H]thymidine. Parenchymal liver cells were isolated by collagenase perfusion and low-speed centrifugation. Nuclei from the isolated cells were prepared for flow cytometry by a treatment with detergent, pepsin and RNase, and stained with ethidium bromide. Parallel samples of cells were incubated with [3 H]thymidine and analysed for rate of incorporation of radioactivity into DNA and for labelling index determination.
The flow cytometric measure of the replicative response, i.e. the presence of cells with S-phase DNA content within the diploid and tetraploid cell populations, was compared with the incorporation of [3 H]thymidine. For each of fourteen animals, including two control rats and twelve partially hepatectomized animals killed either before (at 13 hr after hepatectomy), at the onset (16 and 18 hr) or at the peak (24 hr) of regenerating activity, a fairly good correlation was found between the different methods. Satisfactory resolution of the flow cytometric detection of S-phase cells was indicated by a sorting experiment using an Ortho (system 50-H) cell sorter which demonstrated that after [3 H]thymidine injection in vivo 88% of the diploid and 84% of the tetraploid S-phase nuclei were labelled, while labelling in the G1 -fractions was only 2 and 7%, respectively. 相似文献
The flow cytometric measure of the replicative response, i.e. the presence of cells with S-phase DNA content within the diploid and tetraploid cell populations, was compared with the incorporation of [
5.
G. HARRIS W. A. CRAMP I. OLSEN 《Differentiation; research in biological diversity》1981,20(1-3):92-96
Ionizing radiation inhibited the development of specific haemolysin-producing cells (PFC) and depressed the incorporation of (3 H) thymidine by rabbit spleen explants responding to SRC in the culture medium. In contrast to these effects, the rates of incorporation of precursors for protein and RNA synthesis were much less affected. The depression of (3 H) thymidine incorporation was found to result from a quantitative reduction of new DNA synthesis, without any change in the proportion of labelled cells, at any time after irradiation. The DNA synthesis occurring in these cells preparing to develop antibody-producing capacity was thus radio-sensitive, but the exact nature of the defect resulting from exposure to radiation requires further study. 相似文献
6.
Abstract. [3 H]Thymidine has been extensively used as a selective precursor to DNA in studies on the kinetics of cell proliferation. We have become interested in measuring early inhibition of the DNA synthesis in various organs of intact animals for detecting genotoxic properties of chemicals. Such experiments should, for convenience and to achieve a large capacity, be performed in the simplest way possible.
The present paper deals with some practical aspects on the use of [3 H]thymidine in vivo. [6-3 H]Thymidine was injected intraperitoneally in mice and the uptake of radioactivity was evaluated by using whole-body autoradiography and liquid scintillation spectrometry. Autoradiograms of sections washed with trichloroacetic acid and methanol Were compared with those subjected only to freeze-drying. Liquid scintillation counting was performed of total, non-volatile, acid-insoluble and dNA-associated radioactivities. A rapid increase of the [3 H]thymidine incorporation was seen during the first hour after the injection. Further prolongation of the survival time did not result in any significant increase of the incorporated radioactivity. Moreover, there were only slight differences between the autoradiograms from extracted and non-extracted sections. Radioactivities asśociated with DNA closely eorrelated to those representing acid-insoluble material, indicating that acid-insoluble radioactivity provides a good estimate of the [3 H]thymidine incorporation into DNA. 相似文献
The present paper deals with some practical aspects on the use of [
7.
B. F. Roth-Schechter M. Winterith G. Tholey A. Dierich P. Mandel 《Journal of neurochemistry》1979,33(3):669-676
Abstract— Primary cultures of glial cells prepared from brains of newborn rats were grown for 1 week and then exposed to 5 × 10−4 m -pentobarbital (PB) for 4 weeks. Compared with glial cells grown in drug-free medium throughout, exposure to PB significantly increased hexokinase activity, primarily in its mitochondrial form. Furthermore, cellular protein and RNA concentrations were significantly higher in barbiturate-cultivated than in control cells. Pulse labelling with [3 H]thymidine after 4 weeks of PB exposure resulted in a significant increase (332%) in 3 H incorporation into mitochondrial DNA while 3 H incorporation into nuclear DNA was reduced by 58%. In addition, there was a time dependent increase in the size and number of mitochondria as determined in electron micrographs. These results are interpreted to reflect an increased mitochondrial metabolism in glial cells after chronic exposure to the barbiturate and may constitute a compensatory mechanism to the depressant action of this drug. 相似文献
8.
Abstract. A circadian dependent delay in the incorporation of [3 H]TdR into DNA, presumably due to variations in the intracellular pool of [3 H]TdR derivatives, was found. It seems reasonable to relate this effect to a circadianally varying age distribution of cells in S phase.
At any given time the S phase cells showed large variations in DNA synthesis rate, but it was still possible to identify a mean diurnal variation in the DNA synthesis rate.
Differences in the ability of S phase cells to incorporate [3 H]TdR are also discussed in relation to flow cytometrical measurements, and this contributes to the understanding of the commonly observed phenomenon that flow cytometry estimates of S-fractions are higher than those obtained with autoradiography. 相似文献
At any given time the S phase cells showed large variations in DNA synthesis rate, but it was still possible to identify a mean diurnal variation in the DNA synthesis rate.
Differences in the ability of S phase cells to incorporate [
9.
K. MIYAHARA K. SHIOKAWA K. YAMANA 《Differentiation; research in biological diversity》1982,21(1-3):45-49
Xenopus embryos were dissociated into cells and cultured in Ca2+ -free medium to study the relationship between the cell-to-cell interaction and macromolecular synthesis. Under the conditions, cells did not aggregate at all, and remained isolated even while they were dividing actively. Synthesis of DNA and protein as studied by the incorporation of (3 H)thymidine and (3 H)leucine proceeded as in the aggregating cells. Also, the activity to synthesize rRNA, 5S RNA, and heterogeneous RNA as determined by the incorporation of (3 H)uridine was not impaired. Such an increase in the activity of alkaline phosphatase, as occurs in embryos after the gastrula stage, was found to be inhibited greatly when early-blastula cells were cultured in the non-aggregating conditions. However, we found here that the inhibition was not observed with cells isolated from late-blastulae. Therefore, it appears that the increase in the activity of alkaline phosphatase during post-gastrular stages is dependent on some cellular commitment which may be established by cell-to-cell contact during the blastula stage. 相似文献
10.
SYNOPSIS. DNA of Naegleria gruberi strain NEG, grown in axenic culture, forms a band at a density of 1.6912 in CsCl gradient and has a GC content of 31.8%. Incorporation of [3 H]thymidine into DNA is much reduced in differentiating Naegleria immediately after the stimulation to transforms, primarily because of the reduction in thymidine uptake by differentiating cells. In addition, there is a marked decrease in the rate of incorporation of [3 H]thymidine and [3 H]uracil into DNA at from 45 to 60 min after the stimulation for differentiation. This decrease in the rate of precursor incorporation into DNA appears to be due to the differentiation-dependent cessation of nuclear DNA synthesis. The differentiated phenotype (the flagellate) emerges at ∼ 70 min after the stimulation, and over 90% of the population differentiates within the next 30 min. Synthesis of mitochondrial DNA is detectable until 190 min after the stimulation. Since the S phase of Naegleria lasts ∼ 180 min, some cells in the population must cease synthesizing nuclear DNA in the middle of the S phase. 相似文献
11.
SYNOPSIS. Experiments were designed to investigate the effects of insect juvenile hormone (JH) on the over-all growth and macromolecular synthesis of Crithidia sp. in vitro. Cells grown in the presence of 10−5 M-10−3 M JH showed a concentration-dependent inhibition of growth, which appeared to result from both a prolongation of generation time and a delay in the onset of logarithmic growth. Juvenile hormone (10−3 M) inhibited the incorporation of [3 H]thymidine, [3 H]uridine and [3 H] leucine into logarithmically growing cells by 50, 70 and 40% respectively. The incorporation of [3 H]uridine into acid insoluble material could be stopped within 1 hr of application of the hormone (10−3 M). The inhibitory effect was reversible in terms of cell numbers in subcultures of washed cells but an examination of the reversibility of RNA synthesis inhibition suggested that the resumption of RNA synthesis at an optimal level would require a lag period of at least 1–3 hr. It is suggested that JH may act by interfering with RNA synthesis either directly or indirectly by primarily acting at the level of the plasma membrane. 相似文献
12.
Reynold Spector 《Journal of neurochemistry》1981,36(6):2019-2024
Abstract: The development of the thymidine phosphorylating systems was studied in various regions of brain. Brain slices from cerebellum, brain stem, and forebrain of rabbits 2, 7, 14, 30, 90, 500, and 2500 days of age were incubated for various times in artificial CSF containing 3 nM-[3 H]thymidine at 37°C under 95% O2 -5% CO2 . When slices from all brain regions of 2-day-old rabbits were incubated in [3 H]thymidine for 30 min, tissue-to-medium ratios of 3 H were between 2 and 4 and declined with age, and the percentages of the total 3 H in perchloric acid homogenates of brain slices as [3 H]DNA were 26–29%, declining to low levels with age. However, at all ages and in all regions studied, 41 -88% of the 3 H within the slices was phosphorylated. After homogenization and subcellular fractionation of the brain slices incubated in [3 H]thymidine for 30 min, the highest percentage of [3 H]thymidine phosphates plus [3 H]DNA was present in the nuclear (crude and purified) and mitochondrial fractions of all brain regions. The [3 H]DNA content in the nuclear and mitochondrial fractions declined with age, but the percentage of [3 H]thymidine phosphates did not. Thymidine phosphates were synthesized from thymidine in all brain regions tested throughout the entire life span. 相似文献
13.
Jacob Barg† Mariana Mancheva Belcheva Jan Rowiski Carmine James Coscia 《Journal of neurochemistry》1993,60(4):1505-1511
Abstract: A body of evidence has indicated that μ-opioid agonists can inhibit DNA synthesis in developing brain. We now report that K -selective opioid agonists (U69593 and U50488) modulate [3 H]thymidine incorporation into DNA in fetal rat brain cell aggregates in a dose- and developmental stage-dependent manner. K agonists decreased thymidine incorporation by 35% in cultures grown for 7 days, and this process was reversed by the K -selective antagonist, norbinaltorphimine, whereas in 21-day brain cell aggregates a 3,5-fold increase was evident. Cell labeling by [3 H]thymidine was also inhibited by the K -opioid agonist as shown by autoradiography. In addition, U69593 reduced basal rates of phosphoinositide formation in 7-day cultures and elevated it in 21-day cultures. Control levels were restored by norbin-altorphimine. Pertussis toxin blocked U69593-mediated inhibition of DNA synthesis. The action of K agonists on thymidine incorporation in the presence of chelerythrine, a protein kinase C (PKC) inhibitor, or in combination with LiCl, a noncompetitive inhibitor of inositol phosphatase, was attenuated in both 7- and 21-day cultures. These results suggest that K agonists may inhibit DNA synthesis via the phosphoinositide system with a pertussis toxin-sensitive G protein as transducer. In mixed glial cell aggregates, U50488 increased thymidine incorporation into DNA 3.1-fold, and this stimulation was reversed by the opioid antagonist naltrexone. 相似文献
14.
Metabolism of Deoxyuridine in Rabbit Brain 总被引:1,自引:2,他引:1
Abstract: The metabolism of [3 H]deoxyuridine by rabbit brain was investigated in vitro and in vivo . In vitro , brain slices from various regions of brain and from all age groups accumulated [3 H]deoxyuridine from artificial CSF. Within the slices, a portion of the accumulated [3 H]deoxyuridine was metabolized to [3 H]deoxyuridine phosphate, with subsequent conversion to [3 H]thymidine phosphate, and ultimately [3 H]DNA. The percentage of the [3 H]deoxyuridine phosphorylated and subsequently converted into [3 H]DNA was highest at birth and declined to adult levels in 3-month-old rabbits. Thymidine, when added to the incubation medium with the [3 H]deoxyuridine, was approximately 10 times as potent as unlabeled deoxyuridine in inhibiting the intracellular phosphorylation and conversion of [3 H]deoxyuridine to [3 H]thymidine phosphate in brain slices. In vivo , 2.5 h after intraventricular injection of [3 H]deoxyuridine, over 90% of the [3 H]deoxyuridine was cleared from the central nervous system at all ages. However, in both newborn and 3-month-old rabbits, approximately 40 and 12%, respectively, of the 3 H remaining in brain was phosphorylated and converted to [3 H]thymidine phosphates; and 11 and 4%, respectively, of the 3 H remaining in brain was converted to [3 H]DNA. These results show that both immature and mature rabbit brain is able to incorporate deoxyuridine into DNA. Thus, all the enzymes involved in this conversion, including thymidylate synthetase (EC 2.1.1.45), are present and active in brain throughout life. 相似文献
15.
Abstract We describe a reproducible method for combining tritiated thymidine ([3 H]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 [3 H]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 [3 H]TdR are scored as positive, in particular the most lightly labelled cells, has been quantified.
The inhibition of uptake into DNA of [3 H]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 [3 H]TdR alone are also labelled after simultaneous double labelling, despite the latter producing a lower average grain count. 相似文献
The inhibition of uptake into DNA of [
16.
Abstract— Intracerebral injections of radioactive fucose into developing rats resulted in specific labelling of the brain glycoproteins in their fucose moieties. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate revealed that the radioactive glycoproteins were very heterogeneous with regard to molecular weight. A procedure utilizing [3 H]fucose and [14 C]fucose together with double-label counting techniques was developed for comparing the electrophoretic patterns of newly synthesized glycoproteins from different samples of tissue. By the use of this procedure we showed that the incorporation of radioactive fucose into the glycoproteins of high mol. wt. was relatively greater in the brains of 5-day-old rats than in those of 25-day-old rats. Intracerebral injection of N -[ Ac -3 H]acetyl- d -mannosamine resulted in a high degree of specificity for the labelling of sialic acid moieties in glycoproteins and gangliosides. The ratio of the d.p.m. of N -[3 H]acetylmannosamine incorporated into glycoproteins to the d.p.m. incorporated into gangliosides was higher in 5-day-old rats than in 15- or 25-day-old rats. Experiments in which 15-day-old rats were injected with a mixture of [14 C]fucose and N -[3 H]acetylmannosamine showed that there were differences in the relative degrees of incorporation of the two radioactive precursors into the various glycoproteins. The greatest incorporation of [14 C]fucose relative to that of N- [3 H]acetylmannosamine occurred in some of the glycoproteins of smaller mol. wt. 相似文献
17.
Abstract Incorporation of [3 H]methyl thymidine into bacterial DNA was measured using samples of bacterioplankton from Lake Constance and pure cultures of CO, H2 and CH4 -oxidizing bacteria. Thymidine was incorporated by Pseudomonas carboxydovorans, Paracoccus denitrificans, Methylosinus trichosporium, Methylomonas agile , and by various chemolithotropic or methylotrophic isolates from Lake Constance. Thymidine incorporation by bacterial cultures was stimulated by increasing concentrations of CO or H2 . Increased CH4 concentrations stimulated thymidine incorporation by Ms. trichosporium only if the cells had been starved. In contrast to bacterial cultures, thymidine incorporation by bacterioplankton samples was not stimulated by increasing 相似文献
18.
J. Martinez M. Riera † J. Lalucat † J. Vives-Rego ‡ 《Letters in applied microbiology》1989,8(4):135-138
The incorporation of tritiated ([3 H]-) thymidine into DNA by axenic laboratory cultures of a common marine blue-green algae and two marine eukaryotic algae was measured. Tritiated thymidine was incorporated into the cold TCA fraction of the cyanobacterium and eukaryote algae. However, only in the culture of cyanobacterium was the thymidine consistently incorporated into DNA. Considering the usual algal densities in natural habitats, thymidine concentrations and incubation times, our data do not preclude the use of thymidine incorporation in bacterial production studies in marine environments. 相似文献
19.
Fatty acid composition and microbial activity of benthic marine sediment from McMurdo Sound, Antarctica 总被引:1,自引:0,他引:1
Abstract Signature lipids from the phospholipid esterlinked fatty acids (PELFA) of cell membranes were used to describe benthic microbial communities of 4 Antarctic sediments. Metabolic activities of the communities were determined by incorporation of [3 H]thymidine into bacterial DNA and sodium [14 C]acetate into membrane lipids. Biomass measurements from extractable phospholipid fatty acids per g dry wt. ranged between 6 to 76 nmol, or when converted to number of bacteria, 3.7 × 108 to 4.5 × 109 cells per g dry wt. The West Sound site at New Harbor contained the lowest biomass, while Cape Evans on the East Sound contained the greatest. A marked difference was also noted between sites in their sediment microbial community structure. The East Sound sites at Cape Armitage and Cape Evans contained a greater abundance of diatom marker lipids, whilst both sides of the Sound contained approximately the same relative amounts of bacterial groups distinguished using PELFA. Activity of sediment microorganisms measured by radiolabel incorporation under ambient conditions followed the trends of the biomass measurements. The East Sound sites were more active by an average of 45–73% for [3 H]thymidine and possibly also for sodium [14 C]acetate. 相似文献
20.
Emmanuel Sivvas Georgia Voukelatou Dionissios Papaioannou Alexios J. Aletras Elias D. Kouvelas 《Journal of neurochemistry》1994,63(4):1544-1550
Abstract: The synthesis of (2 S ,3 S ,4 S )-4-[1-(4-azidobenzamidomethyl)ethenyl]-2-carboxy-3-pyrrolidineacetic acid (ABCPA) is described. This novel kainic acid analogue, bearing a photolabile functionality on the isopropenyl side chain, was proven to be a good inhibitor of [3 H]CNQX and [3 H]kainic acid binding on chick cerebellar membranes. [3 H]ABCPA was photoaffinity cross-linked on the membrane fraction of chick cerebellum. Electrophoretic analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two major radioactive bands with apparent molecular masses of 45 and 33.5 kDa. [3 H]ABCPA incorporation in both bands was completely blocked by 2 m M CNQX. When photoaffinity labeling was performed in the presence of 2 m M kainic acid, incorporation of [3 H]ABCPA was blocked by ∼70% in the 45-kDa band and by 18% in the 33.5-kDa band. Incorporation of radioactivity in both bands was blocked by ∼30% with 10 m M glutamate. 相似文献