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1.
The objective was to determine the effect of adrenocorticotropin (ACTH 1-17) on the incorporation of [ 3H]TdR into DNA (DNA synthesis) in the duodenum, colon and rectum of CD2F, mice standardised to 12hr of light alternating with 12hr of darkness. A question asked was whether the difference in times of administration along the 24 -hr time scale influenced any response found. The response was complex as ACTH 1-17 was capable of bringing about statistically significant increases in the incorporation of [ 3H]TdR into DNA at certain times, decreases at other times, or no response at still another time. A generalization that can be made from all these tissues is that ACTH 1-17 had a greater influence in bringing about a decrease in DNA synthesis when it was administered around the time of transition from dark to light. A similar finding was made earlier for the ACTH 1–17 effect upon the tongue, esophagus and stomach.A 2- and 3-way analysis of variance supports our conclusion that the kind-of-treatment, time-of-treatment and the interval-to-kill (Sampling time) as well as their interactions are important factors when determining any response of ACTH 1-17 or placebo. 相似文献
2.
The objective was to determine the effect of adrenocorticotropin (ACTH 1-17) on the incorporation of [3H]TdR into DNA (DNA synthesis) in the tongue, esophagus and stomach of CD2F1 mice standardized to 12 hours of light alternating with 12 hours of darkness. A question asked was whether the time of administration along the 24-hour time scale influenced any response found. The response was complex as ACTH 1-17 was capable of bringing about statistically significant increases in the incorporation of [3H]TdR into DNA at certain times, decreases at other times, or no response at still another time. In general the most marked effects of 20 IU/kg of ACTH 1-17 when compared to controls, was to decrease DNA synthesis of as much as 60% 4 hours after administration at the end of the dark or beginning of the light span. A 2- and 3-way analysis of variance supported the conclusion that the kind-of-treatment, time-of-treatment and the interval-to-kill (Sampling time) as well as their interactions are important factors when determining any response of ACTH 1-17 or placebo. 相似文献
3.
Abstract. The central zone of the rat lens epithelium, extending half way from the centre to the periphery of a whole mount preparation, normally has less than 1% of the cells in the cell cycle at any given time. Mechanical wounding initiates a burst of proliferation in the central zone. DNA synthesis begins 14 hr after wounding followed by mitosis 10 hr later. When [ 3H]TdR was applied at 2 hr prior to S phase, some moderately heavy and some light labelling was observed after the onset of S phase. When [ 3H]TdR was applied 5 hr before S phase (9 hr after wounding), all the cells were lightly labelled. Only small amounts of the label were available to these cells 5 hr after application. It is significant that there was labelling in this group because it indicates the persistence of relatively small intracellular pools of [ 3H]TdR for several hours after the initial 'pulse' labelling of cells. Determinations of the duration of S phase were based on the assumption that pulse labelling may be affected by the persistence of the pools of [ 3H]TdR and consequent light labelling of the cells. 相似文献
4.
We measured the toxicity and mutagenicity induced in human diploid lymphoblasts by various radiation doses of X-rays and two internal emitters. [ 125I]iododeoxyuridine ([ 125I]dUrd) and [ 3H]thymidine ([ 3H]TdR), incorporated into cellular DNA. [ 125I]dUrd was more effective than [ 3H]TdR at killing cells and producing mutations to 6-thioguanine resistance (6TG R). No ouabain-resistant mutants were induced by any of these agents. Expressing dose as total disintegrations per cell (dpc), the D0 for cell killing for [ 125I]dUrd was 28 dpc and for [ 3H]TdR was 385 dpc. The D0 for X-rays was 48 rad at 37°C. The slopes of the mutation curves were approximately 75 × 10 −8 6TG R mutants per cell per disintegration for [ 125I]dUrd and 2 × 10 −8 for [ 3H]TdR. X-Rays induced 8 × 10 −8 6TG R mutants per cell per rad. Normalizing for survival, [ 125I]dUrd remained much more mutagenic at low doses (high survival levels) than the other two agents. Treatment of the cells at either 37°C or while frozen at −70°C yielded no difference in cytotoxicity or mutation for [ 125I]dUrd or [ 3H]TdR, whereas X-rays were 6 times less effective in killing cells at −70°C. Assuming that incorporation was random throughout the genome, the mutagenic efficiencies of the radionuclides could be calculated by dividing the mutation rate by the level of incorporation. If the effective target size of the 6TGR locus is 1000–3000 base pairs, then the mutagenic efficiency of [125I]dUrd is 1.0–3.0 and of [3H]TdR is 0.02–0.06 total genomic mutations per cell per disintegration. 125I disintegrations are known to produce localized DNA double-strand breaks. If these breaks are potentially lethal lesions, they must be repaired, since the mean lethal dose (D0) was 28 dpc. The observations that a single dpc has a high probability of producing a mutation (mutagenic efficiency 1.0–3.0) would suggest, however, that this repair is extremely error-prone. If the breaks need not be repaired to permit survival, then lethal lesions are a subset of or are completely different from mutagenic lesions. 相似文献
5.
Abstract. A circadian dependent delay in the incorporation of [ 3H]TdR into DNA, presumably due to variations in the intracellular pool of [ 3H]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 [ 3H]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
6.
The effect of the deacetylated (amine) metabolite of diamphenethide (DAMD, 10 μg ml −1) on the uptake and incorporation by adult Fasciola hepatica of radioactively labelled precursors of DNA, RNA and protein synthesis ([ 3H]thymidine, [ 3H]uridine and [ 3H] leucine, respectively) was measured by liquid scintillation counting. Comparison was made between the effects of DAMD and those of specific inhibitors of DNA, RNA and protein synthesis, namely, 5-fluorouracil, cordycepin and cycloheximide, respectively. DAMD caused a significant decrease in the overall uptake and incorporation of [ 3H]uridine by F. hepatica, decreased the incorporation of [ 3H] leucine and also caused a significant decrease in the overall protein content of the flukes. The effect of DAMD was similar to that of cycloheximide (1 × 10 −3M), a potent inhibitor of protein synthesis, which also caused a significant decrease in the incorporation of [ 3H] leucine by the fluke and a decrease in the overall protein content of the fluke. Cordycepin (100 μg ml −1) caused a significant decrease in the protein content of the fluke, but had no effect on the uptake or incorporation of [ 3H]uridine. 5-Fluorouracil (1 × 10 −4
) did not affect the uptake or incorporation of [ 3H]thymidine, nor did it decrease the protein content of the fluke. The results indicate that DAMD inhibits protein synthesis by F. hepatica, possibly by inhibiting RNA synthesis. The results are also consistent with previous morphological investigations involving DAMD. 相似文献
7.
Abstract. About twice as much tritiated thymidine ([ 3H]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 ([ 3H]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 [ 3H]UdR, but has no effect on [ 3H]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 [ 3H]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
8.
Abstract. Cellular uptake of [ 3H]thymidine ([ 3H]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 G 1 cells was unmeasurable but rose to maximum values at the border of the G 1-S phase. It decreased again during G 2. The [ 3H]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
9.
Abstract. A method is described for the isolation of enriched populations of crypt cells from the murine small intestine. The method was developed to study the response of cells to various stimuli in vitro . The properties of the isolated cell preparations varied with the state of the intestinal mucosa of the mice from which they were isolated. Thus we could distinguish between cells from lactating and non-lactating mice. Polyamines, which are putative modulators of crypt cell division, failed to stimulate [ 3H]TdR incorporation in vitro . Lymphocyte culture supernatants suppressed [ 3H]TdR incorporation at dilutions of 1:4 to 1:64. Supernatants of 12- O -tetradecanoylphorbol-13-acetate-stimulated EL-4 cells and of mixed lymphocyte cultures failed to stimulate [ 3H]TdR incorporation of any dilution. Supernatants of concanavalin A-stimulated spleen cells gave less suppression of [ 3H]TdR incorporation than those of unstimulated spleen cells and stimulated incorporation at dilutions of 1:64 and 1:128. Phytohaemagglutinin stimulated [ 3H]TdR incorporation at high concentrations, whereas concanavalin A (con A) had no effect. This study shows that the isolated murine crypt cells may have the potential to provide a useful in vitro model for crypt cell responses to stimuli. 相似文献
10.
The importance of administration time along the 24-h scale is shown for a potent corticosteroidogenic adrenocorticotropin analogue, ACTH 1-17 (Synchrodyn 1-17). This molecule affects the incorporation of [3H]TdR into DNA (DNA synthesis) in the thymus, bone marrow and spleen, and total RNA and DNA of spleen in CD2F1 mice, standardized in light alternating with darkness at 12-h intervals. As a function of timing, the same dose of ACTH 1-17 at one time increases, at another time decreases (in each case with statistical significance) and at still another time elicits no response in DNA synthesis or in total RNA and DNA of spleen. Effects upon DNA synthesis are recorded with doses of 0.02 IU/kg body weight. The most marked effect with 20 IU/kg body weight is a decrease of DNA synthesis seen (4 h) after administration of ACTH 1-17 late in the dark span and early in the light span. The effect of ACTH 1-17 on the thymus is more prominent than that on bone marrow and spleen. Time-dependence also characterizes placebo effects by comparison to values in untreated controls. At the cellular level responses to ACTH 1-17 or placebo are characterized by critical interactions of treatment kind with treatment timing as well as interval-to-kill-time. The study documents the need to time-specify, in several ways, responses to ACTH 1-17 and suggests more broadly that 'increases' and 'decreases' may have to be complemented by changes in endpoints of rhythms in all those endocrine studies that involve rhythmic variables and rhythm-dependent effects upon these variables. 相似文献
11.
The enzyme activity of dephosphorylation of thymidine triphosphate was found in microsomal fraction of rat liver. The enzyme activity decreased at the time when [ 3H]thymidine incorporation into DNA of regenerating liver increased. When the [ 3H]thymidine incorporation was suppressed by 1,3-diaminopropane, the enzyme activity remained elevated. These results suggest that the enzyme activity appears to be closely linked to DNA synthesis. 相似文献
12.
Abstract. The proportion of haematopoietic stem cells (CFU-s) engaged in DNA synthesis was determined by means of the [ 3H]-thymidine ([ 3H]TdR) suicide technique during recovery of bone marrow from the damage caused by a sublethal total body irradiation. In contrast with previous reports the [ 3H]TdR suicide rate was not permanently increased. It was observed that CFU-s passed through S phase in synchronous waves, following a dose of irradiation of 1.5 Gy. After a dose of 2.6 Gy, there was only one initial wave of increased CFU-s sensitivity to the action of [ 3H]TdR. Following the depression occurring 26 hr after the irradiation with 2.6 Gy, the proportion of CFU-s killed by the [ 3H]TdR was permanently increased until 5-6 days after irradiation. Thereafter large differences in the [ 3H]TdR suicide data were observed among individual mice. Evidence was obtained that individual mice, which had been irradiated by a dose of 2.6 Gy 8-9 days before, had identical values of the CFU-s [ 3H]TdR suicide rate in the bone marrow from different bones of the lower extremities. the recurrence of the synchronous waves in CFU-s passage through the cell cycle was recorded when the CFU-s population regenerated to only about 10% of its normal value. These waves were obviously not related to a particular time of the day and, consequently, they did not represent the circadian rhythm. It is concluded that the synchronous waves in which CFU-s proliferation occurred reflected the action of the control mechanism on CFU-s proliferation. This mechanism should be endowed with an important systemic component besides locally operating factors. 相似文献
13.
Abstract. The present experiments with [ 14C]-thymidine (TdR) and [ 3H]-bromo-deoxyuridine (BrdU) using mouse jejunal crypt cells show that the upper limit of the tracer dose of TdR is about 0.5 µg g body weight -1 and that of BrdU is about 5·0 µg g body weight -1. Applying these doses, the proportions of the endogenous DNA synthesis attributed to the exogenous DNA precursor are 2% and 9% respectively. For [ 3H]-TdR doses commonly used in cell kinetic studies this proportion is only 0-1-1.0%, a negligible quantity that does not influence the endogenous DNA synthesis. The maximum availability time of tracer doses of TdR as well as BrdU is 40 to 60 min, the majority of the precursors being incorporated after 20 min. The availability time is the same for TdR doses exceeding the tracer dose by a factor of 80, whereas it is prolonged in the case of BrdU doses exceeding the tracer dose by a factor of 50. BrdU is suitable to replace radioactively labelled TdR in short term cell kinetic studies, i.e. determination of the labelling index or of the S phase duration by double labelling. However, more studies are needed to elucidate how far BrdU can replace TdR in long term studies as shown by differences between the fraction of labelled mitoses (FLM) curves of a human renal cell carcinoma measured with BrdU and [ 3H]-TdR. 相似文献
14.
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. 相似文献
15.
Abstract. Continuous exposure of chicken embryo limb bud mesenchyme cells undergoing chondrogenesis in vitro to [ 3H] thymidine ([ 3H]TdR) revealed that more than 90% of the cells synthesized DNA at least once during 120 h of culture. When cells were exposed to [ 3H]TdR for 24 h beginning at various times throughout the culture period, the percentage of cells which incorporated [ 3H]TdR during each period was approximately 92%. However, when the period for incorporation of radioisotope was limited to two hours, the number of cells which incorporated [ 3H]TdR was found to decline during chondrogenesis in vitro. This decline was coincident with the appearance of extracellular matrix material and occurred in those cells which had, and had not, expressed the cartilage phenotype. We conclude from these studies that (1) practically all of the cells continue to proliferate while chondrogenesis is occurring in vitro, (2) there is an increase in the length of the cell cycle during chondrogenesis in vitro, and (3) withdrawal from the cell cycle is not required for differentiation of mesenchyme into cartilage. 相似文献
16.
Abstract. The labelling index (LI) of the individual basal cell positions of the anterior column of mouse tongue filiform papillae was assessed with time after an injection of [ 3H]TdR at 12.00 hours (the minimum point in the circadian LI rhythm). An initial doubling of the LI in the stem cell zone due to cell division was followed by a second rise of 14–16% 16 hr after injection and this occurred even in the presence of vincristine. Although the uptake of [ 3H]TdR and the initial LI doubling were largely prevented by a preceding injection of hydroxyurea, the 14–16% LI rise was still observed. The possible explanations are discussed, the favoured one being that an average of one of the six or seven cells (the stem cell) in each stem cell zone can store [ 3H]TdR in a long-lived precursor pool for at least 16 hr before being utilized for DNA synthesis. This complements previously published work which suggested that one cell in each stem cell zone may selectively segregate DNA at mitosis. 相似文献
17.
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 [ 3H]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 [ 3H]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 [ 3H]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 [ 3H]thymidine injection in vivo 88% of the diploid and 84% of the tetraploid S-phase nuclei were labelled, while labelling in the G 1-fractions was only 2 and 7%, respectively. 相似文献
18.
Abstract The proportion of Langerhans'cells in DNA synthesis in normal mouse skin was assessed by combining tritiated thymidine [ 3H]TdR autoradiography with enzyme histochemistry. After injection of [ 3H]TdR, ear skin was treated in two ways. Epithelial sheet preparations were stained for the presence of non-specific esterase and cytospin preparations of epithelial cell suspensions were stained for beta-glucuronidase activity. The labelling index (p± SE mean) for cytospins, 40 min after injecting [ 3H]TdR, was 1.6 ± 0.15%, doubling to 3–4% from 7–17 days after injection. The sheet preparations showed the proportion of label attributable to paired Langerhans'cells rising from 18% at 40 min after injection, to approximately 45%, on days 1–4 after injection. These results suggest that the proliferation of Langerhans'cells in normal mouse skin might be higher than was previously thought to be the case. 相似文献
19.
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. 相似文献
20.
The acylation of myelin proteolipid protein (PLP) and intermediate protein (IP) was investigated in an in vitro system of tissue slices prepared from actively myelinating rat brainstem. The incorporation of [ 3H]palmitate into the proteins in nine subcellular fractions including myelin and other cellular membranes which are actively involved in the synthesis and intracellular transport of the proteins was measured. More than 80% of [ 3H]palmitate-labeled proteins were recovered in myelin. The incorporation was highest in the heavy myelin and lowest in the light myelin subfraction. Appreciable acylation was also detected in the myelin-like fraction. On the other hand, the remaining fractions comprising a variety of endo- and ectomembranes, which harbored over 90% of newly synthesized PLP and IP as seen from [ 3H]leucine labeling showed practically no [ 3H]palmitate incorporation. The results indicate that the acylation of PLP and IP is a late event in their posttranslational processing and occurs only at their entry into the myelin sheath. 相似文献
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