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1.
In a previous report, we described a new method called FPi analysis to analyze time sequences of DNA histograms taken from a perturbed population of cells. In this paper we utilize the method to analyze the in vivo kinetic response of bone marrow and of lung metastases of the B16 tumor to various chemotherapeutic agents. We show that the technique allows useful kinetic data to be obtained with minimal processing of the raw histograms, thus allowing fast analysis of the data. We also show that, in order to monitor the kinetic response of living tissues, it is essential to collect multiparameter distributions; to monitor only the one dimensional fluorescence histogram can give rise to misleading results. Using these multiparameter histograms, we are also able to monitor the growth fraction of the lung metastases during treatment, allowing discrimination between cell synchrony and cell recruitment from the resting compartment.  相似文献   

2.
A new rapid method for the cell cycle analysis of asynchronously growing cells is presented. The new method is an alternative to the more time consuming and subjective fraction of labeled mitoses (FLM) method. Like the FLM method, all cells in the S phase of the cell cycle are marked by pulse labeling with a radioactive DNA precursor. The subsequent progress of the cohort of cells thus labeled is monitored through a narrow window in the cell cycle. The window is defined by a narrow range of DNA contents corresponding to cells in mid-S phase and is designated Si. The cellular DNA content is measured by flow cytometry and the cells in the window Si are selected by electronic cell sorting. The radioactivity per cell in Si (RCSi) is determined by liquid scintillation counting. The duration of S phase and of the total cycle and the dispersions therein are determined from the oscillation of the RCSi values with time. The complete cell cycle analysis can be accomplished in as little as 1 day following the collection of samples. Exponentially growing Chinese hamster ovary (CHO) cells were analyzed according to the RCSi method and the FLM method. It is demonstrated that the two techniques give essentially the same results.  相似文献   

3.
In flow microfluorometry (FMF) analysis cells stained with a fluorescent dye that binds specifically to DNA are passed through the instrument. The number of cells in the population having a fluorescence intensity is recorded in a single channel of a multichannel pulse height analyzer. The result is a DNA fluorescence histogram for the population.A method for decomposing an FMF histogram into its G1, S and G2 + M components, corresponding to similarly designated phases of the cell cycle is given. This technique can also be applied to find the parameters in all of the previous approaches. The parameters are calculated by iteration which eliminates the need for non-linear optimization procedures.  相似文献   

4.
To help identify components of the blue-light photoreceptor system for phototropism in Phycomyces blakesleeanus Bgff., proteins from a microsomal fraction obtained from synchronous sporangiophores were studied. By two-dimensional gel electrophoresis, two proteins (FP1, FP2) with covalently bound flavins were found. FP1 has a molecular weight of 71 000 and an isoelectric point of 6.6; FP2 has a molecular weight of 59 000 and an isoelectric point of 6.5. These flavoproteins were purified by column chromatography and gel filtration while assaying for flavins by fluorescence. The relative concentrations of FP1 and FP2 were affected by light applied during growth. These flavoproteins are likely components of the blue-light photoreceptor complex mediating phototropism in Phycomyces.Abbreviations 10 k pellet 10 000-g pellet - 100 k pellet 100 000-g pellet - FP1, FP2 proteins with covalently bound flavins having molecular weights of 71 000 and 59 000 and isoelectric points of 6.6 and 6.5, respectively  相似文献   

5.
A numerical method for deriving the fractions of cells in different phases of the cell cycle from a single observed DNA histogram is presented. The observed histogram is regarded as a polluted version (containing allocation errors) of the true histogram. A mathematical model is used to describe the pollution process. A theoretical histogram, representing the true histogram, is constructed so that G1 cells are put into one channel and G2M cells into another; the distribution of S cells in between is approximated with a set of harmonic functions. This theoretical histogram is subsequently disturbed with Gaussian dispersion functions to stimulate the pollution, yielding a predicted histogram. Using a maximum likelihood estimation technique, the model parameters are adjusted iteratively, matching the predicted histogram to the actually observed one. With the final parameter values substituted, the corresponding final theoretical histogram is regarded as a reliable reconstruction of the true histogram. From the latter, the required percentages can be read directly. The advantage of this approach over other mathematical analysis methods is that it allows a wide range of different, continuous distributions for relatively few model parameters (thus featuring flexibility and realism and a diminished risk of encountering computational problems). In addition, estimation errors providing a measure of accuracy can be obtained. To test the method, it was used to analyze various observed histograms from the literature that have been obtained by either simulation or actual flow cytometric measurements. The method appeared to perform well, as compared to the reported results of several other methods of analysis applied to the same data.  相似文献   

6.
Neuronal excitability under stimuli with a complex time course is investigated on the basis of the numerical solution of the Hodgkin-Huxley equations. Each stimulus is composed of 100–1000 unitary excitatory postsynaptic potentials (uEPSP) that start randomly within a definite time window. Probability of initiating a spike [firing probability, FP(W)] as a function of the window width W is calculated by the Monte Carlo method. FP(W) has a step-like shape: it becomes equal to 1 for small W and almost vanishes as W exceeds some value W S. The role of long-lasting somatic inhibition is analysed. W S depends on the inhibition potential, but the step-like shape of FP is preserved. It is concluded that the capability of multisynaptic stimulation to cause a spike can be expressed in terms of temporal coherence between the synaptic inputs. Namely, the spike is initiated if the temporal coherence between active inputs is above a definite threshold. The threshold value can be effectively regulated by varying the inhibition potential.  相似文献   

7.
A method is presented for estimating the rate of DNA synthesis of a cell population by examining the DNA histogram generated by flow cytometry (FCM). The model is based on the use renewal equations to estimate the steady-state fraction of cells in each DNA compartment. The fraction of cells in each compartment is shown to be related to the Laplace transform of the transit time through that compartment. Two methods are introduced for estimating the rate of DNA synthesis utilizing different transit time distributions. One method is shown to be a simplification of the method of Dean and Anderson. The other method allows for variability in the DNA synthesis rate. The effects of quiescent cells are considered and attention is paid to the various assumptions underlying the estimation.  相似文献   

8.
9.
Quantitative methods for interpretation of flow cytometry DNA histograms are required for the widespread clinical use of this technology. The usefulness of a histogram analysis technique in this setting requires that it be operator independent, easy to implement in a clinical laboratory, and provide high sensitivity to the desired information. Additionally, the technique must be tolerant of the relatively low signal-to-noise ratios often found in DNA distributions obtained from clinical samples. Among the factors that have been used to assess the malignant potential of tumors are the presence of an aneuploid population, the proportion of hyperdiploid cells, the width of the G1 peak, the DNA index, and the fraction of cells in S. A computer-based method has been developed for extraction of the above-mentioned features from DNA histograms. The program detects peaks in the histogram and uses straight-line fits to the cumulative frequency distribution to define cell population bounds. A test set of 44 histograms compiled from bladder irrigation specimens obtained from patients with a present or past history of transitional cell carcinoma (TCC) was analyzed by five collaborating laboratories forming a Network sponsored by the National Cancer Institute (NCI). This test set was used to evaluate the performance of the computer-based method by comparing results with those of four expert observers. In this preliminary analysis, perfect agreement was found in the detection of aneuploid cell populations by all observers and the computer-based method. Correlation of percent hyperdiploid cell fraction was also excellent.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

10.
Flow cytometry and cell proliferation kinetics   总被引:1,自引:0,他引:1  
Flow cytometric techniques are presented which allow to determine parameters of cell proliferation kinetics by means of histogram sequences after special manipulations of the cell culture under investigation: (a) In the stathmokinetic method metaphase blocking agents are applied which allow the cells of the population to continue progression through interphase and accumulate at 4C DNA content. The development of DNA specific histograms during this process is analysed as to the G1 phase duration and the fraction of nonproliferating cells. (b) In the BUdR/Hoechst method the suppression of Hoechst fluorescence after BUdR incorporation during S phase is taken as a means for inducing a temporal change of histogram shapes without perturbing the cell cycle progression of the population. This temporal development of histogram shapes is analysed as to phase duration, whole cycle time and fraction of nonproliferating cells. (c) By combining the BUdR/Hoechst technique with a simultanous DNA specific stain and analysing with a two-parametrical flow cytometer, more information is obtained from each histogram after BUdR incorporation: The location of cells in the cycle at the beginning of the experiment, the cycle stage at cell harvest, and from this the distance and velocity of progression through the cycle during drug incubation. By introduction of these dynamic methods flow cytometry has become a powerful tool for the study of cell proliferation kinetics in culture.  相似文献   

11.
Samples of mitotic L-cells were investigated after different preparation and staining procedures using the technique of pulse-cytophotometry. It is shown that most mitotic cells which should appear in the second peak of the DNA histogram are disintegrated or separated into halves by pepsin pretreatment. Hence, the designation‘G2+ M’for the second peak is not correct for this preparative method. This should be taken into account in cell kinetic investigations performed after pepsin pretreatment.  相似文献   

12.
A SIMPLIFIED METHOD OF DNA DISTRIBUTION ANALYSIS   总被引:9,自引:0,他引:9  
Current methods of analysing DNA distributions utilize complex mathematical expressions that require the use of large non-linear curve fitting methods and, consequently, large computers. This paper presents a new method of analysing DNA distributions of asynchronously growing or mildly perturbed cells. The S phase fraction is obtained by fitting a second degree polynomial to that part of the distribution, mid S phase, which is not influenced by either the G1 or the G2+ M peaks. The method is simple and fast, it exceeds the accuracy of other methods and it can be used on a large desk calculator or mini-computer.  相似文献   

13.
Mass-spectrometric measurements of 18O exchange from 13C18O2 were used to follow changes in the intracellular carbonic anhydrase (CA) activity of cells of Chlamydomonas reinhardtii Dang, wild type and the ca-1 mutant during adaptation to air. With intact cells as well as with crude homogenates total intracellular CA activity in wild-type cells increased six to tenfold within 4 h after transferring cells from 5% CO2 (high inorganic carbon, Ci) to ambient air (air adapted). After that time the activity slowly declined to a level similar to that observed with cells which had been continuously grown in air (low-Ci grown). In the ca-1 mutant, total CA was induced to a similar extent during 4 h of adaptation; however, absolute activities were two to three times lower in ca-1 than in the wild type regardless of the CO2 supply. When crude extracts from wild-type cells were separated into soluble and insoluble fractions, each fraction contained about half of the internal CA activity. Within 4 h of adaptation, both forms of CA activity were simultaneously enhanced by nine to tenfold, reaching levels similar to those found in low-Cigrown cells. In contrast, in the ca-1 mutant the soluble CA activity was only enhanced by about eightfold while the level of insoluble CA was very low even in low-Ci cells. After isolation of intact chloroplasts from wild-type cells and further subfractionation, around 70–80% of total chloroplastic CA activity was found to be in the insoluble fraction while 17–20% remained in the soluble fraction. Both chloroplastic CA activities were inducible within the first 4 h of adaptation to air, with each of them being eight to ten times higher than in high-Ci algae. After that time their activities were similar to the corresponding CA values in low-Ci-grown cells. In contrast, plastids from high-Ci cells of the ca-1 mutant showed 40% less insoluble-CA activity compared to the wild type and this insoluble-CA activity was not increased at all by transferring algae to air. In addition, no soluble-CA activity was detected in chloroplasts from high-Ci and air-adapted ca-1 cells. These results indicate the presence of three intracellular CA activities in high-Ci air-adapted and low-Ci cells of the wild type and that two of them are associated with the chloroplasts. All three activities are completely induced within the first 4 h of adaptation to air in wild-type cells. In contrast, it was not possible to induce any of the chloroplastic CA activities in the ca-1 mutant. The possibility that the soluble chloroplastic CA represents a pyrenoid-located CA is discussed.This work is dedicated to Professor A. Wild on the occasion of his 65th birthday  相似文献   

14.
Relative movement methods use the timed progression of the mean fluorescence of cells which have been labeled with monoclonal antibodies against bromodeoxyuridine and displayed with bivariate flow cytometry according to DNA and label content to compute duration of DNA synthesis, TS. The relative movement is the difference of the mean DNA fluorescence of the labeled undivided cells from the G1 channel relative to the difference between the G1 and G2M channels. In this communication, we show how to extend this method to compute the potential doubling time, Tpot, the time required for a population of cells to double, given quiescent cells but no cell loss. A quantity v is introduced that is a function of the fraction of labeled divided cells and the fraction of labeled undivided cells. We show that v is independent of time and is equal to ln(2)Ts/Tpot so that Tpot (equal to ln(2)Ts/v) can be directly found from the information available in computing the relative movement. The method is applied to Chinese hamster ovary cells to demonstrate its utility.  相似文献   

15.
应用真彩色医学图像分析技术, 对90 例甲状腺肿瘤(其中甲状腺腺瘤10 例, 不典型腺瘤15 例,乳头状腺癌25 例, 滤泡癌15 例, 髓样癌15 例, 未分化癌10 例) 细胞核DNA含量进行了分析。结果显示,甲状腺腺瘤组与各型甲状腺癌比较均有显著性差异(P< 001),甲状腺腺瘤组同不典型腺瘤组比较无统计学意义(P> 005)。甲状腺癌随组织分化程度的不同, DNA 含量明显增加, 多为高倍异倍体细胞, DNA直方图明显右移, 峰值主要位于≥5C处; 甲状腺腺瘤组DNA含量较低, 多为低倍整倍体细胞, DNA 直方图峰值位于2C- 4C处; 不典型腺瘤组DNA含量介于上述二者之间, DNA 直方图逐渐右移。表明DNA倍性程度与肿瘤的增殖程度呈正相关, 高倍异倍体细胞随肿瘤恶性程度的增高而增多。作者认为DNA原位图像定量分析可为甲状腺肿瘤的诊断、分级及早期发现癌变趋势提供一个可靠的参考指标  相似文献   

16.
We have found that extracellular phosphomannan is the main phosphate reserve in the yeast Kuraishia capsulata, in contrast to other yeast species effectively absorbing Pi. Under nitrogen starvation, K. capsulata absorbed essentially all Pi from the medium containing 240 mM glucose, 2.5 mM MgSO4, and 11 mM KH2PO4. Inorganic polyphosphate level in the cells was about 14% of the Pi absorbed. Most of the Pi (~60%) was found in the fraction of extracellular phosphomannan that can be used as a carbon and phosphorus source by this yeast in deficient media.  相似文献   

17.
Guanine nucleotide-binding protein (G protein) coupled receptors (GPCRs) are frequently expressed by a baculovirus expression vector system (BEVS). We recently established a novel BEVS using the bacmid system of Bombyx mori nucleopolyhedrovirus (BmNPV), which is directly applicable for protein expression in silkworms. Here, we report the first example of GPCR expression in silkworms by the simple injection of BmNPV bacmid DNA. Human nociceptin receptor, an inhibitory GPCR, and its fusion protein with inhibitory G protein alpha subunit (Giα) were both successfully expressed in the fat bodies of silkworm larvae as well as in the BmNPV viral fraction. Its yield was much higher than that from Sf9 cells. The microsomal fractions including the nociceptin receptor fusion, which are easily prepared by only centrifugation steps, exhibited [35S]GTPγS-binding activity upon specific stimulation by nociceptin. Therefore, this rapid method is easy-to-use and has a high expression level, and thus will be an important tool for human GPCR production.  相似文献   

18.
Joe W.  Gray 《Cell proliferation》1976,9(6):499-516
A mathematical model is presented that permits simulation of a time sequence of DNA distributions with a single set of cell-cycle parameters. The method is particularly suited to the quantitative analysis of sets of sequential DNA distributions from perturbed cell populations. The model permits determination of the durations and associated dispersions of the phases of the cell cycle as well as the point in the cell cycle at which the perturbing agent exerts its effect. The mathematical details of the simulation technique are presented, and the technique is applied to the analysis of DNA distributions from perturbed cell populations. Three cell populations are modeled: CHO-line cells released from a block at the interface of the G1 and S-phases, 3T3 cells released from a G1-phase block produced by serum starvation, and S49 mouse lymphoma cells responding to a block in the G1-phase produced by N6,02′-dibutyryl adenosine 3′:5′-cyclic monophosphate (Bt2cAMP).  相似文献   

19.
The relationship of the DNA sequences from which polyribosomal messenger RNA (mRNA) and heterogeneous nuclear RNA (NRNA) of mouse L cells are transcribed was investigated by means of hybridization kinetics and thermal denaturation of the hybrids. Hybridization was performed in formamide solutions at DNA excess. Under these conditions most of the hybridizing mRNA and NRNA react at values of Dot (DNA concentration multiplied by time) expected for RNA transcribed from the nonrepeated or rarely repeated fraction of the genome. However, a fraction of both mRNA and NRNA hybridize at values of Dot about 10,000 times lower, and therefore must be transcribed from highly redundant DNA sequences. The fraction of NRNA hybridizing to highly repeated sequences is about 1.7 times greater than the corresponding fraction of mRNA. The hybrids formed by the rapidly reacting fractions of both NRNA and mRNA melt over a narrow temperature range with a midpoint about 11°C below that of native L cell DNA. This indicates that these hybrids consist of partially complementary sequences with approximately 11% mismatching of bases. Hybrids formed by the slowly reacting fraction of NRNA melt within 4°–6°C of native DNA, indicating very little, if any, mismatching of bases. Hybrids of the slowly reacting components of mRNA, formed under conditions of sufficiently low RNA input, have a high thermal stability, similar to that observed for hybrids of the slowly reacting NRNA component. However, when higher inputs of mRNA are used, hybrids are formed which have a strikingly lower thermal stability. This observation can be explained by assuming that there is sufficient similarity among the relatively rare DNA sequences coding for mRNA so that under hybridization conditions, in which these DNA sequences are not truly in excess, reversible hybrids exhibiting a considerable amount of mispairing are formed. The fact that a comparable phenomenon has not been observed for NRNA may mean that there is less similarity among the relatively rare DNA sequences coding for NRNA than there is among the rare sequences coding for mRNA.  相似文献   

20.
The toxic and inhibitory properties of hydroxyurea (HU) have been studied in asynchronous and synchronized populations of mouse L-cells. Hydroxyurea is a potent growth inhibitor and appears to be specifically lethal for cells which are in the early part of S phase at the time the compound is introduced. Cells in late S phase, G2, mitosis and G1 appear to progress normally around the cycle in the presence of the compound until they reach the G1/S boundary. There are indications that at least some G1 cells are able to enter the S phase even in the presence of the drug; however their flow into S is much slower than that of control cells and therefore they are killed at a slow rate. Upon prolonged exposure to the drug a second phase of more rapid killing is observed, beginning at about the time division would occur in uninhibited cells. Hydroxyurea exhibits a rapid and marked inhibition on DNA synthesis but its effect on RNA synthesis is much less pronounced and may be a consequence of the inhibition of DNA synthesis. The effects of hydroxyurea on cell viability and DNA synthesis can be partially prevented by the addition of deoxyribonucleosides which in sufficient concentration appear to compete temporarily with the drug. The fact that the protection is only temporary would appear to rule out the hypothesis that the primary mode of action of the drug is the inhibition of the reduction which converts ribonucleotides to deoxyribonucleotides. The data presented in this communication taken together with observations of other workers would appear to suggest that the effect of the drug may be directly on the DNA molecule.  相似文献   

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