ANALYSIS OF THE PROLIFERATION KINETICS OF BURKITT'S LYMPHOMA CELLS

The in vitro proliferation kinetics of a cell line derived from a patient with American Burkitt's lymphoma were investigated at three different growth phases: lag (day 1), exponential (day 3) and plateau (day 5). The growth curve, labeling and mitotic indices, percentage labeled mitosis (PLM) curves and DNA content distributions were determined. The data obtained have been analysed by the previously developed discrete-time kinetic (DTK) model by which a time course of DNA distributions during a 10-day growth period was characterized in terms of other cell kinetic parameters. The mean cell cycle times, initially estimated from PLM curves on days 1, 3 and 5, were further analysed by the DTK model of DNA distributions and subsequently the mean cell cycle times with respect to DNA distributions during the entire growth period were determined. The doubling times were 39·6, 31·2 and 67·2 hr, respectively, at days 1, 3 and 5. The mean cell cycle time increased from 23·0 to 37·7 hr from day 3 to day 5 mainly due to an elongation of the G₁ and G₂ phases. A slight increase in the cell loss rate from 0·0077 to 0·0081 fraction/hr was accompanied by a decrease in the cell production rate from 0·0299 to 0·0184 fraction/hr. This calculated cell loss rate correlated significantly with the number of dead cells determined by trypan blue exclusion. Analysis of the number of dead cells in relation to the cell cycle stage revealed that a majority of cell death occurred in G₁ (r= 0·908; P < 0·0001). There was a good correlation between the in vitro proliferation kinetics at plateau phase of this Burkitt's lymphoma derived cell line and the in vivo proliferation kinetics of African Burkitt's lymphoma (Iversen et al., 1974), suggesting the potential utility of information obtained by in vitro kinetic studies.     

ON THE EXISTENCE OF A Go-PHASE IN THE CELL CYCLE

The model is based on the assumption that the cell cycle contains a G_o-phase which cells leave randomly with a constant probability per unit time, γ. After leaving the G_o-phase, the cells enter the C-phase which ends with cell division. The C-phase and its constituent phases, the‘true’G₁-phase, the S-phase, the G₂-phase and mitosis are assumed to have constant durations of T, T₁T_s, T₂ and T_m, respectively. For renewal tissue it is assumed that the probability per unit time of being lost from the population is a constant for all cells irrespective of their position in the cycle. The labelled mitosis curve and labelling index for continuous labelling are derived in terms of γ, T, and T_s. The model generates labelled mitosis curves which damp quickly and reach a constant value of twice the initial labelling index, if the mean duration of the G_o-phase is sufficiently long. It is shown that the predicted labelled mitosis and continuous labelling curves agree reasonably well with the experimental curves for the hamster cheek pouch if T has a value of about 60 hr. Data are presented for the rat dorsal epidermis which support the assumption that there is a constant probability per unit time of a cell being released from the Go-phase.     

Relationships between opercula girth, maximum girth and total length of fish species caught in gillnet and trammel net selectivity surveys off the Portuguese coast

The relationships of total length (L_t) to girth at opercula (G_ope) and maximum girths (G_max) were estimated for 39 and 22 fish species, respectively, captured off the Portuguese coast between Póvoa do Varzim and Santo André (38°05′54′′–41°15′36′′N and 8°46′54′′–9°31′13′′W). The data were collected between April 1994 and September 1995. The best‐represented family was Sparidae (five species), followed by Soleidae and Triglidae (four species each). G_ope and G_max were found to increase linearly with length for all species, all r² values being statistically significant (P < 0.01) and higher than 0.81. When G_ope and G_max were plotted against length for all species combined, three groups were identified, corresponding to general girth–length relationships: G1 = 1.408 + 0.238 ×L_t, G2 = 2.455 + 0.328 × L_t and G3 = ?0.72 + 0.60 × L_t for opercula girth and G1 = 3.402 + 0.195 × L_t, G2 = 2.211 + 0.374 × L_t and G3 = ‐2.514 + 0.717 × L_t for maximum girth. These groups correspond to different body shapes of fishes: G1‐round, G2‐torpedo‐form and G3‐compressed body.     

GRAIN COUNT THRESHOLD DEPENDENCE OF FLM CURVE PATTERN AND CELL CYCLE PARAMETERS OF HEPATOCYTES IN VIVO

FLM curves from hepatocytes of regenerating rat liver in vivo were compared at different grain count thresholds. Estimates of cell cycle phases derived from curves with thresholds decreasing from 15 to 1 grain (background 0.2 grains per nuclear area) revealed a prolongation of t_s from 6.6 to 9.5 hr, at the expense of t_G2M, and t_G1, whereas t_c remained constant. A similar pattern was observed in FLM curves at various threshold levels for hepatocytes localized in subunits of the liver lobule along the vascular axis from afferent to efferent pole. The shapes of these FLM curves indicated an intralobular gradient of reutilizable labelled material. The use of two different threshold levels is crucial for proper selection of FLM curves to evaluate cell cycle phases in regenerating rat liver: first, a threshold to exclude the autoradiographic background, and a second one to avoid errors due to reutilization of labelled DNA precursors. Each threshold has its own implications for the estimation of cell cycle phases.     

Cell cycle duration in antheridial filaments ofChara spp. (Characeae) with different genome size and heterochromatin content

The relationship between nuclear 1 C DNA content and cell cycle progression throughout successive stages of antheridial filaments were studied among five taxa ofChara: two dioecious species (n = 14):C. aspera (7.2 pg DNA),C. tomentosa (7.4 pg DNA), and three monoecious species (n = 28):C. vulgaris (13.5 pg DNA),C. fragilis (19.3 pg DNA), andC. contraria (19.6 pg DNA). With the use of double³H-thymidine labelling and morphometry a number of characteristics common to all of the investigated species were determined within the proliferative periods preceding spermiogenesis. These include: (1) simplified type of the cell cycle (S + G₂ + M), due to complete lack of G₁ intervals, (2) constant duration of S phase, (3) progressive shortening of G₂ + M periods, and (4) gradual reduction of cell lengths at successive mitotic divisions. Nucleotypic dependence was found between genome size and several time parameters estimated for consecutive stages of antheridial filaments: the higher the DNA C-value, the longer the cell cycles, their component phases, the total duration of the proliferative period, as well as the lower the rate of growth of interphase cells. Differential Giemsa staining of late G₂ phase nuclei revealed that the higher content of C-heterochromatin is connected with prolonged cell cycle durations in species with similar DNA C-values.     

EVOLUTIONARY IMPLICATIONS OF DNA DIVERGENCE IN THE DROSOPHILA OBSCURA GROUP

Using DNA–DNA hybridization, we have determined the degree of single-copy DNA (scDNA) divergence among eight species of the Drosophila obscura group. These include Old World and New World species as well as members of two subgroups. Contrary to classical systematics, members of the affinis subgroup are more closely related to American members of the obscura subgroup than are Old World species. The Old World species are not a monophyletic group. The degree of scDNA divergence among species is not necessarily correlated with morphology, chromosomal divergence, or ability to form hybrids. A unique pattern of hybrid formation was found: species separated by a ΔT_m of 6.5°C can form hybrids whereas species separated by a ΔT_m of 2.5°C cannot. As with other groups of Drosophila, the obscura group has discrete parts of the genome evolving at very different rates. The slow evolving fraction of the nuclear genome is evolving at about the same rate as mitochondrial DNA. The additional scDNA divergence accompanying the step from partial reproductive isolation (between North American pseudoobscura and the isolated Bogotà population) to full isolation is very small. The resolution of the technique was challenged by five closely related taxa with a maximum ΔT_m of 2.5°C separating them; the taxa were unambiguously resolved and the “correct” phylogeny recovered. Finally, there is some indication that scDNA in the obscura group may be evolving considerably slower than in the melanogaster subgroup.     

Studies on interaction between poly(L-lysine) and DNA of varied G + C contents

Interaction between polylysine and DNA's of varied G + C contents was studied using thermal denaturation and circular dichroism (CD). For each complex there is one melting band at a lower temperature t_m, corresponding to the helix–coil transition of free base pairs, and another band at a higher temperature t′_m, corresponding to the transition of polylysine-bound base pairs. For free base pairs, with natural DNA's and poly(dA-dT) a linear relation is observed between the t_m and the G + C content of the particular DNA used. This is not true with poly(dG)·poly(dC), which has a t_m about 20°C lower than the extrapolated value for DNA of 100% G + C. For polylysine-bound base pairs, a linear relation is also observed between the t′_m and the G + C content of natural DNA's but neither poly(dA-dT) nor poly(dG)·poly(dC) complexes follow this relationship. The dependence of melting temperature on composition, expressed as dt_m/dX_G·C, where X_G·C is the fraction of G·C pairs, is 60°C for free base pairs and only 21°C for polylysine-bound base pairs. This reduction in compositional dependence of T_m is similar to that observed for pure DNA in high ionic strength. Although the t′_m of polylysine-poly(dA-dT) is 9°C lower than the extrapolated value for 0% G + C in EDTA buffer, it is independent of ionic strength in the medium and is equal to the t_m⁰ extrapolated from the linear plot of t_m against log Na⁺. There is also a noticeable similarity in the CD spectra of polylysine· and polyarginine·DNA complexes, except for complexes with poly(dA-dT). The calculated CD spectrum of polylysine-bound poly(dA-dT) is substantially different from that of polyarginine-bound poly(dA-dT).     

THE PROLIFERATION KINETICS OF NHIK 1922 CELLS IN VITRO AND IN SOLID TUMOURS IN ATHYMIC MICE

The proliferation kinetics of cells of the line NHIK 1922 grown in vitro and as solid tumours in the athymic mutant nude mouse has been studied. In vitro, growth curves were determined for exponentially growing populations and for populations synchronized by mitotic selection. The phase durations for these populations were determined by flow cytofluorometric measurements of DNA-histograms and pulsed incorporation of [³H]TdR respectively. The generation time and the phase durations for synchronized populations were found to be about equal to those for exponentially growing populations. The duration of the phases G₁, S and G₂+ M was found to be 8·5–9·5, 11·0–12·0 and 6·0–6·5 hr respectively, i.e. the generation time was 26·5–27·0 hr. The proliferation kinetics in vivo were studied by flow cytofluorometry and by the technique of percentage labelled mitoses. The median duration of S-phase and (G₂+ M)-phase in vivo was found to be approximately the same as that observed in vitro, while the median duration of G₁-phase was found to be approximately 5 hr longer in vivo than under the present in vitro growth conditions. The growth fraction in vivo was estimated to be approximately 50%. The non-proliferative compartment of the tumour cells was found to consist mainly of cells with the DNA-content of cells in G₁-phase. It is concluded that the reduced rate of proliferation of NHIK 1922 cells in vivo is correlated with alterations in the duration of G₁-phase and, hence, the proportion of cells in G₁-phase.     

Productivity of high and low density populations ofJaponaria laminata armigera (Diplopoda) in a warm-temperate forest ecosystem

The productivity of the final larval and adult populations of a large sized diplopod, Japonaria laminata armigera were studied at a warm temperate ever-green broad leaf forest in Chiba Japan. The population density was about 200/m² in May 1962, and 7/m² in May 1966, the former being about 30 times as much as the latter. A remarkable growth of animals was seen from June to November, and the growth pattern of individuals in both populations was very similar. The growth and mortality were calculated by the following formulae, where G is growth, M mortality, N_t number at the time t,W_t−1 mean body weight of animals at the time t−1, ΔN decrease in number during the period of time from t−1 to t, ΔW growth in mean body weight during the time from t−1 to t. The constant b in the formula R=aW^b representing the relation between body weight and oxygen consumption of Japonaria laminata was determined at 0.81, and Q₁₀ was estimated at about 2. The amounts of growth and assimilation were calculated. The pattern of dynamics in terms of productivity of the population in the two periods were very similar. But, the amounts of assimilation were computed at 57.8 kcal/m²/from May 1962 to April 1963, and 3.01 kcal from May 1966 to April 1967. The former is 20 times as much as the latter. The growth or ecological efficiencies for the populations were similar.     

PROLIFERATION KINETICS OF AN EXPERIMENTAL ASCITES TUMOUR OF THE MOUSE

The cell cycles of an experimental ascitic tumour of the C3H mouse (NCTC 2472) were determined at various times after the intraperitoneal injection of 10⁶ cells. It was found that, contrary to results in solid NCTC 2472 tumours, obtained with the same NCTC cells, the duration of the cell cycle and its phases lengthened with the age of the tumour while the growth fraction remained relatively constant. G₁ was the first phase to lengthen, while later T_s and T_G2 increased also. The amount of DNA per cell was determined by cytospectrophotometry. This method provides data on the evolution during growth of the relative number of cells in each phase of the cell cycle.     

Predicting sequence-dependent melting stability of short duplex DNA oligomers

Many important applications of DNA sequence-dependent hybridization reactions have recently emerged. This has sparked a renewed interest in analytical calculations of sequence-dependent melting stability of duplex DNA. In particular, for many applications it is often desirable to accurately predict the transition temperature, or t_m, of short duplex DNA oligomers (∼ 20 base pairs or less) from their sequence and concentration. The thermodynamic analytical method underlying these predictive calculations is based on the nearest-neighbor model. At least 11 sets of nearest-neighbor sequence-dependent thermodynamic parameters for DNA have been published. These sets are compared. Use of the nearest-neighbor sets in predicting t_m from the DNA sequence is demonstrated, and the ability of the nearest-neighbor parameters to provide accurate predictions of experimental t_m's of short duplex DNA oligomers is assessed. © 1998 John Wiley & Sons, Inc. Biopoly 44: 217–239, 1997     

CELL-PRODUCTION RATES ESTIMATED BY THE USE OF VINCRISTINE SULPHATE AND FLOW CYTOMETRY I. AN IN VITRO STUDY USING MURINE TUMOUR CELL LINES

A method for the evaluation of cell-production rates is described which combines flow cytometry (FCM) and the stathmokinetic method. By means of FCM it is possible to estimate the distribution of cells with G₁, S and (G₂+ M) DNA content in a population. As this method gives the relative (G₂+ M) DNA content of cells within the cell cycle, it may be possible to evaluate cell-production rates by this technique. In the present study it was found that administration of a metaphase-arresting (stathmokinetic) agent, vincristine sulphate (VS), to asynchronous cell populations of three different murine tumour cell lines in vitro increased the peak representing cells with (G₂+ M) DNA content as the number of mitotic (M) cells increased during the period of treatment. The accumulation of mitotic cells was determined by cell counts on smears under the microscope and compared with the increase in the (G₂+ M) DNA peak measured by FCM as a function of time after the administration of VS. Good agreement was obtained between the cell-production rates as estimated by FCM and by mitotic counts in all three cell lines investigated.     

The time and duration of the premeiotic inteprhase in microsporocytes ofTrillium kamtschaticum

The time and duration of each phase of the premeiotic interphase were determined in microsporocytes of two clones (S and K clones) ofTrillium kamtschaticum. After collectionTrillium plants were stored at 3 C or 7 C prior to completion of premeiotic mitosis in archesporial cells. For autoradiography, cells were explanted in the presence of³H-thymidine to identify the interval of the premeiotic DNA synthesis. Approximate durations of the G₁, S and G₂ phases for the K clone stored at 3 C were estimated to be 12, 12 and 14 days, respectively. The interval of premeiotic development was markedly different between clones. A high degree of synchrony in meiotic development, which is usually observed within anthers up to late meiotic prophase, was confirmed at the S phase, suggesting that synchrony is established during the G₁ interval.     

STUDIES ON THE DIVISION CYCLE OF MAMMALIAN CELLS : V. Modifications of Time Parameters by Different Steady-State Culture Conditions

In cultures of murine neoplastic mast cells, the duration of different phases of the division cycle (G₁, S, G₂, and mitosis [M]) was determined under optimal and several well-defined suboptimal growth conditions. Two methods of evaluation were applied to the same culture system: first, the relative number of G₁, S, G₂, and M cells was determined by pulse labeling of samples with thymidine-³H and subsequent radioautography in conjunction with a microfluorometric technique permitting rapid measurements of cellular DNA content; second, after pulse labeling with thymidine-³H, the variations with time of the mitotic labeling index were analyzed. Suboptimal culture conditions were obtained by reducing the concentration of single essential medium components (leucine, glucose, or serum) or by the addition of specific metabolic inhibitors (actinomycin D, amethopterin). Growth-limiting culture conditions resulted in increased generation times. Even under control conditions, the cell number doubling time exceeded the generation time, and this difference was more pronounced in suboptimal media. Under most of the suboptimal conditions tested, the increase in generation time was attributable primarily to an extended duration of the G₁ phase. Under certain growth-limiting conditions, however, other phases were also prolonged. In addition, the variabilities of the generation time and of certain cell cycle phases were increased under suboptimal culture conditions. Results obtained by the two methods of evaluation were, in general, in good agreement with each other. Some differences were, however, observed and interpreted in terms of cell death and/or asymmetric frequency distributions of cell cycle parameters.     

Helix-coil transitions in nucleoprotein: Computer-generated curves for independent melting of double-stranded regions with fixed ends

Helix-coil transitions in nucleoprotein at low salt concentrations are known to be characterized by two phases of the process: independent melting of uncomplexed “naked” regions without rearrangement of proteins, followed, at higher temperatures, by melting of complexed DNA. Blocking at the ends of these regions increases their thermal stability and three is a shift of 10–20°C in t_m of the melting profiles. In this study the basic assumption is that the loop entropy effect is mainly responsible for such stabilization. Calculations are made using conventional h-c transition theory for a system of independently melted segments with fixed ends. Segments are either homosize or have randomly distributed lengths. Calculated melting curves are used to obtain t_m, and transition width-dependence on segment length (or average length when randomly distributed) and on the nucleation parameter σ. Base-pair heterogeneity is taken into account by averaging over different base-pair distributions in the individual segments, using Gaussian distribution around the overall (G+C)-content. It is shown that this causes only an additional widening of the transition but no additional t_m shift. Comparison is made with similar systems in the literature. The main conclusion drawn is that the treatment proposed may be useful for analysis of the lower temperature melting phase in nucleoprotein at low counterion concentrations. It may be used as an independent method to reveal the features of nucleoprotein structure.     

Phenomenological analysis of the clotting curve

A model-independent (phenomenological) characterization of the clotting curve is proposed. Three parameters are used to encapsulate the main features of the increase in absorbance observed at 350 nm due to the reaction of thrombin with fibrinogen that leads to clot formation: (1) the maximum increase in absorbance per unit time, A _m , at the inflection point of the clotting curve; (2) the time needed to reach the maximum increase in absorbance,t _m ; and (3) the clotting time,t _c , obtained from extrapolation of the slope att _m to the zero absorbance baseline. Clotting curves at low fibrinogen concentrations (0.125 ÷ 0.250 µM), well below the K_m, where thrombin amidase activity is rate-limiting with respect to the subsequent aggregation process, have been measured under a wide variety of experimental conditions, (i.e., as a function of thrombin concentration,pH and temperature) in order to explore the basic response of each parameter to changes in solution conditions. Under all conditions examined in this study we have observed thatt _m andt _c are linked through a linear relationship that appears to be an important invariant property of the clotting curve, regardless of experimental conditions. No such clear relationship exists between A _m andt _c , witht _c being associated with several possible values of A _m and vice versa, depending upon solution conditions. It is proposed thatt _c is strictly dependent on thrombin amidase activity, while A _m reflects properties of the aggregation process leading to clot formation. The clotting time shows apH and temperature dependence that closely resembles that of K_m/V_m for synthetic amide substrates. Futhermore,t _c changes linearly with either the inverse thrombin concentration and the concentration of competitive inhibitors of fibrinogen binding to thrombin, as expected for the ratio K_m/V_m. We show how the analysis of clotting curves obtained at different thrombin and inhibitor concentrations yields a quantitative measure of K_I that is in excellent agreement with the value determined independently from steady-state measurements of thrombin amidase activity.     

Heterozygote deficiency,population substructure and their implications in DNA fingerprinting

Summary Substructured populations exhibit an overall deficiency of heterozygosity whose proportional magnitude depends on the nature of substructuring, i.e., the number of subpopulations (s), their time of divergence (t) from the ancestral population, and the rate of gene flow amongst them (m). Since apparent heterozygote deficiency could be caused by many factors other than population substructuring, one must examine the nature of substructuring that could produce the observed extent of heterozygote deficiency, in order to infer the substructuring from an observed heterozygote deficiency. Using the equivalence of proportional heterozygote deficiency and the coefficient of gene differentiation (G _ST), we can generate isolines of G _ST as functions of s, t (in units of 2N _e generations, N _e being the effective population size) and m. Analytical results suggest that large G _ST values cannot be reached by substructuring alone, unless the number of subpopulations are large and they remain isolated over a long period of time. Application of the theory to population data on six variable number of tandem repeats (VNTR) loci in US Caucasians and US Blacks demonstrates that the observed heterozygote deficiencies at these loci cannot be explained by substructuring within these populations alone. This is so because such large values of G _ST (3%–10%) would require an absence of gene exchange between the subpopulations and a divergence time from each other of at least 25000 years ago, neither of which is compatible with the demography and ethnohistory of US Caucasians and Blacks. In contrast, the inability to detect extreme-sized alleles and/or incomplete resolution of nearly similar-sized alleles following Southern gel electrophoresis could easily explain the observed heterozygote deficiencies. The implications of these results are discussed in the context of the forensic use of DNA-typing data, and justify the employment of population genetic principles in forensic genetics.     

THE LIFE HISTORY OF COLEOCHAETE SCUTATA (CHLOROPHYCEAE) STUDIED BY A FEULGEN MICROSPECTROPHOTOMETRIC ANALYSIS OF THE DNA CYCLE1,2

The life history of Coleochaete scutata Bréb. was analyzed by Feulgen microspectrophotometry, a technique measuring DNA content in individual nuclei. By correlating nuclear DNA content with morphological structures or stages in the life history, changes in ploidy level are revealed. The microspectrophotometric study confirmed the earlier reports of a haploid vegetative thallus with mitotic division restricted primarily to the margin of the thallus. In the mitotic cycle the G₁ (pre-synthesis) phase is longer in duration than the synthesis find G₂ (post-synthesis) phases. Oogamous sexual reproduction results in resistant oospores which attain DNA levels of 2C 8C (1C being the DNA level of gamete nuclei).     

A New Variant for Mathematical Analysis of Pulse Cytophotometric Data

The staining of DNA by specific fluorochromes provides a suitable method of receiving histograms in a short time by means of pulse cytometry. They represent the proliferative structure of cell populations at a high degree of statistical security. A method for quantitative determination of cell cycle phases (G_1-, S- and G₂ + M-phase) is presented which includes the fraction of cell debris in the calculation procedure. The advantages of this method are the elimination of overlapping between the fraction of debris and cell cycle phases and the quantitative determination of the fraction of cell debris offers the opportunity to get information on cytolytic potencies. Apart from the calculation of the various cell cycle phases the method provides criteria on the adaptation of mathematical analysis to primary data.