Matilde H. Navarrete

A model for cell cycle and growth kinetics in roots

A model is proposed which accounts for the cell cycle and growth kinetics in roots growing under steady state conditions. In the model the root is conceived as a single file of cells and the three sequential zones of the root are considered. Nc cells, arranged in a single longitudinal file, make up the meristem zone. The most distal cell of this file is the initial and it has an indefinite cycle capacity, whereas all the others are derived cells with a limited programme of n cycles. The model predicts Nc = 2n. The number (Nc) of meristem cells will allow us to calculate the number of cycles that any derived cell undergoes before arresting division and triggering off its elongation. The cycle kinetics are linear and therefore the distribution of the Nc cells in the cycle compartments is directly proportional to each phase duration, and constant with time. The rate of meristem cell formation is defined by the cell flow (ϕ) as the frequency of cells that pass any point of the cycle per time unit. It also represents the frequency of cells that the meristem supplies to the elongation zone per time unit. The elongation zone is formed by Ntr cells in the process of enlargement. The model predicts that N tr = ϕ . N o . T tr Ttr being the time that cells devote to their enlargement throughout this zone. The equation allows us to estimate this value. Changes in root length will depend only on length changes in the mature zone where G = N o . ϕ . L G being the rate of growth and L the final cell size. During symplastic growth the root may be regarded as a bundle of files with identical growth rates. This leads to quantitative relationships between the growth components of the different kinds of files. Lastly, the fitness of the single file model against the actual growing root is discussed.

G2 repair and aging: influence of donor age on chromosomal aberrations in human lymphocytes

The caffeine effects on chromosomal aberration frequency and mean G2 duration were studied in human lymphocytes in vitro from three age groups of normal donors (I: 1-5 years old; II: 30-40 years old; III: 60-70 years old). Under control conditions, the three age groups showed a similar frequency of chromosomal aberrations. All three age groups exhibited a linear dose response for aberrations with caffeine treatments. However, lymphocytes from aged individuals (groups II and III) showed higher chromosomal aberration frequencies and longer G2 duration than cells from young individuals (group I). The caffeine effect in reducing G2 length was rather similar in every age group. The reversion of caffeine effects by adenosine or niacinamide in lymphocytes from older individuals was higher than in cells from group I. The different caffeine effects and G2 values between lymphocytes from old and young individuals are most likely due to a higher number of DNA lesions reaching G2 phase and/or a decrease of the G2 repair capability of lymphocytes from older individuals.

Effect of vitamin E on chromosomal aberrations in lymphocytes from patients with Down's syndrome

A possible protective effect of vitamin E ( dl‐α‐tocoferol) on chromosomal damage was evaluated in lymphocytes from patients with Down’s syndrome (DS) and from controls. This included the analysis of the basal and G2 chromosomal aberration frequencies in lymphocytes cultured with and without 100 μM vitamin E. The chromosomal damage in G2 was determined by scoring the number of chromosomal aberrations in lymphocyte cultures treated with 5 mM caffeine, 2 h before harvesting.Vitamin E treatment decreased the basal and G2 chromosomal aberrations both in control and DS lymphocytes. In DS cells, this protective effect, expressed as a decrease in the chromosomal damage, was greater (50%) than in controls (30%). These results suggest that the increment in basal and G2 aberrations yield in DS lymphocytes may be related to the increase in oxidative damage reported in these patients.

The positional control of mitosis and cytokinesis in higher-plant cells

Abstract. The present work establishes a correlation between cell length and patterns of mitotic microtubular assemblies in Allium cepa L. root meristems. Binucleate cells were formed by a short caffeine treatment which aborted the formation of the phragmoplast during telophase. The largest binucleate cells (about 50 μm in length) behaved as two contiguous mononucleate cells in their next mitosis: they developed two preprophase bands (PPBs), one around each nucleus, where two spindles and two phragmoplasts were subsequently formed. On the other hand, the shortest binucleate cells (about 36 μm in length) formed a single PPB at the site of the aborted phragmoplast and, in the medium-sized cells (about 44 μm) in which the single PPB formed around the nucleus possessing the largest cytoplasmic environment, the two mitotic spindles and the new phragmoplasts moved to, or were assembled in the position of the phragmoplast that had been aborted one cycle earlier. Some rules derive from these observations. First of all, the aborted phragmoplast left a signal for microtubule positioning which was still operative one cycle later, in two-thirds of the bimitoses. Also, that formation of the PPB is dispensable. Moreover, its development does not always predict the future division plane, because of the presence of competing old signals which are stronger than those shed by the PPB in the same mitosis, but which fade away with distance. Finally, the positional signals were reinforced when the ratio of monomeric to fibrillar actin was increased by cytochalasin D during their shedding. When this drug was given simultaneously with caffeine, the frequency of bimitoses which, one cycle later, developed spindles and phragmoplasts in the positions of the old phragmoplast increased. On the other hand, those frequencies dropped in relation to control when the cytochalasin D treatment took place during bimitosis, indicating that at this time the treatment reinforced the signals produced in bimitosis itself.

Analysis of the interphase accumulation induced by hydroxyurea on proliferating plant cells

Abstract Cell distribution in different compartments of the cell cycle (G1, early, middle and late S, G2 and mitosis) has been studied during continuous treatments with hydroxyurea (HU) in onion root meristems by cytophotometric and autoradiographic methods. A sublethal dosis of HU (0.75 mM) has been chosen to allow a good wave of mitotic synchrony during recovery, with a negligible level of chromosomal aberrations. Proliferating cells begin the S period in the presence of HU and are accumulated in early S, where the maximum value (60%) is reached after 8 h of treatment; at the same time middle and late S are practically empty. In the presence of the drug, residual DNA synthesis allows a slow but continuous progress of cells throughout the S period. Differential sensitivity of S cells to HU can be observed; replication is more affected in early S (85% inhibition) than in the second half of the period (70% inhibition). On the other hand, G1 cells are not apparently affected by HU, while cells in G2 show a delay in their entrance into mitosis.

Partial elimination of G1 and G2 periods in higher plant cells by increasing the S period.

Meristematic cells from Allium cepa L roots can attain a steady-state of growth at both 15 and 25 degrees C in the presence of drugs, hydroxyurea and 5-amino-uracil, which reduce the rate of DNA synthesis. These drugs, at used concentrations, significantly lengthen the S period without altering the cell growth rate, as indicated by the maintenance of the generation time. It has been observed that steady-state populations respond to a gradual increase in S by a reduction of G2 until a minimum value; with larger lengthening of S, both G1 and G2 are reduced. Natural synchronous populations have been used to study cell cycle parameters during transition from the physiological steady-state to the new one created by the presence of the drug. G2 (but not G1) is reduced during transition even in the presence of maximum drug concentrations that do not alter the cell growth rate. Both the S period and the division time are lengthened during transition. These observations support the concept that certain fractions of G1 and G2 are expendable, because they have no role in the DNA-division sequence of cell cycle events. We conclude that cell size regulates the length of these fractions by means of a negative correlation.

Estimation of chromatin patterns at G1, S, and G2 of the cell cycle

Abstract We used a new method based on the study of nuclear areas above certain density thresholds to estimate changes in the condensation of chromatin of a cell. Allium cepa L. root meristematic cells were “labelled” as binucleate by a 1 h treatment with 0.1 % caffeine and were fixed at the middle of each interphase period. The distribution of chromatin densities of Feulgen-stained cells in G1, S and G2 phases was so different that by simply estimating chromatin patterns it would be possible to identify which period of the interphase any cell has reached. G2 nuclei have an increased number of chromatin-dense areas compared with G1 or S nuclei. We postulate that the estimation of chromatin condensation may be useful for the evaluation of intranuclear differentiation at the level of the intact cell.

Defective G2 repair in Down syndrome: effect of caffeine, adenosine and niacinamide in control and X‐ray irradiated lymphocytes

Lymphocytes from both Down syndrome (DS) patients and age‐matched control donors have been investigated to identify a possible disturbance in chromosomal G2 repair. Analyses of caffeine treatments during G2 have shown that the frequency of chromosomal aberrations is higher in DS lymphocytes than in normal lymphocytes. Likewise, G2 duration is longer in DS cells than in normal cells. In both control and DS lymphocytes, caffeine treatments increase the frequencies of chromatid breakages and decrease the average of G2 duration. The reversal of the caffeine potentiation effect by adenosine and niacinamide is higher in DS cells than in normal cells. Furthermore, ATP content per cell in DS lymphocytes is one third of that estimated in normal lymphocytes. The increase of ATP level produced by adenosine or niacinamide generally correlates with the reversal of the caffeine effect on chromosome aberrations. Under the experimental conditions tested, a good negative exponential correlation between ATP level and chromosome aberrations has been detected in both normal and DS lymphocytes which were or were not X‐irradiated. Finally, we postulate a decrease in G2 repair capability of DS lymphocytes caused by a low availability of ATP and/or some other factor correlating with it.

Cell size of proliferating plant cells increases with temperature: Implications in the control of cell division☆

As chemical reactions related to the regulation of cell proliferation are governed by availability, amount, and concentration of relevant molecules, it has been suggested that cell size is an important factor in the control of cell cycle. We have measured the size of proliferating cells of Allium cepa roots in which growth rate was modified by changes in growth temperature. Two independent cell size parameters have been measured by cytophotometry: cell surface area projection and cell protein content. Average cell sizes of both the proliferating cell population and the subpopulation at the end of mitosis show that cell size increases with growth rate. Calculation of cell size at initiation of DNA replication clearly indicates that average cell size at this point is not growth invariant but positively correlated with growth rate.

The probability of G1 cells to enter into S increases with their size while S length decreases with cell enlargement in Allium cepa

The distribution of cell surface area projection (cell size) has been measured at birth and at initiation of DNA synthesis in steady-state populations of Allium cepa root meristems. The conditional probability, P(I/G1), that initiation occurs given that the event of being in G1 also occurs has been estimated from these data. P(I/G1) was found to increase when cells became larger. The distribution of G1 duration has been constructed from indicated cell size distributions. The absolute frequencies of G1 times showed a maximum in the zone of cells with short G1 periods; about 14% of cells appear to enter into S with G1 congruent to 1 h. These results suggest that the increase of P(I/G1) was due to cell enlargement and not to cell aging. By comparing the cell size distribution at initiation of S and at the end of this period, a drastic reduction of cell size variability during DNA replication was observed and both curves were seen as rather similar in shape although they obviously had different modal points. These observations support that there is a negative correlation between the initiation size and the duration of genome duplication, and that cells which initiate DNA synthesis with the same size have a similar replication time. From this hypothesis, a plot of S duration versus cell size at initiation of this period was constructed by comparing the distributions of cell size at start and end of replication; this plot was also consistent with the existence of a negative correlation between cell initiation size and S length.