QUANTITATIVE STUDIES OF THE VEGETATIVE SHOOT APEX OF EQUISETUM SCIRPOIDES

Equisetum scirpoides Michx., propagated from a single clone, was grown in a controlled growth chamber at 24 ± 1 C under a photoperiod of 16 hr light/8 hr darkness. The apical cell of aerial vegetative shoots gives rise to derivatives (merophytes) in a helical sequence. Each newly formed merophyte divides anticlinally to form two superposed cells that are parallel to a lateral face of the apical cell. Radial longitudinal divisions then take place in the two superposed cells. Shoot tips were fixed every 2 hr for 24 hr to determine the mitotic index of the apical cell, six subjacent cells, and the remaining cells above the level of leaf initiation. Average mitotic indices for the 24-hr period were 3.9%, 3.9%, and 7.0%, respectively. The results indicate that the apical cell is quite active mitotically; there was no clear evidence of endopolyploidy in cells of the shoot apex, young leaves or in the developing cortex, based upon cytophotometric measurements of DNA content.     

MITOTIC ACTIVITY IN THE ROOT APEX OF THE WATER FERN MARSILEA VESTITA HOOK. AND GREV.

Roots of Marsilea vestita ranging from 1–120 mm in length, as well as root primordia, were analyzed to determine mitotic activity and ploidy levels in the apical cell, five well-defined regions of the root proper, and two regions in the root cap. The mitotic index of the apical cell tended to be above the overall mean mitotic index for the entire apical meristem. No diurnal rhythm in mitotic index was apparent. The cell-cycle duration of the apical cell ranged from 12.1–25.2 hr, that of other regions of the root from 16.1–41.5 hr. There was no indication of polyploidy in any part of the apical meristem except in a few procambial cells. Thus, the results support the classical concept that the apical cell is the ultimate source of cells in the root.     

CELL CYCLE DURATION IN THE MERISTEM OF NEPHROLEPIS BISERRATA STOLONS: THE ROLE OF THE APICAL CELL

The stolons of Nephrolepis biserrata (sw.) Schott are thin axes that grow rapidly (from 2 to 4 mm per day) in the controlled conditions applied. In the cylindro-conical meristem, three histological zones are defined. Cell cycle duration was determined for each zone by autoradiographic methods after incorporation of tritiated thymidine and confirmed by the colchicine-induced metaphase-accumulation technique. The apical cell and its derivatives (Zone 1) are mitotically more active (cell cycle duration: 80 hr) than the cells of the subapical zones (2 and 3), where cell cycle lengths are 142 hr and 95 hr respectively. These data, compared to previous results, give evidence for the main role played by the relative rate of division of the apical cell compared to that of lateral cells in the organization and the shape of the meristem of pteridophytes. Moreover, the apical cell appears to be unique in having a differentiated cytological aspect not usually associated with an intensely proliferating cell.     

Occurrence of two cell subpopulations with different cell-cycle durations in the central and peripheral zones of the vegetative shoot apex of Sinapis alba L.

The cell-cycle duration and the growth fraction were estimated in the vegetative shoot apical meristem of Sinapis alba L. The length of the cell cycle was about 86 h, i.e. 2.5 times shorter than the cell-doubling time (M. Bodson, 1975, Ann. Bot. 39, 547–554) and the growth fraction was between 32 to 41%. These data demonstrated that the cell population of this meristem was heterogeneous, including one subpopulation of rapidly cycling cells and one subpopulation of non-cycling cells, i.e. cells with a very long cell cycle compared with that of the rapidly cycling cells. Non-cycling cells had no particular localization within the meristem. Both the central and peripheral zones of the meristem were mosaics of rapidly cycling and non-cycling cells.Abbreviations G₁ pre-DNA-synthesis phase - G₂ post-DNA-synthesis phase - GF growth fraction - M mitosis phase - PLM pulse-labelled-mitoses method - S DNA-synthesis phase - T cell-cycle duration - TdR thymidine     

CELL DIVISION KINETICS IN THE SHOOT APICAL MERISTEM OF CERATOPTERIS THALICTROIDES BRONG. WITH SPECIAL REFERENCE TO THE APICAL CELL

The duration of mitosis and the cell cycle were determined for defined cell populations of the shoot apical meristem of Ceratopteris thalictroides Brong. by using the colchicine-induced metaphase accumulation technique. The results indicate that the apical cell is mitotically active and cycles at an apparently greater frequency than the cells of subjacent populations. Duration of mitosis was similar for all cells of the meristem. These results are correlated with mitotic indices of control apices, the geometry of the apex, and the mean number of cells in the meristem. Shoot apices from adult plants were examined to determine mitotic indices within the meristem; mitotic activity was again noted for the apical cell. These results contradict recent proposals that the pteridophyte apical cell serves as a unicellular quiescent center which lacks histogenic potential and offer experimental support for the classical concept of apical cell function in those fern shoot meristems which terminate in a single apical cell.     

MITOTIC ACTIVITY IN THE ROOT APICAL MERISTEM OF AZOLLA FILICULOIDES LAM., WITH SPECIAL REFERENCE TO THE APICAL CELL

The meristematic activity of the apical cell and its derivatives (merophytes) in the unbranched, determinate roots of Azolla filiculoides Lam. was investigated. The plane of division of the apical cell indicates that it is the initial of each merophyte. The division plane of each newly formed merophyte is strictly periclinal to the root surface and provides confirmation that the immediate derivatives of the apical cell cannot be the ultimate root initials. The frequency of cell division as determined by the mitotic index, and by the duration of the cell cycle as determined by the colchicine method, confirmed the meristematic activity of the apical cell. As roots increase in length, the duration of the cell cycle in the total meristem increases, with the apical cell possessing the longest cell cycle, whereas the immediate derivatives maintain approximately the same cycle duration as in shorter roots. In determinate Azolla roots, cell division appears to play a major role up to a certain root length, then increase in length is produced mainly by cell elongation.     

QUANTITATIVE STUDIES OF THE ROOT APICAL MERISTEM OF EQUISETUM SCIRPOIDES

An investigation was made of the meristematic activity of the apical cell, its immediate derivatives (merophytes), and of other selected cell populations of the root of Equisetum scirpoides Michx. The plane of the first division of a derivative of the apical cell is radiallongitudinal, which provides evidence that merophytes immediately adjacent to the apical cell cannot be the ultimate root initials. The apical cell is as active mitotically in roots 20–40 mm long as it is in roots that are 0.25–1 mm in length. The mitotic activity of the apical cell and of other cell populations was determined from the mitotic index, and from determination of the durations of the cell cycle and of mitosis of the apical cell by using the colchicine method of metaphase accumulation. Microspectrophotometric measurements of DNA content indicated that there was no consistent increase in DNA (endopolyploidy) in the apical cell or in the other meristematic cells as roots increased in length. Conclusion: there is no evidence that the apical cell becomes quiescent or undergoes endopolyploidy as a root increases in length.     

CELL ARREST IN Gl AND G2 OF THE MITOTIC CYCLE OF VICIA FABA ROOT MERISTEMS

Experiments were performed with cultured excised primary root tips of Vicia faba ‘Longpod’ to determine: (1) the proportion of meristematic cells arrested in Gl and in G2 during carbohydrate starvation, and to determine if the proportion is fixed or can be varied experimentally; (2) the effect of increased starvation on the ability of arrested cells in Gl and G2 to initiate DNA synthesis and mitosis, respectively, when exogenous sucrose was supplied; and (3) whether puromycin, cycloheximide, or actinomycin D prevented the initiation of DNA synthesis and the onset of mitosis. Microspectrophotometry of nuclear DNA and autoradiographic measurements of incorporated ³H-thymidine showed that 72 hr of starvation immediately after excision produced tissue with more than 70 % of the cells arrested in G2 and less than 30 % in Gl. If cultured for three days and then starved for 72 hr, the tissue had nearly equal numbers of cells arrested in Gl and G2. As the duration of starvation increased, the time required to initiate DNA synthesis and to divide when carbohydrate was replenished also increased. Inhibition of protein synthesis by puromycin and cycloheximide prevented the initiation of DNA synthesis and mitosis, but actinomycin D, an inhibitor of RNA synthesis, did not prevent division of cells from G2 nor DNA synthesis by cells from Gl. The experiments demonstrated that the mitotic cycle of Vicia has two major controls, one in Gl and another in G2, and that other factors determine how many cells are affected by either of these cycle controls.     

Cell kinetic studies of chick brain cells in culture: an autoradiographic study with [3H]- and [14C]-thymidine

Labelling index, S-phase duration and cell-cycle time of proliferating brain cells from 6-day-old chick embryos in culture were investigated autoradiographically after labelling with [3H]- and/or [14C]-thymidine. The dissociated cells were cultured in the absence or in the presence of brain extract from 8-day-old chick embryos. Cultures contained essentially two cell types, which could be easily distinguished by the size of their nuclei: small nuclei identified as belonging to precursor cells of neurons and large nuclei corresponding to astroglial cells. The labelling index of astroglial cells (16.4%) was about 2 times higher than that of the neuronal cells (9.9%). Under the influence of brain extract the labelling index of neuroblasts was nearly doubled while that of the astroglial cells remained nearly unchanged. From double-labelling experiments with [3H]- and [14C]-thymidine, the same S-phase duration of about 7 hr was found for both cell types cultured with or without brain extract. A cell-cycle duration of 39 hr for neuronal and of 29 hr for astroglial cells was found. The cycle times remained constant under the influence of brain extract. From the measured data mentioned above, a growth fraction of 50% (neuroblasts) and 68% (astroglial cells) was calculated in control cultures without brain extract. After addition of brain extract, the growth fraction increased for both cell types (neuroblasts: 92%; astroglial cells: 80%). The results demonstrate that more cells proliferate in the presence of brain extract, but the durations of the S-phase and the cell cycle remain unchanged.     

The response of apical meristems of primary roots of Vicia faba L. to colchicine treatments

The effects of 0.5% and 0.025% solutions of colchicine on the passage of cells through the mitotic cycle in apical meristems of primary roots of Vicia faba have been examined. Both treatments affected cell progression through the mitotic cycle in the same way: S and G1 were shorter, and G2 and mitosis longer, than the corresponding control values. The duration of the various phases of the mitotic cycle were similar to those reported previously for apical meristems of lateral roots though cycle time itself was longer. Recovery of root proliferating tissues from colchicine-induced inhibition of growth is correlated with the presence of quiescent cells. Meristems which have no quiescent cells do not recover from eolchicine treatment, while meristems which contain many quiescent cells recover faster than those which contain few. The growth fraction and the proportion of proliferating cells with a short cycle time are linearly related to the duration of the S period in root meristems.     

MEASUREMENT OF THE DURATION OF DNA SYNTHESIS IN ERYTHROID CELLS OF THE BONE MARROW USING A DOUBLE LABELLING AUTORADIOGRAPHIC TECHNIQUE DESIGNED SPECIFICALLY FOR USE WITH HAEMOPOIETIC TISSUES

This paper describes a double labelling autoradiographic technique for use with haemopoietic tissues. In involves two photographic emulsions separated by a thin piece of mica on which the cells have been smeared. In this way the autoradiographic grains due to tritium and carbon-14 appear above and below the cells respectively. Applying the method to bone marrow normoblasts of young rats, the average duration of DNA synthesis (t_s) for the pro- and early normoblasts taken together is found to be 5.1 hr and the mean cell cycle time (t_c) to be 8.2 hr. For the intermediate normoblasts, the corresponding figures are 6.3 hr and 15.7 hr. Average values for all dividing normoblasts in the bone marrow are 5.8 hr and 12.8 hr respectively for t_s and t_c. The average duration of mitosis is 32 min.     

Light-induced synchrony of cell division in the protonema of the fern,Pteris vittata

Summary In protonemata of Pteris vittata grown for 6 days under red light, which brings about a marked depression of mitotic activity, the first division of the cells was synchronously induced by irradiation with blue light, and subsequent cell divisions were also promoted. The peak of the mitotic index reached a maximum of about 70% at 11.5 hrs, and 90% of all protonemata divided between the 11th and 13th hour after exposure to blue light. When the protonemata were continuously irradiated with blue light, synchronism of the next cell division in the apical cells decreased to a mitotic index of about 30%, and further divisions occurred randomly.The synchronization of cell division was found to be a combined effect of red and blue light. Red light maintained the cells in the early G₁ phase of the cell cycle; blue light caused the cells to progress synchronously through the cell cycle, with an average duration of 12 hr. By using ³H-thymidine, the average duration of the G₁, S, G₂ and M phases was determined to be about 3.5, 5, 2.5 and 1 hr, respectively.Synchronous cell division could be induced in older protonemata grown for 6 to 12 days in red light and even in protonemata having two cells. It could be repeated in the same protonema by reexposure to red light for 24 hrs or more before another irradiation with blue light.     

Cell Kinetic Studies of Chick Brain Cells In Culture: an Autoradiographic Study With [3H]- and [14C]-Thymidine

Labelling index, S-phase duration and cell-cycle time of proliferating brain cells from 6-day-old chick embryos in culture were investigated autoradiographically after labelling with [³H]- and/or [¹⁴C]-thymidine. the dissociated cells were cultured in the absence or in the presence of brain extract from 8-day-old chick embryos. Cultures contained essentially two cell types, which could be easily distinguished by the size of their nuclei: small nuclei identified as belonging to precursor cells of neurons and large nuclei corresponding to astroglial cells. the labelling index of astroglial cells (16.4%) was about 2 times higher than that of the neuronal cells (9.9%). Under the influence of brain extract the labelling index of neuroblasts was nearly doubled while that of the astroglial cells remained nearly unchanged. From double-labelling experiments with [³H]- and [¹⁴C]-thymidine, the same S-phase duration of about 7 hr was found for both cell types cultured with or without brain extract. A cell-cycle duration of 39 hr for neuronal and of 29 hr for astroglial cells was found. the cycle times remained constant under the influence of brain extract. From the measured data mentioned above, a growth fraction of 50% (neuroblasts) and 68% (astroglial cells) was calculated in control cultures without brain extract. After addition of brain extract, the growth fraction increased for both cell types (neuroblasts: 92%; astroglial cells: 80%). the results demonstrate that more cells proliferate in the presence of brain extract, but the durations of the S-phase and the cell cycle remain unchanged.     

Formative and proliferative cell divisions,cell differentiation,and developmental changes in the meristem of Azolla roots

The root of the water fern Azolla is a compact higher-plant organ, advantageous for studies of cell division, cell differentiation, and morphogenesis. The cell complement of A. filiculoides Lam. and A. pinnata R.Br. roots is described, and the lineages of the cell types, all derived ultimately from a tetrahedral apical cell, are characterised in terms of sites and planes of cell division within the formative zone, where the initial cells of the cell files are generated. Subsequent proliferation of the initial cells is highly specific, each cell type having its own programme of divisions prior to terminal differentiation. Both formative and proliferative divisions (but especially the former) occur in regular sequences. Two enantiomorphic forms of root develop, with the dispositions of certain types of cell correlating with the direction, dextrorse or sinistrorse, of the cell-division sequence in the apical cells. Root growth is determinate, the apical cell dividing about 55 times, and its cell-cycle duration decreasing from an initial 10 h to about 4 h during the major phase of root development. Sites of proliferation progress acropetally during aging, but do not penetrate into the zone of formative divisions. The detailed portrait of root development that was obtained is discussed with respect to genetic and epigenetic influences; quantal and non-quantal cell cycles; variation in cell-cycle durations; relationships between cell expansion and cell division: the role of the apical cell; and the limitation of the total number of mitotic cycles during root formation.     

Cell-cycle progression without an intact microtuble cytoskeleton

For mammalian somatic cells, the importance of microtubule cytoskeleton integrity during interphase cell-cycle progression is uncertain. The loss, suppression, or stabilization of the microtubule cytoskeleton has been widely reported to cause a G1 arrest in a variable, and often high, proportion of cell populations, suggesting the existence of a "microtubule damage," "microtubule integrity," or "postmitotic" checkpoint in G1 or G2. We found that when normal human cells (hTERT RPE1 and primary fibroblasts) are continuously exposed to nocodazole, they remain in mitosis for 10-48 hr before they slip out of mitosis and arrest in G1; this finding is consistent with previous reports. To eliminate the persistent effects of prolonged mitosis, we isolated anaphase-telophase cells that were just finishing a mitosis of normal duration, then we rapidly and completely disassembled microtubules by chilling the preparations to 0 degrees C for 10 minutes in the continuous presence of nocodazole or colcemid treatment to ensure that the cells entered G1 without a microtubule cytoskeleton. Without microtubules, cells progressed from anaphase to a subsequent mitosis with essentially normal kinetics. Similar results were obtained for cells in which the microtubule cytoskeleton was partially diminished by lower nocodazole doses or augmented and stabilized with taxol. Thus, after a preceding mitosis of normal duration, the integrity of the microtubule cytoskeleton is not subject to checkpoint surveillance, nor is it required for the normal human cell to progress through G1 and the remainder of interphase.     

DURATION OF CELL CYCLES IN CULTURED ROOTS OF CONVOLVULUS

The durations of the cell cycle in physiologically different regions of the meristem of cultured roots of Convolvulus arvensis were determined by the metaphase-accumulation technique involving colchicine. The cell cycle in the root cap increases from 13 hr in the actively dividing initials of the first tier to 155 hr in the slowly dividing initials of tiers 2–4 to an indeterminate value for derivatives of the initials in the root cap columella. The cycle times for the cells of the central cylinder and cortex are 21 and 27 hr, respectively. The cells of the quiescent center have a cycle of an estimated 420 hr. The duration of the cell cycle in these different regions is discussed in relation to the increased duration of G₁ in slowly or non-dividing cells. The possible regulation of cell division by the synthesis of a cell-division factor in the quiescent center is also discussed.     

Species-specific growth rate of phytoplankton in the West Pacific sector of the Southern Ocean. I.Prorocentrum scuttellum schröder

Phytoplankton samples were collected from the West Pacific Sector of the Southern Ocean to measure the growth rate from November 30 to December 1, 1995.Prorocentrum scuttellum was selected for growth rate measurement using the method of cell cycle analysis. During the 24 hr sampling cycle, cells ofP. scuttellum changed from 2,500 to 5,000 cells/L. The highest abundance was observed at 8:40 AM, December 1, and lowest at 11:40 PM, November 30. Cellular division seemed to occur sometime between 11:40 PM, November 30 and 2:40 AM, December 1. After cell division, DNA fluorescence shifted slowly towards the right, representing the S phase, and the majority of the cells were in S+G2 phases at 8:40 AM, December 1. Between the next six hours, a sharp drop in DNA fluorescence occurred, representing mitosis, and the majority of the cells returned to the G1 phase by 2:40 PM, December 1. We can not determine the duration time of the terminal event from this result However, the growth rate ofP. scuttellum was calculated as 0.43 d^-1 with the help of curve fitting methods. This unexpected result seems to have resulted due to background noise, unsynchronous cell division, unequal sampling, water column unstability, and migrating behavior ofP. scuttellum.     

Lasioglossins LLIII affect the morphogenesis of Candida albicans and reduces the duration of experimental vaginal candidiasis in mice

Lasioglossins are a group of peptides with identified antimicrobial activity. The inhibitory effects of two synthetic lasioglossin derivatives, LLIII and D‐isomeric variant LLIII‐D, on morphological changes in Candida albicans in vitro and the effect of local administration of LLIII during experimental murine candidiasis were investigated. C. albicans blastoconidia were grown in the presence of lasioglossin LLIII or LLIII‐D at concentrations of 11.5 μM and 21 μM, respectively, for 1, 2 and 3 days and their viability determined by flow cytometry using eosin Y staining. Morphological changes were examined by light and fluorescent microscopy. The Candida‐inhibitory effect of daily intravaginal administration of 0.7 or 1.4 μg of LLIII was assessed in mice with experimentally‐induced vaginal candidiasis. LLIII and LLIII‐D lasioglossins exhibited candidacidal activity in vitro (>76% after 24 hr and >84% after 48 hr of incubation). After 72 hr incubation of Candida with low concentration of lasioglossins, an increase in viability was detected, probably due to a Candida antimicrobial peptides evasion strategy. Furthermore, lasioglossins inhibited temperature‐induced morphotype changes toward hyphae and pseudohyphae with sporadic occurrence of atypical cells with two or enlarged nuclei, suggesting interference with mitosis or cytokinesis. Local application of LLIII reduced the duration of experimental candidiasis with no evidence of adverse effects. Lasioglossin LLIII is a promising candidate for development as an antimicrobial drug for treating the vaginal candidiasis.

     

REGENERATIVE PROLIFERATION OF MOUSE EPIDERMAL CELLS FOLLOWING ADHESIVE TAPE STRIPPING

The proliferating cells of mouse epidermis (basal cells) can be separated from the non-proliferating cells (differentiating cells) (Laerum, 1969) and brought into a mono-disperse suspension. This makes it possible to determine the cell cycle distributions (e.g. the relative number of cells in the G^ S and (G₂+ M) phases of the cell cycle) of the basal cell population by means of micro-flow fluorometry. To study the regenerative cell proliferation in epidermis in more detail, changes in cell cycle distributions were observed by means of micro-flow fluorometry during the first 48 hr following adhesive tape stripping. ³H-TdR uptake (LI and grain count distribution) and mitotic rate (colcemid method) were also observed. An initial accumulation of G₂ cells was observed 2 hr after stripping, followed by a subsequent decrease to less than half the control level. This was followed by an increase of cells entering mitosis from an initial depression to a first peak between 5 and 9 hr which could be satisfactorily explained by the changes in the G₂ pool. After an initial depression of the S phase parameters, three peaks with intervals of about 12 hr followed. The cells in these peaks could be followed as cohorts through the G₂ phase and mitosis, indicating a partial synchrony of cell cycle passage, with a shortening of the mean generation time of basal cells from 83-3 hr to about 12 hr. The oscillations of the proportion of cells in G₂ phase indicated a rapid passage through this cell cycle phase. The S phase duration was within the normal range but showed a moderate decrease and the Gj phase duration was decreased to a minimum. In rapidly proliferating epidermis there was a good correlation between change in the number of labelled cells and cells with S phase DNA content. This shows that micro-flow fluorometry is a rapid method for the study of cell kinetics in a perturbed cell system in vivo.     

Synchronization of cell division in eight-cell bovine embryos produced in vitro: Effects of nocodazole

Current methods of arresting and synchronizing cell division have not been very successful and have had few applications in embryo studies. Our objective was to determine the reliability of a metaphase arrest agent, nocodazole, for halting and synchronizing blastomere division in cleavage-stage bovine embryos, and to verify its reversibility and toxicity in vitro. Eight-cell-stage embryos obtained at 58 hr postinsemination were treated with varying concentrations of nocodazole for 12 hr. Treated embryos were assessed for cleavage arrest, chromatin morphology, DNA synthesis, and histone H1 and myelin basic protein (MBP) kinase activity, and were scored for blastocyst formation and hatching rate. They were subsequently fixed to count the number of nuclei. Complete arrest of cell division was observed at concentrations of 0.4 μg ml⁻¹ and above. Removal from nocodazole treatment led to immediate release from cleavage arrest, and was followed by synchronized mitosis, histone H1 kinase deactivation, and reentry into interphase within 3–5 hr. DNA synthesis was reinitiated at 6 hr after release. Although cell numbers and hatching rate decreased, the proportion of embryos reaching blastocyst stage was not significantly affected in nocodazole-treated embryos. It is concluded that nocodazole is a suitable choice for the cell-cycle synchronization of donor embryos for use in studies on the interactions between nucleus and cytoplasm during early embryogenesis. © 1996 Wiley-Liss, Inc.