Mitotic Wave Research Articles

The mixed lymphocyte response of senescent mice: Sensitivity to alloantigen and cell replication time

The age-dependent alteration in the proliferative response of C57B1/6J lymph node cells to stimulation by H-2- and M-locus alloantigens was examined in one-way mixed lymphocyte cultures (MLC). Balb/c (H-2 d, Mls b) and DBA (H-2 d, Mls a) spleen cells served as stimulating cells differing from C57B1/6J (H-2 b, Mls b) at the H-2 and H-2 plus Mls loci, respectively. The day of peak response and the ratio of responder to stimulator cells required for optimal stimulation were the same for all the age groups (3 to 29 months) tested, irrespective of the stimulator strain used. Results obtained in MLC under optimal conditions showed a maximal response to both Balb/c and DBA/2 stimulation at the age of 6 months, followed by a gradual decline in the response with age. In order to determine whether the decline with age in mixed lymphocyte reactivity can be attributed to a reduction in the proliferative capacity of the responding lymphocytes of aged mice, cell cycle analyses were performed. Auto-radiographic studies of MLC containing lymphocytes from CS7B1/6J mice aged 6 and 24 months showed no difference in generation time, S, G 2, G 1, and M phases of the cell cycle. In addition, lymphocytes of both age groups underwent two identical mitotic waves within the period of examination. Our results determine that the functional decline with age in proliferative activity in mixed lymphocyte cultures is attributable to a neither decrease in sensitivity to alloantigen nor to a decrease in generation time or the ability to undergo several mitotic divisions, and suggest that such a decline is caused by fewer cells capable of response in old mice.

Cytokinin as a Possible Component of the Floral Stimulus in Sinapis alba

Results of previous investigations indicated that one of the early and essential events occurring in the apical meristem of Sinapis alba L. during the transition to flowering is the release to mitosis of the G(2) nuclei; the trigger to mitosis is generated in the leaves and its movement out of the leaves begins around 16 hours after the start of the inductive treatment. The mitotic wave in the meristem culminates 10 hours later.In this paper, it is shown that a single application of a cytokinin (benzyladenine or zeatin) at concentrations ranging from 1 to 20 mug/ml directly to the apical bud of vegetative plants, at a time corresponding to the time of movement of the mitotic trigger in induced plants, produces a mitotic wave which is very similar to that found in induced plants. It is thus proposed that the mitotic component of the floral stimulus in Sinapis is a cytokinin. As the cytokinins are completely unable to induce flowering, it appears that there is a multicomponent floral stimulus in this species.

Development of the Drosophila retina, a neurocrystalline lattice

Pattern formation in the Drosophila retina proceeds by the recruitment of cells, along a morphogenetic front, into a lattice. At the advancing front, marked by a dorso-ventral furrow in the eye imaginal disc, cells are organized into ommatidial precursors, each containing cells destined to become photoreceptors 2, 3, 4, 5, and 8. Behind the front, a mitotic wave produces photoreceptors 1, 6, and 7, plus the remaining cells needed to complete the ommatidia. During the third larval instar, the front sweeps anteriorly across the eye disc, leaving a highly ordered pattern in its wake. Preceding the dorso-ventral furrow is a groove that bisects the eye disc into dorsal and ventral halves and presumably plays a role in establishing the equatorial symmetry line. Cell lineage plays little role in pattern formation in the eye. Genetic mosaics show that the cells of each ommatidium are not derived from a single mother cell; the cells appear to be recruited at random at the morphogenetic front. Similarly, the mirror symmetry above and below the equator is not established by a clonal mechanism; a single clone can contribute cells to ommatidia on both sides of the equator.

Mitochondrial functional changes during hepatic hyperplasia and azo dye carcinogenesis

Resting and active-state respiratory velocities, respiratory control, high amplitude volume changes, and latent ATPase activities were examined in hepatic mitochondria from rats fed 3′-methyl-4-dimethylaminoazobenzene (3′MeDAB) for production of liver tumors and from rats in three phases of liver regeneration subsequent to subtotal hepatectomies. Tetrabutylammonium bromide, a lipophilic probe capable of selectively inhibiting phosphorylating oxidation or uncoupling oxidation from phosphorylation, was used to detect subtle alterations in lipophilicity characteristics of the organelles and it was concluded that mitochondria from pre-hyperplastic, hyperplastic, and neoplastic tissues had a higher than normal degree of membrane lipophilicity at specific functional sites. Control of respiration by ADP was markedly augmented in all experimental groups; this behavior, plus depressed sensitivity to swelling agents and energized contraction, were similar in mitochondria from hepatomas and from 3-day regenerating livers. These mitochondrial functions were even more pronounced, however, in cells in prehyperplastic states (6 and 16 h subsequent to partial hepatectomy). Many forms of liver damage result in mitochondrial alterations which elevate the capacity for oxidative phosphorylation. Such changes associated with induction of azo dye oncogenesis are mimicked by the degree of hyperplasia in the tissue following the first mitotic wave of regeneration; implications relevant to hepatocarcinogenesis are discussed.

Mechanism of mitotic synchronization induced by 5-aminouracil

The mechanism of mitotic synchronization induced by 5-aminouracil (5-AU) in onion root meristems has been investigated. We studied the effect of the chemical upon asynchronous meristematic cells, as well as upon a synchronous fraction of them the position of which in the cell cycle was known. The kinetic studies have shown a fairly linear relationship between the treatment time and the induced mitotic wave. Therefore, cell accumulation in a particular zone of interphase appears to be the cause of mitotic synchronization. This zone seems to be located at the end of S period. 5-AU induces maximal mitotic delay on the cells located in the accumulation zone and severely depresses the S-G2 transit while the passage through G 2 remains relatively unaffected. The possibility that preferential inhibition affects the late replicating DNA synthesis is discussed.

Mitosis in the intact and regenerating planarian Dugesia mediterranea n.sp. II. Mitotic studies during regeneration, and a possible mechanism of blastema formation

AbstractIn planarian regeneration mitosis occurs in both anterior and posterior blastemas. The mitotic increase is visible as early as one hour after amputation, reaches a rapid first maximum at 5–12 hours, a relative minimum at 24–30 hours, and a higher and longer second maximum at two to four days depending on body size, level of amputation, and on whether blastema is anterior or posterior. The spatial dynamics of mitosis show a rapid and high mitotic increase in the distal regions but not in the proximal regions. Later, the proliferative zone shifts in sucessive mitotic waves to more proximal regions, paralleling the formation of the head ganglia and nerve cords.These results do not agree with theories of blastema formation through neoblast migration or through cell dedifferentiation, in as much as the local proliferation of neoblasts encountered is sufficiently high and early to account for the number of blastema cells found during period of regeneration. Therefore, we suggest that blastema formation during planarian regeneration occurs mainly through local neoblast proliferation.These results and the data obtained on regeneration rates enable us to suggest that nervous tissue may be one of the factors responsible for the differing mitotic increases found. Since neoblasts are the only planarian cells capable of mitosis, we suggest that neoblast proliferation could be under nervous tissue control through some stimulatory substance (s) released from the nerve terminals. The implications of this hypothesis for the maintenance of axial polarity are also discussed.

The development of ommatidial patterning in metamorphosed eye imaginal discs implants ofDrosophila melanogaster.

The development of the rhabdomeric pattern in the compound eye ofDrosophila has been studied using combined transplantation and electron microscope techniques. In a first series of experiments eye imaginal discs of increasing age were implanted into larvae ready to pupate, thus losing variable amounts of the normal time for development. A sequence of differentiative abilities was found in the metamorphosed test pieces. As far as the photoreceptor cells are concerned, the most prominent steps of this sequence are: ability to form groups with other similar elements, anatomical polarization of microvilli, establishment of the rhabdomeric pattern and formation of an equator line. The stability of determination of the equator line was tested in a second experimental series. Fragment of different topographical origin within the mature eye anlage were brought to metamorphosis by implantation into larvae ready to pupate. It was found that an equator line differentiates only in those pieces which according to the published anlage maps contain the prospective equator region prior to metamorphosis. The mitotic abilities of implanted eye imaginal discs were investigated by means of "in vitro"3H-thymidine pulse-labelling and light microscope autoradiography of the differentiated test pieces. During the third larval stage the eye anlage is traversed by two consecutive mitotic waves, each one of them producing different categories of receptor cells. The first, anterior wave predominantly produces cells oriented toward the poles of the eye within the ommatidia, while the second, posterior wave gives rise to elements exclusively in an equatorial position. The dynamics of this proliferation are discussed in relation to the findings in the implantation experiments. Silver-grain counts support the possibility that at least two successive cell divisions occur in the eye anlage between labeling with tritiated thymidine and beginning of morphological differentiation. The relevance of this finding for the understanding of the concept of acquisition of competence is discussed.

Localization in the cell cycle of the antimitotic action of the pyrrolizidine alkaloid, lasiocarpine and of its metabolite, dehydroheliotridine

The antimitotic action of the pyrrolizidine alkaloid lasiocarpine on rat liver parenchyma was investigated using as the experimental model the wave of mitosis produced in liver by a single dose of thioacetamide. A single low dose of lasiocarpine administered two weeks before the thioacetamide, almost completely inhibited the mitotic wave without inhibiting to the same extent the preceding wave of DNA synthesis. By the use of selective inhibitors and radioisotope labelling, the location of the mitotic block was found to be either in the latter half of the DNA synthetic phase, S, or early in G 2, the post-synthetic phase. The mitotic wave was similarly inhibited by pretreatment of the rats with a single injection of dehydroheliotridine, a pyrrolic metabolite of heliotridine-based pyrrolizidine alkaloids.

Control circuits for determination and transdetermination: interpreting positional information in a binary epigenetic code.

Positional information in an embryo requires interpretation to guide spatially ordered differentiation. Evidence, from dtermination and transdetermination among imaginal disc derivatives in Drosophila, suggests the interpretative commitment, or determination, is carried by entities possissing only two alternate states each. This hypothesis correctly predicts pairs of classes of mutants affecting complementary subsets of imaginal discs. Four such complementary pairs define four boundaries separating the various imaginal discs on the fate map of the egg. Crossing any boundary corresponds to a change in state of the associated determination circuit. Using these boundaries, the model makes 43 predictions about relative transdetermination frequencies. Thirty-four appear correct. This has an a priori probability of less than 10- minus 4. False predictions appear to reflect incompleteness in the theory. The four boundaries on the fate map presumably correspond to threshold levels of spatial gradients constituting positional information; on one side of a threshold one circuit state occurs, on the other, the second state. The last four cleavage divisions in Drosophila melanogaster occur as four mitotic waves with geometries similar to the four boundaries. Disc determination happens at about this time. These waves may be, or reflect, the signalling system carrying positional information. Focus on the interpretive machinery, the determination circuits, suggests two slightly new concepts: (1 the set of possible combinations of states of determination circuits constitutes an epigenetic code, thus raising familiar coding questions concerning redundancy or nonsense words; (2) each circuit may respond to its own gradient or positional signal distributed in two or three dimensions.

Some problems of sampling for chromosomal aberrations from synchronous populations

The problems of obtaining compartmental chromosomal aberration yields from populations synchronized by mitotic shake-off and allowed to run on for sampling during the first post-synchrony mitotic wave are investigated. Using a simple model, where each cell has an independent rate invariant through the cycle, a lognormal developmental age distribution, and the assumption of an “all or nothing” radiosensitivity during 10% of the cell's transit of a compartment, yield-time curves are derived from radiation given in G2 or G1. Two basic methods of sampling are available: I, a single irradiation followed by serial sampling with time through the mitotic wave; II, irradiation given at intervals of time prior to sampling populations at a fixed time from release. Either method gives an infinitude of quite different yield time curves for the same fixed set of population parameters. The key factor is the “age” of the population, i.e. the time from release from synchrony to irradiation or observation. It is concluded that within rather narrow limits, sampling method II can give a reliable indication of the radiosensitivity situation obtaining in the G1 compartment. There is no method of obtaining interpretable and usable quantitative chromosomal aberration data for the G2 compartment.

Experimental Studies on the Mitotic Activity of the Shoot Apical Meristem and Its Relation to Floral Evocation and Morphogenesis in Sinapis alba

Results of a previous investigation indicate that there are two successive increases of mitotic activity in the shoot apical meristem of Sinapis alba plants immediately following a single inductive long day. The evidence presented in this paper indicates that the critical daylength for the first or "early" rise in the mitotic index is 1 h shorter than the critical daylength for floral induction. This early mitotic activation is induced by a stimulus generated in the leaves. In a variety of experimental conditions, induction of flowering was always associated with the early mitotic wave. Available evidence thus indicates that this mitotic wave is an essential component of floral evocation. However, this early cell division process always occurs a long time (usually 36 h) before the initiation of the first flower buds and apparently has no functional relationship to floral morphogenesis. Since this early mitotic rise occurs in plants exposed to a short day given at an unusual time during the 24-h cycle, and in plants subjected to a long day but deprived of CO2 during the second half of this long day, that is, without an extended period of photosynthetic CO2 fixation, the mitotic rise noticed is not due to an increased supply of assimilates. In all cases examined, the second or "late" mitotic rise occurs simultaneously with the initiation of the first flower buds by the meristem, and it appears to be an essential event of floral morphogenesis. Except in plants deprived of CO2 during the second half of an inductive long day, the early mitotic rise always occurs 26-30 h after the start of the effective light period and the late rise 36 h later.

Descriptive and quantitative study of ultrastructural changes in the apical meristem of mustard in transition to flowering: II. the cytoplasm, mitochondria and proplastids

ABSTRACT Transition to flowering was induced in the shoot apical meristem of Sinapis alba (mustard), a long-day species, by subjecting vegetative plants to a single 22-h long day. The ultrastructural changes occurring in the meristematic cells during the complete morphogenetic switch were investigated by quantitative methods. The earliest detectable changes are seen 18 h after the start of the inductive long day. One of these changes is the replacement of the large vacuoles of the cells of vegetative meristems by an increased number of smaller vacuoles in the cells of meristems of plants induced to flower (evoked meristems). The other earliest change is an increase in size of the chondriome. Remarkably enough both changes have in common that they lead to an increase in membrane area and are thus concerned with increased membrane synthesis. In this respect this very early effect of the leaf-generated floral stimulus is very similar to that produced by various animal and plant hormones in their respective target tissues. The large rise in chondriome size is paralleled by an increase in succinic dehydrogenase activity. Both changes certainly reflect a rise in cellular respiratory activity which produces the necessary energy supply for the morphogenetic switch. As the size of the plastidome does not change during the transition to flowering, the plasti dome: chondriome ratio decreases markedly. The size of the cytoplasmic matrix is greater in cells of evoked meristems than in cells of vegetative meristems. A first size maximum is reached at 26 h after the start of the long day and a second at 54 h. These 2 maxima occur just prior to 2 mitotic waves culminating respectively at 26–30 and 62 h. The increases in amount of endoplasmic reticulum (ER) and dictyosome number that are found in evoked meristems collected just prior to or at the time of the second mitotic wave are also probably related to the mitotic activation of the tissue. As the size of the vacuolar apparatus considered as a whole does not change at all and the size of the cytoplasmic matrix increases, the vacuoles:cytoplasm ratio decreases in evoked meristems collected following 26 h after the start of the long day. Also, there is an unexpected decrease in the nucleus:cytoplasm ratio in the cells of evoked meristems after 46 h.

Descriptive and quantitative study of ultrastructural changes in the apical meristem of mustard in transition to flowering. I. The cell and nucleus.

ABSTRACT Transition to flowering was induced in the shoot apical meristem of Sinapis alba (mustard), a long-day species, by subjecting vegetative plants to a single 22-h long day. The ultrastructural changes occurring in the meristematic cells during the complete morphogenetic switch were quantitatively investigated using both the planimetric and the point-counting stereological methods. The 2 methods yield very similar results and are thus equally appropriate for the study. The cell, nucleus, and dispersed chromatin sizes are greater in meristems of plants induced to flower (evoked meristems) than in meristems of control vegetative plants. A first size maximum is reached at 26 h after the start of the inductive long day and a second at 54 h. These 2 maxima occur just prior to 2 mitotic waves culminating respectively at 26–30 and 62 h. Neither the condensed chromatin size nor the number of chromocentre profiles per nucleus section change. Consequently the dispersed chromatin: condensed chromatin ratio increases in evoked meristems. This change is discussed in relation to current views on the differential genetic activity of the 2 kinds of chromatin. There is a late but large increase in size of the nucleolus. Dramatic changes in texture are associated with the enlargement of this organelle. These changes are the disappearance of the segregation of fibrillar and granular components and a loss of compactness due to marked vacuolation. All these changes in size and structure are interpreted as indicating a late increase in nucleolar synthesis of ribosomes in the evoked meristems.

Heterogenous chromosomal radiosensitivity of phytohaemagglutinin-stimulated human blood lymphocytes in culture

Human blood lymphocytes were irradiated with hard X-rays, stimulated with phytohaemagglutinin (PHA), and grown in presence of amethopterin to accumulate the responding cells at the G I/S boundary of the first cell cycle in vitro. After reversal of the G I/S block with exogenous thymidine, the frequencies of asymmetric chromosome exchanges in relation to the position of metaphases within the first generation mitotic wave were compared. Significant differences of aberration yields within replicate culture series were found in several experiments. A gradual increase of aberration frequencies with increasing duration of S + G2 phases was the most constant feature encountered. In addition, in two parallel series of cultures from one donor, the highest frequency of aberrations was found in samples corresponding to the shortest S+G2 phase duration. A significant contribution of selective mitotic delay of aberration-carrying cells to the distribution of aberration frequencies was excluded. Therefore, it was inferred that the results reflect a true variability of radiosensitivity among the PHA-responsive cells, probably of a discontinuos character, ranging over the ratio of two.

The diurnal variation of the first mitotic wave released among rat kidney tubule cells by partial nephrectomy.

Mitotic waves were released in kidney tubules by resecting one kidney and a half in a group of 4 rats at intervals of a few hours during the 24‐hr‐cycle. The course of the mitotic wave appearing on the second day after the operation was followed in each half‐kidney by determining the incidence of mitosis at intervals of 3 hours. The waves showed a distinct diurnal pattern of variation: those initiated by resection between noon and midnight had single peaks, while those initiated by resection between 3 a.m. and 9 a.m. gave waves with 2 peaks. It is assumed that the single as well as the paired waves are based mainly on mitoses in the proximal tubules. The alternation between sequences of single‐ and double‐peaked waves through the 24 hours is interpreted as the result of an interference of an endogenous mitotic rhythm, the mitotic wave starting slightly less than 30 hr after the resection.

Circadian variations of DNA synthesis, mitotic activity, and cell size of hepatocyte population in young immature male mouse growing liver.

Circadian variations in DNA synthesis after tritiated thymidine and autoradiography, mitotic activity with the Colcemid method, and cell size, represented inversely by the number of cells per microscopic field, are reported for the hepatocyte population of the young immature growing liver of the male mouse. The peak of DNA synthesis is at 04:00 hours and that of mitotic activity at 12:00 hours. The 8-hour interval between the two peaks is considered, in agreement with other authors, a good estimation of the lag period between DNA synthesis and the initiation of mitotic activity. The minimum cell number per microscopic field, which is considered to correspond to the maximum cell size, coincides with the starting of the circadian mitotic wave. The significance of this coincidence and the relationships of the circadian curves of the parameters controlled with the feeding pattern and food consummatory curve of standardized mice, are discussed.

Changes in the DNA synthetic period during the phase of rapid growth in cultured diploid fibroblastic cells.

ABSTRACTThe DNA synthetic period in diploid fibroblastic cell populations was studied during the phase of rapid growth in vitro, using the labelled mitotic wave method and quantitative autoradiography.It was found that the length of the DNA synthetic period increased progressively, from early passage levels, with the number of generations in vitro. the increase was mainly in the time taken to synthesize the first 50%, and especially the first 10%, of DNA.The change was detectable despite the complication due to a low level of tritium incorporation after the removal of tritiated thymidine from the cultures.

Effect of length sampling bias on labeled mitotic index waves

The theoretical labeled mitotic index [PLMI( t)] is obtained for the situation where a steady state cell population is subjected to a pulse label experiment. The model used to describe this situation allows for a correlation structure among the durations of the different phases of the cell cycle. It is shown that cells which are labeled have different kinetic properties than those cells in the general population. This unrepresentativeness arises because cells are not randomly labeled but are labeled by a length biasing phenomenon; i.e. cells which have longer DNA synthesis periods have a greater probability of being labeled.

The first mitotic wave released among rat kidney tubule cells by partial nephrectomy.

On the second day after the removal of a part of an adult rat's kidney mass, a mitotic wave is released among the tubule cells of the remaining kidney. This wave proceeds synchronously throughout the cortex and may be followed by a second but much smaller wave on the third day. The removal of increasing amounts of kidney tissue (a half, a whole or a kidney and a half) is paralleled by the release of mitotic waves of growing size. The peak of a larger wave occurs sooner after the resection than that of a smaller wave. The bisection of a kidney initiates more cell divisions than those expected by the diminution of kidney mass alone. This increase of the mitotic wave appears not to be caused by the presence of the transectional wound. As a working hypothesis it is proposed that the bisection leads to a relative increase in blood flow through the diminuted organ, thereby affecting a more effective drainage of growth‐inhibitor(s) and a corresponding enhancement of the mitotic wave. This latter is always preceded by a non‐mitotic period of slightly less than 30 hr. It is proposed that about the first 2/3 of this period may be taken for the inactivation of an organ‐specific growth inhibitor that suppresses the growth of the convoluted tubules of the intact adult rat.

Stimulation of DNA synthesis in cultures of mouse embryo fibroblast-like cells.

AbstractStimulation of the DNA synthesis and mitoses in stationary cultures of mouse embryo fibroblast‐like cells was induced by various agents such as ribonuclease, digitonin, fresh medium and commercial preparations of hyaluronidases. Time sequence of stimulation was similar in experiments with all these agents. Cells were activated to enter S phase from GI phase. The rise of the number of DNA‐synthesizing cells was preceded by a latent period of about 8–12 hours with the maximal number of DNA‐synthesizing cells being observed at 16–24 hours. Mitotic wave was observed after the wave of DNA synthesis. Stimulation of DNA synthesis and mitosis was not preceded by any significant decrease of an average cell density in the culture. The progeny of activated cells had no greater chance than other cells to be activated again when stimulation was repeated.It is concluded that similar proliferative reactions can be induced in stationary cultures by a variety of diverse agents. Possible role of cell surface changes in the induction of these reactions is discussed.