MITOTIC ACTIVITY AT THE SHOOT APEX OF AZOLLA FILICULOIDES

The mosquito fern, Azolla filiculoides Lam., was grown in a growth chamber on a nitrogen-free culture solution at 24 C under the following photoperiod: 16 hr light/8 hr darkness. Shoot tips were fixed every 2 hr for 24 hr to determine the mitotic index for the apical cell, immediate derivatives, and remaining cells to the level of the first leaf or lateral shoot primordium. Mitotic indices were 6.9%, 6.5% and 6.3%, respectively. The colchicine method was employed to determine the cell-cycle durations and duration of mitosis for the same populations of cells. The cell-cycle duration and duration of mitosis of the apical cell were 28.2 hr and 2.8 hr, respectively; for the immediate derivatives, 26.7 hr and 2.5 hr; for the remaining cells, 23.6 hr and 2.1 hr. Conclusions: the apical cell is as mitotically active as its immediate derivatives, and there is no evidence of a quiescent apical cell.     

AUTORADIOGRAPHIC INVESTIGATION ON THE CELL KINETICS OF CRYPT EPITHELIA IN THE JEJUNUM OF THE MOUSE °CONFIRMATION OF STEADY STATE GROWTH WITH CONSTANT FREQUENCY DISTRIBUTION OF CELLS THROUGHOUT THE CYCLE

Cell kinetic parameters of cells in the crypt of the jejunum of the mouse were obtained autoradiographically. A number of different methods used in cell proliferation studies were applied to the same animal strain kept under constant conditions. In order to avoid effects of geometrical factors, squashes of isolated crypts were used.
The generation time was determined by the per cent labelled mitoses method of Quastler, modified by double labelling with ³H- and ¹⁴C-TdR. This modified method permits a more exact determination of the generation time. The duration of the cycle was 14 hr.
Double labelling experiments in which an injection of ³H-TdR was followed by an injection of ¹⁴C-TdR after 1 hr showed that the cell flux was 7.0%/hr at the beginning of the S-phase and 7.68%/hr at the end. Assuming steady state growth a constant cell flux of 7.15%/hr within the whole cycle can be derived from the measured generation time of 14 hr. These results clearly show that the crypt epithelia constitute a steady state system with constant frequency distribution of the cells throughout the cycle.
The per cent labelled mitoses method after a single injection of ³H-TdR as well as double labelling experiments with ³H- and ¹⁴C-TdR give an estimate of the S-phase of 8.0 or 7.4 hr respectively. Double determinations lead to a value of 0.54 or 0.52 hr respectively for the duration of mitosis and to values of 77% and 72%     

CELL CYCLE PROPERTIES OF DIFFERENTIATING SPERMATOGONIA IN ADULT SPRAGUE-DAWLEY RATS

The duration of the mitotic cycle and of its components was analysed for each of the six successive generations of differentiating spermatogonia (A₁, A₂, A₃, A4, intermediate and B), using radioautographed whole mounts of seminiferous tubules from testes of adult Sprague-Dawley rats. Cell cycles were determined from two successive waves of per cent labeled metaphases obtained during the period of 81 hr after a single dose of ³H-thymidine. Except for the A₁ spermatogonia, all spermatogonial types (A₂ to B) had similar cell cycle durations of 41-42.5 hr and comparable pre-DNA synthesis phases (G₁) of 11-13 hr. Although the combined duration of DNA synthesis (S) and the post-synthesis phase (G₂) remained identical for all the cell types including A₁, there was a progressive lengthening of the S period at the expense of G₂ during the process of spermatogonial maturation. This change was most marked during the transition from A₁ to A₃ spermatogonia when the S period increased from 14 hr to 21 hr, and the G₂ phase shortened from 13 hr to 7.5 hr. This feature seems to be unique to germ cells and may be associated with an increasing amount of heterochromatin in the nucleus. Excluding the development of type A₁ cells, the entire process of spermatogonial maturation lasted for 208 hr. Combined data on cell cycle times indicated that every 313 hr or 13 days, a new sequence of spermatogonial differentiation was initiated by the A₁ cells. This was equivalent to the duration of one 'cycle' of the seminiferous epithelium as measured by other techniques.     

VARIATION IN THE DURATION OF MITOSIS IN THE CRYPTS OF LIEBERKUHN OF THE RAT; A CYTOKINETIC STUDY USING VINCRISTINE

The variation in the duration of mitosis ( t _m) with cell position in the small intestinal crypts of the adult rat has been measured by a stathmokinetic technique using vincristine. The value for the whole crypt column was 0.43 hr, or 26 min. At the bottom of the crypt t _m was in excess of 1 hr, but rapidly decreased and throughout the greater part of the proliferative compartment was between 0.40 and 0.50 hr. At the top of the proliferative compartment an increase in t _m was demonstrated.
If the value of 0.43 hr for the whole crypt column is correct, then one argument for postulating the formation of metabolic DNA during differentiation in the small bowel epithelium of the rat becomes invalid. Variations in t _m within the crypt have been shown to increase the values of cell velocity obtained from cumulative birth rate diagrams. Finally further evidence has been presented for the existence of a slowly dividing subpopulation of cells at the base of the crypt. These cells may be important in crypt repopulation after damage with phase specific anti-tumour drugs.     

CYTOKINETICS OF RAT TRACHEAL EPITHELIUM STIMULATED BY MECHANICAL TRAUMA

Mild abrasion of rat tracheal epithelium results in irreversible damage to the superficial cells and stimulates the viable basal cells to participate in a nearly synchronous wave of DNA synthesis and mitosis. For the growth population as a whole, DNA synthesis started at 14 hr after injury and persisted for 16 hr. The duration of S in individual cells was determined autoradiographically by identifying the time at which a second pulse of DNA precursor (¹⁴C-TdR) was no longer incorporated by cells labelled with ³H-TdR at the onset of S. S was found to be 8–9 hr long. It was also determined that cells entering S at later times synthesized DNA for the same 8–9 hr period. T_G2 was calculated to be 21/2–31/2 hr by subtraction of T_s and 1/2T_M from the period from onset of DNA synthesis to metaphase. By making a second denuding lesion adjacent to the first injury, the cells were stimulated through at least another period of S. At the peak of the second wave of DNA synthesis (50 hr after injury) ¹⁴C-TdR was present in the same cells which had incorporated ³H-TdR administered at the mid-point of the preceding synthetic phase. The 28-hr interval between these two peaks of synthesis is the measure of cell cycle duration for these regenerating tracheal epithelial cells.     

NATURAL VARIATION IN THE S-PHASE OF RENEWING EPITHELIA OF THE POUCHLESS OPOSSUM, MARMOSA MITIS

The sites of cell proliferation and the duration of the S-phases in epithelia (tongue, stomach, duodenum, jejunum, ileum and descending colon) of the pouchless opossum, Marmosamitis , have been studied following the injection of tritiated thymidine. the sites of cell proliferation in these epithelia are not significantly different than those reported for rodent tissues. On the other hand, measurements of the mean duration of DNA synthesis revealed great variability in this phase: tongue (12.8 hr), stomach (>14.0 hr), duodenum (8.5 hr), jejunum (8.6 hr), ileum (9.7 hr) and descending colon (11-3 hr). In addition, the values obtained for the mean duration of t₂ (G₂+₂/¹M) are fairly constant among the various epithelia. It is concluded that the times obtained for the average duration of the S-phases are longer and more variable in M. mitis than similar observations reported on renewing epithelia of eutherian mammals.     

DURATION OF PREMEIOTIC DEOXYRIBONUCLEIC ACID SYNTHESIS AND THE STAGES OF PROPHASE I IN RABBIT OOCYTES

To estimate the duration of oocyte DNA synthesis 36, 3-day-old female rabbits received 3, 6, 9, 12, 15, or 18 injections of tritiated thymidine (thy-³H) at hourly intervals. The ovaries, removed at 1, 10, or 20 days after the first injection, were radioautographed. Counts made of the number of silver grains associated with oocyte nuclei in meiotic Prophase I indicate that the duration of DNA synthesis is between 9 and 12 hr. To determine the length of the stages of meiotic Prophase I, a group of 2-3-day-old rabbits was given a single sub-cutaneous injection of thy-³H, and the ovaries were removed at hourly and/or daily intervals after treatment. The minimum duration of leptotene was 3 hr and the maximum duration probably was less than 8 hr. The maximum durations of zygotene, pachytene, and diplotene were estimated to be 44, 216, and 96 hr, respectively. The interval from the end of oogonial DNA synthesis to the beginning ofpremeiotic DNA synthesis (G₂ + Mitosis + G₁) appeared to be less than 6 hr.     

Long duration of mitosis and consequences for the cell cycle concept, as seen in the isthmal cells of the mouse pyloric antrum. II. Duration of mitotic phases and cycle stages, and their relation to one another

Abstract. The kinetics of isthmal cells in mouse antrum were examined in three ways: (a) the duration of cell cycle and DNA-synthesizing (S) stage was measured by the 'fraction of labelled mitoses' method; (b) the duration of interphase and mitotic phases was determined from how frequently they occurred; and (c) mice were killed at various intervals after an intravenous injection of ³H-thymidine to time the acquisition of label by the various phases of mitosis.
The duration of the isthmal cell cycle was found to be 13.8 hr and that of the DNA-synthesizing (S) stage, 5.8 h. Estimates for the duration of the G₁ and G₂ stages were 6.8 and 1.0 hr, respectively.
From the frequency of mitotic phases, defined as indicated in the preceding article (El-Alfy & Leblond, 1987) and corrected for the probability of their occurence, it was estimated that prophase lasted 4.8 hr; metaphase, 0.2 hr; anaphase, 0.06 hr and telophase, 3.3 hr, while the interphase lasted 5.4 hr. In accordance with this, the duration of the whole mitotic process was 8.4 hr.
Ten minutes after an intravenous injection of ³H-thymidine, 38% of labelled isthmal cells were in interphase and 62% in early or mid prophase, while cells in late prophase and other mitotic phases were unlabelled. After 60 min, label was in late prophase, after 120 min, in mid telophase and after 180 min, in late telophase.
We conclude that there is overlap between some mitotic phases and cycle stages. Thus, while nuclei are at interphase during the early third of S, they are in prophase during the late two-thirds as well as during G₂. Also, nuclei are in telophase during the early half of G₁ but at interphase during the late half. Differences in nuclear diameter show that subdivision of both S and G₁ into early and late periods is practical.     

Growth pattern of the human promyelocytic leukaemia cell line HL60

Abstract. In order to characterize the growth pattern of the human promyelocytic leukaemia cell line HL60, its kinetic parameters were studied. The doubling time was calculated from serial cell counts, the duration of the various cell cycle phases from the analysis of the labelled mitoses curve, and quiescent population from continuous labelling experiments. Proliferation in culture was exponential up to a saturation density of about 3.0 × 10⁶ cells/ml, with a doubling time of 34.0 hr. The cell cycle duration was 24.3 ± 4.1 hr (SD), and that of the cell cycle phases was: G₁, 3.8 ± 2.2 hr; S, 15.1 ± 3 hr; and G₂, 5.4 ± 1.2 hr. The growth fraction was 0.85, and cell loss was restricted to the quiescent cells. The HL60 cell line, with fully characterized kinetics, provides a useful tool for the in vitro study of substances which may affect human leukaemic myelopoietic proliferation.     

THE HEAT RESISTANCE OF ESCHERICHIA COLI CELLS FROM CULTURES OF DIFFERENT AGES

SUMMARY: From the mortality curves of Escherichia coli cells heated at 55° in Ringer's solution both Decimal Reduction Times (DRTs) and 99–9% mortality times were obtained. In terms of these measures of heat resistance, cells harvested from broth cultures 0–8 hr old were more susceptible than those from more mature cultures. The time of commencement and the approximate duration of the logarithmic phase of growth of the organism in broth were determined from growth curves, and it was observed that the heat resistance was minimal during that phase.
Death rates were not always uniform for the whole of a given population. Particularly among young cultures, a period of rapid death was frequently followed by the slower death of a relatively small number of survivors. In one instance only was an initial period of slow death followed by one of more rapid death.     

Proliferation kinetics of rabbit articular chondrocytes in primary culture and at the first passage

The in-vitro proliferation kinetics of young rabbit articular chondrocytes were compared in primary culture and at the first passage. The growth curves labelling and mitotic indices, percentage labelled mitosis (PLM) curves and DNA content distributions by flow-microfluorometric analysis during a 7-day growth period were determined in both cases. The length of the cell cycle and the doubling time calculated from the exponential part of the growth curve were quite similar: Tc = 19 hr and Td = 20 hr for the primary culture, Tc = 17 X 3 hr and Td = 20 hr for the first passage. However, the growth curve and the DNA distribution during the 7-day period showed some differences. The duration of the lag period studied by the growth curve was longer in the primary culture than at the first passage. This phenomenon was also observed using the FCM analysis. The growth fraction determination on the second day of culture was in accordance with the lower proliferation capacity of the cells in primary culture. These data suggest that it would be better to study growth kinetics and drug modifications in articular chondrocytes at the first passage than in primary culture.     

EFFECTS OF SPREADING CORTICAL DEPRESSION ON THE INCORPORATION OF [14C]LEUCINE INTO PROTEINS OF RAT BRAIN

Abstract— Incorporation of dl -[1-¹⁴C]leucine into proteins of the cerebral cortex of the rat was measured during spreading cortical depression (CSD) evoked by a single topical application of 25% (w/v) KCI. Maximal inhibition (42 per cent) of the rate of incorporation occurred 1 hr after application of KCI. Spreading depression of 2–3 hr duration was associated with 22 per cent and 13 per cent decreases, respectively, of incorporation of labelled leucine. Specific activity of the free pool leucine was not decreased during CSD but appeared to be higher than controls at 20 min after initiation of CSD. The specific activity of the total free pool amino acids was also increased at 10, 20, 60 and 120 min after application of KCI.
The inhibitory effect of CSD on incorporation of leucine into proteins was uniformly distributed among the crude mitochondrial, microsomal and soluble subcellular fractions from brains of adult animals, while in fractions from 25-day old animals there appeared to be relatively more inhibition in the crude mitochondrial fraction.     

EEG power density during nap sleep: reflection of an hourglass measuring the duration of prior wakefulness

The relation between the duration of prior wakefulness and EEG power density during sleep in humans was assessed by means of a study of naps. The duration of prior wakefulness was varied from 2 to 20 hr by scheduling naps at 1000 hr, 1200 hr, 1400 hr, 1600 hr, 1800 hr, 2000 hr, and 0400 hr. In contrast to sleep latencies, which exhibited a minimum in the afternoon, EEG power densities in the delta and theta frequencies were a monotonic function of the duration of prior wakefulness. The data support the hypothesis that EEG power density during non-rapid eye movement sleep is only determined by the prior history of sleep and wakefulness and is not determined by clock-like mechanisms.     

INHIBITION OF EHRLICH TUMOR GROWTH IN MICE BY ELECTRIC INTERFERENCE THERAPY (IN VIVO STUDIES)

A study of solid tumor growth retardation by employing extremely low frequency (ELF) electric fields has been carried out. ELF electric fields were generated in tumor tissue in mice by the interference of two high frequency sinusoidal waves with the beat frequency centered at the tumor core. The results indicated a pronounced decrease in tumor growth rate in animals exposed to a 5-Hz interferential frequency for 1 hr daily. The 1 hr/day treatment produced a greater retardation effect than the 1 hr/week treatment. This indicates that treatment duration at the applied field frequency appears to play an important role in tumor growth delay. The dielectric properties of the tumor cells showed higher permittivity and conductivity values than homologous normal tissue. The permittivity of tumor cells treated daily with 5 Hz reaches nearly the same value as control tissue. Moreover, histological studies show that tumor tissues treated daily with the same frequency undergo partial regression and shrinkage of the aggregates of neoplastic cells leaving very little of them. We conclude that this new interferential technique is promising for tumor treatment in which a resonating electric field affects cell-to-cell communication.     

THE EFFECT OF ACUTE RENAL FAILURE ON MITOTIC DURATION OF MOUSE ILEAL EPITHELIUM

Previous percentage labelled mitoses studies in acutely uraemic mice have demonstrated a lengthening of the cell cycle and the DNA synthetic phase of ileal epithelium. The mitotic index was unaltered. Further studies have been performed to obtain an estimate of mitotic duration. Acute renal failure was produced by urinary outflow obstruction in male mice. Controls were subjected to sham operation. The mean number of cells per crypt cell column, number of mitoses present per crypt section and differential mitotic stage count were assessed 18 hr after operation for uraemic and control mice. The mean number of metaphases accumulated per crypt section over a 2 hr interval following colchicine injection was obtained in other groups of mice and the mitotic duration calculated. The mean number of mitoses per crypt section was 1.30 ± 0.46 for the controls and 1.48 ± 0.66 for the uraemic group. No evidence for a block in mitosis was indicated by the differential mitotic stage count. After applying Tannock's correction factor the mitotic duration was estimated to be 0.91 ± 0.18 hr for the control group and 2.81 ± 0.89 hr for the uraemic group. The difference in duration between the groups, 1.90 ± 0.91 hr, was significant (P≤0.05). Reduction in cell proliferation may explain the development of uraemic lesions in the gastrointestinal tract.     

CELL POPULATION KINETICS OF PLUCKED AND UNPLUCKED MOUSE SKIN I. UNIRRADIATED SKIN

A detailed study of the cellular proliferation kinetics in interfollicular plucked and unplucked mouse skin has been made in Swiss albino mice, using tritiated thymidine autoradiography. Diurnal variations in mitotic and labelling indices were demonstrated in both systems.
The mean cell cycle times for unplucked and plucked skin were estimated by four different methods and found to be 100 ± 10 and 47 ± 3 hr respectively. Most of the difference was due to the shortening of G₁ phase after plucking. Repeated labelling at intervals shorter than the DNA synthesis times resulted in all the basal layer cells becoming labelled, so that the growth fraction was unity, in unplucked and plucked skin.
A well-defined second wave of labelled mitoses was seen at about 100 hr after labelling the unplucked (i.e. normal) mouse skin.
A double labelling technique using ¹⁴C-TdR and ³H-TdR with a single layer of emulsion gave reasonable values for the duration of the DNA synthesis phase.     

AUTORADIOGRAPHIC CONFIRMATION OF THE MITOTIC DIVISION OF EVERY MOUSE JEJUNAL CRYPT CELL LABELLED WITH 3H-THYMIDINE

The question was investigated of whether for crypt epithelia of the jejunum of the mouse all cells labelled after a single injection of ³H-TdR subsequently divide or whether cells exist in the crypt which synthesize metabolic DNA and, therefore, do not undergo division after labelling.
A double labelling experiment was performed with a first injection of ³H-TdR followed 1 hr later by an injection of ¹⁴C-TdR. Then from double emulsion autoradiographs of isolated squashed crypts the number of ³H-only, ¹⁴C-only and double labelled cells and mitoses were counted.
The double labelling produced a narrow, 1 hr wide sub-population of ³H-only labelled cells. This subpopulation of S cells completed its division before labelled cells were lost from the crypts by migration onto the villi. The results showed that this subpopulation of ³H-only cells completely doubled within 3 hr and then remained constant through 6 hr. From this result it was concluded that every cell labelled after a single injection of ³H-TdR divides.
From the same autoradiographs the flow rate through the end of mitosis was measured. From the flow rate and the mitotic index a mitotic duration of 0·5 hr was determined. The agreement of this measured mitotic time with the value calculated from the labelling index, mitotic index and S duration is also strong evidence that every labelled cell divides.
Both experiments show that the intestinal crypt does not contain cells synthesizing metabolic DNA.     

CYTOKINETIC ANALYSIS OF THE JB-1 ASCITES TUMOUR AT DIFFERENT STAGES OF GROWTH

The cell kinetics of the murine JB-1 ascites tumour have been investigated on days 4, 7 and 10 after transplantation of 2·5 × 10⁶ cells. The experimental data, growth curve, percentage of labelled mitoses curves, continuous labelling curves and cytophotometric determination of single-cell DNA content have been analysed by means of a mathematical model for the cell kinetics. The important result was the existence of 8% non-cycling cells with G₂ DNA content in the 10-day tumour, while only 0·2 and 0% were observed in the 7- and 4-day tumours, respectively. The doubling times determined from the growth curve were 22·8, 70 and 240 hr, respectively, in the 4-, 7- and 10-day tumours. Growth fractions of 76, 67 and 44% were calculated for the same tumour ages. The mean cell cycle time increased from 14 to 44 hr from day 4 to 7 due to a proportional increase in the mean transit time of all phases in the cell cycle. In the 10-day tumour, the mean cell cycle changed to 41 hr and T _G1 decreased to 0·5 hr. The cell production rate was 4·3%/hr in the 4-day tumour, 1·2%/hr in the 7-day tumour and 1·0%/hr in the 10-day tumour. The cell loss rates in the same tumours were 1·3, 0·2 and 0·7%/hr, respectively. The analysis made it probable that the mode of cell loss was an age-specific elimination of non-cycling cells with postmitotic DNA content.     

INCORPORATION OF H3-THYMIDINE IN LEAF NUCLEI OF XANTHIUM PENNSYLVANICUM

The basic kinetics and the pattern of incorporation of H³-thymidine was studied in the leaf lamina of Xanthium pennsylvanicum. A method of foliar absorption was used to incorporate the radioisotope into leaf nuclei. The autoradiographic techniques employed provided data on the amount of the isotope incorporated. It was determined that 10 μc/ml (sp. act. 6.7 c/mmole) of H³-thymidine with 1–8 hr of isotopic growth and 4 hr of postisotopic growth gave the most satisfactory results. The percent of labelled nuclei and the number of grains per nucleus were presented as functions of isotopic and postisotopic growth periods. Distribution of grains in the nuclei approximated the Poisson distribution at 1 hr of isotopic growth. Increased time of isotopic growth changed the pattern of grain distribution. No deleterious effects were observed using an 8-hr period of isotopic growth, but prolonged incubation time significantly decreased the proportion of mitotic figures in the lamina. The amount of incorporation of the DNA precursor expressed as percent of labelled nuclei was linear to about 16 hr of isotopic growth and thereafter decreased gradually. As indicated by the average number of grains per nucleus, H³-thymidine incorporation increased to about 16 hr, and soon after reached a saturation level. The percent of labelled nuclei and the number of grains per nucleus decreased as a function of the postisotopic growth period. However, they were significantly greater in the lamina near the vein than in the lamina region at some distance from the vein. The radioactive precursor was initially absorbed by the cells of the lamina and was subsequently translocated into the vascular system. There it was circulated and made available to the dividing cells near veins of the lamina. This region may be a metabolically distinct part of the lamina with significantly higher rates of incorporation and mitotic turnover.     

DIURNAL VARIATION IN THE LABELING INDEX OF MOUSE EPIDERMIS: A DOUBLE ISOTOPE AUTORADIOGRAPHIC DEMONSTRATION OF CHANGING FLOW RATES

A diurnal rhythmicity in the labeling index was observed in the epidermis of hairless mice, injected with either ¹⁴C- or ³H-thymidine, at different times during a 24 hr period. A modified autoradiographic technique, using ¹⁴C- and ³H-thymidine and two overlying emulsion layers, makes it possible to clearly differentiate synthesizing cells which are singly labeled with either carbon-14 or tritium, and cells labeled with both isotopes. At various times during a 24 hr period, hairless mice were injected with thymidine-2-¹⁴C and colcemid, followed at 2 or 3 hr by a second injection of ³H-thymidine. The labeling indices were calculated for the ¹⁴C- and ³H-thymidine injection times. These labeling indices were consistent with the control, single isotope, labeling indices and exhibited the same diurnal rhythm. Cells singly labeled with ³H- or ¹⁴C-thymidine have either started or completed DNA synthesis during the interval between the two injections. Flow rates into and out of DNA synthesis, throughout the 24 hr period, can be calculated from these singly labeled cells. The flow rates varied rhythmically throughout the day and paralleled changes in the labeling indices. The influx and efflux flow rates, at all times measured, were not equal. The influx flow rate was reflected in the efflux rate at a time later equal to the duration of S. By means of these flow rates, the per cent of cells in DNA synthesis was calculated for each hour during a 24 hr period. The resulting labeling index curve matches the observed 24 hr diurnal rhythm in labeling indices. By extension of these flow rates through mitosis, the resulting mitotic index curve is comparable to the reported 24 hr diurnal rhythm in mitotic indices.