Comparison of growth and cell proliferation kinetics during mouse molar odontogenesis in vivo and in vitro

Abstract. The growth of embryonic first lower mouse molars in vitro was slow and reduced in comparison with in vivo development: the volume of teeth removed on day 15 and 16 of gestation and cultured for 6 days did not exceed the volume reached at day 18 in vivo . The volume of teeth removed on day 17 and 18 and cultured for 6 days either remained constant or decreased. The appearance of post-mitotic odontoblasts and ameloblasts was delayed in vitro . This behaviour might be correlated with a lengthening of the cell cycle ( T _c). In vitro , the average durations of T _c (established by the percentage labelled mitoses technique) were 17.4–20.2 hr and 19.1–19.4 hr for pre-odontoblasts and pre-ameloblasts respectively. In vivo , the corresponding T _c values were 13.9 hr and 13.5 hr. The coordination of mitotic activities of pre-odontoblasts and pre-ameloblasts existing in vivo was maintained in vitro , and therefore seemed to require intra-dental control mechanisms. Non-specific extra-dental serum factors may affect the duration of T _c.     

THE KINETICS OF GRANULOSA CELLS IN DEVELOPING FOLLICLES IN THE MOUSE OVARY

This investigation describes the kinetics of the granulosa cells in medium-sized follicles type 3b, 4 and 5a in ovaries of 28-day-old Bagg mice. the method of labelling with ³H-thymidine followed by high resolution autoradiography is used in the experimental work, which consist of determining percentage labelled mitosis (PLM-) and continuous labelling (CL-) curves. In order to analyse the data by computer two alternative hypotheses A and B are set up. Both include the assumptions of no cell loss, exponential growth and a resting compartment Q. In hypothesis A cells from Q re-enter the mitotic cycle via the normal DNA-synthesis compartment S_p. Hypothesis B includes beside compartment S_p a special DNA-synthesis compartment S_q where only cells from Q are synthesizing DNA, and these cells re-enter the mitotic cycle via the G₂ compartment. the mean transit time in S_q is considered to be longer than the mean transit time in S_q. On the basis of the hypothesis mathematical expressions for the PLM- and CL-curves are obtained, and by means of a computer the theoretical curves are fitted to the experimental values: thereby all relevant cell kinetical parameters are estimated. Hypothesis B seems to give the best fit between the theoretical and experimental curves. the estimated parameters are: mean cycle times, μ_c= (56.1 hr, 56.1 hr and 22.3 hr for type 3b, 4 and 5a respectively), doubling times, T _D= (96.4 hr, 118.6 hr and 59.1 hr) and the proportion of cells in Q, p _Q = (0.60, 0.71 and 0.69).     

KINETICS OF THE INNER ENAMEL EPITHELIUM IN THE ADULT RAT INCISOR DURING ACCELERATED ERUPTION

Inner enamel epithelial (IEE) cell production was compared in accelerated and normal eruption (control). Each group consisting of thirty rats received 1 μCi/g tritiated thymidine. The animals were sacrificed at short time intervals up to 14 hr after injection. The excised incisors were cut mid-sagittally and processed autoradiographically.
The fast growing incisor produces twice as many cells as the control. Increased cell production is achieved in two ways: proliferative pool expansion (by 25.5%) and generation time ( t _c) shortening to 16 hr ( t _c= 23 hr in the control). Generation time shortening resulted mainly from a diminution in t _g1= 8.6 hr ( t _g1= 14.1 hr in the control) and t _s which equaled 5.5 hr ( t _s= 7 hr in control). Mitotic times which equaled 0.4 hr and t _g2, 1.5 hr were identical in both groups.
The eruption/IEE cell production ratio equals 1.1 in both groups.     

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.     

Involvement of Calcium Influx in Muscarinic Cholinergic Regulation of Phospholipase C in Cerebellar Granule Cells

Abstract: Inositol phosphate accumulation on carbachol stimulation of rat cerebellar granule cells shows a marked dependence on factors affecting cytosolic Ca²⁺ concentration ([Ca²⁺]_c). After 5 min, potassium depolarisation caused a modest accumulation of inositol phosphates but augmented the response to carbachol by a factor of 2–3. These effects of potassium were dependent on an extracellular source of calcium and could be partially blocked by specific (nifedipine) and nonspecific (verapamil) calcium channel blockers. Measurements of [Ca²⁺]_c under a range of stimulatory conditions demonstrated a close correlation between the elevation of [Ca²⁺]_c and agonist-stimulated phospholipase C (PLC) activity. The maximal potentiation of carbachol-stimulated inositol phosphate accumulation was achieved using 20 m M KCl, which increased [Ca²⁺]_c from ∼20 to ∼75 n M , indicating the involvement of relatively low threshold Ca²⁺ channels and the high sensitivity of the relevant PLC to small changes in [Ca²⁺]_c. By contrast, increases in [Ca²⁺]_c induced by the Ca²⁺ ionophore ionomycin were associated with more modest and less potent effects on agonist-stimulated PLC. These results demonstrate a cooperative interaction between a receptor/G protein-regulated PLC and voltage-stimulated elevations of [Ca²⁺]_c, which may function to integrate ionotropic and metabotropic signalling mechanisms in cerebellar granule cells.     

Measurement of c-myc protein content and cell cycle kinetics of normal and spontaneously transformed murine mastocytes by bivariate flow cytometry

Abstract. Progressive in vitro culturing of interleukin-3 (IL-3) dependent normal murine mastocytes (PB-3) resulted in a variant cell line (PB-1) able to grow without exogenous IL-3 and which was tumorogenic in syngenic mice. Bivariate flow cytometry was used to evaluate the c-myc protein and DNA content of PB-3 and PB-1 cells. The c-myc protein was detected by specific monoclonal antibodies. Kinetic characteristics of PB-3 and PB-1 cell lines, namely, the duration of the G¹, S and G₂+ M cell cycle phases were also evaluated using the bromodeoxyuridine (BrdU) pulse-chase method and BrdU/DNA flow cytometry. Levels of c-myc protein in PB-1 cells were about twofold higher than those of PB-3 cells in all cell cycle phases. Mean duration of the cell cycle ( T _c) was 15-3 h for PB-3 cells and 12-4 h for PB-1 cells. Shortening in T _c for the transformed cells was due to a decrease of nearly 30% in mean duration of the G ₁ phase (from 8 h to 5.7 h). No significant differences were found in the duration of the S and G₂+ M phases. These results indicate that acquired IL-3 independency in vitro and tumorogenicity of PB-1 cells were accompanied by a doubling of c-myc protein level and by a parallel shortening, or bypass, of the regulatory events within the G¹ phase of the cell cycle.     

Pharmacological evidence that stalk cell differentiation involves increases in the intracellular Ca(2+) and H(+) concentrations in Dictyostelium discoideum

Differentiation-inducing factors (DIFs) are required for stalk cell formation in Dictyostelium discoideum . In the present study, in order to support our hypothesis that DIFs may function via increases in [Ca²⁺]_c and [H⁺]_c, we investigated the combined effects of 5,5-dimethyl-2,4-oxazolidinedione (DMO, a [H⁺]_c-increasing agent), thapsigargin (Tg) and BHQ ([Ca²⁺]_c-increasing agents) on in vitro stalk cell formation in several strains. DMO, in combination with Tg or BHQ, induced stalk cell formation in a DIF-deficient mutant HM44. Although the rates of stalk cell induction by the drugs were low in the presence of cerulenin (an inhibitor of endogenous DIF production) in HM44 and V12M2 (a wild-type strain), the drugs succeeded in inducing sufficient stalk cell formation when a small amount of DIF-1 was supplied. Furthermore, co-addition of DMO, BHQ and a small amount of DIF-1 also induced sufficient stalk cell formation in AX-4 (an axenic strain) and HM1030 ( dmtA ⁻) but not in CT15 ( dimA ⁻). The drugs suppressed spore formation and promoted stalk cell formation in both HM18 (a sporogenous mutant) and 8-bromo-cAMP-stimulated V12M2. The present results suggest that DIFs function, at least in part, via increases in [Ca²⁺]_c and [H⁺]_c in D. discoideum .     

Epidermal conductance, stomatal density and stomatal size among genotypes of Sorghum bicolor (L.) Moench

Abstract. The ability of a plant to survive severe water deficits depends on its ability to restrict water loss through the leaf epidermis after stomata attain minimum aperture. At this stage, the rate of water loss is regulated by the epidermal conductance (g_c). Low g_c would be a useful selection criterion to identify genotypes with enhanced survival capability. Consequently, variation in g_c among Sorghum bicolor (L.) Moench genotypes was evaluated. Since there is little conclusive evidence linking g _c with leaf waxiness, alternative hypotheses relating g _c to stomatal trails were also examined. Epidermal conductance varied from 6.3 to 17.6mmol m⁻² s⁻¹ among sorghum genotypes. It was unrelated to stomatal pore length which varied with genotype and to pore depth which was similar for all genotypes measured. However, g _c, increased with increasing stomatal density. This indicates that stomatal density plays a direct role in water loss even at very low conductances. The association of low stomatal density with low g _c is consistent with the hypothesis that at the smallest stomata aperture, water loss from the epidermis above guard cell teichodes becomes a significant source of leaf water loss. Since low g _c is directly related to crop survival under severe water deficits, it is recommended that genotypes with low g _c. be selected using the selection criterion of stomatal density.     

甲胎蛋白在体外对人肝癌细胞生长的影响

本文用MTT比色法观察了甲胎蛋白(AFP)在体外对人肝癌细胞生长的影响。结果表明,AFP能促进SMMC-7721人肝癌细胞的生长。当AFP与AFP抗体合用时,AFP抗体能减弱AFP对SMMC-7721细胞生长的促进作用;AFP抗体单用对此种细胞的生长亦有抑制作用。另一方面,在相同的实验条件下,AFP和AFP抗体对HL-60人白血病细胞的生长无明显影响;提示AFP的促生长作用具有一定的肿瘤细胞特异性,并非一种蛋白质对培养细胞的非特异性营养作用。此外,AFP亦能促进MCF-7人乳腺癌细胞的生长,AFP抗体对此种细胞的生长有抑制作用。由于MCF-7细胞存在功能性AFP受体,也能合成和分泌AFP。这就提示,人肝癌细胞中的AFP很可能与其受体特异性结合,产生促生长效应。确切机制尚待进一步阐明。     

THE LYMPHOCYTES OF CHRONIC LYMPHOCYTIC LEUKEMIA: THEIR PROLIFERATION AND CELL CYCLE KINETICS

Lymphocytes obtained from CLL patients exhibited a delayed and reduced response to PHA when cultured in diffusion chambers. DNA synthesis (8–10 hr) and general time (15–19 hr) of the late-developing CLL blasts were consistent with normal values ( T _s: 8–10 hr; T _c: 14–17 hr). However, the G₂ period of CLL blasts seemed more variable, and their mitotic index during the response at 5–6 days was 30–50% of the values determined for normal blasts during their peak response at 2–3 days.     

ON THE RESTING STAGES OF THE JB-1 ASCITES TUMOUR

Cytophotometric determination of single-cell DNA after repeated ³H-thymidine labelling of the JB-1 ascites tumour in the plateau phase of growth showed a massive accumulation of unlabelled cells with both G₁ and G₂ content. Autoradiography combined with cytophotometry or colcemid block demonstrated that some of these unlabelled cells were rapidly triggered into the cell cycle when plateau tumours were transferred to new hosts. This indicated that tumour cells may be held up in non-cycling stages corresponding to both the G₁ and the G₂ phase of the cell cycle.     

The critical-period concept for juvenile survival and its relevance for population regulation in young sea trout, Salmo trutta

An example of density-dependent regulation is provided by a long-term investigation (1966-present) of a population of migratory trout (estuarine and sea trout), Salmo trutta L., in a Lake District stream. Evidence for the concept of a critical period for the survival of young fish is briefly reviewed and found to be rather equivocal. The concept is, however, relevant to the trout population. Loss rates were high before but low after a critical survival time ( t_c days after fry emergence) that varied between year-classes (range 33-70 days) and was inversely density-dependent on egg density. Survivor density and loss rates were strongly density-dependent on egg density before t _c, but proportionate survival with stable loss-rates occurred after t _c. Some trout established feeding territories soon after emergence and the number of fish without territories decreased from a high initial value to a negligible value at t _c. Fish size at t_c was not constant but increased as t _c increased. The range of t _c for the different year-classes was similar to that for survival times of unfed fry in the laboratory. A new stock-recruitment model, incorporating t _c, has been developed for the trout population and shown to be related to the model (Ricker curve) used in the long-term study. The critical time can also be regarded as the critical age for survival in young trout; this concept may be relevant to other fish species.     

Modulation of Ion Gradients and Glutamate Release in Cultured Cerebellar Granule Cells by Ouabain

Abstract: Upon addition of the cardiac glycoside ouabain to cultured cerebellar granule cells, an immediate increase in intracellular free sodium is evoked mediated by two pathways, a voltage-sensitive channel blocked by tetrodotoxin and a channel sensitive to flunarizine. Ouabain induces a steady plasma membrane depolarization in low Ca²⁺ medium; whereas in the presence of Ca²⁺, a distinct discontinuity is observed always preceded by a large increase in intracellular free Ca²⁺ ([Ca²⁺]_c). The plateau component of the increase can be inhibited additively by the L-type Ca²⁺ channel antagonist nifedipine, the spider toxin Aga-Gl, and the NMDA receptor antagonist MK-801. Single-cell imaging reveals that the [Ca²⁺]_c increase occurs asynchronously in the cell population and is not dependent on a critical level of extracellular glutamate or synaptic transmission between the cells. A prolonged release of glutamate is also observed that is predominantly Ca²⁺ dependent for the first 6–10 min after the evoked increase in [Ca²⁺]_c. This release is four times as large as that observed with 50 m M KCl and is predominantly exocytotic because release was inhibited by tetanus toxin, the V-type ATPase inhibitor bafilomycin, and Aga-Gl. It is proposed, therefore, that ouabain induces a period of membrane excitability culminating in a sustained exocytosis above that observed upon permanent depolarization with KCl.     

The role of photosynthesis/light relationships in determining lower depth limits of Characeae in South Island, New Zealand lakes

1. The maximum depth of colonization of aquatic macrophytes ( Z _c) was investigated in eighteen South Island, New Zealand lakes. The downward attenuation coefficient for photosynthetically active radiation ( K _d(PAR)) was calculated and the spectral characteristics of the lakes determined with a spectroradiometer.
2. Characean algae dominated the deepest communities in sixteen of the study lakes.
3. Z _c was significantly related to K _d(PAR) by the relationship Z _c = 4.5/ K _d– 2.2.
4. From measurements of the photosynthetic properties of Chara corallina (Kl. ex Willd., em R.D.W.) and incident radiation over the course of a year we calculated the depth at which daily net photosynthesis would be equal to zero for each day of the year. An annual average of this depth was significantly related to Z _c with an r 2 of 0.86.
5. Correcting K _d(PAR) for spectral quality and taking into account the potential absorption spectrum of a characean meadow did not improve the relationships.
6. We suggest that relationships established between K _d(PAR) and Z _c of characean algae in South Island, New Zealand lakes can be explained to a great extent by light limitation of photosynthesis.     

The role of photosynthesis/light relationships in determining lower depth limits of Characeae in South Island, New Zealand lakes

1. The maximum depth of colonization of aquatic macrophytes ( Z _c) was investigated in eighteen South Island, New Zealand lakes. The downward attenuation coefficient for photosynthetically active radiation ( K _d(PAR)) was calculated and the spectral characteristics of the lakes determined with a spectroradiometer.
2. Characean algae dominated the deepest communities in sixteen of the study lakes.
3. Z _c was significantly related to K _d(PAR) by the relationship Z _c = 4.5/ K _d– 2.2.
4. From measurements of the photosynthetic properties of Chara corallina (Kl. ex Willd., em R.D.W.) and incident radiation over the course of a year we calculated the depth at which daily net photosynthesis would be equal to zero for each day of the year. An annual average of this depth was significantly related to Z _c with an r 2 of 0.86.
5. Correcting K _d(PAR) for spectral quality and taking into account the potential absorption spectrum of a characean meadow did not improve the relationships.
6. We suggest that relationships established between K _d(PAR) and Z _c of characean algae in South Island, New Zealand lakes can be explained to a great extent by light limitation of photosynthesis.     

Light and oxygen stress in Spirulina platensis (cyanobacteria) grown outdoors in tubular reactors

As the effects of light and oxygen stress in algae on mass culture has not been intensively studied, we investigated them in Spirulina platensis under outdoor conditions in controlled tubular reactors where the respective roles of each stress can be distinguished. It was observed that exposure of this cyanobacterium at two oxygen concentrations (ca 20 and 53 mg 1⁻¹) caused very little change in the ratio between variable and maximum fluorescence (F_v/F_m) during the day even when the culture was grown at higher oxygen concentration (about 7% lower in the evening than in the morning). Vice-versa, when the photochemical efficiency of PSII (photon yield, Φ_c) was measured, a reduction of about 20% was observed. Neither the F_v/F_m ratio nor the Φ_c of the culture grown at the lower oxygen concentration changed significantly during the day. The daily productivity of the culture exposed to the higher oxygen concentration was reduced by about 20%. Laboratory cultures bubbled with air or pure oxygen under continuous light showed a similar response; i.e., a smaller decrease in F_v/F_m (17%) than in the Φ_c (56%) after 4 h. After 32 h of culture in pure oxygen, a total lysis of the cells occurred. Our results support the hypothesis that photoinhibition and photooxidation, two traditionally linked terms, although often closely associated under similar environmental conditions, may comprise two types of stress with different sites of inhibition.     

DNA SYNTHESIS TIME IN LEUKAEMIC CELLS AS MEASURED BY THE DOUBLE LABELLING AND THE PERCENTAGE LABELLED MITOSES METHODS

Values of T _s provided by the double labelling method have been compared with those given by the percentage labelled mitoses curve for blast cells in the peripheral blood of a patient with plasma cell leukaemia and of rats bearing a transferable acute leukaemia. the double labelling method was carried out giving the first label (³H-thymidine) in vivo and the second label (¹⁴C-thymidine) in vitro with several values for the interval between the two labels. T _s was calculated by fitting regression lines to the results obtained. Data for percentage labelled mitoses were analysed by computer. For the plasma cell leukaemia values of T _s= 17.1 ± 7.0 hr and T _s= 19.8 ± 3.4 hr, and for the rat leukaemia values of 8.7 ± 1.7 hr and 9.0 ± 1.7 hr (7.1 hr corrected for exponential growth) were obtained from the percentage labelled mitoses and double labelling methods respectively. It is concluded that the double labelling method is valid for the study of cell proliferation in leukaemic blast cells.     

EFFECTS OF ENVIRONMENTAL FACTORS ON MECHANICAL PROPERTIES OF THE CELL WALL IN RICE COLEOPTILES

Cell wall properties determined by the stress-relaxation technique were studied with coleoptiles of rice seedlings grown under different environmental conditions. The cell wall was simulated by a viscoelastic model consisting of either four or an infinite number of Maxwell components. Reciprocal of relaxation time for the first component in the former model (1/τ₁) and minimum and maximum relaxation times (T_o and T_m) in the latter, in addition to the stress/strain ratio, were parameters representing cell wall properties. Parameters changed depending on the ages and regions of the coleoptilles used and 011 the environmental conditions under which rice seedlings were grown. Effects on cell wall properties of aeration during submerged growth, excision of the coleoptile tip, and exposure to small doses of red and/or far-red light were examined. In most cases, high values of 1/τ₁ and of T_m and small values of T_o were consistent with the growth potentiality of cells, while the stress/strain ratio seemed to be a consequence of elongation growth.     

CELL PROLIFERATION IN THE CASTRATE MOUSE SEMINAL VESICLE IN RESPONSE TO TESTOSTERONE PROPIONATE

The cell proliferation kinetics following induced DNA synthesis in the mouse seminal vesicle were measured after treatment with testosterone propionate. Fraction labelled mitosis curves at 24, 48 and 72 hr after injection gave t ₂ values of 1·5, 2·0 and 1·8 hr respectively, and t _s values of 10·5, 8·0 and 8·0 hr. T _c measured 48 hr after stimulation was 17·5 hr. Growth fraction rose from 0·14 at 24 hr to 0·64 at 48 hr, and fell to 0·32 by 72 hr. A simple model is proposed in which the rise and fall of mitotic index and labelled index is determined by the 'cell distribution ratio'.     

GROWTH AND PHOTOSYNTHESIS OF MARINE SYNECHOCOCCUS (CYANOPHYCEAE) UNDER IRON STRESS

Prokaryotic picoplankton such as Synechococcus are relatively abundant in putatively Fe-limited high-nutrient, low-chlorophyll (HNLC) regions of the oceans. The physiology of Synechococcus under Fe stress has been studied less than eukaryotic algae. Recent evidence suggests that although biomass and growth rates of Synechococcus are not typically Fe limited in situ, cells may still exhibit symptoms of Fe stress. We grew Synechococcus A2169 and WH7803 in laboratory batch cultures in the artificial medium Aquil and enriched natural seawater, at a series of Fe concentrations and Fe:macronutrient ratios, and with either nitrate or ammonium as the sole nitrogen source. Cell yields, and in some experiments exponential specific growth rate (μ), were more readily Fe limited in the Atlantic isolate WH7803 than in the equatorial Pacific isolate A2169. In both strains, final cell yields spanned about an order of magnitude and decreased continuously with Fe concentration from 900 to 3.6 nM (150 μM N, 10 μM P), whereas μ decreased much less and only at Fe concentrations below 90 nM. Synechococcus yield was controlled by both absolute Fe concentration and Fe:macronutrient ratio, but μ was determined primarily by absolute Fe concentration. Contrary to theoretical predictions, neither yield nor μ was higher in Fe-limited cells grown in ammonium compared to nitrate. Under severe Fe stress, cellular chlorophyll (Chl) content and light-saturated gross photosynthetic capacity (P^cell_m) decreased proportionately, and dark respiration (R^cell_d) increased, such that net P^cell_m was extremely low but gross P^Chl_m was unchanged. This is the first report of an absolute increase in R^cell_d under Fe stress in phytoplankton.