Influence of 3HTdR on the circadian rhythm—a model analysis for mouse epidermis

A mathematical model for the normal circadian rhythm in epidermis is formulated. It reproduces the experimental data for mice if the duration of either the G₁ or the S phase oscillates. As a second step, the model is generalized to account for the influence of ³HTdR on the circadian rhythm. A simultaneous interpretation of experimental curves for LI, PLM, the mitotic rate (MR) and the phase indices G₁I, SI, G₂I and MI measured by micro-spectrophotometry or flow cytometry, can be given by the following hypothesis. (a) Of the S phase cells (as measured by DNA content), only the most mature fraction is labelled. Some of these labelled cells die (or loose their label) within a few hours. The free label is then reutilized. (b) For about 12 hr the flux of unlabelled cells from G₁ into S phase is accelerated. These cells stay correspondingly longer in S so that their cell cycle time is scarcely affected. (c) The normal circadian triggering is disturbed for at least 36 hr after labelling. The implications of this hypothesis for double labelling experiments are discussed.     

Specific expression of proteins and phosphoproteins in 3T3 T mesenchymal stem cells at distinct growth arrest and differentiation states

Abstract. Murine mesenchymal stem cells can be induced to arrest their growth at a series of growth and differentiation states in the G₁ phase of the cell cycle. These include the predifferentiation arrest state (G_D) at which the integrated control of proliferation and differentiation is mediated, the growth factor/serum deficiency arrest state (G_S), and the nutrient deficiency arrest state (G_N). Cells at states of reversible nonterminal differentiation (G_D?) and irreversible terminal differentiation (TD) can also be isolated. In this paper we have employed 1- and 2-dimensional (D) gel electrophoresis to evaluate changes in specific proteins that occur during the various growth and differentiation states of 3T3 T mesenchymal stem cells. The protein composition of membrane, microsome and cytosol preparations of cells arrested at G_D, G_S and G_N states was determined by 2-D gel electrophoresis. More than 50 distinct polypeptides could be identified for each arrest state in gels analysed by a silver staining procedure or by autoradiography following [³⁵S]-methionine labelling. A second series of studies established that a more limited number of differences could be identified if phosphoproteins were analysed by 1-D gel electrophoresis in cells at the G_S, G_D, G_D?. and TD states. These results established that one distinct 37 kD phosphoprotein is present in all growth arrested cells and that two distinct differentiation-associated phosphoproteins with molecular weights of 29 kD and 72 kD are present in cells at the G_D? and TD states. Thus, the composition of proteins and phosphoproteins in mesenchymal stem cells serves to characterize different states of growth arrest and differentiation. The identification of differential protein expression provides an opportunity to test their functional role in growth and differentiation control.     

An improved mathematical method to estimate DNA synthesis time of bromodeoxyuridine-labelled cells, using FCM-derived data

Abstract. Chinese hamster ovary cells in vitro were pulse-labelled with bromodeoxyuridine (BrdUrd and were then allowed to progress through the cell cycle. Every half hour after labelling, cells were harvested and prepared for simultaneous flow cytometric determination of DNA content and incorporated BrdUrd, with the intercalating dye propidium iodide and with a monoclonal antibody against incorporated BrdUrd, respectively. The relative movement (RM), i.e. the relative mean DNA content of the moving cohort of BrdUrd-labelled cells in relation to that of G₁ and G₂ cells, was calculated. RM was then used to calculate DNA synthesis time (T_S), at all post-labelling times (t). Since labelled cells in G₂ and mitosis (M) in addition to S phase cells, are included in the cohort of moving labelled cells, and since the time of G₂ and M (T_g2+M) phases is finite, a non-linear relationship exists between RM and post-labelling time. Because of this, the use of a linear formula in the calculation of T_S yields results that are affected by t. We found that RM data can be corrected with regard to T_G2+M resulting in the derivation of a non-linear T_S formula. This non-linear T_S formula gave results that were nearly independent of t. Moreover, windows were set in the mid DNA distributions for G₁, S and G₂+ M cells in the bivariate DNA v. BrdUrd cytograms, to estimate the fraction of BrdUrd-labelled cells in each window at every post-labelling time. Plots of the fraction of BrdUrd-labelled cells v. post-labelling time were then made for each window. T_S obtained in this way was in agreement with T_S obtained with the corrected RM method. In conclusion, we present a method to calculate T_s which theoretically first makes the determination of RM independent of T_G2+M, and secondly compensates for the non-linear function of RM with post-labelling time caused by accumulation of BrdUrd-labelled cells in G₂+ M.     

Changes in thioacidolysis products of lignin in wheat straw as affected by SO2 treatment and passage through the gastro-intestine of sheep

Sheep were fed two rations based on untreated (WS) and SO₂-treated (SO₂–WS) wheat straw. The neutral detergent fiber (NDF) preparations and water soluble lignins (WSL) isolated from straws, rumen liquor and feces were subjected to thioacidolysis, to study the effect of treatment and passage through the gastro-intestine on monolignol composition of the lignins. SO₂ treatment affected guaiacyl (G) and syringyl (S) contents of the straw cell walls with major effect on S, reducing G to 66% and S to 26% of their original concentrations, thus changing the S/G ratio in the CW fraction from 1.6 in WS to 0.64 in SO₂–WS. Concomitantly, as a reflection of the above-mentioned there was an increase in the S/G ratio of the WSL, from 0.54 in WS to 1.27 in SO₂–WS. Fecal CW residues were richer in p-hydroxyphenyl (H), G and S as compared with the original straw cell walls; the gap being particularly large in the SO₂–WS treatment in which the concentrations of monolignols in CW residues were increased by more than ten-fold. The concentration of WSL in the rumen of sheep fed the SO₂–WS ration was about eight-fold that found in the rumen of the WS sheep, most likely a result of the combined chemical (SO₂) and digestive (rumen microorganisms) effects. Most G and S were found in the CW fraction of the untreated straw, whereas in the SO₂-treated straw, the majority of G and S were in the water soluble lignin fraction. In the untreated wheat straw ration (WS), 25.7% of G and 36.6% of S disappeared from the digestive tract. Feed CW-G was higher by 18% and feed CW-S by 60% than the respective fecal CW components in the WS treatment, accompanied by only modest quantitative changes between feed and feces, in the G and S components of WSL. In the SO₂–WS treatment, CW-G was increased from 3.48 to 6.26 g/day (by 80%) and CW-S from 2.58 to 6.56 g/day (by 154%) between feed and feces. The digestibility of total acetyl bromide lignin (ABL) was in accord with that of the monolignol data namely, a small proportion (11.4%) of total ABL disappeared from the gastro-intestine of the WS sheep, but a minor, almost negligible percentage (2.94%) of the ABL was digested by the SO₂–WS sheep.     

Analytical expressions for PLM(t), CL(t) and CLM(t) without use of Laplace transforms

Based on the age density functions for each phase of the cell life cycle (G₁, S, G₂ and M) in an exponentially growing steady state population derived by Trucco &; Brockwell (1968), the expressions for the percentage labeled mitoses curve [PLM(t)], the continuous labeling curve [CL(t)] and the continuous labeled mitotic curve [CLM(t)] are obtained explicitly without use of Laplace transforms. This approach is useful in describing the cell population when the steady state is disturbed due to, for example, irradiation. The mitotic index [MI(t)] for this case is considered.     

Autophagy is required for G1/G0 quiescence in response to nitrogen starvation in Saccharomyces cerevisiae

In response to starvation, cells undergo increased levels of autophagy and cell cycle arrest but the role of autophagy in starvation-induced cell cycle arrest is not fully understood. Here we show that autophagy genes regulate cell cycle arrest in the budding yeast Saccharomyces cerevisiae during nitrogen starvation. While exponentially growing wild-type yeasts preferentially arrest in G₁/G₀ in response to starvation, yeasts carrying null mutations in autophagy genes show a significantly higher percentage of cells in G₂/M. In these autophagy-deficient yeast strains, starvation elicits physiological properties associated with quiescence, such as Snf1 activation, glycogen and trehalose accumulation as well as heat-shock resistance. However, while nutrient-starved wild-type yeasts finish the G₂/M transition and arrest in G₁/G₀, autophagy-deficient yeasts arrest in telophase. Our results suggest that autophagy is crucial for mitotic exit during starvation and appropriate entry into a G₁/G₀ quiescent state.     

Synaptic Electrogenesis in Eel Electroplaques

Whether evoked by neural or by chemical stimulation, the synaptic membrane of eel electroplaques contributes a depolarizing electrogenesis that is due to an increased conductance for Na and K. The reversal potential (E_S) is the same for the two modes of synaptic activation. It is inside-positive by about 30–60 mv, or about midway between the emf's of the ionic batteries for Na (E_Na) and K(E_K). The total conductance contributed by synaptic activity (G_S) varied over a fivefold range, but the individual ionic branches, G_SSNa, and G_SSK, change nearly equally so that the ratio G_SSNa:G_SSK is near unity. G_SSK increases independently of the presence or absence of Na in the bathing medium, and independently of the presence or absence of the electrically excitable G_K channels. When activated, the synaptic membrane appears to be slightly permeable to Ca and Mg. When the membrane is depolarized into inside positivity the conductance of the synaptic components decreases and approaches zero for large inside-positive values. Thus, the synaptic components become electrically excitable when the potential across the membrane becomes inside-positive, responding as do the nonsynaptic components, with depolarizing inactivation.     

Model analysis of circadian rhythms in mouse epidermal basal cell proliferation

Computer simulations were made of circadian variations in six observed cell kinetic variables in the basal cell layer of hairless mouse epidermis. Different mathematical models were used and simultaneously fitted to all observed variables by an automatic method. The analysis shows that the origin of the circadian variations in cell kinetics in hairless mouse epidermis is not in the G₁ phase or at the $\text{G}{}_1\text{S}$ transition alone as concluded from other studies, but must result from circadian variations in the duration of S and G₂ phases. The results show that the data are compatible with the existence of circadian variations in the G₁- and M-phase durations, although such variations, unlike the S and G₂ variations, was not necessary to obtain a good fit of the model to the data. The results also indicate that the data are compatible with the existence of transient resting states of limited duration in S- and G₂-phases.     

A Model for the Action of Vinblastine in Vivo

A model for the action of vinblastine (VLB) on cells multiplying exponentially in vivo with a generation time, T_G, has been derived. It is based on the assumption that cells attempting to pass through mitosis in the presence of VLB lose their proliferative capacity and that this lethal effect occurs only when the cells are exposed to a concentration of VLB which is above a critical value, C_k. The model leads to two predictions. First, that the percentage of cells surviving at any time after exposure to a dose, D, of VLB is 100% if D < D_k and decreases to 0% after a time, T_G, following a dose D ≥ D_k·2^{T G/T1/2}, where D_k represents the dose of VLB required to produce the concentration C_k, and T_1/2 is the half-life of the VLB in vivo. Second, that the time, T_G, at which the percentage of cells surviving an exposure to VLB, at doses greater than D_k·2^{U G/T1/2}, decreases to zero should be equal to the generation time of the cells. Both of these predictions were confirmed experimentally which indicates that the model adequately explains the action of VLB in vivo.     

Modified minimal model for effect of physical exercise on insulin sensitivity and glucose effectiveness in type 2 diabetes and healthy human

The Bergman’s minimal model of glucose and insulin plasma levels is commonly used to analyse the results of glucose tolerance tests in humans. In this paper, we present the modified minimal model with plasma insulin compartment under the assumption that if the plasma glucose compartment drops below the basal glucose levels, the rate of insulin entering the plasma glucose compartment is zero. Insulin is cleared from the plasma insulin compartment at a rate proportional to the amount of insulin in the plasma insulin compartment. The modified minimal model was used to study the effect of physical exercise via parameters of a mathematical model to qualitative the magnitude of changes in insulin sensitivity (S _I) and glucose effectiveness (S _G) in response to exercise in type 2 diabetes and healthy human. The short-term effects of physical exercise in type 2 diabetes did not improve S _G, but markedly improved the low S _I values found in type 2 diabetes, indicating that the effects of exercise on S _I are quantitatively important in the interpretation of training-related S _I changes and may even be therapeutically useful in type 2 diabetes patients. Physical exercise is indicated either to prevent or delay the onset of type 2 diabetes or to assure a good control of type 2 diabetes by increasing insulin sensitivity.     

Responses of Water and Salt Parameters to Groundwater Levels for Soil Columns Planted with Tamarix chinensis

Groundwater is the main water resource for plant growth and development in the saline soil of the Yellow River Delta in China. To investigate the variabilities and distributions of soil water and salt contents at various groundwater level (G_L), soil columns with planting Tamarix chinensis Lour were established at six different G_L. The results demonstrated the following: With increasing G_L, the relative soil water content (RWC) declined significantly, whereas the salt content (S_C) and absolute soil solution concentration (C_S) decreased after the initial increase in the different soil profiles. A G_L of 1.2 m was the turning point for variations in the soil water and salt contents, and it represented the highest G_L that could maintain the soil surface moist within the soil columns. Both the S_C and C_S reached the maximum levels in these different soil profiles at a G_L of 1.2 m. With the raise of soil depth, the RWC increased significantly, whereas the S_C increased after an initial decrease. The mean S_C values reached 0.96% in the top soil layer; however, the rates at which the C_S and RWC decreased with the G_L were significantly reduced. The RWC and S_C presented the greatest variations at the medium (0.9–1.2 m) and shallow water levels (0.6 m) respectively, whereas the C_S presented the greatest variation at the deep water level (1.5–1.8 m).The RWC, S_C and C_S in the soil columns were all closely related to the G_L. However, the correlations among the parameters varied greatly within different soil profiles, and the most accurate predictions of the G_L were derived from the RWC in the shallow soil layer or the S_C in the top soil layer. A G_L at 1.5–1.8 m was moderate for planting T. chinensis seedlings under saline groundwater conditions.     

THE RELATION BETWEEN DNA SYNTHESIS AND CHROMOSOME STRUCTURE AS RESOLVED BY X-RAY DAMAGE

Vicia faba root tip cells were treated for short periods with tritiated thymidine, either immediately before or after exposure of roots to x-rays, and autoradiograph preparations were analysed in an attempt to test the hypothesis that chromatid type (B') aberrations are induced only in those chromosome regions that have synthesized DNA prior to x-irradiation, whereas chromosome type (B') aberrations are induced only in unduplicated chromosome regions. Studying the relation between presence or absence of label at loci involved in aberrations, in cells irradiated at different development stages, and the pattern of labelling in cells carrying both types of aberration leads to the conclusion that B' aberrations are induced only in unreplicated chromosome regions. Following replication, only B' aberrations are induced, but these aberrations are also induced in chromosome regions preparing to incorporate DNA. It is suggested that the doubled response of the chromosome to x-rays prior to DNA incorporation might reflect a physical separation of replicating units prior to replication. The aberration yields in damaged cells which were irradiated in G₁ S, and early G₂ were in the ratio of 1.0:2.0:3.2. The data indicate that the increased yield of B' in early G₂ relative to S cells may be a consequence of changes in the spatial distribution of the chromosomes within the nucleus.     

Microfluorometry analysis II. A Bayesian approach.

In flow microfluorometry (FMF) analysis cells stained with a fluorescent dye that binds specifically to DNA are passed through the instrument. The number of cells in the population having a fluorescence intensity is recorded in a single channel of a multichannel pulse height analyzer. The result is a DNA fluorescence histogram for the population.A method for decomposing an FMF histogram into its G₁, S and G₂ + M components, corresponding to similarly designated phases of the cell cycle is given. This technique can also be applied to find the parameters in all of the previous approaches. The parameters are calculated by iteration which eliminates the need for non-linear optimization procedures.     

The R* rule and energy flux in a plant-nutrient ecosystem

The R* rule predicts that the species that can survive in steady state at the lowest level of limiting resource, R*, excludes all other species. Simple models indicate that this concept is not necessarily consistent with Lotka's conjecture that an ecological system should evolve towards a state of maximum power, Max(G), where G is the power, or rate of biomass production of the system. To explore the relationship in detail, we used a published model of a plant-nutrient system in which a plant can use various strategies, S, of allocation of energy between foliage, roots, and wood. We found that the allocation strategy, S_MinR*, that leads to , where is a limiting nutrient in soil pore water in our model (and equivalent to R* in Tilman's notation), is the same as the strategy, S_{MaxG_root}, for which energy flux to roots is maximized. However, that allocation strategy is different from the strategy, S_MaxG, that produces maximum power, or maximum photosynthetic rate, for the plant system, Max(G). Hence, we conclude that and Max(G) should not necessarily co-occur in an ecological system. We also examined which strategy, S_fit, was fittest; that is, eliminated any other strategies, when allowed to compete. The strategy S_fit differed from S_MinR*, S_MaxG, and S_{MaxG_root}, which we demonstrated mathematically. We also considered the feasible situation in which a plant is able to positively influence external nutrient input to the system. Under such conditions, the strategy, S_{MaxG_root}, that maximizes energy flux to roots was the same as the strategy, S_MaxR*, that leads to maximum concentration of available nutrient in soil pore water, , and not same as S_MinR*, for .     

CELL PROLIFERATION AND SPECIALIZATION DURING ENDOCHONDRAL OSTEOGENESIS IN YOUNG RATS

Endochondral osteogenesis was studied autoradiographically in ribs and tibiae of 32 Long-Evans rats injected with 1 µc/gm H³-thymidine at 6 days of age and sacrificed at intervals between 1 hour and 2 weeks later. Proliferation and specialization of bone cells were studied by analyses of (a) the percentage of mitoses which were labeled, (b) the percentage of labeled nuclei in bone cells, and (c) grain counts. The following conclusions were derived: The various types of bone cells represent different functional states of the same cell. Cell division is usually restricted to cells in the morphologically unspecialized "osteoprogenitor" state. Specialized bone cells arise by modulation of osteoprogenitor cells. The average cell generation time is shortest in the metaphysis, longest in the periosteum, and intermediate in the endosteum. The average duration of DNA synthesis is relatively constant (about 8 hours). With increasing length of generation time there is a slight increase in G₂ + mitosis, but the major change is a lengthening of G₁. After dividing, cells in the osteoprogenitor state may remain within the progenitor pool or undergo modulation of cell type, specializing as osteoblasts or becoming incorporated in osteoclasts.     

Inhibition of non-muscle myosin II leads to G0/G1 arrest of Wharton's jelly-derived mesenchymal stromal cells

Background aimsMesenchymal stromal cells (MSCs) have remarkable clinical potential for cell-based therapy. Wharton's jelly-derived mesenchymal stromal cells (WJ-MSCs) from umbilical cord share unique properties with both embryonic and adult stem cells. MSCs are found at low frequency in vivo, and their successful therapeutic application depends on rapid and efficient large-scale expansion in vitro. Non-muscle myosin II (NMII) has pivotal roles in different cellular activities, such as cell division, migration and differentiation. We performed this study to understand the role of NMII in proliferation and cell cycle progression in WJ-MSCs.MethodsWJ-MSCs were cultured in the presence of blebbistatin, and cell cycle analysis was performed using flow cytometry, proliferation kinetics, senescence assay and gene expression profile using polymerase chain reaction array.ResultsWhen cultured in the presence of blebbistatin, an inhibitor of NMII adenosine triphosphatase activity, WJ-MSCs exhibited dose-dependent reduction in proliferative potential along with increase in cell size and induction of early senescence. Inhibition of NMII activity also affected cell cycle progression in WJ-MSCs and led to an increase in the percentage of cells in G₀/G₁ phase with a corresponding reduction in the percentage of cells in G₂/M phase. Blebbistatin-induced G₀/G₁ arrest of WJ-MSCs was further associated with up-regulation of cell cycle inhibitory genes CDKN1A, CDKN2A and CDKN2B and down-regulation of numerous genes related to progression through S and M phases of the cell cycle.ConclusionsOur study demonstrates that inhibition of NMII activity in WJ-MSCs leads to G₀/G₁ arrest and alteration in the expression levels of certain key cell cycle-related genes.     

5-AMINOURACIL TREATMENT : A Method for Estimating G2

Treatment of Vicia faba lateral roots with a range of concentrations of 5-aminouracil (5-AU) indicate that cells are stopped at a particular point in interphase. The timing of the fall in mitotic index suggests that cells are held at the S - G₂ transition. When cells are held at this point, treatments with 5-AU can be used to estimate the duration of G₂ + mitosis/2 of proliferating cells. Treatment with 5-AU can also be used to demonstrate the presence of subpopulations of dividing cells that differ in their G₂ duration. Using this method, 5-AU-induced inhibition, we have confirmed that in V. faba lateral roots there are two populations of dividing cells: (a) a fast-dividing population, which makes up ~85% of the proliferating cell population and has a G₂ + mitosis/2 duration of 3.3 hr, and (b) a slow-dividing population, which makes up ~15% of dividing cells and has a G₂ duration in excess of 12 hr. These estimates are similar to those obtained from percentage labeled mitosis (PLM) curves after incorporation of thymidine-³H.     

Design and synthesis of new antitumor agents with the 1,7-epoxycyclononane framework. Study of their anticancer action mechanism by a model compound

This article describes the design, synthesis and biological evaluation of a new family of antitumor agents having the 1,7-epoxycyclononane framework. We have developed a versatile synthetic methodology that allows the preparation of a chemical library with structural diversity and in good yield. The synthetic methodology has been scaled up to the multigram level and can be developed in an enantioselective fashion. The study in vitro of a model compound, in front of the cancer cell lines HL-60 and MCF-7, showed a growth inhibitory effect better than that of cisplatin. The observation of cancer cells by fluorescence microscopy showed the presence of apoptotic bodies and a degradation of microtubules. The study of cell cycle and mechanism of death of cancer cells by flow cytometry indicates that the cell cycle arrested at the G₀/G₁ phase and that the cells died by apoptosis preferably over necrosis. A high percentage of apoptotic cells at the subG₀/G₁ level was observed. This indicates that our model compound does not behave as an antimitotic agent like nocodazole, used as a reference, which arrests the cell cycle at G₂/M phase. The interaction of anticancer agents with DNA molecules was evaluated by atomic force microscopy, circular dichroism and electrophoresis on agarose gel. The results indicate that the model compound has not DNA as a target molecule. The in silico study of the model compound showed a potential good oral bioavailability.     

Phreatophytes under stress: transpiration and stomatal conductance of saltcedar (Tamarix spp.) in a high-salinity environment

Background and aims

We sought to understand the environmental constraints on an arid-zone riparian phreatophtye, saltcedar (Tamarix ramosissima and related species and hybrids), growing over a brackish aquifer along the Colorado River in the western U.S. Depth to groundwater, meteorological factors, salinity and soil hydraulic properties were compared at stress and non-stressed sites that differed in salinity of the aquifer, soil properties and water use characteristics, to identify the factors depressing water use at the stress site.

Methods

Saltcedar leaf-level transpiration (E_L), LAI, and stomatal conductance (G_S) were measured over a growing season (June–September) with Granier and stem heat balance sensors and were compared to those for saltcedar at the non-stress site determined in a previous study. Transpiration on a ground-area basis (E_G) was calculated as E_L?×?LAI. Environmental factors were regressed against hourly and daily E_L and G_S at each site to determine the main factors controlling water use at each site.

Results

At the stress site, mean E_G over the summer was only 30 % of potential evapotranspiration (ET_o). G_S and E_G peaked between 8 and 9 am then decreased over the daylight hours. Daytime G_S was negatively correlated with vapor pressure deficit (VPD) (P?<?0.05). By contrast, E_G at the non-stress site tracked the daily radiation curve, was positively correlated with VPD and was nearly equal to ET_o on a daily basis. Depth to groundwater increased over the growing season at both sites and resulted in decreasing E_G but could not explain the difference between sites. Both sites had high soil moisture levels throughout the vadose zone with high calculated unsaturated conductivity. However, salinity in the aquifer and vadose zone was three times higher at the stress site than at the non-stress site and could explain differences in plant E_G and G_S.

Conclusions

Salts accumulated in the vadose zone at both sites so usable water was confined to the saturated capillary fringe above the aquifer. Existence of a saline aquifer imposes several types of constraints on phreatophyte E_G, which need to be considered in models of plant water uptake. The heterogeneous nature of saltcedar E_G over river terraces introduces potential errors into estimates of ET by wide-area methods.