Maintenance of mitotic homeostasis in somatic cell populations

Recent theoretical and experimental studies have indicated that the rate of proliferative activity in somatic cell populations can be modulated by regulation of the transmembrane potential level (E_m). These studies have shown that such modulation is accomplished through control of DNA synthesis preparations, presumably through variations in the intracellular ionic conditions which accompany transmembrane potential changes. This capability for modulating mitotic activity appears to provide a potential mechanism for maintenance of mitotic homeostasis in somatic cell populations. The present paper, after a consideration of the primary kinetic requirement for homeostasis attainment, proceeds to develop two independent concepts of E_m-modulated homeostatic control: (1) “statistical” or “passive” homeostasis obtained by maintenance of a particular, constant E_m level at which proliferation rate equals death rate for the cells under study; and (2) “modulated” or “active” homeostasis obtained by local induction of cellular depolarization, from a high level where mitosis is blocked to a low level where mitosis is stimulated, by loss of intimate surface contact or chemical interaction with a dying cell. The problem of hormone-activated periodic or cyclic proliferative homeostasis is also interpreted in terms of E_m variation.     

Discrimination between closed and open forms of lipases using electrophoretic techniques

The enhanced catalytic activity of lipases is often associated with structural changes. The three-dimensional (3D) structures showed that the covalently inhibited lipases exist under their open conformations, in contrast to their native closed forms. We studied the inhibition of various lipases--human and dog gastric lipases, human pancreatic lipase, and Humicola lanuginosa lipase--by the octyl-undecyl phosphonate inhibitor, and we measured the subsequent modifications of their respective electrophoretic mobility. Furthermore, the experimental values of the isoelectric points found for the native (closed) and inhibited (open) lipases are in agreement with theoretical calculations based on the electrostatic potential. We concluded that there is a significant difference in the isoelectric points between the closed (native) and open (inhibited) conformations of the four lipases investigated. Thus, analysis of the electrophoretic pattern is proposed as an easy experimental tool to differentiate between a closed and an open form of a given lipase.     

Variants of asynchronous DNA synthesis and mitoses in multinucleate cells induced by cytochalasin B

Cases of asynchronous progression with separate nuclei of S-period and initial mitotic stages in multinucleate cells were discovered in Chinese hamster cell cultures during a prolonged action of cytochalasin B (7 days) and after its stopping (7 days of cell cultivation without drug). The interphase asynchrony under experimental conditions vary in value corresponding to the level of interphase asynchrony in spontaneous multinucleate cells in control cultures. So, the interphase asynchrony in cytochalasin B-induced multinucleate cells is suggested not to be connected with the drug action. Fusion of heterophase cells and a high level of proliferation activity of multinucleate cells seem to be the main reason of interphase asynchrony both in control cultures and in experimental conditions. Unlike the interphase asynchrony, the appearance of the mitotic asynchrony in multinucleate cells is shown to be connected with the action of cytochalasin B. The high level of the mitotic asynchrony remains after the stopping of drug action. A conclusion is made that mitotic asynchrony of nuclei, along with multipolar mitosis and cytokinesis inhibition, is one more display of the cytotoxic action of cytochalasin B on mitosis.     

Transport theory for growing cell populations

The partial differential equation that describes the growth of cell populations whose maturation rate is random is developed. The equation resembles that used in classical transport theory but mitotic boundary conditions and the restriction of the maturation rate to non-negative values brings out new features and new problems. This is a generalization of a previously published formulation in which cells could make transitions at random between only two maturation velocities: a characteristic velocity and zero. Growth rates, cycle time distributions and pulsed labeled mitotic curves are calculated for a simple choice of parameters. A numerical algorithm that is suited to the solution of the transport equation is given.     

Kinetics and thermodynamics of protein adsorption: a generalized molecular theoretical approach

The thermodynamics and kinetics of protein adsorption are studied using a molecular theoretical approach. The cases studied include competitive adsorption from mixtures and the effect of conformational changes upon adsorption. The kinetic theory is based on a generalized diffusion equation in which the driving force for motion is the gradient of chemical potentials of the proteins. The time-dependent chemical potentials, as well as the equilibrium behavior of the system, are obtained using a molecular mean-field theory. The theory provides, within the same theoretical formulation, the diffusion and the kinetic (activated) controlled regimes. By separation of ideal and nonideal contributions to the chemical potential, the equation of motion shows a purely diffusive part and the motion of the particles in the potential of mean force resulting from the intermolecular interactions. The theory enables the calculation of the time-dependent surface coverage of proteins, the dynamic surface tension, and the structure of the adsorbed layer in contact with the approaching proteins. For the case of competitive adsorption from a solution containing a mixture of large and small proteins, a variety of different adsorption patterns are observed depending upon the bulk composition, the strength of the interaction between the particles, and the surface and size of the proteins. It is found that the experimentally observed Vroman sequence is predicted in the case that the bulk solution is at a composition with an excess of the small protein, and that the interaction between the large protein and the surface is much larger than that of the smaller protein. The effect of surface conformational changes of the adsorbed proteins in the time-dependent adsorption is studied in detail. The theory predicts regimes of constant density and dynamic surface tension that are long lived but are only intermediates before the final approach to equilibrium. The implications of the findings to the interpretation of experimental observations is discussed.     

The influence of the peptide bond on the conformation of amino acids: a theoretical and FT-IR matrix-isolation study of N-acetylproline

A combined experimental matrix-isolation FT-IR and theoretical study has been performed to investigate the conformational behavior of N-acetylproline. The conformational landscape of N-acetylproline was explored using successively higher computational methods, i.e. HF, DFT(B3LYP) and finally MP2. The exploration resulted in 10 conformations with a relative energy difference smaller than 22 kJ.mol(-1) at the HF/3-21G level of theory. These conformations led to six different conformations after DFT(B3LYP) optimizations. Further optimization at the MP2/6-31++G** level of theory resulted in the same six conformations, all of them with an energy difference smaller than 11.5kJ.mol(-1). One conformation with an intramolecular H-bond was found which was energetically the most favorable conformation. The vibrational and thermodynamical features were calculated using the DFT and MP2 methodologies. In the experimental matrix-isolation FT-IR spectrum, the most stable conformation was dominant and at least two non-H-bonded conformations could be identified. An experimental rotamerization constant between the H-bonded and the other non-H-bonded conformations was estimated and appeared to agree reasonably well with the theoretical MP2 predictions. Some new spectral features of N-acetylproline compared to proline were discovered which might be used to discriminate between the acetylated and non-acetylated form.     

Regulation of mitotic activity in rat vaginal epithelium: relationships between the level of its inhibitor (G2-chalone) and estrogens.

The level of a tissue-specific inhibitor of mitotic activity (G2-chalone) and mitotic activity in the vaginal mucosa of cycling rats of varying age and castrated rats were studied. A direct correlation between the level of the inhibitor and mitotic index is found in cycling animals. Both parameters are maximal during estrus and minimal in proestrus, when estrogen level in blood circulation is the highest. The undulating variations in G2 inhibitor level during estrous cycle are less pronounced and the concentrations of the inhibitor in relevant phases are significantly lower in aged females than in adult rats. Administration of estradiol benzoate (1 microgram/100 g) to castrated female rats was followed by a significant decrease in mitotic inhibitor level in vaginal mucosa within 12 hrs. This, in turn, was followed by a rise in mitotic activity 18 hr after estrogen administration. Therefore, the estrogen exerts its effect on mitotic activity in target tissue after it has induced a decrease in the level of the antimitotic factor (G2-chalone).     

Frequency Spectrum of Transepithelial Potential Difference Reveals Transport-Related Oscillations

How epithelia transport fluid is a fundamental issue that is unresolved. Explanations offered include molecular engines, local transcellular osmosis, local paracellular osmosis, and paracellular fluid transport. On the basis of experimental and theoretical work done on corneal endothelium, a fluid transporting epithelium, we suggest electroosmotic coupling at the level of the intercellular junctions driven by the transendothelial electrical potential difference as an explanation of paracellular fluid transport. We collect frequency spectra of that potential difference in real-time. For what we believe is the first time for any epithelium, we report that, unexpectedly, the potential difference displays oscillations at many characteristic frequencies. We also show that on both stimulating cell activity and inhibiting ion transport mechanisms, there are corresponding changes in the oscillations amplitudes that mirror changes known previously in rates of fluid transport. We believe these findings provide a novel tool to study the kinetics of electrogenic elements such as channels and transporters, which from this evidence would give rise to current oscillations with characteristic periods going from 150 ms to 8 s.     

A multivalent combination of experimental antituberculosis DNA vaccines based on Ag85B and regions of difference antigens

Two candidate DNA vaccines based on the proteins CFP10 and CFP21 encoded by regions of difference (RDs) of Mycobacterium tuberculosis were evaluated individually and in multivalent combination with the immunodominant protein Ag85B for induction of protective immune responses against experimental tuberculosis. Experimental DNA vaccines induced substantial levels of cell-mediated immune responses as indicated by marked lymphocyte proliferation, significant release of the Th1 cytokines IFN-gamma and IL-12 (p40), and predominant cytotoxic T cell activity. High levels of antigen-specific IgG1 and IgG2a antibodies observed in the sera of immunized mice depicted strong humoral responses generated by DNA vaccine constructs. The multivalent combination of three DNA vaccine constructs induced maximal T cell and humoral immune responses. All the experimental vaccines imparted significant protection against challenge with M. tuberculosis H(37)Rv (in terms of colony-forming unit reduction in lungs and spleen) as compared to vector controls. The level of protection exhibited by multivalent DNA vaccine formulation was found to be equivalent to that of Mycobacterium bovis BCG observed both at 4 and 8 weeks post-challenge. These results show the protective potential of the multivalent DNA vaccine formulation used in this study.     

Symmetry breaking and convergent extension in early chordate development

The initiation of axis, polarity, cell differentiation, and gastrulation in the very early chordate development is due to the breaking of radial symmetry. It is believed that this occurs by an external signal. We suggest instead spontaneous symmetry breaking through the agency of the Turing-Child field. Increased size or decreased diffusivity, both brought about by mitotic activity, cause the spontaneous loss of stability of the homogeneous state and the evolution of the metabolic pattern during development. The polar metabolic pattern is the cause of polar gene expression, polar morphogenesis (gastrulation), and polar mitotic activity. The Turing-Child theory explains not only the spontaneous formation of the invagination in gastrulation but also the coherent cell movement observed in convergence and extension during gastrulation and neurulation. The theory is demonstrated with respect to experimental observations on the early development of fish, amphibian, and the chick. The theory can explain a multitude of experimental details. For example, it explains the splayed polar progression of reduction in the fish blastoderm. Reduction starts on that side of the blastoderm margin, which will initiate invagination several hours later. It progresses toward the blastoderm center and somewhat laterally from this future "dorsal lip". This is precisely as predicted by a Turing-Child system in a circle. And for a fish like zebrafish with a blastoderm that is slightly oval, reduction is observed to progress along the long axis of the ellipse, which is what Turing-Child theory predicts. In general the shape and the chemical nature of the experimental patterns are the same as predicted by the Turing couple (cAMP, ATP). Embryological polarity and convergent extension are based on polar eigenfunction and saddle-shaped eigenfunction, respectively.     

The pattern of cell division in the early development of the sea urchin,Paracentrotus lividus

In the sea urchin Paracentrotus lividus, the first three cleavages are synchronous in all blastomeres. Upon the segregation of the micromeres at the fourth cleavage, a vegetal-animal gradient of cell division begins; i.e., the closer the cells are to the micromeres, the earlier they enter mitosis. The phase difference between mitotic cells along the vegetal-animal axis increases concurrently with the increase in cell number. At the blastula stage, mitoses appear organized in clusters. From the mesenchyme-blastula stage onward, the clusters become smaller and most of the mitotic cells are solitary. The size of the clusters increases upon treatment with colchicine, thus suggesting the existence of pacemaker units of mitotic activity. We confirm that the division of the micromeres is out of phase with respect to the other blastomeres. Of the eight cells originating from the first division of the micromeres, only the four outer ones continue to cleave; the four inner ones appear to have lost the ability to divide. The overall mitotic activity is high during cleavage and suddenly drops to very low levels around hatching. An interpretation of these results is presented in terms of a nonlinear oscillators theory.     

Contact inhibition of division: involvement of the electrical transmembrane potential

Measurements of simultaneous mitotic activity, electrical transmembrane potential (E_m), and cell density levels in both 3T3 and Chinese hamster ovary (CHO) cell cultures reveal that a 5- to 6-fold increase in the E_m level is associated with development of mitotic arrest at saturation densities. This rise occurs both in confluent monolayers and in interior areas of isolated colonies, and is independent of the rate at which confluence is attained. The E_m rise is accompanied by a substantial decrease in intracellular Na. Electron microscopy of saturated CHO monolayer sections shows from 46 to 63% of the cell surfaces to be in close apposition (<300 Å spacing). These results for contact inhibited cultures support the hypothesis that mitotic activity may be functionally coupled with the E_m level and associated ionic concentration levels. It is suggested that contact inhibition of mitosis may result from a reduction in synthesis of mitogenically essential RNA following a decrease in intracellular Na produced by contact-induced alteration of surface ion-transport activity.     

THE EFFECT OF A MEDIUM DOSE (430 ROENTGENS) OF X-RAY IRRADIATION ON RESTING CELLS OF THE LIVER

The mitotic activity of regenerating liver cells after a single dose (430 r) of x-ray irradiation was studied. In every group of the experimental animals (white rats), the mitotic activity (mitotic index) and the number of abnormal mitotic figures were determined. The results indicated that resting cells irradiated a short time before mitotic activity showed reactions similar to those of cells irradiated during mitotic activity. The disturbances in the irradiated mitotically active cells were only quantitatively different from those in the irradiated resting cells. The disturbances in the irradiated resting cells depended upon the time interval between the irradiation and the beginning of mitotic activity stimulated by partial hepatectomy. It was found that the shorter the time interval, the more pronounced were the disturbances and the more similar they became to those of irradiated mitotically active cells. Conversely, the longer the time interval between the irradiation and the beginning of mitotic activity, the less pronounced were the disturbances and the more similar they became to those of the non-irradiated control cells. A discussion is presented as to whether or not the lesions of resting cells caused by a single medium dose of x-ray irradiation are reversible, and whether such lesions are only brought to light by the process of mitosis or whether the process of mitosis renders it possible for these lesions to develop.     

Enrichment and stability: a phenomenological coupling of energy value and carrying capacity

Simple predator-prey models with a prey-dependent functional response predict that enrichment (increased carrying capacity) destabilizes community dynamics: this is the 'paradox of enrichment'. However, the energy value of prey is very important in this context. The intraspecific chemical composition of prey species determines its energy value as a food for the potential predator. Theoretical and experimental studies establish that variable chemical composition of prey affects the predator-prey dynamics. Recently, experimental and theoretical approaches have been made to incorporate explicitly the stoichiometric heterogeneity of simple predator-prey systems. Following the results of the previous experimental and theoretical advances, in this article we propose a simple phenomenological formulation of the variation of energy value at increased level of carrying capacity. Results of our study demonstrate that coupling the parameters representing the phenomenological energy value and carrying capacity in a realistic way, may avoid destabilization of community dynamics following enrichment. Additionally, under such coupling the producer-grazer system persists for only an intermediate zone of production--a result consistent with recent studies. We suggest that, while addressing the issue of enrichment in a general predator-prey model, the phenomenological relationship that we propose here might be applicable to avoid Rosenzweig's paradox.     

Proliferation kinetics of mouse tongue epithelium under normal conditions and following single dose irradiation

Epithelial proliferation in the ventral surface of mouse tongue follows a pronounced circadian rhythm with a peak in mitotic activity at 10.00 a.m., preceded by a wave of DNA synthesis 8 h earlier. Nearly all cells (85%) pass through G2 and mitosis immediately after the S-phase; they subsequently divide again, usually after 2 or 3 days, indicating cohorts of cells with different G1-duration. The fraction of all nucleated cells comprised in one daily proliferation wave is about 20%, indicating a turnover time of the nucleated cell compartment of about 5 days. Cytotoxic injury by a single radiation dose of 20 Gy causes a steep decrease in cell counts, leading to complete denudation after 9–13 days. The difference between the latent period before ulceration and the tissue turnover time is explained by a marked proliferative activity of the doomed cells. The mitotic index increases steeply after day 1 to three times the control level, but most mitotic figures display gross abnormalities such as multipolar spindles or chromosome clumping. As a consequence cells with abnormal or multiple nuclei appear in the basal layers 3 days post irradiation and subsequently migrate to the upper layers. After denudation the epithelium rapidly becomes restored, with a phase of transient hyperplasia on days 13–14. Normal architecture is regained by day 15. Over the whole healing period the mitotic index remains at a high level, with most of the mitoses appearing histologically normal.     

Thermoosmosis through charged membranes. theoretical analysis of concentration dependence

A theoretical equation for thermoosmosis through charged membranes in electrolyte solutions is derived from nonequilibrium thermodynamics. The theory shows that the volume flux through the membrane is proportional to the temperature difference across the membrane. The proportionality constant, i.e., the thermoosmotic coefficient is a function of electrolyte concentration. The electrolyte concentration dependence of the thermoosmotic coefficient calculated is compared with our previous experimental results. Agreement between theory and experiments is satisfactory.     

Proliferation kinetics of mouse tongue epithelium under normal conditions and following single dose irradiation.

Epithelial proliferation in the ventral surface of mouse tongue follows a pronounced circadian rhythm with a peak in mitotic activity at 10.00 a.m., preceded by a wave of DNA synthesis 8 h earlier. Nearly all cells (85%) pass through G2 and mitosis immediately after the S-phase; they subsequently divide again, usually after 2 or 3 days, indicating cohorts of cells with different G1-duration. The fraction of all nucleated cells comprised in one daily proliferation wave is about 20%, indicating a turnover time of the nucleated cell compartment of about 5 days. Cytotoxic injury by a single radiation dose of 20 Gy causes a steep decrease in cell counts, leading to complete denudation after 9-13 days. The difference between the latent period before ulceration and the tissue turnover time is explained by a marked proliferative activity of the doomed cells. The mitotic index increases steeply after day 1 to three times the control level, but most mitotic figures display gross abnormalities such as multipolar spindles or chromosome clumping. As a consequence cells with abnormal or multiple nuclei appear in the basal layers 3 days post irradiation and subsequently migrate to the upper layers. After denudation the epithelium rapidly becomes restored, with a phase of transient hyperplasia on days 13-14. Normal architecture is regained by day 15. Over the whole healing period the mitotic index remains at a high level, with most of the mitoses appearing histologically normal.     

试验设计中的重复试验次数的确定

研究单因素、双因素的方差分析,提出因素水平差异显著与否,主要取决于试验效应的平方和（或试验效应方差）与随机误差.引入平均相对试验效应,在其已知前提下,给出保证一定概率的因素水平差异显著的最小总试验次数、或每水平的最小平均重复试验次数.     

Fit, efficiency, and biology: some thoughts on judging food web models

Revealing the processes that determine who eats whom, and thereby the structure of food webs, is a long running challenge in ecological research. Recent advances include development of new methods for measuring fit of models to observed food web data, and thereby testing which are the ‘best’ food web models. The best model could be considered the most efficient with relatively few parameters and high explanatory power. Another recent advance involves adding some additional biology to food web models in the form of foraging theory based on maximisation of energy intake as the predictor of species' diets in food webs. While it is interesting to compare efficiency among food web models, we believe that such comparisons at least should be interpreted with caution, since they do not account for any differences in motivation, formulation, and potential that might also exist among models. Furthermore, we see an important but somewhat neglected role for experimental tests of models of food web structure.     

Peculiarities of mitosis and nucleolar characteristics of the birch plantlets under antropogenous pollution

A study was made of some cytogenetic characteristics (mitotic activity, the level and spectrum of pathological mitosis, nucleolar features in root tip cells) in birch plantlets. The seeds were collected in four districts of Voronezh and in the ecologically clean territory. The index of mitotic activity has a considerable resistance to anthropogenous pollution. In the experimental areas, the level and spectrum of pathological mitosis increase. In contaminated areas we observed changes of nucleolar characteristics (the increased surface area of nucleoli and their higher number in cells, the increased number of cells with highly active types of nucleoli, the appearance of residual nucleoli). These changes can be considered as possible mechanisms of adaptation to stress due to antropogenous pollution. It is suggested that the use of such indices as single nucleolar surface area or the level of pathological mitosis may be perspective for cytogenetic monitoring of the environment, and for prognostification of environmental conditions suitable or unsuitable for the human health.