Pore disappearance in a cell after electroporation: theoretical simulation and comparison with experiments.

The process of pore disappearance after cell electroporation is analyzed theoretically. On the basis of the kinetic model, in which the formation and annihilation of a metastable hydrophilic pore are considered as random one-step processes, a distribution function of cell resealing times, Fr(t), is derived. Two cases are studied: 1) the rate of pore resealing, k(r), is significantly greater than the rate of pore formation, k(f); and 2) the rate of pore formation, k(f), is comparable with k(r). It is determined that the shape of the distribution function depends on the initial number of pores in a cell, n(i). If in the absence of an external electric field the rate of pore formation, k(f), is significantly less than the rate of pore resealing, k(r) (case 1), pores disappear completely, whereas when k(f) approximately k(r) (case 2), the cell achieves a steady state in which the number of pores is equal to k(f)/k(r). In case 1, when n(i) = 1, the distribution function Fr(t) is exponential. The developed theory is compared with experimental data available in the literature. Increasing the time of incubation at elevated temperature increases the fraction of resealed cells. This indicates that the time necessary for the resealing varies from cell to cell. Although the shape of experimental relationships depends on the electroporation conditions they can be described by theoretical curves quite well. Thus it can be concluded that the disappearance of pores in the cell membrane after electroporation is a random process. It is shown that from the comparison of presented theory with experiments, the following parameters can be estimated: the average number of pores, n(i), that appeared in a cell during an electric pulse; the rate of pore disappearance, k(r); the ratio k(f)/k(r); and the energy barrier to pore disappearance deltaWr(0). Estimated numerical values of the parameters show that increasing the amplitude of an electric pulse increases either the apparent number of pores created during the pulse (the rate of pore resealing remains the same) or the rate of pore resealing (the average number of pores remains the same).     

Influenza viruses as lymphocyte mitogens. I. B cell mitogenesis by influenza A viruses of the H2 and H6 subtypes is controlled by the I-E/C subregion of the major histocompatibility complex

Influenza A viruses of the H2, H3, and H6 subtypes function as T cell-independent B cell mitogens for lymphocytes from BALB/c mice. Lymphocytes from C57BL/10 mice, however, undergo mitogenesis only in response to H3 viruses. The failure of C57BL/10 lymphocytes to respond to H2 and H6 viruses was shown not to reflect a difference in dose-response profile or kinetics of the response, nor was it due to the activity of suppressor T cells. Experiments with congenic and recombinant strains of mice established that mitogenic responsiveness to H2 and H6 viruses is linked to the major histocompatibility complex, and is controlled by a gene located in the I-E/C subregion. Furthermore, responsiveness was shown to correlate with the expression of surface I-E antigen, being positive for mouse strains that express I-E antigen (haplotypes a, d, k, p, r) and negative for strains that do not (haplotypes b, f, q, s). The data suggest that influenza A viruses of the H2 and H6 subtypes may interact directly with I-E molecules on the surface of B cells or possibly on an accessory cell. Because mitogenesis by H3 viruses is not I-E dependent, it appears that influenza A viruses stimulate B cell mitogenesis by at least two different mechanisms.     

Modalities of synthesis of Ki67 antigen during the stimulation of lymphocytes

The antibody Ki67 is currently used to evaluate the proliferative fraction of solid tumors and some hematological malignancies. We have used phytohemagglutinin (PHA)-stimulated peripheral blood lymphocytes as a model to study the entry of quiescent cells into cell cycle and to follow their progress to the next cycle. Flow cytometric analysis of lymphocyte samples stained with the antibody Ki67 and a DNA marker has allowed us to follow the expression of Ki67 antigen (Ki67 Ag) as a function of the position of the cells in the cell cycle. The use of drugs blocking the stimulated lymphocytes in different phases of the cell cycle permitted us to demonstrate that Ki67 Ag expression started from the beginning of the first S phase. The level of Ki67 Ag increased during S phase until mitosis, when its expression was maximal. After division, the cells in G1 phase showed a decrease in Ki67 Ag expression (possibly corresponding to degradation) until they reentered S phase, when the level of Ki67 Ag increased again. The results confirm that the expression of Ki67 Ag is related to the proliferative state of the cells and suggest that it may be used to determine the proliferative cell fraction in hematopoietic tissues.     

Atoms-in-molecules study of the genetically encoded amino acids. III. Bond and atomic properties and their correlations with experiment including mutation-induced changes in protein stability and genetic coding

This article presents a study of the molecular charge distributions of the genetically encoded amino acids (AA), one that builds on the previous determination of their equilibrium geometries and the demonstrated transferability of their common geometrical parameters. The properties of the charge distributions are characterized and given quantitative expression in terms of the bond and atomic properties determined within the quantum theory of atoms-in-molecules (QTAIM) that defines atoms and bonds in terms of the observable charge density. The properties so defined are demonstrated to be remarkably transferable, a reflection of the underlying transferability of the charge distributions of the main chain and other groups common to the AA. The use of the atomic properties in obtaining an understanding of the biological functions of the AA, whether free or bound in a polypeptide, is demonstrated by the excellent statistical correlations they yield with experimental physicochemical properties. A property of the AA side chains of particular importance is the charge separation index (CSI), a quantity previously defined as the sum of the magnitudes of the atomic charges and which measures the degree of separation of positive and negative charges in the side chain of interest. The CSI values provide a correlation with the measured free energies of transfer of capped side chain analogues, from the vapor phase to aqueous solution, yielding a linear regression equation with r2 = 0.94. The atomic volume is defined by the van der Waals isodensity surface and it, together with the CSI, which accounts for the electrostriction of the solvent, yield a linear regression (r2 = 0.98) with the measured partial molar volumes of the AAs. The changes in free energies of transfer from octanol to water upon interchanging 153 pairs of AAs and from cyclohexane to water upon interchanging 190 pairs of AAs, were modeled using only three calculated parameters (representing electrostatic and volume contributions) yielding linear regressions with r2 values of 0.78 and 0.89, respectively. These results are a prelude to the single-site mutation-induced changes in the stabilities of two typical proteins: ubiquitin and staphylococcal nuclease. Strong quadratic correlations (r2 approximately 0.9) were obtained between DeltaCSI upon mutation and each of the two terms DeltaDeltaH and TDeltaDeltaS taken from recent and accurate differential scanning calorimetry experiments on ubiquitin. When the two terms are summed to yield DeltaDeltaG, the quadratic terms nearly cancel, and the result is a simple linear fit between DeltaDeltaG and DeltaCSI with r2 = 0.88. As another example, the change in the stability of staphylococcal nuclease upon mutation has been fitted linearly (r2 = 0.83) to the sum of a DeltaCSI term and a term representing the change in the van der Waals volume of the side chains upon mutation. The suggested correlation of the polarity of the side chain with the second letter of the AA triplet genetic codon is given concrete expression in a classification of the side chains in terms of their CSI values and their group dipole moments. For example, all amino acids with a pyrimidine base as their second letter in mRNA possess side-chain CSI < or = 2.8 (with the exception of Cys), whereas all those with CSI > 2.8 possess an purine base. The article concludes with two proposals for measuring and predicting molecular complementarity: van der Waals complementarity expressed in terms of the van der Waals isodensity surface and Lewis complementarity expressed in terms of the local charge concentrations and depletions defined by the topology of the Laplacian of the electron density. A display of the experimentally accessible Laplacian distribution for a folded protein would offer a clear picture of the operation of the "stereochemical code" proposed as the determinant in the folding process.     

Circadian gene expression in individual fibroblasts: cell-autonomous and self-sustained oscillators pass time to daughter cells

The mammalian circadian timing system is composed of a central pacemaker in the suprachiasmatic nucleus (SCN) of the brain and subsidiary oscillators in most peripheral cell types. While oscillators in SCN neurons are known to function in a self-sustained fashion, peripheral oscillators have been thought to damp rapidly when disconnected from the control exerted by the SCN. Using two reporter systems, we monitored circadian gene expression in NIH3T3 mouse fibroblasts in real time and in individual cells. In conjunction with mathematical modeling and cell co-culture experiments, these data demonstrated that in vitro cultured fibroblasts harbor self-sustained and cell-autonomous circadian clocks similar to those operative in SCN neurons. Circadian gene expression in fibroblasts continues during cell division, and our experiments unveiled unexpected interactions between the circadian clock and the cell division clock. Specifically, the circadian oscillator gates cytokinesis to defined time windows, and mitosis elicits phase shifts in circadian cycles.     

A hydrodynamic mechanism for the disintegration of Saccharomyces cerevisiae in an industrial homogenizer

Amongst the commercial type of homogenizers the Manton-GaulinAPV homogenizer (APV Company Ltd., Crawley, Surrey, England) which is generally being used for other purposes than cell disintegration processes, has recently been proved to be effective for the breakage of yeast cells. To understand fully the disintegration process occurring in such machines it becomes necessary to describe their functions through mathematical expressionsbased on a realistic hydrodynamic model. A mathematical expression describing the protein release at an applied pressure has been derived from an energy balance in the homogenizer combined with the size distribution function of yeast cell population. This expression has been confirmed experimentally under conditions where it shows that turbulence is the controlling factor in the system. Furthermore it indicates the area where more investigations are needed to improve the efficiency of the process of disintegration.     

The efficiency of adenovirus transformation of rodent cells is inversely related to the rate of viral E1A gene expression.

While the products of the type 5 adenovirus E1A and E1B genes can initiate pathways leading to a transformed rodent cell, little is known about how the rate of viral early gene expression influences the efficiency of this process. An adenovirus mutant [E1a(r) virus] that expresses its viral E1A and E1B genes at as much as a 100-fold-reduced rate relative to wild-type virus in infected CREF or HeLa cells transforms CREF cells at an 8-fold-higher efficiency than wild-type virus. Additional studies show that the reduction in viral E1A gene expression is solely responsible for this transformation phenotype, and at this low rate of viral E1A gene expression both E1A gene products must be expressed. Unlike previously characterized viruses which transform CREF cells at frequencies greater than wild-type virus, the foci obtained following E1a(r) virus infection were indistinguishable from those arising from wild-type virus by several criteria (morphological characteristics and anchorage-independent growth). Surprisingly, an analysis of viral early gene expression from a panel of wild-type- and E1a(r) virus-transformed CREF cell lines showed similar average rates of both viral E1A and E1B gene expression. By using an adenovirus-transformed cell line that is cold-sensitive for maintenance of the transformed cell phenotype, we show that both wild-type and the E1a(r) viruses can transform these cells at equally high efficiencies at the nonpermissive temperature of 32 degrees C. Our findings suggest that the process leading to a fully transformed cell involves multiple stages, with an early stage being facilitated by a reduced rate of viral E1A gene expression.     

In vivo measure of average bacterial cell size from a polarized light scattering function.

A particular combination of elements of the Mueller matrix for scattering of polarized light given by (S34 + S14)/(S11 + S13) identical to (S34/S11)++ is measured vs angle at a wavelength of 633 nm for randomly oriented suspensions of several species of bacteria in different stages of growth. (This combination of elements is dominated in the present measurements by the behavior of the normalized S34 matrix element, as is indicated by the notation defined on the right side of the equation.) The resulting graph in each case shows an oscillating function of angle. This function is compressed toward smaller angles when the bacteria are in the exponential phase of growth in comparison with results for a suspension of the same bacteria in the stationary (starving-smaller cells) phase of growth. Microscopic measurements were made to determine, for each case, the average dimensions of the bacterial population. Graphs were then plotted of the peak positions from the Mueller matrix function plots vs either cell length or cell diameter. The function was shown to be strongly correlated with cell diameter under the conditions of this experiment and poorly correlated with cell length. The measurements were shown to have a sensitivity to changes in average diameter of about 20 nm.     

Short-term information processing, long-term responses: Insights by mathematical modeling of signal transduction. Early activation dynamics of key signaling mediators can be predictive for cell fate decisions

How do cells interpret information from their environment and translate it into specific cell fate decisions? We propose that cell fate is already encoded in early signaling events and thus can be predicted from defined signal properties. Specifically, we hypothesize that the time integral of activated key signaling molecules can be correlated to cellular behavior such as proliferation or differentiation. The identification of these decisive key signal mediators and their connection to cell fate is facilitated by mathematical modeling. A possible mechanistic linkage between signaling dynamics and cellular function is the directed control of gene regulatory networks by defined signals. Targeted experiments in combination with mathematical modeling can increase our understanding of how cells process information and realize distinct cell fates.     

Sur L'interprétation de la Loi Logistique de Croissance: Une Re-lecture de la Relation Entre Autocatalyse et Croissance On The Interpretation of the Logistic Law of Growth: A New Reading of the Relationships between Autocatalysis and Growth

The logistic function now constitutes the most widely used model for there presentation of growth kinetics of the continuous monotonous type in biological systems (populations, organisms, organs, ...). This ubiquity led to consider logistics from a phenomenological rather than mechanistic viewpoint. Whence the question : can logistics be given an interpretation, a signification which confers the rank of an "explicative" model to it? This Note presents some critical comments on the relationships between logistics and three types of biological systems : population demography, environmental resources, autocatalyzed reactions. The so-called functional (in the mathematical meaning) interpretation, which is then discussed, is based upon a variational principle : the occurrence of a minimum of a function associated with the logistic law. Its present limitation to the only simple logistics of Verhulst and the difficulties of its expression in biological terms are then pointed out. The problem is then examined within the framework of Delattre's transformation system theory which affords a new reading of there lationships between growth and autocatalysis (without requiring reference to a particular reactional chemical analogue). The resulting new model constitutes an extension of Verhulst's logistics which is quite different from the well-known Richards-Nelder function. In addition to its theoretical background, one feature of the new model is the generation of varied growth kinetics, particularly a non-monotonous variation of the specific growth rate r = (1/y)(dy/dt). This property, which is often neglected, is the more valuable as a number of biological growths are characterized by a rate r which is not continuously decreasing. This specific characteristic is not predicted by the usual growth functions.     

Developmental Analysis of the Achaete-Scute System of DROSOPHILA MELANOGASTER

The interpretation of the wild-type function of a gene depends on our knowledge of the phenotype caused by its absence. We have first defined the genetic extent of the achaete-scute system by studying the phenotype of different terminal and intercalary deficiencies including these genes. When these deficiencies were lethal, we have defined the phenoeffective phase of lethality and studied their phenotype in genetic mosaics (gynandromorphs and mitotic recombination clones). The achaete-scute system affects two functions, one necessary for the differentiation of the embryonic (central?) nervous system and the other necessary for the differentiation of peripheral nervous elements of the chaetes and sensillae of the adult cuticle. The possibility that these functions correspond to differential expression of a single mechanism is discussed.     

A simple expression for quantifying bacterial chemotaxis using capillary assay data: application to the analysis of enhanced chemotactic responses from growth-limited cultures.

An individual cell-based mathematical model of Rivero et al. provides a framework for determining values of the chemotactic sensitivity coefficient chi 0, an intrinsic cell population parameter that characterizes the chemotactic response of bacterial populations. This coefficient can theoretically relate the swimming behavior of individual cells to the resulting migration of a bacterial population. When this model is applied to the commonly used capillary assay, an approximate solution can be obtained for a particular range of chemotactic strengths yielding a very simple analytical expression for estimating the value of chi 0, [formula: see text] from measurements of cell accumulation in the capillary, N, when attractant uptake is negligible. A0 and A infinity are the dimensionless attractant concentrations initially present at the mouth of the capillary and far into the capillary, respectively, which are scaled by Kd, the effective dissociation constant for receptor-attractant binding. D is the attractant diffusivity, and mu is the cell random motility coefficient. NRM is the cell accumulation in the capillary in the absence of an attractant gradient, from which mu can be determined independently as mu = (pi/4t)(NRM/pi r2bc)2, with r the capillary tube radius and bc the bacterial density initially in the chamber. When attractant uptake is significant, a slightly more involved procedure requiring a simple numerical integration becomes necessary. As an example, we apply this approach to quantitatively characterize, in terms of the chemotactic sensitivity coefficient chi 0, data from Terracciano indicating enhanced chemotactic responses of Escherichia coli to galactose when cultured under growth-limiting galactose levels in a chemostat.     

The rate of cellular hydrogen peroxide removal shows dependency on GSH: mathematical insight into in vivo H2O2 and GPx concentrations

Although its concentration is generally not known, glutathione peroxidase-1 (GPx-1) is a key enzyme in the removal of hydrogen peroxide (H2O2) in biological systems. Extrapolating from kinetic results obtained in vitro using dilute, homogenous buffered solutions, it is generally accepted that the rate of elimination of H2O2 in vivo by GPx is independent of glutathione concentration (GSH). To examine this doctrine, a mathematical analysis of a kinetic model for the removal of H2O2 by GPx was undertaken to determine how the reaction species (H2O2, GSH, and GPx-1) influence the rate of removal of H2O2. Using both the traditional kinetic rate law approximation (classical model) and the generalized kinetic expression, the results show that the rate of removal of H2O2 increases with initial GPx(r), as expected, but is a function of both GPx(r) and GSH when the initial GPx(r) is less than H2O2. This simulation is supported by the biological observations of Li et al. Using genetically altered human glioma cells in in vitro cell culture and in an in vivo tumour model, they inferred that the rate of removal of H2O2 was a direct function of GPx activity x GSH (effective GPx activity). The predicted cellular average GPx(r) and H2O2 for their study are approximately GPx(r) < or =1 microm and H2O2 approximately 5 microm based on available rate constants and an estimation of GSH. It was also found that results from the accepted kinetic rate law approximation significantly deviated from those obtained from the more generalized model in many cases that may be of physiological importance.     

Analysis of vagally induced sinus arrhythmias.

Vagal stimulation at precise times in successive cardiac cycles can elicit sinus arrhythmias. Two mechanisms have been identified that can, but do not necessarily, cause these vagally induced sinus arrhythmias. First, changes in cycle length elicited by a given concentration of acetylcholine (ACh) depend on the phase of the pacemaker cell action potential when the ACh binds to muscarinic receptors. Second, acetylcholinesterase degrades ACh rapidly enough for the mean concentration of ACh per cardiac cycle to vary from cycle to cycle. We used a mathematical model of the underlying cellular physiology, to examine whether these mechanisms are responsible for arrhythmogenesis. Computer simulation showed that both mechanisms contribute to the vagally induced sinus arrhythmias.     

In silico simulation of the effect of hypoxia on MCF-7 cell cycle kinetics under fractionated radiotherapy

The treatment outcome of a given fractionated radiotherapy scheme is affected by oxygen tension and cell cycle kinetics of the tumor population. Numerous experimental studies have supported the variability of radiosensitivity with cell cycle phase. Oxygen modulates the radiosensitivity through hypoxia-inducible factor (HIF) stabilization and oxygen fixation hypothesis (OFH) mechanism. In this study, an existing mathematical model describing cell cycle kinetics was modified to include the oxygen-dependent G1/S transition rate and radiation inactivation rate. The radiation inactivation rate used was derived from the linear-quadratic (LQ) model with dependence on oxygen enhancement ratio (OER), while the oxygen-dependent correction for the G1/S phase transition was obtained from numerically solving the ODE system of cyclin D-HIF dynamics at different oxygen tensions. The corresponding cell cycle phase fractions of aerated MCF-7 tumor population, and the resulting growth curve obtained from numerically solving the developed mathematical model were found to be comparable to experimental data. Two breast radiotherapy fractionation schemes were investigated using the mathematical model. Results show that hypoxia causes the tumor to be more predominated by the tumor subpopulation in the G1 phase and decrease the fractional contribution of the more radioresistant tumor cells in the S phase. However, the advantage provided by hypoxia in terms of cell cycle phase distribution is largely offset by the radioresistance developed through OFH. The delayed proliferation caused by severe hypoxia slightly improves the radiotherapy efficacy compared to that with mild hypoxia for a high overall treatment duration as demonstrated in the 40-Gy fractionation scheme.     

Cell cycle-regulated inactivation of endothelial NO synthase through NOSIP-dependent targeting to the cytoskeleton

Nitric oxide (NO) plays a key role in vascular function, cell proliferation, and apoptosis. Proper subcellular localization of endothelial NO synthase (eNOS) is crucial for its activity; however, the role of eNOS trafficking for NO biosynthesis remains to be defined. Overexpression of NOS-interacting protein (NOSIP) induces translocation of eNOS from the plasma membrane to intracellular compartments, thereby impairing NO production. Here we report that endogenous NOSIP reduces the enzymatic capacity of eNOS, specifically in the G(2) phase of the cell cycle by targeting eNOS to the actin cytoskeleton. This regulation is critically dependent on the nucleocytoplasmic shuttling of NOSIP and its cytoplasmic accumulation in the G(2) phase. The predominant nuclear localization of NOSIP depends on a bipartite nuclear localization sequence (NLS) mediating interaction with importin alpha. Mutational destruction of the NLS abolishes nuclear import and interaction with importin alpha. Nuclear export is insensitive to leptomycin B and hence different from the CRM1-dependent default mechanism. Inhibition of NOSIP expression by RNA interference completely abolishes G(2)-specific cytoskeletal association and inhibition of eNOS. These findings describe a novel cell cycle-dependent modulation of endogenous NO levels that are critical to the cell cycle-related actions of NO such as apoptosis or cell proliferation.     

Tissue distribution of human minor histocompatibility antigens. Ubiquitous versus restricted tissue distribution indicates heterogeneity among human cytotoxic T lymphocyte-defined non-MHC antigens.

We determined the tissue distribution of 7 human minor histocompatibility (H) Ag. Each of these Ag is defined by one or more MHC class I-restricted CTL clones, previously generated from PBL primed against minor H Ag by HLA-identical bone marrow transplantation (BMT). CTL-mediated lysis of tissue-derived cells and cultured cell lines was used as an in vitro assay for minor H Ag expression of several human tissues. The Ag HA-3 (HLA-A1-restricted), HA-4 (HLA-A2 restricted), HA-6 and HA-7 (HLA-B7 restricted), and the male-specific Ag H-Y (HLA-A2 and B7 restricted) were found to be expressed on cells of all tissues tested. In contrast, the HLA-A2-restricted Ag HA-1 and HA-2 were demonstrated on PHA-blasts, EBV-BLCL, purified T cells, B cells, monocytes, and immature thymocytes, but could not be demonstrated on skin-derived cultured fibroblasts, keratinocytes, melanocytes, cultured epithelial cells of kidney proximal tubili, and umbilical cord vein-derived endothelial cells. Incubation of the latter cell lines with rIFN-gamma, rTNF-alpha, and/or rIL-1 alpha, in concentrations shown to maximally increase their susceptibility to lysis by allo-MHC class I CTL, did not induce recognition by HA-1- and HA-2-specific CTL in vitro. These results indicate an ubiquitous tissue expression of the minor H Ag HA-3, -4, -6, -7 and H-Y in contrast to a to the hemopoietic cell lineage-restricted expression for HA-1 and HA-2. The heterogeneity in tissue expression of T cell-defined, class I-restricted non-MHC Ag implies that they might be derived from intracellular proteins with either an ubiquitous or a more specialized cell type-specific function.