Dynamics of a Laterally Evolving Community of Ribozyme-Like Agents as Studied with a Rule-Based Computing System

The very early forms of life probably comprised ribozyme-like agents that were able to catalyze reactions and serve as templates for their own replication. The early evolution has also been suggested to occur mainly horizontally between proto-cells or inorganic compartments rather than vertically from parent cell to their dividing siblings. In order to study the evolutionary dynamics of such a community a rule-based computing system entitled as PrimordialEvo was developed. The system simulates a three dimensional matrix of compartments in which replicators, resource collectors and various other actors thrive. Horizontal movement between compartments may be due to genetically induced vesicle formation or random drift. Analysis of the simulation experiments suggests that active sharing of innovations between compartments is important for the overall reproductive success of life. The capability of natural selection to favor genes in the system was also tested, and, for example, the frequency of anti-parasites was observed to increase when parasites were allowed to emerge.     

Stochastic theory of compartments: An open,two compartment,reversible system with independent poisson arrivals

This paper discusses two compartment models with interaction allowed between the compartments. The total number of particles in the system at any time is discussed along with the number to the found in each separate compartment. An interesting result is that the number of particles in each of the two compartments areindependent random variables. Some asymptotic results are also given. The paper is a continuation of some earlier work by the author.     

THE CELL POPULATION KINETICS OF NEUTROPHILIC CELLS

Autoradiographic data for the entry of tritiated thymidine labelled cells into the post-proliferative neutrophilic cell compartments following a single injection of isotope have been analysed in terms of two cell kinetic models which differ in the assumed relationships between cell maturation and division. Comparisons with the experimental data were made in an attempt to assess the validities of the models, and kinetic parameters for the compartments of recognizable neutrophilic cells were estimated. Control mechanisms which have been proposed for the granulocyte system are discussed in terms of the kinetic models which were chosen in their determination. Although it was not possible to make a clear choice between the proposed models, preference was established for a random model which did not involve cell loss.     

A simpler way of comparing the labelling densities of cellular compartments illustrated using data from VPARP and LAMP-1 immunogold labelling experiments

Quantitative immunoelectron microscopy of gold label in intracellular compartments often involves calculating labelling densities (LDs). These are related to antigen concentrations and usually refer gold particle counts to the sizes of compartments on sections (for example, golds per microm(2) of organelle profile area or per microm of membrane trace length). Here, we show how LD values can be estimated more simply (without estimating areas or lengths) and also how observed and expected LD values can be used to calculate a relative labelling index (RLI) for each compartment and then test statistically for preferential (non-random) labelling. For random labelling, RLI=1. Compartment size is estimated stereologically by superimposing random test points (which hit organelle profiles in proportion to their area) or test lines (which intersect membrane traces in proportion to their length). By this means, the observed LD of a compartment (LD(obs)) can be expressed simply as golds per test point (organelles) or per intersection (membranes). Furthermore, the LD obtained by dividing total golds (on all compartments) by total points or intersections (on all compartments) is the value to be expected (LD(exp)) when compartments label randomly. For each compartment, RLI=LD(obs)/LD(exp). Statistical analysis is undertaken by comparing observed distributions of golds with predicted random distributions (calculated from point or intersection counts). A compartment is preferentially labelled if two criteria are met: (1) its RLI>1 (i.e. LD(obs) is greater than LD(exp)) and (2) its partial chi-squared value makes a substantial contribution to total chi-squared value. This approach provides a simple and efficient way of comparing LDs in different compartments. Its utility is illustrated using data from VPARP and LAMP-1 labelling experiments.     

Non-linear compartmental systems: extensions of S. R. Bernard's urn model

One of the limitations of stochastic, linear compartmental systems is the small degree of variability in the contents of compartments. S. R. Bernard's (1981) urn model (S. R. Bernardet al., Bull. math. Biol. 43, 33–45.) which allows for bulk arrivals and departures from a one-compartment system, was suggested as a way of increasing content variability. In this paper, we show how the probability distribution of the contents of an urn model may be simply derived by studying an appropriate set of exchangeable random variables. In addition, we show how further increases in variability may be modeled by allowing the size of arrivals and departures to be random. Supported by NSF Grant No. MCS 8102215-01.     

Secretory Vesicle Pools and Rate and Kinetics of Single Vesicle Exocytosis in Neurosecretory Cells

Secretory vesicles are localized in specific compartments within neurosecretory cells. Morphometric, cytochemical and electrophysiological techniques have allowed the definition of secretory vesicle compartments. These are different pools in which vesicles are in various states of releasability. The transit of vesicles between compartments is not random, but an event controlled and regulated by Ca²⁺ and the cortical F-actin network. Cortical F-actin disassembly, a Ca²⁺-dependent event, controls the transit of secretory vesicles from the reserve compartment to the release-ready vesicle pool. Furthermore, the recent development of new technical approaches (patch-clamp membrane capacitance, electrochemical detection of amines with carbon-fibre microelectrodes) has now permitted us to understand the kinetics of single vesicle exocytosis.     

Stochastic analysis of environmental fluctuations in a compartmental system

In this paper the general two-compartment system with environmental stochasticity is investigated. The transfer rates and outputs are assumed to be random telegraph processes. The Laplace-transform of the mean-value function of the amount of substance present in the two compartments is evaluated. The Gaussian white noise limit case is also discussed and the stability of the system is examined. It is shown that while the deterministic model is stable, environmental stochasticity may induce in the mean-value function all sorts of behaviour — stable, unstable and oscillatory. This is in contrast to the intrinsic stochasticity in linear models wherefor the mean-value function is the same as the solution of the deterministic counterpart.     

Morphological intergration between development compartments in the Drosophila wing

Abstract. Developmental integration is the covariation among morphological structures due to connections between the developmental processes that built them. Here we use the methods of geometric morphometrics to study integration in the wing of Drosophila melanogaster . In particular, we focus on the hypothesis that the anterior and posterior wing compartments are separate developmental units that vary independently. We measured both variation among genetically diverse individuals and random differences between body sides of single individuals (fluctuating asymmetry, FA). For both of these sources of variation, the patterns of variation identified by principal component analyses all involved landmarks in both the anterior and posterior compartments simultaneously. Analyses focusing exclusively on the covariation between the anterior and posterior compartments, by the partial least-squares method, revealed pervasive integration of the two compartments, for both individual variation and FA. These analyses clearly indicate that the anterior and posterior compartments are not separate units of variation, but that the covariation between compartments is sufficient to account for nearly all the variation throughout the entire wing. We conclude that variation among individuals as well as the developmental perturbations responsible for FA generate shape variation primarily through developmental processes that are integrated across both compartments. In contrast, much less of the shape variation in our sample can be attributed to the localized processes that establish the identity of particular wing veins.     

DNA display of biologically active proteins for in vitro protein selection

In vitro display technologies are powerful tools for screening peptides with desired functions. We previously proposed a DNA display system in which streptavidin-fused peptides are linked with their encoding DNAs via biotin labels in emulsion compartments and successfully applied it to the screening of random peptide libraries. Here we describe its application to functional and folded proteins. By introducing peptide linkers between streptavidin and fused proteins, we achieved highly efficient (>95%) formation of DNA-protein conjugates. Furthermore, we successfully enriched a glutathione-S-transferase gene by a factor of 20-30-fold per round on glutathione-coupled beads. Thus, DNA display should be useful for rapidly screening or evolving proteins based on affinity selection.     

A stochastic model for then-compartment irreversible system

This paper deals with a stochasticn-compartment irreversible system with a non-homogeneous Poisson input and arbitrary residence time for each of the compartments. Results relating to the number of particles present in each of the compartments as well as the total number of particles present in the system at any time are derived. Further, explicit expressions for the auto covariance function for each compartment and the cross-covariance function between any two compartments with a given time lag are obtained. As a particular case, then-compartment irreversible system is analyzed with homogeneous Poisson input and exponential residence time distribution for each of the compartments. The possible applications of the model are discussed.     

Quantifying immunogold localization on electron microscopic thin sections: a compendium of new approaches for plant cell biologists

A review is presented of recently developed methods for quantifying electron microscopical thin sections on which colloidal gold-labelled markers are used to identify and localize interesting molecules. These efficient methods rely on sound principles of random sampling, event counting, and statistical evaluation. Distributions of immunogold particles across cellular compartments can be compared within and between experimental groups. They can also be used to test for co-localization in multilabelling studies involving two or more sizes of gold particle. To test for preferential labelling of compartments, observed and expected gold particle distributions are compared by χ(2) analysis. Efficient estimators of gold labelling intensity [labelling density (LD) and/or relative labelling index (RLI)] are used to analyse volume-occupying compartments (e.g. Golgi vesicles) and/or surface-occupying compartments (e.g. cell membranes). Compartment size is estimated by counting chance events after randomly superimposing test lattices of points and/or line probes. RLI=1 when there is random labelling and RLI >1 when there is preferential labelling. Between-group comparisons do not require information about compartment size but, instead, raw gold particle counts in different groups are compared by combining χ(2) and contingency table analyses. These tests may also be used to assess co-distribution of different sized gold particles in compartments. Testing for co-labelling involves identifying sets of compartmental profiles that are unlabelled and labelled for one or both of two gold marker sizes. Numbers of profiles in each labelling set are compared by contingency table analysis and χ(2) analysis or Fisher's exact probability test. The various methods are illustrated with worked examples based on empirical and synthetic data and will be of practical benefit to those applying single or multiple immunogold labelling in their research.     

The effects of interaction compartments on stability for competitive systems

The interactions between species are unlikely to be randomly arranged, and there is increasing evidence that most interactions occur within small species sub-groups, or compartments, that do not strongly interact with one another. We examine whether arranging the interactions of a competitive system into compartments influences the system properties of linear stability, feasibility, reactivity, and biomass stability, thereby altering the likelihood of species persistence. Model Lotka-Volterra systems of diffuse competition were analysed with interactions arranged randomly and in compartments. It was found, using a variety of dynamical measures, that arranging interactions into compartments enhances the likelihood of species persistence. Since many natural competitive systems appear to have interactions arranged within compartments, this may be an outcome of the positive attributes that this form of organization offers.     

Applications of an efficient method for comparing immunogold labelling patterns in the same sets of compartments in different groups of cells

Quantitative immunoelectron microscopy often involves determining the distributions of gold label in different intracellular compartments and then drawing comparisons between compartments in the same sample of cells or between experimental groups of cells. In the case of within-group comparisons, recent developments in the estimation of relative labelling index and labelling density make it possible to test whether or not particular compartments are preferentially labelled. These methods are ideally suited to analysing gold label restricted to volume (organelle) or surface (membrane) compartments but may be modified to analyse label localised in mixtures of both. Here, a simple and efficient approach to drawing between-group comparisons for label associated with organelles and/or membranes is presented. The method relies on multistage random sampling of specimens (via blocks and microscopic fields) followed by simply counting gold particles associated with different compartments. The distributions of raw gold counts in different groups are then compared by contingency table analysis with statistical degrees of freedom for chi-squared values being determined by the number of compartments and the number of experimental groups of cells. Compartmental chi-squared values making substantial contributions to the total chi-squared values then identify where the main between-group differences reside. The method requires no information about compartment size (for example, organelle profile area or membrane trace length) and does not even depend critically on standardising between-group magnification. Its application is illustrated using datasets from immunolabelling studies designed to localise the KDEL receptor, phosphatidyl-inositol 4,5-bisphosphate, GLUT4 and rab4 at the electron microscopic level.     

Discrete and continuous mathematical models of DNA branch migration

DNA junctions, known as Holliday junctions, are intermediates in genetic recombination between DNAs. In this structure, two double-stranded DNA helices with similar sequence are joined at a branch point. The branch point can move along these helices when strands with the same sequence are exchanged. Such branch migration is modeled as a random walk. First, we model this process discretely, such that the motion of the branch is represented as transfer between discrete compartments. This is useful in analysing the results of DNA branch migration on junction comprised of synthetic oligonucleotides. The limit in which larger numbers of smaller steps go to continuous motion of the branch is also considered. We show that the behavior of the continuous system is very similar to that of the discrete system when there are more than just a few compartments. Thus, even branch migration on oligonucleotides can be viewed as a continuous process. One consequence of this is that a step size must be assumed when determining rate constants of branch migration.We compare migration where forward and backward movements of the branch are equally probable to biased migration where one direction is favored over the other. In the latter case larger differences between the discrete and continuous cases are predicted, but the differences are still small relative to the experimental error associated with experiments to measure branch migration in oligonucleotides.     

Mesophyll freezing and effects of freeze dehydration visualized by simultaneous measurement of IDTA and differential imaging chlorophyll fluorescence

Infrared differential thermal analysis (IDTA) and differential imaging chlorophyll fluorescence (DIF) were employed simultaneously to study the two-dimensional pattern of ice propagation in leaves and mesophyll freeze dehydration as detected by a significant increase of basic chlorophyll fluorescence (F(0)). IDTA and DIF technique gave different insights into the freezing process of leaves that was highly species-specific. IDTA clearly visualized the freezing process consisting of an initial fast spread of ice throughout the vascular system followed by mesophyll freezing. While mesophyll freezing was homogeneously in Poa alpina, Rhododendron ferrugineum and Senecio incanus as determined by IDTA, DIF showed a distinct pattern only in S. incanus, with the leaf tips being affected earlier. In Cinnamomum camphora, a mottled freezing pattern of small mesophyll compartments was observed by both methods. In IDTA images, a random pattern predominated, while in DIF images, compartments closer to lower order veins were affected earlier. The increase of F(0) following mesophyll freezing started after a species-specific time lag of up to 26 min. The start of the F(0) increase and its slope were significantly enhanced at lower temperatures, which suggest a higher strain on mesophyll protoplasts when freezing occurs at lower temperatures.     

A three-symbol code for organized proteomes based on cyclical imaging of protein locations.

BACKGROUND: A major challenge in the post genomic era is to map and decipher the functional molecular networks of proteins directly in a cell or a tissue. This task requires technologies for the colocalization of random numbers of different molecular components (e.g. proteins) in one sample in one experiment. METHODS: Multi-epitope-ligand-"kartographie" (MELK) was developed as a microscopic imaging technology running cycles of iterative fluorescence tagging, imaging, and bleaching, to colocalize a large number of proteins in one sample (morphologically intact routinely fixed cells or tissue). RESULTS: In the present study, 18 different cell surface proteins were colocalized by MELK in cells and tissue sections in different compartments of the human immune system. From the resulting sets of multidimensional binary vectors the most prominent groups of protein-epitope arrangements were extracted and imaged as protein "toponome" maps providing direct insight in the higher order topological organization of immune compartments uncovering new tissue domains. The data sets suggest that protein networks, topologically organized in proteomes in situ, obey a unique protein-colocation and -anticolocation code describable by three symbols. CONCLUSION: The technology has the potential to colocalize hundreds of proteins and other molecular components in one sample and may offer many applications in biology and medicine.     

Rate Constants and the Variation of Random Concentration Curves in a 2-Compartment System

In biology and medicine many substances and drugs enter the system not at regular time intervals but rather according to a random process. In the present article a situation is investigated where input enters a 2-compartment system according to a Poisson process. The arising two random concentration curves y(t), one for the central and one for the peripheral compartment are discussed (shot noise). The equations for E[y(t)] and Var [y(t)] are derived. The dependence of E[y(t)] and Var [y(t)] and of the index of dispersion ID[y(t)] on the rate parameters is analysed and discussed in both compartments. The arising calculations were considerably simplified by means of “Mathematica”, a computer program which allows to perform symbolic calculations.     

High-pressure freezing combined with in vivo-DAB-cytochemistry: A novel approach for studies of endocytic compartments

Methods for fine structural and functional analyses of complex and dynamic cell compartments must ensure high temporal resolution together with an excellent fine structural preservation. High-pressure freezing followed by freeze-substitution, and resin embedding is state of the art but its use is limited in combination with preembedding cytochemical techniques. Here we show a new approach for the exploration of compartments of the endocytosis system, which combines high-pressure freezing with peroxidase-catalyzed cytochemistry, thus using the potencies of both synergistically. Uptake of horseradish peroxidase-labeled molecules is followed by in vivo-staining and immobilization of endocytic compartments by generation of diaminobenzidine precipitates. Subsequently, the specimens are high pressure frozen, freeze-substituted, and embedded in resin. The excellent fine structural preservation, together with the high temporal resolution, and differentiating visualization of endocytic compartments qualify the new approach for morpho-functional studies of the complex and dynamic components of the endocytosis system involved in physiologic and pathologic cellular traffic, and in routes utilized in drug targeting strategies. The distinct appearances of membranes and reactive compartments provide optimal conditions for 3D-analyses by electron tomography allowing to discern subtle details of the complex 3D-structures of endocytic compartments.