Metastable equilibrium with random local fluctuations: Simulations of dynamic receptor pattern generation in a fluid mosaic membrane

The generation of receptors in the animal cell's membrane was simulated by a model consisting of units in four possible states within a hexagonal area (playboard) ofn units of a triangular network. The state of each unit was determined by the previous state or itself and of its six nearest neighbours, as regulated by a set of transition rules, which kept the mean relative frequency (m.r.f.) of each state constant. The transition rules were applied to the system exactlyn times, regardedless whether this involved selection of a unit on 0, 1, 2 or more occasions (programme random selection with repeat; RS-R). Comparison to previous results obtained by other ways of application of the rules has shown that the RS-R programme accounted for the highest m.r.f. of quiet (Q) units and Q clusters (sub-patterns), and also for the longest survival of Q configurations through several generations. Functioning of the model under the RS-R programme simulates an integrated system in metastable equilibrium with random local fluctuations, such as the cytoplasmic membrane is imagined to be in standardized environmental conditions. The formation-persistence-disintegration cycle of the sub-patterns is believed to simulate the dynamic generation of transitory receptor configurations in the cell membrane.     

Attenuation of photobleaching in two-photon excitation fluorescence from green fluorescent protein with shaped excitation pulses

The two-photon excitation fluorescence (TPEF) process of an enhanced green fluorescent protein (EGFP) for fluorescence signals was adaptively controlled by the phase-modulation of femtosecond pulses. After the iteration of pulse shaping, a twofold increase in the ratio of the fluorescence signal to the laser peak power was achieved. Compared with conventional pulses optimized for peak power, phase-optimized laser pulses reduced the bleaching rate of EGFP by a factor of 4 while maintaining the same intensity of the fluorescence signal. Our method will provide a powerful solution to various problems confronting researchers, such as the photobleaching of dyes in two-photon excitation microscopy.     

High-energy ion generation by short laser pulses

This paper reviews the many recent advances at the Center for Ultrafast Optical Science (CUOS) at the University of Michigan in multi-MeV ion beam generation from the interaction of short laser pulses focused onto thin foil targets at intensities ranging from 10¹⁷ to 10¹⁹ W/cm². Ion beam characteristics were studied by changing the laser intensity, laser wavelength, target material, and by depositing a well-absorbed coating. We manipulated the proton beam divergence using shaped targets and observed nuclear transformation induced by high-energy protons and deuterons. Qualitative theoretical approaches and fully relativistic two-dimensional particle-in-cell simulations modeled energetic ion generation. Comparison with experiments sheds light on ion energy spectra for multi-species plasma, the dependences of ion-energy on preplasma scale length and solid density plasma thickness, and laser-triggered isotope yield. Theoretical predictions are also made with the aim of studying ion generation for high-power lasers with the energies expected in the near future, and for the relativistic intensity table-top laser, a prototype of which is already in operation at CUOS in the limits of several-cycle pulse duration and a single-wavelength spot size.     

Rapid generation of random mutant libraries

A simple and efficient method utilizing in vivo recombination to create recombinant libraries incorporating the products of PCR amplification is described. This will be especially useful for generating large pools of randomly mutagenized clones after error-prone PCR mutagenesis. Here we investigate various parameters to optimize this approach and we demonstrate that as little as 1 pmole of PCR fragment can generate a library with greater than 104 clones in a single transformation without ligation.     

Cell membrane permeability during the cell generation cycle in Chinese hamster ovary cells

Summary Changes in the permeability of the cell membrane in cultured Chinese hamster ovary cells at different stages of the cell cycle were investigated. These were followed by measuring the intracellular retention of fluorescein molecules produced by the enzymatic hydrolysis of fluoresceindiacetate in the cytoplasm of CHO cells. Rate constants for the permeation of fluorescein have been calculated.     

Cell communication by periodic cyclic-AMP pulses.

At the surface of aggregating cells of the slime mould, Dictyostelium discoideum, two different sites interacting with extracellular cAMP are detectable: binding sites and cycl-nucleotide phosphodiesterase. Both sites are developmentally regulated. An adequate stimulus for the chemoreceptor system in D. discoideum is the change of cAMP concentration in time, rather than concentration per se: long-term binding of cAMP causes only short-term response. The system is, consequently, adapted to the recognition of pulses rather than to steady-state concentrations of cAMP. The ce,lls are, nevertheless, able to sense stationary spatial gradients and to respond to them by chemotactic orientation. The possibility is discussed that they do so by transforming spatial concentration changes into temporal ones, using extending pseudopods as sensors. The cAMP recognition system is part of a molecular network involved in the generation of spatio-temporal patterns of cellular activities. This system controls the periodic formation of chemotactic signals and their propagation from cell to cell. The phosphodiesterase limits the duration of the cAMP pulses and thus sharply separates the periods of signalling; the binding sites at the cell surface are supposed to be the chemoreceptors. The control of cellular activities via cAMP receptors can be studied with biochemical techniques with cell suspensions in which spatial inhomogeneities are suppressed by intense stirring, whereas the temporal aspect of the spatiotemporal pattern is preserved. Under these conditions it can be shown that the extracellular cAMP concentration changes periodically, and that the phase of the cellular oscillator can be shifted by external pulses of cAMP. It can also be shown that small cAMP pulses induce a high output of cAMP, which demonstrates signal amplification, a function necessary for a cellular relay system.     

DNA-based random number generation in security circuitry

DNA-based circuit design is an area of research in which traditional silicon-based technologies are replaced by naturally occurring phenomena taken from biochemistry and molecular biology. This research focuses on further developing DNA-based methodologies to mimic digital data manipulation. While exhibiting fundamental principles, this work was done in conjunction with the vision that DNA-based circuitry, when the technology matures, will form the basis for a tamper-proof security module, revolutionizing the meaning and concept of tamper-proofing and possibly preventing it altogether based on accurate scientific observations. A paramount part of such a solution would be self-generation of random numbers. A novel prototype schema employs solid phase synthesis of oligonucleotides for random construction of DNA sequences; temporary storage and retrieval is achieved through plasmid vectors. A discussion of how to evaluate sequence randomness is included, as well as how these techniques are applied to a simulation of the random number generation circuitry. Simulation results show generated sequences successfully pass three selected NIST random number generation tests specified for security applications.     

Cell culture of infantile digital fibromatosis

Two cell cultures were obtained from excised tumors of two cases of infantile digital fibromatosis (IDF). The cells had eosinophilic cytoplasmic inclusion bodies characteristic of IDF. Although the rate of cells bearing the inclusion bodies was high at the earlier passage levels, it was reduced to zero by the 15th passage of one of the cultures, but the cells of the other culture continued to produce the inclusion bodies even at the 30th passage. Chromosome analysis revealed both cultures to have tetraploid cells in approximately 8 to 12% in late passage levels. No viruslike particles were found in electron microscopy. No tumors developed when the cells were inoculated into athymic, nude mice subcutaneously. These cell cultures will be valuable for characterizing the eosinophilic inclusion bodies and determining the origin of the tumors.     

Permeabilization of cell membrane by programmed form of electrical pulses

The role of the shape of electric pulses of cell permeabilization and lysis was studied using the newly developed DPS electroporator. The effects of bipolar pulses, steep rising and falling edges in the pulses, delays between pulses, and shapes of DC signals between the edges on the lysis of bovine oocytes and the permeabilization of their cell membranes were investigated. Comparing the permeabilization rates with the lysis rates revealed a number of correlations, which make it possible to optimize the pulse shapes for achieving maximum permeabilization rates while keeping the lysis rates low. The optimization of pulse shape is essential for improving the procedure of electroporation in mammalian cloning technology.     

Prospore membrane formation: how budding yeast gets shaped in meiosis

During meiosis in Saccharomyces cerevisiae four daughter cells, called spores, are generated within the boundaries of the mother cell. This cell differentiation process requires de novo synthesis of prospore membranes (PSMs), which are the precursors of the spore plasma membranes. Assembly of these membranes is initiated at the spindle pole bodies (SPBs) during meiosis II. At this stage of the cell cycle, 4 SPBs are present. Two different meiosis-specific structures are known to be required for PSM formation. At the SPBs, specialized attachments, called the meiotic plaques, provide the required functionality necessary for the recruitment and assembly of the membranes. During subsequent membrane elongation, a second structure becomes important. This proteinaceous assembly forms a coat, called the leading edge protein coat (LEP coat), which covers the boundaries of the membranes. Assembly of the coat occurs at sites next to the SPBs, whereas its disassembly is concomitant to the closure of the membranes. This mini review discusses our current understanding of how the meiotic plaque and the LEP coat might function during biogenesis of the prospore membrane.     

Modeling the membrane potential generation of bacteriorhodopsin

The archaeon Halobacterium salinarum can grow phototrophically with only light as its energy source. It uses the retinal containing and light-driven proton pump bacteriorhodopsin to enhance the membrane potential which drives the ATP synthase. Therefore, a model of the membrane potential generation of bacteriorhodopsin is of central importance to the development of a mathematical model of the bioenergetics of H. salinarum. To measure the current produced by bacteriorhodopsin at different light intensities and clamped voltages, we expressed the gene in Xenopus laevis oocytes. We present current-voltage measurements and a mathematical model of the current-voltage relationship of bacteriorhodopsin and its generation of the membrane potential. The model consists of three intermediate states, the BR, L, and M states, and comparisons between model predictions and experimental data show that the L to M reaction must be inhibited by the membrane potential. The model is not able to fit the current-voltage measurements when only the M to BR phase is membrane potential dependent, while it is able to do so when either only the L to M reaction or both reactions (L to M and M to BR) are membrane potential dependent. We also show that a decay term is necessary for modeling the rate of change of the membrane potential.     

Simulate the diffusion of hydrated ions by nanofiltration membrane process with random walk

We propose a new diffusion model describing the diffusion behaviours of hydrated ions in the process of nanofiltration (NF) based on the random walk (RW) theory when the NF membrane is uncharged or low charged. In this model, the hydration of ions and their deformation capacity are considered. The structure of the membrane is idealised into a lozenge shape and the diameter of membrane pore is defined as gapsize. A computer program named RW system in chemistry is developed to simulate based on this model. Six familiar ions Li⁺, Na⁺, Mg²⁺, Al³⁺, K⁺ and Ca²⁺ are investigated. Their characteristics are calculated by Gaussian 03, Pople, Inc., Wallingford, CT. The diffusivities of hydrated ions are calculated and discussed. The results show that the hydration of ions cannot be ignored in NF process when the membrane pore size is near the dimensions of the hydrated ions.     

Optimizing the generation of random amplified polymorphic DNAs in chrysanthemum

Many conditions of the RAPD reaction procedure may influence the result. This paper presents rapid detection of influential factors with a fractional factorial experiment. A more extensive study of these factors is also presented. Polymerase brand, thermal cycler brand, annealing temperature, and primer, are important factors in obtaining good DNA yields and optimal fragment patterns. Each primer has its optimal annealing temperature, and this is not correlated with the GC content of the primer. Optimal species-primer combinations have to be found by trial and error.     

A massively connected subthalamic nucleus leads to the generation of widespread pulses.

A composite model of the subthalamic nucleus is developed from physiological and anatomical considerations. First, study of a geometric model of the anatomical arrangements of projection neurons within the nucleus indicates that they form a massively connected network. Second, given the excitatory nature of these neurons, their threshold and peak firing rates, a simple model of neuron responses reveals that large regions of this highly interconnected nucleus can respond to excitatory input in the form of a wide-spread uniform pulse. Such widespread pulses of activity may act as a braking signal that resets the major basal ganglia output nuclei.     

The Remorin C-terminal Anchor was shaped by convergent evolution among membrane binding domains

StREM1.3 Remorin is a well-established plant raftophilic protein, predominantly associated with sterol- and sphingolipid-rich membrane rafts. We recently identified a C-terminal domain (RemCA) required and sufficient for StREM1.3 anchoring to the plasma membrane. Here, we report a search for homologs and analogs of RemCA domain in publicly available protein sequence and structure databases. We could not identify RemCA homologous domains outside the Remorin family but we identified domains sharing bias in amino-acid composition and predicted structural fold with RemCA in bacterial, viral and animal proteins. These results suggest that RemCA emerged by convergent evolution among unrelated membrane binding domain.     

Cell motility through plasma membrane blebbing

Plasma membrane blebs are dynamic cytoskeleton-regulated cell protrusions that have been implicated in apoptosis, cytokinesis, and cell movement. Influencing Rho-guanosine triphosphatase activities and subsequent actomyosin dynamics appears to constitute a core component for bleb formation. In this paper, we discuss recent evidence in support of a central role of nonapoptotic membrane blebbing for cell migration and cancer cell invasion as well as advances in our understanding of the underlying molecular mechanisms. Based on these studies, we propose that in a physiological context, bleb-associated cell motility reflects a cell's response to reduced substratum adhesion. The importance of blebbing as a functional protrusion is underscored by the existence of multiple molecular mechanisms that govern actin-mediated bleb retraction.