A computer program for on-line measurement, storage, analysis and retrieval of urodynamic data

A computer program is presented which allows for direct connection of a minicomputer to a urodynamic set-up. The program stores measured pressure and flow data in a random access disc file with minimal intervention of the urodynamicist, and enables the direct application of a number of methods of analysis to the data. The program is modular, and other analysis methods are easily added. Results of analyses are stored in the same disc file, and both results and measured data can be quickly and easily retrieved. The program is written in FORTRAN; hardware-dependent functions (analog input, graphics display, and random access disc storage) are implemented in subroutines (partly assembler) which can easily be replaced.     

Arrangement of Photosystem II and ATP Synthase in Chloroplast Membranes of Spinach and Pea

We used cryoelectron tomography to reveal the arrangements of photosystem II (PSII) and ATP synthase in vitreous sections of intact chloroplasts and plunge-frozen suspensions of isolated thylakoid membranes. We found that stroma and grana thylakoids are connected at the grana margins by staggered lamellar membrane protrusions. The stacking repeat of grana membranes in frozen-hydrated chloroplasts is 15.7 nm, with a 4.5-nm lumenal space and a 3.2-nm distance between the flat stromal surfaces. The chloroplast ATP synthase is confined to minimally curved regions at the grana end membranes and stroma lamellae, where it covers 20% of the surface area. In total, 85% of the ATP synthases are monomers and the remainder form random assemblies of two or more copies. Supercomplexes of PSII and light-harvesting complex II (LHCII) occasionally form ordered arrays in appressed grana thylakoids, whereas this order is lost in destacked membranes. In the ordered arrays, each membrane on either side of the stromal gap contains a two-dimensional crystal of supercomplexes, with the two lattices arranged such that PSII cores, LHCII trimers, and minor LHCs each face a complex of the same kind in the opposite membrane. Grana formation is likely to result from electrostatic interactions between these complexes across the stromal gap.     

Brownian dynamics simulation of the lateral distribution of charged membrane components

Brownian dynamics simulations were performed to study the contribution of electric interactions between charged membrane components to their lateral distribution in a two-dimensional viscous liquid (bilayer lipid membrane). The electrostatic interaction potential was derived from an analytical solution of the linearized Poisson-Boltzmann equation for point charges in an electrolyte solution — membrane — electrolyte solution system. Equilibrium as well as dynamic quantities were investigated. The lateral organization of membrane particles, modelled by mobile cylinders in a homogeneous membrane separating two electrolyte solutions was described by spatial distribution functions, diffusion coefficients and cluster statistics. Disorder, local order and crystal-like arrangements were observed as a function of the particle charge, the closest possible distances between the charges and the particle density. The simulations revealed that the system is very sensitive to the position of the charges with respect to the electrolyte solution — membrane interface. Electrostatic interactions of charges placed directly on the membrane surface were almost negligible, whereas deeper charges demonstrated pronounced interaction. Biologically relevant parameters corresponded at most to local and transient ordering. It was found that lateral electric forces can give rise to a preferred formation of clusters with an even number of constituents provided that the closest possible charge-charge distances are small. It is concluded that lateral electrostatic interactions can account for local particle aggregations, but their impact on the global arrangement and movement of membrane components is limited. Correspondence to: D. Walther     

Large-Area Two-Dimensional Plasmonic Meta-Glasses and Meta-Crystals: a Comparative Study

The geometrical arrangement of metallic nanoparticles plays a crucial role on the optical response of nanoplasmonic samples due to particle-particle interactions. In this work, large-area, two-dimensional meta-glasses (random arrangements) and meta-crystals (periodic arrangements) made of identical metallic nanoparticles are investigated for three different particle densities of 5, 10, and 15 discs/μm². A direct comparison between random and periodically ordered arrays is presented. The comparison clearly shows that the particle density has the largest influence on the extinction spectra for both periodic and random samples, and that for equal densities, the optical response away from diffraction effects is strikingly similar in both cases. The role of the radial density function and minimum particle distance is also determined. This study elucidates the role of the particle-particle interactions on the response of plasmonic nanoparticles and indicates how to control position and shape of the plasmonic resonance.     

Stochastic simulation of activation in the G-protein cascade of phototransduction.

Activation of the G-protein cascade underlying phototransduction has been modeled by simulating the two-dimensional diffusional interactions that occur at the rod disc membrane between the three reacting protein species, which are the activated rhodopsin (R*), the G-protein (G), and the effector protein (E, the phosphodiesterase, PDE). The stochastic simulations confirm the main predictions of a simplified analytical model (Lamb, T. D., and E. N. Pugh, 1992, Journal of Physiology 449:719-758), and extend that treatment to more complicated cases, where there is a finite probability of reaction or a finite time for reaction. The simulations also provide quantitative estimates of the efficiency of coupling from activated G-protein (G*) to activated effector (E*) in terms of the concentrations, lateral diffusion coefficients, and binding rate constants of the participating molecules; the efficiency of coupling from G* to E* is found to be not as high as in the previous simplified analytical theory. The findings can be extended to other G-protein cascades, provided that the physical parameters of those cascades are specified.     

A simple model for the function of proteoglycans and collagen in the response to compression of the intervertebral disc.

The nucleus pulposus of the intervertebral disc exerts a pressure which enables it to support axial compression when contained by the annulus fibrosus. The disc was modelled as a thick-walled cylindrical pressure vessel in which the nucleus was contained radially by the annulus. As a result, the stress in the annulus had radial (compressive) as well as tangential (tensile) components. The radial stress at a given point in the annulus was considered to be balanced by the internal pressure which is expected to arise from the attraction of water by proteoglycans. There was a reasonable agreement between the calculated radial stress distribution and published results on the distribution of water within the annulus. As the internal pressure is expected to be isotropic, the annulus was expected to contribute to the axial resistance to compression of the disc; this contribution would be equal, in magnitude, to the radial stress. Predictions of the pressure distribution within the annulus were similar to published experimental measurements made in the radial and axial directions. The tangential stress within the annulus was considered to arise from the restoring stress in its strained collagen fibrils.     

Semiparametric models and inference for biomedical time series with extra-variation

Biomedical trials often give rise to data having the form of time series of a common process on separate individuals. One model which has been proposed to explain variations in such series across individuals is a random effects model based on sample periodograms. The use of spectral coefficients enables models for individual series to be constructed on the basis of standard asymptotic theory, whilst variations between individuals are handled by permitting a random effect perturbation of model coefficients. This paper extends such methodology in two ways: first, by enabling a nonparametric specification of underlying spectral behaviour; second, by addressing some of the tricky computational issues which are encountered when working with this class of random effect models. This leads to a model in which a population spectrum is specified nonparametrically through a dynamic system, and the processes measured on individuals within the population are assumed to have a spectrum which has a random effect perturbation from the population norm. Simulation studies show that standard MCMC algorithms give effective inferences for this model, and applications to biomedical data suggest that the model itself is capable of revealing scientifically important structure in temporal characteristics both within and between individual processes.     

Ultrastructure of the peritrophic membrane-secreting cells in the cardia of the blowfly, Lucilia cuprina

Scanning and transmission electron microscopy are used to reveal the internal anatomy and ultrastructure of the cardia which is the source of the triple layered peritrophic membrane in the blowfly Lucilia cuprina. Within the cardia, rings of secretory cells (formation zones) and non-secretory tissue (valvula cardiaca) interlock to secrete and mould the layers of membrane. Formation zone cells have abundant rough endoplasmic reticulum, Golgi and secretory vesicles. A portion of midgut just posterior to the formation zone is covered by close-packed microvilli connected by septate-like junctions. The cuticle-lined valvula cardiaca is rich in smooth endoplasmic reticulum, glycogen and microtubules. The oesophageal cuticle is unusual in containing tubular structures. The ultrastructural features of the separate components of the cardia are discussed in terms of their secretory and non-secretory roles; modified midgut cells secrete chitin and protein whereas modified foregut tissue (valvula cardiaca) appears to be adapted to provide structural integrity (extensive junctions, microtubules), movement (muscles, possibly microtubules), a store of energy (glycogen deposits) and possibly a lipidic secretion (from smooth endoplasmic reticulum) to lubricate the passage of the membranes.     

Mechanical properties that influence antimicrobial peptide activity in lipid membranes

Antimicrobial peptides are small amphiphilic proteins found in animals and plants as essential components of the innate immune system and whose function is to control bacterial infectious activity. In order to accomplish their function, antimicrobial peptides use different mechanisms of action which have been deeply studied in view of their potential exploitation to treat antibiotic-resistant bacterial infections. One of the main mechanisms of action of these peptides is the disruption of the bacterial membrane through pore formation, which, in some cases, takes place via a monomer to oligomer cooperative transition. Previous studies have shown that lipid composition, and the presence of exogenous components, such as cholesterol in model membranes or carotenoids in bacteria, can affect the potency of distinct antimicrobial peptides. At the same time, considering the membrane as a two-dimensional material, it has been shown that membrane composition defines its mechanical properties which might be relevant in many membrane-related processes. Nevertheless, the correlation between the mechanical properties of the membrane and antimicrobial peptide potency has not been considered according to the importance it deserves. The relevance of these mechanical properties in membrane deformation due to peptide insertion is reviewed here for different types of pores in order to elucidate if indeed membrane composition affects antimicrobial peptide activity by modulation of the mechanical properties of the membrane. This would also provide a better understanding of the mechanisms used by bacteria to overcome antimicrobial peptide activity.     

Characterization and Simulation of Uncertain Frequency Distributions: Effects of Distribution Choice,Variability, Uncertainty,and Parameter Dependence

We use bootstrap simulation to characterize uncertainty in parametric distributions, including Normal, Lognormal, Gamma, Weibull, and Beta, commonly used to represent variability in probabilistic assessments. Bootstrap simulation enables one to estimate sampling distributions for sample statistics, such as distribution parameters, even when analytical solutions are not available. Using a two-dimensional framework for both uncertainty and variability, uncertainties in cumulative distribution functions were simulated. The mathematical properties of uncertain frequency distributions were evaluated in a series of case studies during which the parameters of each type of distribution were varied for sample sizes of 5, 10, and 20. For positively skewed distributions such as Lognormal, Weibull, and Gamma, the range of uncertainty is widest at the upper tail of the distribution. For symmetric unbounded distributions, such as Normal, the uncertainties are widest at both tails of the distribution. For bounded distributions, such as Beta, the uncertainties are typically widest in the central portions of the distribution. Bootstrap simulation enables complex dependencies between sampling distributions to be captured. The effects of uncertainty, variability, and parameter dependencies were studied for several generic functional forms of models, including models in which two-dimensional random variables are added, multiplied, and divided, to show the sensitivity of model results to different assumptions regarding model input distributions, ranges of variability, and ranges of uncertainty and to show the types of errors that may be obtained from mis-specification of parameter dependence. A total of 1,098 case studies were simulated. In some cases, counter-intuitive results were obtained. For example, the point value of the 95th percentile of uncertainty for the 95th percentile of variability of the product of four Gamma or Weibull distributions decreases as the coefficient of variation of each model input increases and, therefore, may not provide a conservative estimate. Failure to properly characterize parameter uncertainties and their dependencies can lead to orders-of-magnitude mis-estimates of both variability and uncertainty. In many cases, the numerical stability of two-dimensional simulation results was found to decrease as the coefficient of variation of the inputs increases. We discuss the strengths and limitations of bootstrap simulation as a method for quantifying uncertainty due to random sampling error.     

ESR spin-label studies of lipid-protein interactions in membranes

Lipid spin labels have been used to study lipid-protein interactions in bovine and frog rod outer segment disc membranes, in (Na+, K+)-ATPase membranes from shark rectal gland, and in yeast cytochrome oxidase-dimyristoyl phosphatidylcholine complexes. These systems all display a two component ESR spectrum from 14-doxyl lipid spin-labels. One component corresponds to the normal fluid bilayer lipids. The second component has a greater degree of motional restriction and arises from lipids interacting with the protein. For the phosphatidylcholine spin label there are effectively 55 +/- 5 lipids/200,000-dalton cytochrome oxidase, 58 +/- 4 mol lipid/265,000 dalton (Na+, K+)-ATPase, and 24 +/- 3 and 22 +/- 2 mol lipid/37,000 dalton rhodopsin for the bovine and frog preparations, respectively. These values correlate roughly with the intramembrane protein perimeter and scale with the square root of the molecular weight of the protein. For cytochrome oxidase the motionally restricted component bears a fixed stoichiometry to the protein at high lipid:protein ratios, and is reduced at low lipid:protein ratios to an extent which can be quantitatively accounted for by random protein-protein contacts. Experiments with spin labels of different headgroups indicate a marked selectivity of cytochrome oxidase and the (Na+, K+)-ATPase for stearic acid and for cardiolipin, relative to phosphatidylcholine. The motionally restricted component from the cardiolipin spin label is 80% greater than from the phosphatidylcholine spin label for cytochrome oxidase (at lipid:protein = 90.1), and 160% greater for the (Na+, K+)-ATPase. The corresponding increases for the stearic acid label are 20% for cytochrome oxidase and 40% for (Na+, K+)-ATPase. The effective association constant for cardiolipin is approximately 4.5 times greater than for phosphatidylcholine, and that for stearic acid is 1.5 times greater, in both systems. Almost no specificity is found in the interaction of spin-labeled lipids (including cardiolipin) with rhodopsin in the rod outer segment disc membrane. The linewidths of the fluid spin-label component in bovine rod outer segment membranes are consistently higher than those in bilayers of the extracted membrane lipids and provide valuable information on the rate of exchange between the two lipid components, which is suggested to be in the range of 10(6)-10(7) s-1.     

Jumping mode atomic force microscopy on grana membranes from spinach

The thylakoid membrane system is a complex membrane system that organizes and reorganizes itself to provide plants optimal chemical energy from sunlight under different and varying environmental conditions. Grana membranes are part of this system and contain the light-driven water-splitting enzyme Photosystem II (PSII) and light-harvesting antenna complexes. Here, we present a direct visualization of PSII complexes within grana membranes from spinach. By means of jumping mode atomic force microscopy in liquid, minimal forces were applied between the scanning tip and membrane or protein, allowing complexes to be imaged with high detail. We observed four different packing arrangements of PSII complexes, which occur primarily as dimers: co-linear crystalline rows, nanometric domains of straight or skewed rows, and disordered domains. Upon storing surface-adhered membranes at low temperature prior to imaging, large-scale reorganizations of supercomplexes between PSII and light-harvesting complex II could be induced. The highest resolution images show the existence of membrane domains without obvious topography extending beyond supercomplexes. These observations illustrate the possibility for diffusion of proteins and smaller molecules within these densely packed membranes.     

Electron crystallography of biomolecules: mysterious membranes and missing cones

One of the major challenges facing structural biologists today is the determination of high-resolution 3D structures of membrane proteins. The requirement for detergent molecules to be present makes X-ray crystallography particularly difficult, coupled with the added problems of isolating sufficient (viable) protein samples at high enough concentrations to yield 3D crystals. One technique that enables structural determination with fewer constraints is electron crystallography of two-dimensional crystals, in which small amounts of membrane proteins can be studied in native form in lipid bilayers.     

Three-dimensional modeling of the brain's ECS by minimum configurational energy packing of fluid vesicles

The extracellular space of the brain is the heterogeneous porous medium formed by the spaces between the brain cells. Diffusion in this interstitial space is the mechanism by which glucose and oxygen are delivered to the brain cells from the vascular system. It is also a medium for the transport of certain informational substances between the cells (called volume transmission), and for drug delivery. This work involves three-dimensional modeling of the extracellular space as void space in close-packed arrays of fluid membrane vesicles. These packings are generated by minimizing the configurational energy using a Monte Carlo procedure. Both regular and random packs of vesicles are considered. A random walk algorithm is then used to compute the geometric tortuosities, and the results are compared with published experimental data. For the random packings, it is found that although the absolute values for the tortuosities differ, the dependence of the tortuosity on pore volume fraction is very similar to that observed in experiment. The tortuosities we measure are larger than those computed in previous studies of packings of convex polytopes, and modeling improvements, which require higher resolution studies and an improved modeling of brain cell shapes and mechanical properties, could help resolve remaining discrepancies between model simulations and experiment. It is also shown that the specular reflection scheme is the appropriate technique for implementing zero-flux boundary conditions in random walk simulations commonly encountered in diffusion problems.     

Preferred orientations of LDL in vitreous ice indicate a discoid shape of the lipoprotein particle

The structure of the human low-density lipoprotein (LDL) was analyzed in vitreous ice using cryo-electron microscopy (cryo-EM). In relatively thick cryo-EM preparations, random orientation of LDL particles produced various types of projections on the microscope screen, including circular projections with a high-density ring and rectangular projections with two high-density bands. However, in especially thin preparations, preferred, non-random orientations of the LDL particle produced only circular projections of the lipoprotein structure. In preparations with high LDL concentrations, ordered two-dimensional arrays, including hexagonal arrangements of circular projections and short stacks of rectangular projections, were observed. These observations are consistent with a discoid shape of the LDL particle, and suggest that surface tension forces may influence orientation of the LDL disc in thin aqueous films. Face-on orientation of LDL in especially thin cryo-EM preparations may explain earlier difficulties in identifying discoid features of the lipoprotein particle, and illustrates that some caution is warranted when attempts are made to reconstruct the three-dimensional structure of LDL from cryo-electron micrographs.     

A long-lived fusogenic state is induced in erythrocyte ghosts by electric pulses

Treatment of erythrocyte ghosts in random positions in a suspension with membrane fusion-inducing direct current electric field pulses causes the membranes to become fusogenic. Significant fusion yields are observed if the membranes are dielectrophoretically aligned into membrane-membrane contact with a weak alternating electric field as much as 5 min after the application of the pulses. This demonstrates that a long-lived membrane structural alteration is involved in this fusion mechanism. Other experiments indicate that the areas on the membrane which become fusogenic after treatment with the pulses may be very highly localized. The locations of these fusogenic areas coincide with where the trans-membrane electric field strength was greatest during the pulse. The fusogenic membrane alteration, or components thereof, in these areas laterally diffuses very slowly or not at all, or, to be fusogenic, must be present at concentrations in the membrane above a certain threshold. The loss of soluble 0.9-3-nm-diameter fluorescent probes from resealed cytoplasmic compartments of randomly positioned erythrocyte ghosts occurs through electric field pulse-induced pores only during a pulse but not between pulses or after a train of pulses if the probe diameter is 1.2 nm or greater. For a given pulse treatment of membranes in random positions in suspensions, an increase in ionic strength of the medium results in (a) a decrease in loss during the pulse, (b) no difference in loss between pulses, and (c) an increase in fusion yield when membrane-membrane contact is established. The latter two results (b and c) are incompatible with a fusion mechanism that proposes a simple relationship between electric field-induced pores and fusion.     

Comparative proteomics using 2-D gel electrophoresis and mass spectrometry as tools to dissect stimulons and regulons in bacteria with sequenced or partially sequenced genomes

We propose two-dimensional gel electrophoresis (2-DE) and mass spectrometry to define the protein components of regulons and stimulons in bacteria, including those organisms where genome sequencing is still in progress. The basic 2-DE protocol allows high resolution and reproducibility and enables the direct comparison of hundreds or even thousands of proteins simultaneously. To identify proteins that comprise stimulons and regulons, peptide mass fingerprint (PMF) with matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI-TOF-MS) analysis is the first option and, if results from this tool are insufficient, complementary data obtained with electrospray ionization tandem-MS (ESI-MS/MS) may permit successful protein identification. ESI-MS/MS and MALDI-TOF-MS provide complementary data sets, and so a more comprehensive coverage of a proteome can be obtained using both techniques with the same sample, especially when few sequenced proteins of a particular organism exist or genome sequencing is still in progress.     

Two-dimensional polyacrylamide-gel electrophoresis of the proteins and glycoproteins of the human erythrocyte membrane.

A two-dimensional polyacrylamide gel electrophoresis technique has been developed, improving the analytical separation of some proteins and glycoproteins of the human erythrocyte membrane. Freshly prepared membranes are totally solubilized, subjected to dodecylsulfate--polyacrylamide gel electrophoresis in the first dimension, followed by electrophoresis in the second dimension, using a detergent-free polyacrylamide gradient gel. By this method the proteins of the human erythrocyte membrane could be resolved into a two-dimensional pattern, which has been shown to be highly reproducible with respect to various blood-groups and within one blood-group from specimen to specimen. The method enables especially the investigation of the hydrophobic and very likely integrated membrane proteins and glycoproteins. Thus, band III[Fairbanks, G., Steck, Th. & Wallach, D. F. H., Biochemistry, 10, 2606--2617 (1971)] could be shown to consist of five proteins, one of them being the major glycoprotein of the human erythrocyte membrand. The two spectrin bands differed considerably in their two-dimensional patterns. The value of the given method for the investigation of membrane defects, which may be linked with various diseases of human erythrocytes, could be demonstrated in the case of two patients suffering from congenital dyserythropoetic anaemia.     

Control of the erythrocyte membrane shape: recovery from the effect of crenating agents

Intact erythrocytes become immediately crenated upon addition of 2,4- dinitrophenol (DNP) or pyrenebutyric acid (PBA). However, when cells are incubated at 37 degrees C in the presence of the crenating agents with glucose, they gradually (4--8 h) recover the normal biconcave disc form. The recovery process does not reflect a gradual inactivation of DNP or PBA since fresh cells are equally crenated by the supernatant from the recovered cells. Further, after recovery and removal of the crenating agents, cells are found to be desensitized to the readdition of DNP as well as to the addition of PBA, but they are more sensitive to cupping by chlorpromazine. This alteration in the cell membrane responsiveness was reversible upon further incubation in the absence of DNP. Recovery is dependent upon cellular metabolic state since an energy source is needed and incubation with guanosine but not adenosine will accelerate conversion to the disc shape. It is suggested that the conversion of cells from crenated to disc shape in the presence of the crenators, represents an alteration or rearrangement of membrane components rather than a redistribution of the crenators within the membrane. This shape recovery process may be important for erythrocyte shape preservation as well as shape control in other cells.