"Micro-lead" technic for recording ion currents through the membranes of individual myocardial cells

Patch-voltage-clamp method has been used for registration of the fast inward current in single heart muscle cells. Ionic current were measured on a small patch of membrane which is of about 1/200-1/300 of the total cell surface (diameter of patch is of about 5 micrometer). To correct the distortions of raw signal caused by the stray capacity in feedback of current-voltage converter and adjustable increasing of high-frequency gain was used. This allows one to shorten the net time of transient process in response to switching the command potential up to 30 microseconds. The channel population in membrance patch of micrometer in diameter is, on the one hand, large enough to record directly the integral ionic current and is, on the other hand, small enough for fluctuations of ionic current to be pronounced. This circumstance permits the method to be applied for ionic current investigations both by the classical methods and by statistical analysis.     

Quantifying the effects of distance and conspecifics on colonization: experiments and models using the loosestrife leaf beetle, Galerucella calmariensis

The ability of an insect to disperse to new habitat patches is difficult to quantify, but key to the establishment and persistence of populations. In this study, we examined dispersal of the phytophagous chrysomelid beetle, Galerucella calmariensis, which is currently being introduced into North America for the biological control of purple loosestrife (Lythrum salicaria), an aggressive wetland weed. We used a mark, release, and recapture approach to determine how rates of colonization of host patches by this beetle are influenced by the distance of the patch from the source of dispersers, and by the presence of conspecifics at the patch. We released color-coded beetles at six distances from a long, linear patch of purple loosestrife that was divided into segments with and without conspecifics. We observed initial flight directions as beetles left the release points and collected all beetles that settled at the target patch. We found a bias in initial flight toward the target for distances up to 50 m. Over the 7 days of the experiment, beetles arrived at the target from all release points, including the farthest release point, 847 m away. G. calmariensis was strongly attracted to conspecifics when settling after dispersal; 86% of the 582 recovered beetles came from the segments inhabited by conspecifics. The probability of an individual arriving at the patch declined steeply with release distance. This relationship fits a model in which beetles move in a random direction and stop if they intercept the target patch, and where beetles are lost at a constant rate with distance travelled. The dispersal and patch-colonizing behavior of G. calmariensis is likely to have important consequences for the biological control program against purple loosestrife. Received: 23 January 1996 / Accepted:30 September 1996     

基于粒度反推法的景观生态安全格局优化——以海口市秀英区为例

景观生态安全格局优化是改善生态环境、促进人与自然和谐发展的有效途径。为给海口市秀英区生态环境建设提供科学依据和景观生态安全格局优化方法改进提供参考,提出了粒度反推法和生态阻力面综合构建法,结合GIS技术和最小耗费距离模型探讨了海口市秀英区景观生态安全格局优化途径。结果表明:(1)400m粒度生态景观组分是秀英区生态源地选取的合适参照,秀英区有生态源地18块、生态廊道17条、生态节点11个,新增生态源地建设的参考规模为38.5hm2,生态源地的生态服务极限距离为800m,需将现有的11块非生态景观类型斑块转换为生态景观类型,总面积20.26 hm2。(2)粒度反推法能为生态源地选取提供客观参考,在生态源地选择方面比传统方法具有更强的理论基础和客观性,解决了客观选取生态源地的问题。(3)综合生态阻力面能对生态阻力的实际情况进行较好地模拟。显、隐性生态阻力面之间存在显著差异,能反映生态系统中潜在的生态薄弱点,这些生态薄弱点往往是容易被忽略而又需要重点建设的区域。     

In situ estimates of annual net nitrogen mineralization and nitrification in a subarctic watershed

Summary Annual estimates of surface soil nitrogen transformations were determined using an in situ method in four different subarctic vegetation types within a watershed in southwestern Alaska. The net nitrogen mineralization estimates were 22.5, 0.5, 4.7, and 2.7 kg-N ha^-1 yr^-1 for the alder, dry tundra, moist tundra, and white spruce sites, respectively. Only the soil from the alder site showed net nitrification (about 10 kg-N ha^-1 yr^-1). Annual inogranic nitrogen flux from the overlying organic layer to the mineral soil was almost seven times greater than net N production in the surface mineral soil in the alder site, indicating that the alder forest floor is potentially a substantial source for plant-available N. Rates of mobilization of N from the surface organic layers of the other sites were similar to net N production rates in surface mineral soils. In situ rates of N transformations showed a similar trend among sites as did laboratory estimates conducted in a previous study, suggesting a strong substrate control of N transformations in these soils.     

A moorable,automated plankton sampler

A moorable, automated plankton sampler is described, designed to obtain series of plankton samples over extended time periods in the absence of a surface vessel. The sampler consists of one or more net-containing boxes, a unit for generating as well as measuring water flow through the net boxes, a programmeable control unit, and a frame. Each net box contains several nets on a vinyl strip, used to move the nets from the storage chamber into the fishing position and then into the preservative chamber. Preservation of samples is in a formalin-brine mixture.Net boxes can be added and the number of nets per box changed; the prototype is described with 1 net box with 10 nets. Volume of water to be filtered, time between sample collections, number of net boxes and the number of nets in each box is programmed into the control unit before sampler deployment. Collections made with the sampler are compared with those made with a SCOR net and a pump.     

Extinction conditions for isolated populations affected by environmental stochasticity

We determine the critical patch size below which extinction occurs for populations living in one-dimensional habitats surrounded by completely hostile environments in the presence of environmental fluctuations. The population dynamics is reformulated in terms of a stochastic reaction–diffusion equation and is reduced to a deterministic equation that incorporates the systematic contributions of the noise. We obtain bifurcation diagrams and relations for the mean population density at the stationary state, the critical patch size, and the mean number of individuals in the habitat. The effect of the noise differs, depending on whether it affects the net growth rate or the intraspecific competition term. Fluctuations in the net growth rate decrease the critical patch size, whereas fluctuations in the competition term do not change the critical patch size. We compare our analytical results with numerical solutions of the stochastic partial differential equations and show that our procedure proves useful in dealing with reaction–diffusion equations with multiplicative noise.     

基于斑块的红树林空间演变机理分析方法

现有红树林空间动态分析多是从整体的角度分析其面积的变化情况及其影响因素,着重于面积变化的定量分析和影响因素的定性分析,对其变化的发生途径缺乏深入分析,也不研究其斑块数量的变化动态。由于一定区域范围内的红树林由成百上千甚至更多空间上相互分离的斑块组成,各斑块边界和面积变化动态构成了区域红树林整体的空间分布变化动态,因此,只有深入摸清各个斑块的变化情况及其影响因素,才能对区域红树林整体变化情况作出全面、详细和准确的分析评估。提出了基于斑块的红树林空间演变机理分析方法,首先通过两期高空间分辨率遥感图像提取红树林空间分布信息,在GIS支持下采用叠置分析方法,根据前、后两期各个斑块的空间位置、形状和面积变化情况以及图像表征,逐一分析确定每个斑块变化的主要驱动因子(即变化原因,包括自然过程、围垦、养殖塘和盐田建设、工程建设和人工造林5种)和变化途径(即变化类型,包括稳定、扩张、萎缩、碎化、消失和新增6种),在此基础上构建斑块数量和面积变化的驱动因子-变化途径状态矩阵,通过总驱动量、总驱动率、净驱动量、净驱动率、趋势驱动率、总流量、总流率、净流量、净流率、趋势净流率和作用力等系列指标定量地评估红树林斑块数量和面积的变化动态。该方法不但能够定量地表达了各个驱动因子对红树林斑块数量和面积变化的影响程度,而且能够准确地阐明了红树林斑块数量和面积发生变化的途径,并且还能够准确地反映了每个驱动因子通过何种途径影响斑块数量和面积的变化,实现了红树林空间动态变化分析的定位化、定量化和精确化。     

Application of a Mouse Ligated Peyer’s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed to them and occasionally to pathogens. The gut-associated lymphoid tissue (GALT) play a key role for induction of the mucosal immune response to these microbes^{1, 2}. To initiate the mucosal immune response, the mucosal antigens must be transported from the gut lumen across the epithelial barrier into organized lymphoid follicles such as Peyer''s patches. This antigen transcytosis is mediated by specialized epithelial M cells^{3, 4}. M cells are atypical epithelial cells that actively phagocytose macromolecules and microbes. Unlike dendritic cells (DCs) and macrophages, which target antigens to lysosomes for degradation, M cells mainly transcytose the internalized antigens. This vigorous macromolecular transcytosis through M cells delivers antigen to the underlying organized lymphoid follicles and is believed to be essential for initiating antigen-specific mucosal immune responses. However, the molecular mechanisms promoting this antigen uptake by M cells are largely unknown. We have previously reported that glycoprotein 2 (Gp2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane ⁵. Here, we present a method for the application of a mouse Peyer''s patch intestinal loop assay to evaluate bacterial uptake by M cells. This method is an improved version of the mouse intestinal loop assay previously described ^{6, 7}. The improved points are as follows: 1. Isoflurane was used as an anesthetic agent. 2. Approximately 1 cm ligated intestinal loop including Peyer''s patch was set up. 3. Bacteria taken up by M cells were fluorescently labeled by fluorescence labeling reagent or by overexpressing fluorescent protein such as green fluorescent protein (GFP). 4. M cells in the follicle-associated epithelium covering Peyer''s patch were detected by whole-mount immunostainig with anti Gp2 antibody. 5. Fluorescent bacterial transcytosis by M cells were observed by confocal microscopic analysis. The mouse Peyer''s patch intestinal loop assay could supply the answer what kind of commensal or pathogenic bacteria transcytosed by M cells, and may lead us to understand the molecular mechanism of how to stimulate mucosal immune system through M cells.     

Improved estimates of incident radiation and heat load using non‐ parametric regression against topographic variables

Question: Can non‐parametric multiplicative regression (NPMR) improve estimates of potential direct incident radiation (PDIR) and heat load based on topographic variables, as compared to least‐squares multiple regression against trigonometric transforms of the predictors? Methods: We used a multiplicative kernel smoothing technique to interpolate between tabulated values of PDIR, using a locally linear model and a Gaussian kernel, with slope, aspect, and latitude as predictors. Heat load was calculated as a 45 degree rotation of the PDIR response surface. Results: This method yielded a fit to a complex response surface with R² > 0.99 and eliminated the areas of poor fit given by a previously published method based on least squares multiple regression with trigonometric functions of the predictors. Conclusions: Improved estimates of PDIR and heat load based on topographic variables can be obtained by using non‐parametric multiplicative regression (NPMR). The main drawback to the method is that it requires reference to the data tables, since those data are part of the model.     

Spline-based framework for interactive segmentation in biomedical imaging

Active contours constitute a computationally attractive framework for image segmentation. In this paper, we describe a fully parametric design that relies on B-spline bases and is specified by control points on the image. Our technique yields successful segmentation results even for challenging datasets where object contours are not well-defined. We can achieve this because our parametric approach uses few parameters and allows us to constrain the topology of the curve. We provide the details for an efficient implementation and discuss different design alternatives. This work comes with a companion software that gives the end user full control over the snakes. We demonstrate the features of our software which includes an interactive interface where the user can manipulate the position of control points by simple mouse actions.     

Analytically defined surfaces to analyze molecular interaction properties

Molecular surfaces are widely used for characterizing molecules and displaying and quantifying their interaction properties. Here we consider molecular surfaces defined as isocontours of a function (a sum of exponential functions centered on each atom) that approximately represents electron density. The smoothness is advantageous for surface mapping of molecular properties (e.g., electrostatic potential). By varying parameters, these surfaces can be constructed to represent the van der Waals or solvent-accessible surface of a molecular with any accuracy. We describe numerical algorithms to operate on the analytically defined surfaces. Two applications are considered: (1) We define and locate extremal points of molecular properties on the surfaces. The extremal points provide a compact representation of a property on a surface, obviating the necessity to compute values of the property on an array of surface points as is usually done; (2) a molecular surface patch or interface is projected onto a flat surface (by introducing curvilinear coordinates) with approximate conservation of area for analysis purposes. Applications to studies of protein-protein interactions are described.     

Texture mapping parametric molecular surfaces

Texture mapping is an increasingly popular technique in molecular modeling. It is particularly effective in representing high-resolution surface detail using a low-resolution polygonal model. We describe how texture mapping can be used with parametric molecular surfaces represented as expansions of spherical harmonic functions. We define analytically the texture image and its transformation to a parametric surface. Unlike most methods of texture mapping, this transformation defines a one-to-one correspondence between the surface and the texture; texture coordinates are derived from the location of the surface point and not from physical properties at the surface point. This has advantates for the interactive visualization of surface data. We control the interactive response time by lowering the resolution of the polygon mesh while retaining the high-resolution detail of the texture, or we can lower the resolution of the texture image with the same polygonal model. By using a well-defined convention for texture coordinates, we can use the same image for the original surface or its parametric representation, and we can rapidly switch between images that represent different surface properties without recomputing the texture coordinates. Parametric surfaces allow new flexibility for the visualization of molecular surface data.     

Exploration of compact protein conformations using the guided replication monte carlo method

We have studied the use of a new Monte Carlo (MC) chain generation algorithm, introduced by T. Garel and H. Orland[(1990) Journal of Physics A, Vol. 23, pp. L621–L626], for examining the thermodynamics of protein folding transitions and for generating candidate C^αbackbone structures as starting points for a de now protein structure paradigm. This algorithm, termed the guided replication Monte Carlo method, allows a rational approach to the introduction of known “native” folded characteristics as constraints in the chain generation process. We have shown this algorithm to be computationally very efficient in generating large ensembles of candidate C^αchains on the face centered cubic lattice, and illustrate its use by calculating a number of thermodynamic quantities related to protein folding characteristics. In particular, we have used this static MC algorithm to compute such temperature-dependent quantities as the ensemble mean energy, ensemble mean free energy, the heat capacity, and the mean-square radius of gyration. We also demonstrate the use of several simple “guide fields” for introducing protein-specific constraints into the ensemble generation process. Several extensions to our current model are suggested, and applications of the method to other folding related problems are discussed. © 1995 John Wiley & Sons, Inc.     

A surgical method for accessing the plasmamembrane ofChara australis

Summary Progress in patch-clamping the plasmalemma ofChara australis has been slow because of the difficulty in obtaining a clean membrane surface upon which obtain a high resistance seal with the patch pipette. A surgical method is described here for cutting an access window in the cell wall of internodal cells fromChara australis. The method has a high success rate and also has the advantage of obviating the need for enzyme treatments. The patch-clamp technique is applied to theChara plasmalemma. Five types of channel activity are observed of which four of these are selective for Cl^– over K⁺.     

The effects of non-identifiability on testing for detailed balance in aggregated Markov models for ion-channel gating

Aggregated Markov models are a widely used tool to model patch clamp data measured from single ion channels. These channels must obey the principle of detailed balance in thermodynamic equilibrium; otherwise, the channel is driven by an external source of energy. We investigate the power of a likelihood ratio test for detailed balance for a number of data points which is in the order of magnitude of patch clamp experiments. We show that for certain models with nearly equal dwell times, a test for detailed balance suffers from a loss of power to detect violations of detailed balance which is due to the non-identifiability of the transition rates for models with equal dwell times.     

The weak combined magnetic field,tuned to ion parametric resonance for Ca<Superscript>2+</Superscript>, stimulates dystrophin synthesis in the skeletal muscles of mdx mice subjected to cell therapy

The possibility of using bone marrow stem cells for treatment of Duchenne muscular dystrophy is intensely studied. Mdx mice are the most widely used laboratory model of Duchenne muscular dystrophy. One approach of cell therapy of muscular dystrophy is substitution of bone marrow in mdx mice after their X-ray irradiation. However, this method does not allow one to increase significantly dystrophin synthesis in muscular fibers of mdx mice. To improve the effect of transplanted cells on muscle regeneration, we additionally treated mdx mice subjected to transplantation of bone marrow cells with a weak combined magnetic field tuned to ion parametric resonance for Ca²⁺ (Ca²⁺-CMF). We found that, in irradiated chimeric 3 and 5 Gy mdx mice, additional treatment with Ca²⁺-CMF for 1 month resulted in significant increases in the portions of dystrophin-positive muscle fibers, by 15.8 and 18.3%, respectively, as compared to the control groups. Furthermore, the share of muscle fibers without centrally located nuclei also increased. We suggest that the magnetic field with these parameters may stimulate functioning of nuclei of donor cells, which were incorporated into muscle fibers.     

A new approach for surface fitting method of articular joint surfaces

The application of joint contact mechanics requires a precise configuration of the joint surfaces. B-Spline, and NURBS have been widely used to model joint surfaces, but because these formulations use a structured data set provided by a rectangular net first, then a grid, there is a limit to the accuracy of the models they can produce. However new imaging systems such as 3D laser scanners can provide more realistic unstructured data sets. What is needed is a method to manipulate the unstructured data. We created a parametric polynomial function and applied it to unstructured data sets obtained by scanning joint surfaces. We applied our polynomial model to unstructured data sets from an artificial joint, and confirmed that our polynomial produced a smoother and more accurate model than the conventional B-spline method. Next, we applied it to a diarthrodial joint surface containing many ripples, and found that our function's noise filtering characteristics smoothed out existing ripples. Since no formulation was found to be optimal for all applications, we used two formulations to model surfaces with ripples. First, we used our polynomial to describe the global shape of the objective surface. Minute undulations were then specifically approximated with a Fourier series function. Finally, both approximated surfaces were superimposed to reproduce the original surface in a complete fashion.     

Surface potential determination in planar lipid bilayers: A simplification of the conductance-ratio method

One of the methods available for the measurement of surface potentials of planar lipid bilayers uses the conductance ratio between a charged and a neutral bilayer doped with ionophores to calculate the surface potential of the charged bilayer. We have devised a simplification of that method which does not require the use of an electrically neutral bilayer as control. The conductance of the charged bilayer is measured before and after the addition of divalent cations (Ba²⁺) to the bathing solution. Ba²⁺ ions screen fixed surface charges, decreasing the surface potential. If the membrane is negatively charged the screening has the effect of decreasing the membrane conductance to cations. The resulting conductance ratio is used to calculate the surface potential change, which is fed into an iterative computer program. The program generates pairs of surface potential values and calculates the surface charge density for the two conditions. Since the surface charge density remains constant during this procedure, there is only one pair of surface potentials that satisfies the condition of constant charge density.Applying this method to experimental data from McLaughlin et al. [McLaughlin, S.G.A., Szabo, G. and Eisenman, G., Divalent ions and the surface potential of charged phospholipid membranes, J. Gen. Physiol., 58 (1971) 667–687.] we have found very similar results. We have also successfully used this method to determine the effect of palmitic acid on the surface potential of asolectin membranes.     

Hepatocyte adhesion to carbohydrate-derivatized surfaces. I. Surface topography of the rat hepatic lectin

The rat hepatic lectins, galactose- and N-acetylgalactosamine-binding proteins found on the hepatocyte cell surface, mediate adhesion of isolated primary rat hepatocytes to artificial galactose-derivatized polyacrylamide gels. Biochemical and immunohistochemical techniques were used to examine the topographical redistribution of the rat hepatic lectins in response to galactose-mediated cell adhesion. Hepatocytes isolated from rat liver by collagenase perfusion had an average of 7 x 10(5) cell surface lectin molecules per cell, representing 30-50% of the total lectin molecules per cell, the remainder residing in intracellular pools. Hepatocytes incubated on galactose-derivatized surfaces, whether at 0-4 degrees C or 37 degrees C, rapidly lost greater than 80% of their accessible cell surface lectin binding sites into an adhesive patch of characteristic morphology. The kinetics of rat hepatic lectin disappearance were used to estimate a lateral diffusion coefficient greater than 9 x 10(-9) cm2/s at 37 degrees C, suggesting rapid and unimpeded lectin diffusion in the plane of the membrane. Indirect immunofluorescence labeling of adherent cells using antihepatic lectin antibody revealed a structured ring of receptors surrounding an area of exclusion (patch) of reproducible size and shape which represented approximately 8% of the hepatocyte cell surface. Notably, adherent cells, which had lost greater than 80% of their accessible surface binding sites, still endocytosed soluble galactose-terminated radioligand at greater than 50% of the rate of nonadherent control cells. No net movement of rat hepatic lectin from intracellular pools to the cell surface was found on cells recovered after adhesion to galactose-derivatized surfaces at 37 degrees C, suggesting that the physical size and/or lectin density of the patch was restricted by kinetic or topological constraints.     

Biological activities as patchiness driving forces in wetlands of northern Belize

Patchiness in wetlands is a common and well documented phenomenon. Oligotrophic wetlands of northern Belize display noticeable vegetation heterogeneity at both large and small scales. In this paper, we document the small scale patches in herbaceous wetlands, describe differences between patches and surrounding wetland habitats and explain patch formation and sustenance. We conducted a survey of patches and confirmed their occurrence by spatial analysis. Patches were distinguished from a surrounding wetland by denser and taller vegetation, higher amount of empty snail shells and elevated soil phosphorus (P). Plants in patches had higher tissue nitrogen (N) and P content and there was also higher total N and P per m² incorporated in plant biomass. In terms of stable isotopes, plants in patches were enriched in ¹⁵N; patch soils were depleted in ¹³C. Observations of focal individuals of Aramus guarauna, limpkin, a wading bird feeding almost exclusively on snails, revealed the origin of the snail shell piles frequently found in patches. An adult limpkin captured on average 18 snails daily, of these 80% were handled in patches and birds often repeatedly used the same patch. Experimental patch creation by adding chicken manure or P to 1 m² plots resulted in higher and denser vegetation with values increasing in order: control, P, manure plots. The effect was significant at both experimental locations six months after the treatment and at one location even 40 months after the treatment. We present a simple mechanistic explanation for nutrient redistribution in wetlands and their eventual accumulation in patches. Both nutrient and isotopic differences result from animal input into patches, e.g. bird droppings or prey remnants. Foraging activity of Aramus guarauna is most likely responsible for patch formation. A positive feedback (repeated use of a suitable patch) is apparently the mechanism sustaining patches in these marsh environments.