Characterization of esophageal desalination in the seawater eel,Anguilla japonica

To characterize mechanisms of esophageal desalination, osmotic water permeability and ion fluxes were measured in the isolated esophagus of the seawater eel. The osmotic permeability coefficient in the seawater eel esophagus was 2·10^-4 cm·s^-1. This value was much lower than those in tight epithelial, although the eel esophagus is a leaky epithelium with a tissue resistance of 77 ohm·cm^-2. When the esophagus was bathed in normal Ringer solutions on both sides no net ion and water fluxes were observed. However, when mucosal NaCl concentration was increased by a factor of 3, Na⁺ und Cl^- ions were transferred from mucosa to serosa (desalination). If only Na⁺ or Cl^- concentration in the mucosal fluid was increased by a factor of 3, net Na⁺ and Cl^- fluxes were reduced to 30–40%, indicating that 60–70% of the net Na⁺ and Cl^- fluxes are coupled mutually. The coupled NaCl transport seems to be effective in desalting the luminal high NaCl. The remaining 30–40% of the total Na⁺ and Cl^- fluxes seems to be due to a simple diffusion, because these components are independent of each other and follow their electrochemical gradients, and also because these fluxes remain even after treatment with NaCN or ouabain. A half of the coupled NaCl transport could be explained by a Na⁺/H⁺–Cl^-/HCO ₃ ^- double exchanger on the apical membrane of the esophageal epithelium, because mucosal amiloride and 4.4-diisothiocyanatostilbene-2,2-disulphonic acid inhibited the net Na⁺ and Cl^- fluxes by approximately 30%. The other half of the coupled NaCl transport, which follows their electrochemical gradients, still remains to be explained.Abbreviations DIDS 4,4-diisothiocyanatostilbene-2,2-disulphonic acid - NMDG N-methyl-d-glucosamine - P _Cl Cl^- permeability coefficient - PD transepithelial potential difference - P _Na Na⁺ permeability coefficient - P _osm osinotic permeability coefficient - TALH thick ascending limb of Henle's loop     

Numerical Models for the Simulation of Flexible Artificial Heart Valves: Part I - Computational Methods

Abstract

A numerical model of the coupled motion of a flexing surface in a high Reynolds number flow is presented for the simulation of flexible polyurethane heart valves in the aortic position. This is achieved by matching a Lagrangian dynamic leaflet model with a panel method based flow solver. The two models are coupled via the time-dependent pressure field using the unsteady Bernoulli equation.

Incorporation of sub-cycling in the dynamic model equations and fast pre conditioning techniques in the panel method solver yields efficient convergence and near real-time simulations of valve motion. The generality of dynamic model allows different material properties and/or geometries to be studied easily and interactively. This interactivity is realized by embedding the models within a design environment created using the software IRIS Explorer ^TM.

Two flow domains are developed, an infinite domain and an internal domain using conformal mapping theory. In addition bending stress on the valve is computed using a simple stress model based on spline and circle equation techniques.     

Analysis of agonism by dopamine at the dopaminergic D 2 G-protein coupled receptor based on comparative modelling of rhodopsin

A number of theoretical dopaminergic D 2 models were constructed using comparative modelling techniques. The models constructed were based on the recently published crystal structure of rhodopsin. D 2 models were constructed without any ligands bound in the active site and also with the endogenous agonist, dopamine, bound in the active site. Comparison of the bound and unbound models revealed the importance of the chi angle of serine 197 and the effect this has on the bending of helix 5 when an agonist is present in the binding site.     

Two continuous coupled assays for ornithine-δ-aminotransferase

We have developed two new continuous coupled assays for ornithine-δ-aminotransferase (OAT) that are more sensitive than previous methods, measure activity in real time, and can be carried out in multiwell plates for convenience and high throughput. The first assay is based on the reduction of Δ¹-pyrroline-5-carboxylate (P5C), generated from ornithine by OAT, using human pyrroline 5-carboxylate reductase 1 (PYCR1), which results in the concomitant oxidation of NADH (nicotinamide adenine dinucleotide, reduced form) to NAD⁺ (nicotinamide adenine dinucleotide, oxidized form). This procedure was found to be three times more sensitive than previous methods and is suitable for the study of small molecules as inhibitors or inactivators of OAT or as a method to determine OAT activity in unknown samples. The second method involves the detection of l-glutamate, produced during the regeneration of the cofactor pyridoxal 5’-phosphate (PLP) of OAT by an unamplified modification of the commercially available Amplex Red l-glutamate detection kit (Life Technologies). This assay is recommended for the determination of the substrate activity of small molecules against OAT; measuring the transformation of l-ornithine at high concentrations by this assay is complicated by the fact that it also acts as a substrate for the l-glutamate oxidase (GluOx) reporter enzyme.     

A spectrophotometric coupled enzyme assay to measure the activity of succinate dehydrogenase

Respiratory complex II (succinate:ubiquinone oxidoreductase) connects the tricarboxylic acid cycle to the electron transport chain in mitochondria and many prokaryotes. Complex II mutations have been linked to neurodegenerative diseases and metabolic defects in cancer. However, there is no convenient stoichiometric assay for the catalytic activity of complex II. Here, we present a simple, quantitative, real-time method to detect the production of fumarate from succinate by complex II that is easy to implement and applicable to the isolated enzyme, membrane preparations, and tissue homogenates. Our assay uses fumarate hydratase to convert fumarate to malate and uses oxaloacetate decarboxylating malic dehydrogenase to convert malate to pyruvate and to convert NADP⁺ to NADPH; the NADPH is detected spectrometrically. Simple protocols for the high-yield production of the two enzymes required are described; oxaloacetate decarboxylating malic dehydrogenase is also suitable for accurate determination of the activity of fumarate hydratase. Unlike existing spectrometric assay methods for complex II that rely on artificial electron acceptors (e.g., 2,6-dichlorophenolindophenol), our coupled assay is specific and stoichiometric (1:1 for succinate oxidation to NADPH formation), so it is suitable for comprehensive analyses of the catalysis and inhibition of succinate dehydrogenase activities in samples with both simple and complex compositions.     

Synthesis of 4-methylumbelliferyl α-d-mannopyranosyl-(1→6)-β-d-mannopyranoside and development of a coupled fluorescent assay for GH125 exo-α-1,6-mannosidases

Certain bacterial pathogens possess a repertoire of carbohydrate processing enzymes that process host N-linked glycans and many of these enzymes are required for full virulence of harmful human pathogens such as Clostridium perfringens and Streptococcus pneumoniae. One bacterial carbohydrate processing enzyme that has been studied is the pneumococcal virulence factor SpGH125 from S. pneumoniae and its homologue, CpGH125, from C. perfringens. These exo-α-1,6-mannosidases from glycoside hydrolase family 125 show poor activity toward aryl α-mannopyranosides. To circumvent this problem, we describe a convenient synthesis of the fluorogenic disaccharide substrate 4-methylumbelliferone α-d-mannopyranosyl-(1→6)-β-d-mannopyranoside. We show this substrate can be used in a coupled fluorescent assay by using β-mannosidases from either Cellulomonas fimi or Helix pomatia as the coupling enzyme. We find that this disaccharide substrate is processed much more efficiently than aryl α-mannopyranosides by CpGH125, most likely because inclusion of the second mannose residue makes this substrate more like the natural host glycan substrates of this enzyme, which enables it to bind better. Using this sensitive coupled assay, the detailed characterization of these metal-independent exo-α-mannosidases GH125 enzymes should be possible, as should screening chemical libraries for inhibitors of these virulence factors.     

Is the somitogenesis clock really cell-autonomous? A coupled-oscillator model of segmentation

A striking pattern of oscillatory gene expression, related to the segmentation process (somitogenesis), has been identified in chick, mouse, and zebrafish embryos. Somitogenesis displays great autonomy, and it is generally assumed in the literature that somitogenesis-related oscillations are cell-autonomous in chick and mouse. We point out in this article that there would be many biological reasons to expect some mechanism of coupling between cellular oscillators, and we present a model with such coupling, but which also has autonomous properties. Previous experiments can be re-interpreted in light of this model, showing that it is possible to reconcile both autonomous and non-autonomous aspects. We also show that experimental data, previously interpreted as supporting a purely negative-feedback model for the mechanism of the oscillations, is in fact more compatible with this new model, which relies essentially on positive feedback.     

A continuous coupled enzyme assay for bacterial malonyl-CoA:acyl carrier protein transacylase (FabD)

Bacterial malonyl-CoA:acyl carrier protein transacylase catalyzes the transfer of a malonyl moiety from malonyl-CoA to the free thiol group of the phosphopantetheine arm of acyl carrier protein. Malonyl-ACP, the product of this enzymatic reaction, is the key building block for de novo fatty acid biosynthesis. Here, we describe a continuous enzyme assay based on the coupling of the malonyl-CoA:acyl carrier protein transacylase reaction to alpha-ketoglutarate dehydrogenase (KDH). KDH-dependent consumption of the coenzyme A generated by malonyl-CoA:acyl carrier protein transacylase is accompanied by a reduction of nicotinamide adenine dinucleotide, oxidized (NAD(+)) to nicotinamide adenine dinucleotide, reduced. The rate of NAD(+) reduction is continuously monitored as a change in fluorescence using a microtiter plate reader. We show that this coupled enzyme assay is amenable to routine chemical compound screening.     

Models of central pattern generators for quadruped locomotion I. Primary gaits

In this paper we continue the analysis of a network of symmetrically coupled cells modeling central pattern generators for quadruped locomotion proposed by Golubitsky, Stewart, Buono, and Collins. By a cell we mean a system of ordinary differential equations and by a coupled cell system we mean a network of identical cells with coupling terms. We have three main results in this paper. First, we show that the proposed network is the simplest one modeling the common quadruped gaits of walk, trot, and pace. In doing so we prove a general theorem classifying spatio-temporal symmetries of periodic solutions to equivariant systems of differential equations. We also specialize this theorem to coupled cell systems. Second, this paper focuses on primary gaits; that is, gaits that are modeled by output signals from the central pattern generator where each cell emits the same waveform along with exact phase shifts between cells. Our previous work showed that the network is capable of producing six primary gaits. Here, we show that under mild assumptions on the cells and the coupling of the network, primary gaits can be produced from Hopf bifurcation by varying only coupling strengths of the network. Third, we discuss the stability of primary gaits and exhibit these solutions by performing numerical simulations using the dimensionless Morris-Lecar equations for the cell dynamics.     

Local and global pyrogeographic evidence that indigenous fire management creates pyrodiversity

Despite the challenges wildland fire poses to contemporary resource management, many fire‐prone ecosystems have adapted over centuries to millennia to intentional landscape burning by people to maintain resources. We combine fieldwork, modeling, and a literature survey to examine the extent and mechanism by which anthropogenic burning alters the spatial grain of habitat mosaics in fire‐prone ecosystems. We survey the distribution of Callitris intratropica, a conifer requiring long fire‐free intervals for establishment, as an indicator of long‐unburned habitat availability under Aboriginal burning in the savannas of Arnhem Land. We then use cellular automata to simulate the effects of burning identical proportions of the landscape under different fire sizes on the emergent patterns of habitat heterogeneity. Finally, we examine the global extent of intentional burning and diversity of objectives using the scientific literature. The current distribution of Callitris across multiple field sites suggested long‐unburnt patches are common and occur at fine scales (<0.5 ha), while modeling revealed smaller, patchy disturbances maximize patch age diversity, creating a favorable habitat matrix for Callitris. The literature search provided evidence for intentional landscape burning across multiple ecosystems on six continents, with the number of identified objectives ranging from two to thirteen per study. The fieldwork and modeling results imply that the occurrence of long‐unburnt habitat in fire‐prone ecosystems may be an emergent property of patch scaling under fire regimes dominated by smaller fires. These findings provide a model for understanding how anthropogenic burning alters spatial and temporal aspects of habitat heterogeneity, which, as the literature survey strongly suggests, warrant consideration across a diversity of geographies and cultures. Our results clarify how traditional fire management shapes fire‐prone ecosystems, which despite diverse objectives, has allowed human societies to cope with fire as a recurrent disturbance.