Sequential transport of protein between the endoplasmic reticulum and successive Golgi compartments in semi-intact cells.

The vectorial transport of vesicular stomatitis virus (VSV) G protein between the ER and the cis and medial Golgi compartments has been reconstituted using semi-intact (perforated) cells. The transport of VSV-G protein between successive compartments is measured by the sequential processing of the two N-linked oligosaccharide chains present on VSV-G protein to the endoglycosidase (endo) H-resistant structures which have unique electrophoretic mobilities during sodium dodecyl sulfate-gel electrophoresis. The appearance of a form of VSV-G which contains only one endo H-resistant oligosaccharide chain (GH1) is kinetically and biochemically indistinguishable from the appearance of the Man5, endo D-sensitive form (GD), the latter being a processing reaction diagnostic of transport from the ER to the cis Golgi compartment. These results provide evidence that the cis Golgi compartment may contain in addition to alpha-1,2-mannosidase I, both N-acetylglueosamine transferase I and alpha-1,2-mannosidase II. VSV-G protein is subsequently processed to the form which contains two endo H-resistant oligosaccharides (GH2) after a second wave of vesicular transport. Processing of GH1 to GH2 in vitro occurs only after a lag period following the appearance of GH1; processing is sensitive to N-ethylmaleimide, guanosine-5'-O-(3-thiotriphosphate), and a synthetic peptide homologous to the rab1 protein effector domain, and processing is inhibited in the absence of free Ca2+ (in the presence of EGTA), reagents which potently inhibit ER to cis Golgi transport. These results suggest that VSV-G protein proceeds through at least two rounds of vesicular transport from the ER to the medial Golgi compartment for processing to the GH2 form, providing a model system to study the regulation of the vectorial membrane fission and fusion events involved in vesicular trafficking and organelle dynamics in the early stages of the secretory pathway.     

Evaluation of a Voxelized Model Based on DCE-MRI for Tracer Transport in Tumor

Recent advances in the treatment of cancer involving therapeutic agents have shown promising results. However, treatment efficacy can be limited due to inadequate and uneven uptake in solid tumors, thereby making the prediction of drug transport important for developing effective therapeutic strategies. In this study, a patient-specific computational porous media model (voxelized model) was developed for predicting the interstitial flow field and distribution of a systemically delivered magnetic resonance (MR) visible tracer in a tumor. The benefits of a voxel approach include less labor and less computational time (approximately an order of magnitude reduction compared to the traditional computational fluid dynamics (CFD) approach developed earlier by our group). The model results were compared with that obtained from a previous approach based on unstructured meshes along with MR-measured tracer concentration data within tumors, using statistical analysis and qualitative representations. The statistical analysis indicated the similarity between the structured and unstructured models' results with a low root mean square error (RMS) and a high correlation coefficient. The voxelized model captured features of the flow field and tracer distribution such as high interstitial fluid pressure inside the tumor and the heterogeneous distribution of the tracer. Predictions of tracer distribution by the voxelized approach also resulted in low RMS error when compared with MR-measured data over a 1?h time course. The similarity in the voxelized model results with experiment and the nonvoxelized model predictions were maintained across three different tumors. Overall, the voxelized model serves as a reliable and swift alternative to approaches using unstructured meshes in predicting extracellular transport within tumors.     

Virtual models of the HLA class I antigen processing pathway

Antigen recognition by cytotoxic CD8 T cells is dependent upon a number of critical steps in MHC class I antigen processing including proteosomal cleavage, TAP transport into the endoplasmic reticulum, and MHC class I binding. Based on extensive experimental data relating to each of these steps there is now the capacity to model individual antigen processing steps with a high degree of accuracy. This paper demonstrates the potential to bring together models of individual antigen processing steps, for example proteosome cleavage, TAP transport, and MHC binding, to build highly informative models of functional pathways. In particular, we demonstrate how an artificial neural network model of TAP transport was used to mine a HLA-binding database so as to identify HLA-binding peptides transported by TAP. This integrated model of antigen processing provided the unique insight that HLA class I alleles apparently constitute two separate classes: those that are TAP-efficient for peptide loading (HLA-B27, -A3, and -A24) and those that are TAP-inefficient (HLA-A2, -B7, and -B8). Hence, using this integrated model we were able to generate novel hypotheses regarding antigen processing, and these hypotheses are now capable of being tested experimentally. This model confirms the feasibility of constructing a virtual immune system, whereby each additional step in antigen processing is incorporated into a single modular model. Accurate models of antigen processing have implications for the study of basic immunology as well as for the design of peptide-based vaccines and other immunotherapies.     

Coupling satellite data with in situ measurements and numerical modeling to study fine suspended-sediment transport: a study for the lagoon of New Caledonia

This paper investigates the potential of remotely sensed data to map turbidity in a coral reef lagoon and to calibrate a numerical model of fine suspended-sediment transport. Simultaneous measurements of turbidity depth-profile and above-water spectral reflectance integrated according Landsat 7 ETM+ band 2 spectral sensitivity provide a linear regression relationship for the southwest lagoon of New Caledonia (r²=0.95, n=40). This relationship is applied to an empirically atmospherically corrected Landsat ETM+ image of the lagoon acquired on October 23, 2002. A comparison between Landsat estimates of turbidity and concurrent measurements at 14 stations indicates that the mean standard error in the satellite-estimated turbidity is 17.5%. The numerical model introduced in Douillet et al. (2001) is used to simulate the transport of fine suspended sediments in the lagoon in October 2002. A calibration of the erosion rate coefficient required by the model is proposed using in situ turbidity profiles and the remotely sensed turbidity field. In situ data are used to tune locally the erosion rate coefficient, while satellite data are used to determine its spatial zonation. We discuss necessary improvements in coupled studies of fine-sediment transport in coastal zones, namely relationships between turbidity and sediment concentration, integration of wave influence in the model, and correction of bottom reflection in satellite data processing.     

Kinetics of precursor interactions with the bacterial Tat translocase detected by real-time FRET

The Escherichia coli twin-arginine translocation (Tat) system transports fully folded and assembled proteins across the inner membrane into the periplasmic space. Traditionally, in vitro protein translocation studies have been performed using gel-based transport assays. This technique suffers from low time resolution, and often, an inability to distinguish between different steps in a continuously occurring translocation process. To address these limitations, we have developed an in vitro FRET-based assay that reports on an early step in the Tat translocation process in real-time. The natural Tat substrate pre-SufI was labeled with Alexa532 (donor), and the fluorescent protein mCherry (acceptor) was fused to the C terminus of TatB or TatC. The colored Tat proteins were easily visible during purification, enabling identification of a highly active inverted membrane vesicle (IMV) fraction yielding transport rates with NADH almost an order of magnitude faster than previously reported. When pre-SufI was bound to the translocon, FRET was observed for both Tat proteins. FRET was diminished upon addition of nonfluorescent pre-SufI, indicating that the initial binding step is reversible. When the membranes were energized with NADH, the FRET signal was lost after a short delay. These data suggest a model in which a Tat cargo initially associates with the TatBC complex, and an electric field gradient is required for the cargo to proceed to the next stage of transport. This cargo migration away from the TatBC complex requires a significant fraction of the total transport time.     

A simulation of the distribution of Acartia clausi during Oregon Upwelling, August 1973

The distribution of the estuarine copepod Acartia clausi incoastal waters off Oregon during an upwelling period in August1973 is simulated. A time dependent, two dimensional (x, z,t) model relates maximum offshore extent of the copepod's fourlife stages (egg, nauplius, copepodite, and adult) to intensityof the wind stress driving the upwelling circulation, stagedevelopment time, and mortality. Realistic solutions are obtainedby using actual intermittent wind forcing recorded by an anemometerat Newport. Offshore transport is overestimated when the circulationmodel is driven by theoretical continuous winds, suggestingzooplankton may be washed out of coastal upwelling zones (e.g.off Northwest Africa) which undergo periods of prolonged upwelling. With an accurate model of offshore transport and stage developmenttime, the mismatch between predicted and observed distributionsmay be used to estimate field mortality of the various stages.     

Biophysical coarse-grained modeling provides insights into transport through the nuclear pore complex

The nuclear pore complex (NPC) is the gatekeeper of the nucleus, capable of actively discriminating between the active and inert cargo while accommodating a high rate of translocations. The biophysical mechanisms underlying transport, however, remain unclear due to the lack of information about biophysical factors playing role in transport. Based on published experimental data, we have established a coarse-grained model of an intact NPC structure to examine nucleocytoplasmic transport with refined spatial and temporal resolutions. Using our model, we estimate the transport time versus cargo sizes. Our findings suggest that the mean transport time of cargos smaller than 15 nm is independent of size, while beyond this size, there is a sharp increase in the mean transport time. The model confirms that kap-FG hydrophobicity is sufficient for active cargo transport. Moreover, our model predicts that during translocation, small and large cargo-complexes are hydrophobically attached to FG-repeat domains for 86 and 96% of their transport time, respectively. Inside the central channel FG-repeats form a thick layer on the wall leaving an open tube. The cargo-complex is almost always attached to this layer and diffuses back and forth, regardless of the cargo size. Finally, we propose a plausible model for transport in which the NPC can be viewed as a lubricated gate. This model incorporates basic assumptions underlying virtual-gate and reduction-of-dimensionality models with the addition of the FG-layer inside the central channel acting as a lubricant.     

Golgi’s way: a long path toward the new paradigm of the intra-Golgi transport

The transport of proteins and lipids is one of the main cellular functions. The vesicular model, compartment (or cisterna) maturation model, and the diffusion model compete with each other for the right to be the paradigm within the field of the intra-Golgi transport. These models have significant difficulties explaining the existing experimental data. Recently, we proposed the kiss-and-run (KAR) model of intra-Golgi transport (Mironov and Beznoussenko in Int J Mol Sci 13(6):6800–6819, 2012), which can be symmetric, when fusion and fission occur in the same location, and asymmetric, when fusion and fission take place at different sites. Here, we compare the ability of main models of the intra-Golgi transport to explain the existing results examining the evidence in favor and against each model. We propose that the KAR model has the highest potential for the explanation of the majority of experimental observations existing within the field of intracellular transport.     

Expanding the MiniAp‐4 BBB‐shuttle family: Evaluation of proline cis‐trans ratio as tool to fine‐tune transport

Venoms have recently emerged as a promising field in drug discovery due to their good selectivity and affinity for a wide range of biological targets. Among their multiple potential applications, venoms are a rich source of blood‐brain barrier (BBB) peptide shuttles. We previously described a short nontoxic derivative of apamin, MiniAp‐4, which can transport a wide range of cargoes across the BBB. Here, we have studied the conformation of the proline residue of a range of MiniAp‐4 analogues by high‐field NMR techniques, with the aim to identify whether there is a direct relation between the cis/trans population and a range of features, such as the capacity to transport molecules across a human‐based cellular model and stability in various media. The most promising candidate showed improved transport properties for a relevant small fluorophore.     

A new view of convective-diffusive transport processes in the arterial intima

In this paper a new theoretical framework is presented for analyzing the filtration and macromolecular convective-diffusive transport processes in the intimal region of an artery wall with widely dispersed macromolecular cellular leakage sites, as proposed in the leaky junction-cell turnover hypothesis of Weinbaum et al. In contrast to existing convection-diffusive models, which assume that the transport is either 1-D, or convection is primarily in a direction normal to the endothelial surface, the present model considers for the first time the nonuniform subendothelial pressure field that arises from the different hydraulic resistances of normal and leaky endothelial clefts and the special role of the internal elastic lamina (IEL) in modulating the horizontal transport of macromolecules after they have passed through the leaky clefts of cells that are either in mitosis or demonstrate IgG labeling. The new theory is able to quantitatively explain the growing body of recent experiments in which an unexpectedly rapid early-time growth of the leakage spot has been observed and the longer time asymptotic behavior in which the leakage spot appears to approach an equilibrium diameter. The new theory also predicts the observed doubling in macromolecular permeability between EBA labeled blue and white areas when the frequency of leakage sites is doubled. This frequency for doubling of permeability, however, is an order of magnitude smaller than predicted by the author's previous model, Tzeghai et al., in which only convection normal to the endothelial surface was considered and the pressure was uniform in the intima. The longer time model predictions are used to explain the time scale for the formation of liposomes in subendothelial tissue matrix in animal feeding experiments where it has been observed that the extracellular lipid concentration rises sharply prior to the entry of monocytes into the intima.     

Model of the mass transport to the surface of animal cells cultured in a rotating bioreactor operated in micro gravity

A mathematical model is used to investigate the transport of dissolved oxygen from the bulk fluid to the surface of aggregates of animal cells cultured in a rotating bioreactor. These aggregates move through different regions of the bioreactor with a local flow field and concentration distribution that vary with time. The time variation of the Sherwood number and the surface concentration for a range of parameters typical of a cell science experiment executed in the Rotating Wall Perfused Vessel (RWPV) bioreactor in space are investigated. The Reynolds numbers experienced by the aggregate are generally low (Re < 1) and the Peclet numbers range from O(1) to O(100). Comparison of the results from the numerical solution of the mathematical model with those from a quasi-steady model, using a steady-state correlation for mass transport on a sphere, indicate that the quasi-steady assumption is not a good model to compute the instantaneous Sherwood number. This indicates a significant history effect in the Sherwood number response to the variations of acceleration of the aggregates in the bioreactor. A high resistance to the mass transport from the bulk fluid to the surface of the aggregate exists for the bioreactor operated in micro gravity. The difference between the surface concentration and the free stream concentration was as high as 30% for aggregates larger than 3 mm. Diffusion reduces the variations of the free stream concentration resulting in a nearly constant value for the concentration at the surface of the aggregates.     

A Study of Vadose Zone Transport Model VLEACH

Application of vadose zone transport models has been hampered by lack of model validation. Difficulties to validate vadose zone models using field data not only come from model assumptions that are uncertain to the subsurface transport processes but also from the uncertainties associated with soil contaminants’ release time and quantity, soil sampling, sample transport, and analytical procedures. This article first conducts a test of a popularly used vadose zone transport VLEACH by comparing model results with a set of laboratory soil column infiltration and volatilization study data. The comparison shows a close agreement between the VLEACH model results and the laboratory data. Second, the sorption coefficient K_d calculated in VLEACH is compared with field data. The comparison indicates that VLEACH may overestimate the mass leached from soil to groundwater. The article also discusses the selection of the model simulation timestep, the vertical dimension increment, the Courant criterion, and the lower boundary condition using the sensitivity analysis method based on a case study of soil remediation for trichloroethylene. The procedures presented in this paper are important to practical model application and modification. This level of work should be routinely conducted for any new or modified version of vadose zone models.     

GGA1 overexpression attenuates amyloidogenic processing of the amyloid precursor protein in Niemann-Pick type C cells

Alzheimer’s disease (AD) and a rare inherited disorder of cholesterol transport, Niemann-Pick type C (NPC) share several similarities including aberrant APP processing and increased Aβ production. Previously, we have shown that the AD-like phenotype in NPC model cells involves cholesterol-dependent enhanced APP cleavage by β-secretase and accumulation of both APP and BACE1 within endocytic compartments. Since retrograde transport of BACE1 from endocytic compartments to the trans-Golgi network (TGN) is regulated by the Golgi-localized γ-ear containing ADP ribosylation factor-binding protein 1 (GGA1), we analyzed in this work a potential role of GGA1 in the AD-like phenotype of NPC1-null cells. Overexpression of GGA1 caused a shift in APP processing towards the non-amyloidogenic pathway by increasing the localization of APP at the cell surface. However, the observed effect appear to be independent on the subcellular localization and phosphorylation state of BACE1. These findings show that the AD-like phenotype of NPC model cells can be partly reverted by promoting a non-amyloidogenic processing of APP through the upregulation of GGA1 supporting its preventive role against AD.     

Modelling Pb and Cd dynamics in the Seine estuary

The Seine estuary (France) is currently one of the world’s most contaminated estuaries, due in particular to its high cadmium and lead content. Proper understanding of contaminant transfer, transformation and retention mechanisms throughout the estuary and up to the adjacent marine zone require a range of studies involving data collections, experiments, and modeling tool. A multivariable transport model (SiAM-3D) was used to simulate dissolved and particulate transport and it is applied to several calculation grids; a speciation model (MOCO) was used to select key species and obtain a schematic system representation. The coupled model for Cd and Pb was compared with field measurements. The complementary character of various tools (model applied to annual time scale, in situ measurements and experiments) allowed to explore and quantify various hypotheses on the high dissolved cadmium concentrations observed during low river flow. The target is to achieve a compromise between acceptable computing times and adequate result accuracy. Although particle and contaminant behaviour is globally well-reproduced by the coarse grid, calculation errors relating to bayward fluxes and stocks deposited inside the estuary were highlighted after comparison with the fine grid.     

The vacuolar (H+)-ATPases--nature's most versatile proton pumps

The pH of intracellular compartments in eukaryotic cells is a carefully controlled parameter that affects many cellular processes, including intracellular membrane transport, prohormone processing and transport of neurotransmitters, as well as the entry of many viruses into cells. The transporters responsible for controlling this crucial parameter in many intracellular compartments are the vacuolar (H+)-ATPases (V-ATPases). Recent advances in our understanding of the structure and regulation of the V-ATPases, together with the mapping of human genetic defects to genes that encode V-ATPase subunits, have led to tremendous excitement in this field.     

Electrostatic coupling of ion pumps.

In this paper the electrostatic interactions between membrane-embedded ion-pumps and their consequences for the kinetics of pump-mediated transport processes have been examined. We show that the time course of an intrinsically monomolecular transport reaction can become distinctly nonexponential, if the reaction is associated with charge translocation and takes place in an aggregate of pump molecules. First we consider the electrostatic coupling of a single dimer of ion-pumps embedded in the membrane. Then we apply the treatment to the kinetic analysis of light-driven proton transport by bacteriorhodopsin which forms two-dimensional hexagonal lattices. Finally, for the case of nonordered molecules, we also consider a model in which the pumps are randomly distributed over the nodes of a lattice. Here the average distance is equal to that deduced experimentally and the elemental size of the lattice is the effective diameter of one single pump. This latter model is applied to an aggregate of membrane-embedded Na, K- and Ca-pumps. In all these cases the electrostatic potential considered is the exact solution calculated from the method of electrical images for a plane membrane of finite thickness immersed in an infinite aqueous solution environment. The distributions of charges (ions or charged binding sites) are considered homogeneous or discrete in the membrane and/or in the external solution. In the case of discrete distributions we compare the results from a mean field approximation and a stochastic simulation.     

电场作用条件与作物种子生长的相关性

在综合评价电场强度及处理时间对作物生物效应影响时,如何确定生物效应指标之间的权重一直是困扰生物信息综合评价的核心问题。比如叶绿素增长1mg·L–1的价值相当于多少厘米根长的增加,这是一个难以回答的问题。同时,通过指标加权合成的方法获得综合评价的结果也存在信息丢失的缺陷。针对电场条件对作物生物效应的影响,利用数据包络分析的基本思想,给出了用于综合评价电场强度及处理时间对作物生物效应影响的非参数综合分析方法和相应的数学模型,该方法不仅可以克服上述缺点,而且还为分析不同外部条件对作物综合生物效应的影响程度提供了一种有效的分析工具。应用该方法综合评价了不同电场强度和不同处理时间对小麦（Triticumaestivum）种子幼苗株高和根长的影响。结果表明：不同电场强度和处理时间对小麦幼苗生长产生不同影响,在0.5–6.0kV·cm–1场强范围内,随电场强度增加,生理指标呈现振荡性变化,当处理时间为5分钟时,1.0和2.0kV·cm–1场强为最佳处理条件;处理时间为10分钟时,2.5kV·cm–1场强为最佳处理条件。     

黄酮化合物色谱保留时间与其三维结构的关系研究

利用比较分子相似性指数分析（CoMSIA）方法,结合黄酮类化合物含有较多羟基、易形成较强分子内氢键的特点,建立了黄酮类化合物色谱保留时间与其三维结构的关系模型,以探讨黄酮类化合物色谱保留时间预测的新方法。模型交叉验证相关系数q2值为0．705,非交叉验证相关系数r2为0．981,表明模型具有较好的预测能力。该研究结果对进一步开展黄酮类化合物液相色谱保留参数与三维结构关系的研究提供了思路和方法。