Transport model of the human Na+-coupled L-ascorbic acid (vitamin C) transporter SVCT1

Vitamin C (L-ascorbic acid) is an essential micronutrient that serves as an antioxidant and as a cofactor in many enzymatic reactions. Intestinal absorption and renal reabsorption of the vitamin is mediated by the epithelial apical L-ascorbic acid cotransporter SVCT1 (SLC23A1). We explored the molecular mechanisms of SVCT1-mediated L-ascorbic acid transport using radiotracer and voltage-clamp techniques in RNA-injected Xenopus oocytes. L-ascorbic acid transport was saturable (K(0.5) approximately 70 microM), temperature dependent (Q(10) approximately 5), and energized by the Na(+) electrochemical potential gradient. We obtained a Na(+)-L-ascorbic acid coupling ratio of 2:1 from simultaneous measurement of currents and fluxes. L-ascorbic acid and Na(+) saturation kinetics as a function of cosubstrate concentrations revealed a simultaneous transport mechanism in which binding is ordered Na(+), L-ascorbic acid, Na(+). In the absence of L-ascorbic acid, SVCT1 mediated pre-steady-state currents that decayed with time constants 3-15 ms. Transients were described by single Boltzmann distributions. At 100 mM Na(+), maximal charge translocation (Q(max)) was approximately 25 nC, around a midpoint (V(0.5)) at -9 mV, and with apparent valence approximately -1. Q(max) was conserved upon progressive removal of Na(+), whereas V(0.5) shifted to more hyperpolarized potentials. Model simulation predicted that the pre-steady-state current predominantly results from an ion-well effect on binding of the first Na(+) partway within the membrane electric field. We present a transport model for SVCT1 that will provide a framework for investigating the impact of specific mutations and polymorphisms in SLC23A1 and help us better understand the contribution of SVCT1 to vitamin C metabolism in health and disease.

A newly developed in vitro model of the human epithelial airway barrier to study the toxic potential of nanoparticles

The potential health effects of inhaled engineered nanoparticles are almost unknown. To avoid and replace toxicity studies with animals, a triple cell co-culture system composed of epithelial cells, macrophages and dendritic cells was established, which simulates the most important barrier functions of the epithelial airway. Using this model, the toxic potential of titanium dioxide was assessed by measuring the production of reactive oxygen species and the release of tumour necrosis factor alpha. The intracellular localisation of titanium dioxide nanoparticles was analyzed by energy filtering transmission electron microscopy. Titanium dioxide nanoparticles were detected as single particles without membranes and in membrane-bound agglomerates. Cells incubated with titanium dioxide particles showed an elevated production of reactive oxygen species but no increase of the release of tumour necrosis factor alpha. Our in vitro model of the epithelial airway barrier offers a valuable tool to study the interaction of particles with lung cells at a nanostructural level and to investigate the toxic potential of nanoparticles.

Structural model of the CopA copper ATPase of Enterococcus hirae based on chemical cross-linking

The CopA copper ATPase of Enterococcus hirae belongs to the family of heavy metal pumping CPx-type ATPases and shares 43% sequence similarity with the human Menkes and Wilson copper ATPases. Due to a lack of suitable protein crystals, only partial three-dimensional structures have so far been obtained for this family of ion pumps. We present a structural model of CopA derived by combining topological information obtained by intramolecular cross-linking with molecular modeling. Purified CopA was cross-linked with different bivalent reagents, followed by tryptic digestion and identification of cross-linked peptides by mass spectrometry. The structural proximity of tryptic fragments provided information about the structural arrangement of the hydrophilic protein domains, which was integrated into a three-dimensional model of CopA. Comparative modeling of CopA was guided by the sequence similarity to the calcium ATPase of the sarcoplasmic reticulum, Serca1, for which detailed structures are available. In addition, known partial structures of CPx-ATPase homologous to CopA were used as modeling templates. A docking approach was used to predict the orientation of the heavy metal binding domain of CopA relative to the core structure, which was verified by distance constraints derived from cross-links. The overall structural model of CopA resembles the Serca1 structure, but reveals distinctive features of CPx-type ATPases. A prominent feature is the positioning of the heavy metal binding domain. It features an orientation of the Cu binding ligands which is appropriate for the interaction with Cu-loaded metallochaperones in solution. Moreover, a novel model of the architecture of the intramembranous Cu binding sites could be derived.

Wnt activation of immortalized brain endothelial cells as a tool for generating a standardized model of the blood brain barrier in vitro

Reproducing the characteristics and the functional responses of the blood-brain barrier (BBB) in vitro represents an important task for the research community, and would be a critical biotechnological breakthrough. Pharmaceutical and biotechnology industries provide strong demand for inexpensive and easy-to-handle in vitro BBB models to screen novel drug candidates. Recently, it was shown that canonical Wnt signaling is responsible for the induction of the BBB properties in the neonatal brain microvasculature in vivo. In the present study, following on from earlier observations, we have developed a novel model of the BBB in vitro that may be suitable for large scale screening assays. This model is based on immortalized endothelial cell lines derived from murine and human brain, with no need for co-culture with astrocytes. To maintain the BBB endothelial cell properties, the cell lines are cultured in the presence of Wnt3a or drugs that stabilize β-catenin, or they are infected with a transcriptionally active form of β-catenin. Upon these treatments, the cell lines maintain expression of BBB-specific markers, which results in elevated transendothelial electrical resistance and reduced cell permeability. Importantly, these properties are retained for several passages in culture, and they can be reproduced and maintained in different laboratories over time. We conclude that the brain-derived endothelial cell lines that we have investigated gain their specialized characteristics upon activation of the canonical Wnt pathway. This model may be thus suitable to test the BBB permeability to chemicals or large molecular weight proteins, transmigration of inflammatory cells, treatments with cytokines, and genetic manipulation.

Identification of erosional mechanisms during past glaciations based on a bedrock surface model of the central European Alps

The bedrock topography beneath the Quaternary cover provides an important archive for the identification of erosional processes during past glaciations. Here, we combined stratigraphic investigations of more than 40,000 boreholes with published data to generate a bedrock topography model for the entire plateau north of the Swiss Alps including the valleys within the mountain belt. We compared the bedrock map with data about the pattern of the erosional resistance of Alpine rocks to identify the controls of the lithologic architecture on the location of overdeepenings. We additionally used the bedrock topography map as a basis to calculate the erosional potential of the Alpine glaciers, which was related to the thickness of the LGM ice. We used these calculations to interpret how glaciers, with support by subglacial meltwater under pressure, might have shaped the bedrock topography of the Alps. We found that the erosional resistance of the bedrock lithology mainly explains where overdeepenings in the Alpine valleys and the plateau occur. In particular, in the Alpine valleys, the locations of overdeepenings largely overlap with areas where the underlying bedrock has a low erosional resistance, or where it was shattered by faults. We also found that the assignment of two end-member scenarios of erosion, related to glacial abrasion/plucking in the Alpine valleys, and dissection by subglacial meltwater in the plateau, may be adequate to explain the pattern of overdeepenings in the Alpine realm. This most likely points to the topographic controls on glacial scouring. In the Alps, the flow of LGM and previous glaciers were constrained by valley flanks, while ice flow was mostly divergent on the plateau where valley borders are absent. We suggest that these differences in landscape conditioning might have contributed to the contrasts in the formation of overdeepenings in the Alpine valleys and the plateau.

Short-Term Plasticity in a Computational Model of the Tail-Withdrawal Circuit in Aplysia.

The tail-withdrawal circuit of Aplysia provides a useful model system for investigating synaptic dynamics. Sensory neurons within the circuit manifest several forms of synaptic plasticity. Here, we developed a model of the circuit and investigated the ways in which depression (DEP) and potentiation (POT) contributed to information processing. DEP limited the amount of motor neuron activity that could be elicited by the monosynaptic pathway alone. POT within the monosynaptic pathway did not compensate for DEP. There was, however, a synergistic interaction between POT and the polysynaptic pathway. This synergism extended the dynamic range of the network, and the interplay between DEP and POT made the circuit responded preferentially to long-duration, low-frequency inputs.

A model of the roles of essential kinases in the induction and expression of late long-term potentiation.

The induction of late long-term potentiation (L-LTP) involves complex interactions among second-messenger cascades. To gain insights into these interactions, a mathematical model was developed for L-LTP induction in the CA1 region of the hippocampus. The differential equation-based model represents actions of protein kinase A (PKA), MAP kinase (MAPK), and CaM kinase II (CAMKII) in the vicinity of the synapse, and activation of transcription by CaM kinase IV (CAMKIV) and MAPK. L-LTP is represented by increases in a synaptic weight. Simulations suggest that steep, supralinear stimulus-response relationships between stimuli (e.g., elevations in [Ca(2+)]) and kinase activation are essential for translating brief stimuli into long-lasting gene activation and synaptic weight increases. Convergence of multiple kinase activities to induce L-LTP helps to generate a threshold whereby the amount of L-LTP varies steeply with the number of brief (tetanic) electrical stimuli. The model simulates tetanic, -burst, pairing-induced, and chemical L-LTP, as well as L-LTP due to synaptic tagging. The model also simulates inhibition of L-LTP by inhibition of MAPK, CAMKII, PKA, or CAMKIV. The model predicts results of experiments to delineate mechanisms underlying L-LTP induction and expression. For example, the cAMP antagonist RpcAMPs, which inhibits L-LTP induction, is predicted to inhibit ERK activation. The model also appears useful to clarify similarities and differences between hippocampal L-LTP and long-term synaptic strengthening in other systems.

Cryo-EM model of the bullet-shaped vesicular stomatitis virus.

Vesicular stomatitis virus (VSV) is a bullet-shaped rhabdovirus and a model system of negative-strand RNA viruses. Through direct visualization by means of cryo-electron microscopy, we show that each virion contains two nested, left-handed helices: an outer helix of matrix protein M and an inner helix of nucleoprotein N and RNA. M has a hub domain with four contact sites that link to neighboring M and N subunits, providing rigidity by clamping adjacent turns of the nucleocapsid. Side-by-side interactions between neighboring N subunits are critical for the nucleocapsid to form a bullet shape, and structure-based mutagenesis results support this description. Together, our data suggest a mechanism of VSV assembly in which the nucleocapsid spirals from the tip to become the helical trunk, both subsequently framed and rigidified by the M layer.