Structural analysis of endothelial projections from mesenteric arteries.

OBJECTIVE: Endothelial and smooth muscle cells must communicate with one another to regulate arterial diameter. A key structure driving heterocellular communication is the endothelial projection, a thin extension that crosses the internal elastic lamina (IEL) making contact with smooth muscle. This study sought to define the precise structural composition of endothelial projections in the mesenteric circulation. METHODS: Third- and fourth-order mesenteric arteries from hamster were prepared for electron microscopy. Electron tomographic approaches were used to generate 3-D compositional models of endothelial projections. RESULTS: Endothelial projections were categorized based upon their proximity to smooth muscle or how many projections projected through an IEL hole. Irrespective of the initial categorization, endothelial projections were largely devoid of organelles except for sparse membranous structures observed near the tip, close to potential smooth muscle contact sites. Unexpectedly, it was the base of projections which were rich with organelles including the endoplasmic reticulum, ribosomes, vesicles, caveolae, and mitochondria. CONCLUSIONS: Electron tomographic techniques suggest that the base of endothelial projections is likely a dynamic site for signal regulation and contractile control. As projections are largely devoid of membranous organelles, their principal function appears to ensure electrical contact between the two cell layers.

Redirecting soluble antigen for MHC class I cross-presentation during phagocytosis.

Peptides presented by MHC class I molecules are mostly derived from proteins synthesized by the antigen-presenting cell itself, while peptides presented by MHC class II molecules are predominantly from materials acquired by endocytosis. External antigens can also be presented by MHC class I molecules in a process referred to as cross-presentation. Here, we report that mouse dendritic cell (DC) engagement to a phagocytic target alters endocytic processing and inhibits the proteolytic activities. During phagocytosis, endosome maturation is delayed, shows less progression toward the lysosome, and the endocytosed soluble antigen is targeted for MHC class I cross-presentation. The antigen processing in these arrested endosomes is under the control of NAPDH oxidase associated ROS. We also show that cathepsin S is responsible for the generation of the MHC class I epitope. Taken together, our results suggest that in addition to solid structure uptake, DC phagocytosis simultaneously modifies the kinetics of endosomal trafficking and maturation. As a consequence, external soluble antigens are targeted into the MHC class I cross-presentation pathway.

Selective Adsorption of Thiols Using Gold Nanoparticles Supported on Metal Oxides.

Selective capture of thiols from a synthetic hydrogen sulfide containing mixture using supported nanogold materials has been explored for the potential removal of thiols from sour gas production fluids. In this research, TiO2-, Al2O3-, SiO2-, and ZnO-supported gold nanoparticles have been studied for their usage as regeneratable adsorbents to capture CH3SH, C2H5SH, and i-C3H7SH. Au/TiO2 and Au/Al2O3 showed promising properties for removing the thiols efficiently from a gas-phase mixture; however, Au/Al2O3 did catalyze some undesirable side reactions, e.g., carbonyl sulfide formation. It was found that a mild temperature of T = 200 °C was sufficient for regeneration of either Au/TiO2 or Au/Al2O3 adsorbent. The metal oxide mesopores played an important role for accommodating gold particles and chemisorption of the thiols, where smaller pore sizes were found to inhibit the agglomeration/growth of gold particles. The nature of thiol adsorption and the impact of multiple adsorption-desorption cycles on the adsorbents have been studied using electron microscopy, XPS, XRD, GC, and physi/chemiadsorption analyses.

Ca(V)3.2 channels and the induction of negative feedback in cerebral arteries.

RATIONALE: T-type (CaV3.1/CaV3.2) Ca(2+) channels are expressed in rat cerebral arterial smooth muscle. Although present, their functional significance remains uncertain with findings pointing to a variety of roles. OBJECTIVE: This study tested whether CaV3.2 channels mediate a negative feedback response by triggering Ca(2+) sparks, discrete events that initiate arterial hyperpolarization by activating large-conductance Ca(2+)-activated K(+) channels. METHODS AND RESULTS: Micromolar Ni(2+), an agent that selectively blocks CaV3.2 but not CaV1.2/CaV3.1, was first shown to depolarize/constrict pressurized rat cerebral arteries; no effect was observed in CaV3.2(-/-) arteries. Structural analysis using 3-dimensional tomography, immunolabeling, and a proximity ligation assay next revealed the existence of microdomains in cerebral arterial smooth muscle which comprised sarcoplasmic reticulum and caveolae. Within these discrete structures, CaV3.2 and ryanodine receptor resided in close apposition to one another. Computational modeling revealed that Ca(2+) influx through CaV3.2 could repetitively activate ryanodine receptor, inducing discrete Ca(2+)-induced Ca(2+) release events in a voltage-dependent manner. In keeping with theoretical observations, rapid Ca(2+) imaging and perforated patch clamp electrophysiology demonstrated that Ni(2+) suppressed Ca(2+) sparks and consequently spontaneous transient outward K(+) currents, large-conductance Ca(2+)-activated K(+) channel mediated events. Additional functional work on pressurized arteries noted that paxilline, a large-conductance Ca(2+)-activated K(+) channel inhibitor, elicited arterial constriction equivalent, and not additive, to Ni(2+). Key experiments on human cerebral arteries indicate that CaV3.2 is present and drives a comparable response to moderate constriction. CONCLUSIONS: These findings indicate for the first time that CaV3.2 channels localize to discrete microdomains and drive ryanodine receptor-mediated Ca(2+) sparks, enabling large-conductance Ca(2+)-activated K(+) channel activation, hyperpolarization, and attenuation of cerebral arterial constriction.

Resolving the In Situ Three-Dimensional Structure of Fly Mechanosensory Organelles Using Serial Section Electron Tomography.

Mechanosensory organelles (MOs) are specialized subcellular entities where force-sensitive channels and supporting structures (e.g., microtubule cytoskeleton) are organized in an orderly manner. The delicate structure of MOs needs to be resolved to understand the mechanisms by which they detect forces and how they are formed. Here, we describe a protocol that allows obtaining detailed information about the nanoscopic ultrastructure of fly MOs by using serial section electron tomography (SS-ET). To preserve fine structural details, the tissues are cryo-immobilized using a high-pressure freezer followed by freeze-substitution at low temperature and embedding in resin at room temperature. Then, sample sections are prepared and used to acquire the dual-axis tilt series images, which are further processed for tomographic reconstruction. Finally, tomograms of consecutive sections are combined into a single larger volume using microtubules as fiducial markers. Using this protocol, we managed to reconstruct the sensory organelles, which provide novel molecular insights as to how fly mechanosensory organelles work and are formed. Based on our experience, we think that, with minimal modifications, this protocol can be adapted to a wide range of applications using different cell and tissue samples. Key features • Resolving the high-resolution 3D ultrastructure of subcellular organelles using serial section electron tomography (SS-ET). • Compared with single-axis tilt series, dual-axis tilt series provides a much wider coverage of Fourier space, improving resolution and features in the reconstructed tomograms. • The use of high-pressure freezing and freeze-substitution maximally preserves the fine structural details.

Endothelial Ca2+ wavelets and the induction of myoendothelial feedback.

When arteries constrict to agonists, the endothelium inversely responds, attenuating the initial vasomotor response. The basis of this feedback mechanism remains uncertain, although past studies suggest a key role for myoendothelial communication in the signaling process. The present study examined whether second messenger flux through myoendothelial gap junctions initiates a negative-feedback response in hamster retractor muscle feed arteries. We specifically hypothesized that when agonists elicit depolarization and a rise in second messenger concentration, inositol trisphosphate (IP(3)) flux activates a discrete pool of IP(3) receptors (IP(3)Rs), elicits localized endothelial Ca(2+) transients, and activates downstream effectors to moderate constriction. With use of integrated experimental techniques, this study provided three sets of supporting observations. Beginning at the functional level, we showed that blocking intermediate-conductance Ca(2+)-activated K(+) channels (IK) and Ca(2+) mobilization from the endoplasmic reticulum (ER) enhanced the contractile/electrical responsiveness of feed arteries to phenylephrine. Next, structural analysis confirmed that endothelial projections make contact with the overlying smooth muscle. These projections retained membranous ER networks, and IP(3)Rs and IK channels localized in or near this structure. Finally, Ca(2+) imaging revealed that phenylephrine induced discrete endothelial Ca(2+) events through IP(3)R activation. These events were termed recruitable Ca(2+) wavelets on the basis of their spatiotemporal characteristics. From these findings, we conclude that IP(3) flux across myoendothelial gap junctions is sufficient to induce focal Ca(2+) release from IP(3)Rs and activate a discrete pool of IK channels within or near endothelial projections. The resulting hyperpolarization feeds back on smooth muscle to moderate agonist-induced depolarization and constriction.

Synthesis, structure, chemical stability, and electrical properties of Nb-, Zr-, and Nb-codoped BaCeO3 perovskites.

We report the effect of donor-doped perovskite-type BaCeO(3) on the chemical stability in CO(2) and boiling H(2)O and electrical transport properties in various gas atmospheres that include ambient air, N(2), H(2), and wet and dry H(2). Formation of perovskite-like BaCe(1-x)Nb(x)O(3±Î´) and BaCe(0.9-x)Zr(x)Nb(0.1)O(3±Î´) (x = 0.1; 0.2) was confirmed using powder X-ray diffraction (XRD) and electron diffraction (ED). The lattice constant was found to decrease with increasing Nb in BaCe(1-x)Nb(x)O(3±Î´), which is consistent with Shannon's ionic radius trend. Like BaCeO(3), BaCe(1-x)Nb(x)O(3±Î´) was found to be chemically unstable in 50% CO(2) at 700 °C, while Zr doping for Ce improves the structural stability of BaCe(1-x)Nb(x)O(3±Î´). AC impedance spectroscopy was used to estimate electrical conductivity, and it was found to vary with the atmospheric conditions and showed mixed ionic and electronic conduction in H(2)-containing atmosphere. Arrhenius-like behavior was observed for BaCe(0.9-x)Zr(x)Nb(0.1)O(3±Î´) at 400-700 °C, while Zr-free BaCe(1-x)Nb(x)O(3±Î´) exhibits non-Arrhenius behavior at the same temperature range. Among the perovskite-type oxides investigated in the present work, BaCe(0.8)Zr(0.1)Nb(0.1)O(3±Î´) showed the highest bulk electrical conductivity of 1.3 × 10(-3) S cm(-1) in wet H(2) at 500 °C, which is comparable to CO(2) and H(2)O unstable high-temperature Y-doped BaCeO(3) proton conductors.

Association of Plasmodium berghei With the Apical Domain of Hepatocytes Is Necessary for the Parasite's Liver Stage Development.

Plasmodium parasites undergo a dramatic transformation during the liver stage of their life cycle, amplifying over 10,000-fold inside infected hepatocytes within a few days. Such a rapid growth requires large-scale interactions with, and manipulations of, host cell functions. Whereas hepatocyte polarity is well-known to be critical for liver function, little is presently known about its involvement during the liver stage of Plasmodium development. Apical domains of hepatocytes are critical components of their polarity machinery and constitute the bile canalicular network, which is central to liver function. Here, we employed high resolution 3-D imaging and advanced image analysis of Plasmodium-infected liver tissues to show that the parasite associates preferentially with the apical domain of hepatocytes and induces alterations in the organization of these regions, resulting in localized changes in the bile canalicular architecture in the liver tissue. Pharmacological perturbation of the bile canalicular network by modulation of AMPK activity reduces the parasite's association with bile canaliculi and arrests the parasite development. Our findings using Plasmodium-infected liver tissues reveal a host-Plasmodium interaction at the level of liver tissue organization. We demonstrate for the first time a role for bile canaliculi, a central component of the hepatocyte polarity machinery, during the liver stage of Plasmodium development.

First-time electrical characterization of nanotubular ZrO2 films for micro-solid oxide fuel cell applications.

In this work, anodically grown ZrO2 nanotubes (NTs) are examined for the first time for use in micro solid oxide fuel cell (µ-SOFC) applications. This is due to their high surface area to volume ratio and useful nanoscale morphological features (e.g., 5-100 nm thick NT bases that could serve as the electrolyte layer). To understand their full potential for these applications, the determination of their electrical properties is necessary. Therefore, ZrO2 NTs, in the form of a uniform and crack-free film, were obtained by the two-step anodization of Zr foil in aqueous solutions. The films exhibited excellent adhesion to the Zr substrate, which facilitated impedance spectroscopy analyses, used for the first time to obtain the resistivity of the nanotubular array separately from the contact resistances. This gave a conductivity of the ZrO2 NTs of 1.6 × 10(-6) S cm(-1) at 600 °C in N2, approximately twice that reported for dense ZrO2 films measured at the same temperature in air, and also a very reasonable activation energy of 0.90 eV in the 400-600 °C temperature range.

Chemically stable proton conducting doped BaCeO3 -no more fear to SOFC wastes.

Development of chemically stable proton conductors for solid oxide fuel cells (SOFCs) will solve several issues, including cost associated with expensive inter-connectors, and long-term durability. Best known Y-doped BaCeO3 (YBC) proton conductors-based SOFCs suffer from chemical stability under SOFC by-products including CO2 and H2O. Here, for the first time, we report novel perovskite-type Ba0.5Sr0.5Ce0.6Zr0.2Gd0.1Y0.1O3-δ by substituting Sr for Ba and co-substituting Gd + Zr for Ce in YBC that showed excellent chemical stability under SOFC by-products (e.g., CO2 and H2O) and retained a high proton conductivity, key properties which were lacking since the discovery of YBCs. In situ and ex- situ powder X-ray diffraction and thermo-gravimetric analysis demonstrate superior structural stability of investigated perovskite under SOFC by-products. The electrical measurements reveal pure proton conductivity, as confirmed by an open circuit potential of 1.15 V for H2-air cell at 700°C, and merits as electrolyte for H-SOFCs.