Synaptic vesicle transport and synaptic membrane transporter sites in excitatory amino acid nerve terminals in Alzheimer disease

The apparent L-[H-3]glutamate uptake rate (v') was measured in synaptic vesicles isolated from cerebral cortex synaptosomes prepared from autopsied Alzheimer and non-Alzheimer dementia cases, and age-matched controls. The initial synaptosome preparations exhibited similar densities of D-[H-3]aspartate membrane binding sites (B-MAX values) in the three groups. In control brain the temporal cortex D-[H-3]aspartate B-MAX was 132% of that in motor cortex, parallel with the L- [H-3]glutamate v' values (temporal = 139% of motor; NS). Unlike D- [H-3]aspartate B-MAX values, L- [H-3]glutamate v' values were markedly and selectively lower in Alzheimer brain preparations than in controls, particularly in temporal cortex. The difference could not be attributed to differential effects of autopsy interval or age at death. Non-Alzheimer dementia cases resembled controls. The selective loss of vesicular glutamate transport is consistent with a dysfunction in the recycling of transmitter glutamate.

Improving the ionic conductivity of yttria-stabilised zirconia electrolyte materials

Low-temperature anneals (1200 degreesC for 40 h) of 8 mol% yttria-stabilised zirconia, prior to the samples being sintered at 1500 degreesC, had the effect of improving the ionic conductivity of the specimens. The presence Of SiO2 in the specimens was shown to be detrimental, however. Irrespective of the SiO2 content, this type of heat treatment also leads to improvements in conductivity. Extensive microstructural analysis provided indication of the mechanism of this phenomenon. This included selective formation of zircon, relief of sintering strain leading to the formation of coherent grain boundaries and segregation effects. (C) 2002 Elsevier Science B.V All rights reserved.

Ex vivo analysis of T-cell responses to Epstein-Barr virus-encoded oncogene latent membrane protein 1 reveals highly conserved epitope sequences in virus isolates from diverse geographic regions

Epstein-Barr virus (EBV)-encoded oncogene latent membrane protein (LMP) 1, which is consistently expressed in multiple EBV-associated malignancies, has been proposed as a potential target antigen for any future vaccine designed to control these malignancies. However, the high degree of genetic variation in the LMP1 sequence has been considered a major impediment for its use as a potential immunotherapeutic target for the treatment of EBV-associated malignancies. In the present study, we have employed a highly efficient strategy, based on ex vivo functional assays, to conduct an extensive sequence-wide analysis of LMP1-specific T-cell responses in a large panel of healthy virus carriers of diverse ethnic origin and nasopharyngeal carcinoma patients. By comparing the frequencies of T cells specific for overlapping peptides spanning LMP1, we mapped a number of novel HLA class I- and class II-restricted LMP1 T-cell epitopes, including an epitope with dual HLA class I restriction. More importantly, extensive sequence analysis of LMP1 revealed that the majority of the T-cell epitopes were highly conserved in EBV isolates from Caucasian, Papua New Guinean, African, and Southeast Asian populations, while unique geographically constrained genetic variation was observed within one HLA A2 supertype-restricted epitope. These findings indicate that conserved LMP1 epitopes should be considered in designing epitope-based immunotherapeutic strategies against EBV-associated malignancies in different ethnic populations.

Rab23, a negative regulator of hedgehog signaling, localizes to the plasma membrane and the endocytic pathway

The regulation of hedgehog signaling by vesicular trafficking was exemplified by the finding that Rab23, a Rab-GTPase vesicular transport protein, is mutated in open brain mice. In this study, the localization of Rab23 was analyzed by light and immunoelectron microscopy after expression of wild-type (Rab23-GFP), constitutively active Rab23 (Rab23Q68L-GFP), and inactive Rab23 (Rab23S23N-GFP) in a range of mammalian cell types. Rab23-GFP and Rab23Q68L-GFP were predominantly localized to the plasma membrane but were also associated with intracellular vesicular structures, whereas Rab23S23N-GFP was predominantly cytosolic. Vesicular Rab23-GFP colocalized with Rab5Q79L and internalized transferrin-biotin, but not with a marker of the late endosome or the Golgi complex. To investigate Rab23 with respect to members of the hedgehog signaling pathway, Rab23-GFP was coexpressed with either patched or smoothened. Patched colocalized with intracellular Rab23-GFP but smoothened did not. Analysis of patched distribution by light and immunoelectron microscopy revealed it is primarily localized to endosomal elements, including transferrin receptor-positive early endosomes and putative endosome carrier vesicles and, to a lesser extent, with LBPA-positive late endosomes, but was excluded from the plasma membrane. Neither patched or smoothened distribution was altered in the presence of wild-type nor mutant Rab23-GFP, suggesting that despite the endosomal colocalization of Rab23 and patched, it is likely that Rab23 acts more distally in regulating hedgehog signaling.

The trans-membrane protein p25 forms highly specialized domains that regulate membrane composition and dynamics

Trans-membrane proteins of the p24 family are abundant, oligomeric proteins predominantly found in cis-Golgi membranes. They are not easily studied in vivo and their functions are controversial. We found that p25 can be targeted to the plasma membrane after inactivation of its canonical KKXX motif (KK to SS, p25SS), and that p25SS causes the co-transport of other p24 proteins beyond the Golgi complex, indicating that wild-type p25 plays a crucial role in retaining p24 proteins in cis-Golgi membranes. We then made use of these observations to study the intrinsic properties of these proteins, when present in a different membrane context. At the cell surface, the p25SS mutant segregates away from both the transferrin receptor and markers of lipid rafts, which are enriched in cholesterol and glycosphingolipids. This suggests that p25SS localizes to, or contributes to form, specialized membrane domains, presumably corresponding to oligomers of p25SS and other p24 proteins. Once at the cell surface, p25SS is endocytosed, together with other p24 proteins, and eventually accumulates in late endosomes, where it remains confined to well-defined membrane regions visible by electron microscopy. We find that this p25SS accumulation causes a concomitant accumulation of cholesterol in late endosomes, and an inhibition of their motility - two processes that are functionally linked. Yet, the p25SS-rich regions themselves seem to-exclude not only Lamp1 but also accumulated cholesterol. One may envision that p25SS accumulation, by excluding cholesterol from oligomers, eventually overloads neighboring late endosomal membranes with cholesterol beyond their capacity (see Discussion). In any case, our data show that p25 and presumably other p24 proteins are endowed with the intrinsic capacity to form highly specialized domains that control membrane composition and dynamics. We propose that p25 and other p24 proteins control the fidelity of membrane transport by maintaining cholesterol-poor membranes in the Golgi complex.

Ultrasonic absorption in polymer gel dosimeters

Ultrasonic absorption in polymer gel dosimeters was investigated. An ultrasonic interferometer was used to study the frequency (f) dependence of the absorption coefficient (alpha) in a polyacrylamide gel dosimeter (PAG) in the frequency range 5-20 MHz. The frequency dependence of ultrasonic absorption deviated from that of an ideal viscous fluid. The presence of relaxation mechanisms was evidenced by the frequency dependence of alpha/f(2) and the dispersion in ultrasonic velocity. It was concluded that absorption in polymer gel dosimeters is due to a number of relaxation processes which may include polymer-solvent interactions as well as relaxation due to motion of polymer side groups. The dependence of ultrasonic absorption on absorbed dose and formulation was also investigated in polymer gel dosimeters as a function of pH and chemical composition. Changes in dosimeter pH and chemical composition resulted in a variation in ultrasonic dose response curves. The observed dependence on pH was considered to be due to pH induced modifications in the radiation yield while changes in chemical composition resulted in differences in polymerisation kinetics. (C) 2003 Elsevier B.V. All rights reserved.

Contextual binding of p120ctn to E-cadherin at the basolateral plasma membrane in polarized epithelia

E-cadherin-catenin complexes mediate cell-cell adhesion on the basolateral membrane of epithelial cells. The cytoplasmic tail of E-cadherin supports multiple protein interactions, including binding of beta-catenin at the C terminus and of p120(ctn) to the juxtamembrane domain. The temporal assembly and polarized trafficking of the complex or its individual components to the basolateral membrane are not fully understood. In Madin-Darby canine kidney cells at steady state and after treatment with cycloheximide or temperature blocks, E-cadherin and beta-catenin localized to the Golgi complex, but p120ctn was found only at the basolateral plasma membrane. We previously identified a dileucine sorting motif (Leu(586)-Leu(587), termed S1) in the juxtamembrane domain of E-cadherin and now show that it is required to target full-length E-cadherin to the basolateral membrane. Removal of S1 resulted in missorting of E-cadherin mutants (EcadDeltaS1) to the apical membrane; beta-catenin was simultaneously missorted and appeared at the apical membrane. p120(ctn) was not mistargeted with EcadDeltaS1, but could be recruited to the E-cadherin-catenin complex only at the basolateral membrane. These findings help define the temporal assembly and sorting of the E-cadherin-catenin complex and show that membrane recruitment of p120(ctn) in polarized cells is contextual and confined to the basolateral membrane.

The t-SNARE syntaxin 4 is regulated during macrophage activation to function in membrane traffic and cytokine secretion

Activation of macrophages with lipopolysaccharide (LPS) induces the rapid synthesis and secretion of proinflammatory cytokines, such as tumor necrosis factor (TNFalpha), for priming the immune response [1, 2]. TNFalpha plays a key role in inflammatory disease [3]; yet, little is known of the intracellular trafficking events leading to its secretion. In order to identify molecules involved in this secretory pathway, we asked whether any of the known trafficking proteins are regulated by LPS. We found that the levels of SNARE proteins were rapidly and significantly up- or downregulated during macrophage activation. A subset of t-SNAREs (Syntaxin 4/SNAP23/Munc18c) known to control regulated exocytosis in other cell types [4, 5] was substantially increased by LPS in a temporal pattern coinciding with peak TNFalpha secretion. Syntaxin 4 formed a complex with Munc18c at the cell surface of macrophages. Functional studies involving the introduction of Syntaxin 4 cDNA or peptides into macrophages implicate this t-SNARE in a rate-limiting step of TNFalpha secretion and in membrane ruffling during macrophage activation. We conclude that in macrophages, SNAREs are regulated in order to accommodate the rapid onset of cytokine secretion and for membrane traffic associated with the phenotypic changes of immune activation. This represents a novel regulatory role for SNAREs in regulated secretion and in macrophage-mediated host defense.

GAIP participates in budding of membrane carriers at the trans-Golgi network

Galpha interacting protein (GAIP) is a regulator of G protein signaling protein that associates dynamically with vesicles and has been implicated in membrane trafficking, although its specific role is not yet known. Using an in vitro budding assay, we show that GAIP is recruited to a specific population of trans-Golgi network-derived vesicles and that these are distinct from coatomer or clathrin-coated vesicles. A truncation mutant (NT-GAIP) encoding only the N-terminal half of GAIP is recruited to trans -Golgi network membranes during the formation of vesicle carriers. Overexpression of NT-GAIP induces the formation of long, coated tubules, which are stabilized by microtubules. Results from the budding assay and from imaging in live cells show that these tubules remain attached to the Golgi stack rather than being released as carrier vesicles. NT-GAIP expression blocks membrane budding and results in the accumulation of tubular carrier intermediates. NT-GAIP-decorated tubules are competent to load vesicular stomatitis virus protein G-green fluorescent protein as post-Golgi, exocytic cargo and in cells expressing NT-GAIP there is reduced surface delivery of vesicular stomatitis virus protein G-green fluorescent protein. We conclude that GAIP functions as an essential part of the membrane budding machinery for a subset of post-Golgi exocytic carriers derived from the trans-Golgi network.

Influence of electrospinning parameters on Poly(hydroxybutyrate) electrospun membranes fiber size and distribution

Poly(hydroxybutyrate) (PHB) obtained from sugar cane was dissolved in a blend of chloroform and dimethylformamide (DMF) and electrospun at 40 ºC. By adding DMF to the solution, the electrospinning process for the PHB polymer becomes more stable, allowing complete polymer crystallization during the jet travelling between the tip and the grounded collector. The influence of processing parameters on fiber size and distribution was systematically studied. It was observed that an increase of tip inner diameter promotes a decrease of the fiber average size and a broader distribution. On the other hand, an increase of the electric field and flow rate produces an increase of fiber diameter until a maximum of ~2.0 m, but for electric fields higher than 1.5 kV.cm-1, a decrease of the fiber diameter was observed. Polymer crystalline phase seems to be independent of the processing conditions and a crystallinity degree of 53 % was found. Moreover, thermal degradation of the as-spun membrane occurs in single step degradation with activation energy of 91 kJ/mol. Furthermore, MC-3T3-E1 cell adhesion was not inhibited by the fiber mats preparation, indicating their potential use for biomedical applications.

Polymer indentation with mesoscopic molecular dynamics

Indentation tests are used to determine the hardness of a material, e.g., Rockwell, Vickers, or Knoop. The indentation process is empirically observed in the laboratory during these tests; the mechanics of indentation is insufficiently understood. We have performed first molecular dynamics computer simulations of indentation resistance of polymers with a chain structure similar to that of high density polyethylene (HDPE). A coarse grain model of HDPE is used to simulate how the interconnected segments respond to an external force imposed by an indenter. Results include the time-dependent measurement of penetration depth, recovery depth, and recovery percentage, with respect to indenter force, indenter size, and indentation time parameters. The simulations provide results that are inaccessible experimentally, including continuous evolution of the pertinent tribological parameters during the entire indentation process.

Modeling Carbon Nanotube Electrical Properties in CNT/Polymer Composites

In this work it is demonstrated that the capacitance between two cylinders increases with the rotation angle and it has a fundamental influence on the composite dielectric constant. The dielectric constant is lower for nematic materials than for isotropic ones and this can be attributed to the effect of the filler alignment in the capacitance. The effect of aspect ratio in the conductivity is also studied in this work. Finally, based on previous work and by comparing to results from the literature it is found that the electrical conductivity in this type of composites is due to hopping between nearest fillers resulting in a weak disorder regime that is similar to the single junction expression.

Multi-scale modeling of the mechanical behavior of polymer-based materials

Polymers have become the reference material for high reliability and performance applications. In this work, a multi-scale approach is proposed to investigate the mechanical properties of polymeric based material under strain. To achieve a better understanding of phenomena occurring at the smaller scales, a coupling of a Finite Element Method (FEM) and Molecular Dynamics (MD) modeling in an iterative procedure was employed, enabling the prediction of the macroscopic constitutive response. As the mechanical response can be related to the local microstructure, which in turn depends on the nano-scale structure, the previous described multi-scale method computes the stress-strain relationship at every analysis point of the macro-structure by detailed modeling of the underlying micro- and meso-scale deformation phenomena. The proposed multi-scale approach can enable prediction of properties at the macroscale while taking into consideration phenomena that occur at the mesoscale, thus offering an increased potential accuracy compared to traditional methods.

Modelling the Mechanical Behavior of Polymer-Based Nanocomposites

Molecular dynamics simulations were employed to analyze the mechanical properties of polymer-based nanocomposites with varying nanofiber network parameters. The study was focused on nanofiber aspect ratio, concentration and initial orientation. The reinforcing phase affects the behavior of the polymeric nanocomposite. Simulations have shown that the fiber concentration has a significant effect on the properties, with higher loadings resulting in higher stress levels and higher stiffness, matching the general behavior from experimental knowledge in this field. The results also indicate that, within the studied range, the observed effect of the aspect ratio and initial orientation is smaller than that of the concentration, and that these two parameters are interrelated.