Exogenous α-synuclein hinders synaptic communication in cultured cortical primary rat neurons.

Amyloid aggregates of the protein α-synuclein (αS) called Lewy Bodies (LB) and Lewy Neurites (LN) are the pathological hallmark of Parkinson's disease (PD) and other synucleinopathies. We have previously shown that high extracellular αS concentrations can be toxic to cells and that neurons take up αS. Here we aimed to get more insight into the toxicity mechanism associated with high extracellular αS concentrations (50-100 µM). High extracellular αS concentrations resulted in a reduction of the firing rate of the neuronal network by disrupting synaptic transmission, while the neuronal ability to fire action potentials was still intact. Furthermore, many cells developed αS deposits larger than 500 nm within five days, but otherwise appeared healthy. Synaptic dysfunction clearly occurred before the establishment of large intracellular deposits and neuronal death, suggesting that an excessive extracellular αS concentration caused synaptic failure and which later possibly contributed to neuronal death.

Wafer-scale thin encapsulated two-dimensional nanochannels and its application toward visualization of single molecules.

We present a new and simple approach to fabricate wafer-scale, thin encapsulated, two-dimensional nanochannels by using conventional surface-micromachining technology and thin-film evaporation. The key steps to the realization of two-dimensional nanochannels are a fine etching of a sacrificial layer to create underetching spaces at the nanometer regime, and an accurate thin-film evaporation for encapsulation. Well-defined cross-sectional, encapsulated nanochannel arrays with dimensions as small as 20 nm in both width and height have been realized at the wafer-scale. The fabricated nanochannels with a channel length of 10mm have been used as a suitable fluidic platform for confining a solution containing nanomolar concentrations of Alexa fluorescent molecules. Initial results toward visualization of single Alexa molecules in the confined solution are reported.

Analysis of single quantum-dot mobility inside 1D nanochannel devices.

We visualized individual quantum dots using a combination of a confining nanochannel and an ultra-sensitive microscope system, equipped with a high numerical aperture lens and a highly sensitive camera. The diffusion coefficients of the confined quantum dots were determined from the experimentally recorded trajectories according to the classical diffusion theory for Brownian motion in two dimensions. The calculated diffusion coefficients were three times smaller than those in bulk solution. These observations confirm and extend the results of Eichmann et al (2008 Langmuir 24 714-21) to smaller particle diameters and more narrow confinement. A detailed analysis shows that the observed reduction in mobility cannot be explained by conventional hydrodynamic theory.

Occurrence and a possible mechanism of penetration of natural killer cells into K562 target cells during the cytotoxic interaction.

The cytotoxic interaction between cloned human Natural Killer (NK) cells and K562 target cells was studied using confocal laser scanning microscopy (CLSM) and conventional fluorescence microscopy. We observed, using fixed as well as living cells, the occurrence of (pseudo) emperipolesis during the interaction. About 30% of conjugated NK cells penetrated, partly or completely, into the target cells (in-conjugation). Virtually all in-conjugated target cells exhibited polymerized actin. Killer cells of in-conjugates were frequently seen approaching the target cell nucleus or aligning along it. If the cytotoxic process was inhibited by the absence of calcium neither actin polymerization nor in-conjugation were observed. A kinetic study showed that in-conjugation starts somewhat later than actin polymerization but still within a few minutes after addition of calcium to conjugates previously formed in the absence of calcium. The presence of cytochalasin D (an inhibitor of actin polymerization) completely inhibited in-conjugation and partly reduced the cytotoxic activity. Zinc ions (endonuclease inhibition) inhibited in-conjugation and decreased the total number of target cells with polymerized actin in a concentration dependent manner. Cytotoxic activity was also reduced but not as efficiently as in-conjugation. Our study demonstrates that in-conjugation represents a significant fraction of the cytotoxic interaction. The results indicate that it may be a consequence of an actin polymerization and endonuclease activity dependent part of a cytotoxic mechanism.

Changes in actin organization during the cytotoxic process.

Changes in organization of F-actin during the cytotoxic process between NK and K562 cells have been observed and studied using confocal laser scanning microscopy and quantitative fluorescence microscopy. An increase in F-actin content and orientation of F-actin towards the target cell have been observed in conjugated NK cells. The increase in F-actin content probably reflects activation of the NK cell for the killing process. An increase in F-actin content in the conjugated K562 cell, occurring simultaneously with the appearance of filamentous actin structures that often originated/ended at the contact place with the NK cell, was also observed. These changes were delayed compared to the increase in F-actin content in the NK cell and were accompanied by increasing cytotoxic activity. This indicates that they were results of the interaction of the K562 cell with the activated NK cell. The possible role of target cell microfilaments in the cytotoxic process is addressed.

Identification and characterization of plasma cells in normal human bone marrow by high-resolution flow cytometry.

The low frequency of plasma cells and the lack of specific cell surface markers has been a major obstacle for a detailed characterization of plasma cells in normal human bone marrow. Multiparameter flow cytometry enabled the identification of plasma cells in normal bone marrow aspirates. The plasma cells were located in a unique position in the correlation of forward light scattering, orthogonal light scattering, and immunofluorescent-labeled CD38. The identity of the sorted cell populations was confirmed by microscopic examination of Wright's stained slides and slides stained for cytoplasmic immunoglobulin using polyclonal antibodies reactive with light chains; ie, anti-kappa fluorescein isothiocyanate and anti lambda phycoerythrin (PE). The purity of the sorted plasma cells was greater than 97% (n = 4). The average frequency of plasma cells in normal bone marrow aspirates was low--0.25% of the nucleated cells (n = 7)--but surprisingly consistent between individuals (SD = .05; range 0.14% to 0.30%). A detailed analysis showed two distinct populations of plasma cells: (1) A population relatively smaller by forward light scattering expressed CD22, CD35, and sigE and was identified as early plasma cells (ie, lymphoplasmacytoid), and (2) a population larger by forward light scattering lacked these markers and was identified as mature plasma cells. The antigenic profile of the normal plasma cells was determined in two-color immunofluorescence studies. The expression of cell surface immunoglobulin G (IgG), IgA, IgE, IgD, IgM, and the cell surface antigens CD10, CD11b, CD13, CD11c, CD14, CD15, CD16, CD19, CD22, CD20, CD33, CD35, CD45, and HLA-DR was determined on the plasma cells. A significant heterogeneity in cell surface antigen expression was observed within the plasma cell population. Unexpectedly, myeloid-specific cell surface antigens such as CD33 and CD13 and the early B-cell antigen identified by CD10 were expressed on a proportion of plasma cells. These observations imply that the association of myeloid and early B-cell markers described in multiple myeloma may not be associated with the neoplasia but is a normal phenomenon.