Parametric measurements on a doped CO2TEA laser

Parametric measurements have been performed on a CO2TEA laser with a discharge volume of 6.5 × 6.5 × 45 cm3. The effect of a low ionization seed gas, tri-n-propylamine, upon amplification, power output and voltage-current characteristics has been measured. A small-signal gain of 4.8%/cm has been measured in a 1:1:3 mixture and a power output of 60J/ℓ in a 3:11:21 mixture

A FRAP model to investigate reaction-diffusion of proteins within a bounded domain: a theoretical approach

Temporally and spatially resolved measurements of protein transport inside cells provide important clues to the functional architecture and dynamics of biological systems. Fluorescence Recovery After Photobleaching (FRAP) technique has been used over the past three decades to measure the mobility of macromolecules and protein transport and interaction with immobile structures inside the cell nucleus. A theoretical model is presented that aims to describe protein transport inside the nucleus, a process which is influenced by the presence of a boundary (i.e. membrane). A set of reaction-diffusion equations is employed to model both the diffusion of proteins and their interaction with immobile binding sites. The proposed model has been designed to be applied to biological samples with a Confocal Laser Scanning Microscope (CLSM) equipped with the feature to bleach regions characterised by a scanning beam that has a radially Gaussian distributed profile. The proposed model leads to FRAP curves that depend on the on- and off-rates. Semi-analytical expressions are used to define the boundaries of on- (off-) rate parameter space in simplified cases when molecules move within a bounded domain. The theoretical model can be used in conjunction to experimental data acquired by CLSM to investigate the biophysical properties of proteins in living cells.Comment: 25 pages. Abstracts Proceedings, The American Society for Cell Biology, 46th Annual Meeting, December 9-13, 2006, San Dieg

Varicellovirus UL 49.5 proteins differentially affect the function of the transporter associated with antigen processing, TAP

Cytotoxic T-lymphocytes play an important role in the protection against viral infections, which they detect through the recognition of virus-derived peptides, presented in the context of MHC class I molecules at the surface of the infected cell. The transporter associated with antigen processing (TAP) plays an essential role in MHC class I–restricted antigen presentation, as TAP imports peptides into the ER, where peptide loading of MHC class I molecules takes place. In this study, the UL49.5 proteins of the varicelloviruses bovine herpesvirus 1 (BHV-1), pseudorabies virus (PRV), and equine herpesvirus 1 and 4 (EHV-1 and EHV-4) are characterized as members of a novel class of viral immune evasion proteins. These UL49.5 proteins interfere with MHC class I antigen presentation by blocking the supply of antigenic peptides through inhibition of TAP. BHV-1, PRV, and EHV-1 recombinant viruses lacking UL49.5 no longer interfere with peptide transport. Combined with the observation that the individually expressed UL49.5 proteins block TAP as well, these data indicate that UL49.5 is the viral factor that is both necessary and sufficient to abolish TAP function during productive infection by these viruses. The mechanisms through which the UL49.5 proteins of BHV-1, PRV, EHV-1, and EHV-4 block TAP exhibit surprising diversity. BHV-1 UL49.5 targets TAP for proteasomal degradation, whereas EHV-1 and EHV-4 UL49.5 interfere with the binding of ATP to TAP. In contrast, TAP stability and ATP recruitment are not affected by PRV UL49.5, although it has the capacity to arrest the peptide transporter in a translocation-incompetent state, a property shared with the BHV-1 and EHV-1 UL49.5. Taken together, these results classify the UL49.5 gene products of BHV-1, PRV, EHV-1, and EHV-4 as members of a novel family of viral immune evasion proteins, inhibiting TAP through a variety of mechanisms

Visualizing the mobility and distribution of chlorophyll proteins in higher plant thylakoid membranes: effects of photoinhibition and protein phosphorylation

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Curvature-coupling dependence of membrane protein diffusion coefficients

We consider the lateral diffusion of a protein interacting with the curvature of the membrane. The interaction energy is minimized if the particle is at a membrane position with a certain curvature that agrees with the spontaneous curvature of the particle. We employ stochastic simulations that take into account both the thermal fluctuations of the membrane and the diffusive behavior of the particle. In this study we neglect the influence of the particle on the membrane dynamics, thus the membrane dynamics agrees with that of a freely fluctuating membrane. Overall, we find that this curvature-coupling substantially enhances the diffusion coefficient. We compare the ratio of the projected or measured diffusion coefficient and the free intramembrane diffusion coefficient, which is a parameter of the simulations, with analytical results that rely on several approximations. We find that the simulations always lead to a somewhat smaller diffusion coefficient than our analytical approach. A detailed study of the correlations of the forces acting on the particle indicates that the diffusing inclusion tries to follow favorable positions on the membrane, such that forces along the trajectory are on average smaller than they would be for random particle positions.Comment: 16 pages, 8 figure

Alternative Macroautophagic Pathways

Macroautophagy is a bulk degradation process that mediates the clearance of long-lived proteins, aggregates, or even whole organelles. This process includes the formation of autophagosomes, double-membrane structures responsible for delivering cargo to lysosomes for degradation. Currently, other alternative autophagy pathways have been described, which are independent of macroautophagic key players like Atg5 and Beclin 1 or the lipidation of LC3. In this review, we highlight recent insights in indentifying and understanding the molecular mechanism responsible for alternative autophagic pathways

The ubiquitin proteasome system in glia and its role in neurodegenerative diseases

The ubiquitin proteasome system (UPS) is crucial for intracellular protein homeostasis and for degradation of aberrant and damaged proteins. The accumulation of ubiquitinated proteins is a hallmark of many neurodegenerative diseases, including amyotrophic lateral sclerosis, Alzheimer's, Parkinson's, and Huntington's disease, leading to the hypothesis that proteasomal impairment is contributing to these diseases. So far, most research related to the UPS in neurodegenerative diseases has been focused on neurons, while glial cells have been largely disregarded in this respect. However, glial cells are essential for proper neuronal function and adopt a reactive phenotype in neurodegenerative diseases, thereby contributing to an inflammatory response. This process is called reactive gliosis, which in turn affects UPS function in glial cells. In many neurodegenerative diseases, mostly neurons show accumulation and aggregation of ubiquitinated proteins, suggesting that glial cells may be better equipped to maintain proper protein homeostasis. During an inflammatory reaction, the immunoproteasome is induced in glia, which may contribute to a more efficient degradation of disease-related proteins. Here we review the role of the UPS in glial cells in various neurodegenerative diseases, and we discuss how studying glial cell function might provide essential information in unraveling mechanisms of neurodegenerative diseases

Advanced Fluorescence Microscopy Techniques-FRAP, FLIP, FLAP, FRET and FLIM

Fluorescence microscopy provides an efficient and unique approach to study fixed and living cells because of its versatility, specificity, and high sensitivity. Fluorescence microscopes can both detect the fluorescence emitted from labeled molecules in biological samples as images or photometric data from which intensities and emission spectra can be deduced. By exploiting the characteristics of fluorescence, various techniques have been developed that enable the visualization and analysis of complex dynamic events in cells, organelles, and sub-organelle components within the biological specimen. The techniques described here are fluorescence recovery after photobleaching (FRAP), the related fluorescence loss in photobleaching (FLIP), fluorescence localization after photobleaching (FLAP), Forster or fluorescence resonance energy transfer (FRET) and the different ways how to measure FRET, such as acceptor bleaching, sensitized emission, polarization anisotropy, and fluorescence lifetime imaging microscopy (FLIM). First, a brief introduction into the mechanisms underlying fluorescence as a physical phenomenon and fluorescence, confocal, and multiphoton microscopy is given. Subsequently, these advanced microscopy techniques are introduced in more detail, with a description of how these techniques are performed, what needs to be considered, and what practical advantages they can bring to cell biological research