Hemispheric asymmetry in the maturation of the extra striate checkerboard onset evoked potential

Recently we have shown that the single positive deflection in the checkerboard onset evoked potential (EP) of young children of striate origin develops into a negative-positive complex. However, also an early positive peak becomes apparent in the checkerboard onset EP. To determine the origin and development of the activity underlying this early positive deflection we studied the checkerboard onset EPs in children of 9¿16 years of age. It was found that for the children in this age group two different dipole sources are responsible for the activity underlying the pattern onset EP. One of the dipoles corresponds to the activity generated in the striate cortex, whereas a second dipole of extrastriate origin is responsible for the appearance of the early positive deflection. This extrastriate activity shows hemispheric asymmetry, i.e. the strength of the right hemispheric extrastriate source exceeds the strength of the left hemispheric source. These results are in accordance with histological studies of Conel (1939¿1963) [The postnatal development of the human cerebral cortex (Vols 1¿8). Cambridge, Mass.: Harvard Univ. Press] which show that the maturation of the extrastriate areas of the left hemisphere is delayed with respect to the right hemisphere

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

The mating-specific Gα interacts with a kinesin-14 and regulates pheromone-induced nuclear migration in budding yeast

As a budding yeast cell elongates toward its mating partner, cytoplasmic microtubules connect the nucleus to the cell cortex at the growth tip. The Kar3 kinesin-like motor protein is then thought to stimulate plus-end depolymerization of these microtubules, thus drawing the nucleus closer to the site where cell fusion and karyogamy will occur. Here, we show that pheromone stimulates a microtubule-independent interaction between Kar3 and the mating-specific Gα protein Gpa1 and that Gpa1 affects both microtubule orientation and cortical contact. The membrane localization of Gpa1 was found to polarize early in the mating response, at about the same time that the microtubules begin to attach to the incipient growth site. In the absence of Gpa1, microtubules lose contact with the cortex upon shrinking and Kar3 is improperly localized, suggesting that Gpa1 is a cortical anchor for Kar3. We infer that Gpa1 serves as a positional determinant for Kar3-bound microtubule plus ends during mating. © 2009 by The American Society for Cell Biology

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

Interfering with DNA Decondensation as a Strategy Against Mycobacteria

Tuberculosis is once again a major global threat, leading to more than 1 million deaths each year. Treatment options for tuberculosis patients are limited, expensive and characterized by severe side effects, especially in the case of multidrug-resistant forms. Uncovering novel vulnerabilities of the pathogen is crucial to generate new therapeutic strategies. Using high resolution microscopy techniques, we discovered one such vulnerability of Mycobacterium tuberculosis. We demonstrate that the DNA of M. tuberculosis can condense under stressful conditions such as starvation and antibiotic treatment. The DNA condensation is reversible and specific for viable bacteria. Based on these observations, we hypothesized that blocking the recovery from the condensed state could weaken the bacteria. We showed that after inducing DNA condensation, and subsequent blocking of acetylation of DNA binding proteins, the DNA localization in the bacteria is altered. Importantly under these conditions, Mycobacterium smegmatis did not replicate and its survival was significantly reduced. Our work demonstrates that agents that block recovery from the condensed state of the nucleoid can be exploited as antibiotic. The combination of fusidic acid and inhibition of acetylation of DNA binding proteins, via the Eis enzyme, potentiate the efficacy of fusidic acid by 10 and the Eis inhibitor to 1,000-fold. Hence, we propose that successive treatment with antibiotics and drugs interfering with recovery from DNA condensation constitutes a novel approach for treatment of tuberculosis and related bacterial infections

Cell-Specific Monitoring of Protein Synthesis In Vivo

Analysis of general and specific protein synthesis provides important information, relevant to cellular physiology and function. However, existing methodologies, involving metabolic labelling by incorporation of radioactive amino acids into nascent polypeptides, cannot be applied to monitor protein synthesis in specific cells or tissues, in live specimens. We have developed a novel approach for monitoring protein synthesis in specific cells or tissues, in vivo. Fluorescent reporter proteins such as GFP are expressed in specific cells and tissues of interest or throughout animals using appropriate promoters. Protein synthesis rates are assessed by following fluorescence recovery after partial photobleaching of the fluorophore at targeted sites. We evaluate the method by examining protein synthesis rates in diverse cell types of live, wild type or mRNA translation-defective Caenorhabditis elegans animals. Because it is non-invasive, our approach allows monitoring of protein synthesis in single cells or tissues with intrinsically different protein synthesis rates. Furthermore, it can be readily implemented in other organisms or cell culture systems

An In Situ Autologous Tumor Vaccination with Combined Radiation Therapy and TLR9 Agonist Therapy

PURPOSE:Recent studies have shown that a new generation of synthetic agonist of Toll-like receptor (TLR) 9 consisting a 3'-3'-attached structure and a dCp7-deaza-dG dinucultodie shows more potent immunostimulatory effects in both mouse and human than conventional CpG oligonucleotides. Radiation therapy (RT) provides a source of tumor antigens that are released from dying, irradiated, tumor cells without causing systemic immunosuppression. We, therefore, examined effect of combining RT with a designer synthetic agonist of TLR9 on anti-tumoral immunity, primary tumor growth retardation and metastases in a murine model of lung cancer. METHODS:Grouped C57BL/6 and congenic B cell deficient mice (B(-/-)) bearing footpad 3LL tumors were treated with PBS, TLR9 agonist, control oligonucelotide, RT or the combination of RT and TLR9 agonist. Immune phenotype of splenocytes and serum IFN-γ and IL-10 levels were analyzed by FACS and ELISA, 24 h after treatment. Tumor growth, lung metastases and survival rate were monitored and tumor specific antibodies in serum and deposition in tumor tissue were measured by ELISA and immunofluorescence. RESULTS:TLR9 agonist expanded and activated B cells and plasmacytoid dendritic cells in wild-type mice and natural killer DCs (NKDCs) in B cell-deficient (B(-/-)) mice bearing ectopic Lewis lung adenocarcinoma (3LL). Combined RT with TLR9 agonist treatment inhibited 3LL tumor growth in both wild type and B(-/-) mice. A strong tumor-specific humoral immune response (titer: 1/3200) with deposition of mouse IgG auto-antibodies in tumor tissue were found in wildtype mice, whereas the number of tumor infiltrating NKDCs increased in B(-/-) mice following RT+ TLR9 agonist therapy. Furthermore, mice receiving combination therapy had fewer lung metastases and a higher survival than single treatment cohorts. CONCLUSIONS:Combination therapy with TLR9 agonist and RT induces systemic anti-tumoral humoral response, augments tumoral infiltration of NKDCs, reduces pulmonary metastases and improves survival in a murine model of 3LL cancer

Proteasome Nuclear Import Mediated by Arc3 Can Influence Efficient DNA Damage Repair and Mitosis in Schizosaccharomyces Pombe

Proteasomes must efficiently remove their substrates throughout the cells in a timely manner as many of these proteins can be toxic. This study shows that proteasomes can do so efficiently because they are highly mobile. Furthermore this study uncovers that proteasome mobility requires functional Arc3, a subunit of the Arp2/3 complex