Structural determinants of subunit -subunit interactions and subcellular localization of the calcium /calmodulin -dependent protein kinase II

The multifunctional Ca$\sp{2+}$/calmodulin-dependent protein kinase II (CaM kinase) is a Ser/Thr directed protein kinase that participates in diverse Ca$\sp{2+}$ signaling pathways in neurons. The function of CaM kinase depends upon the ability of subunits to form oligomers and to interact with other proteins. Oligomerization is required for autophosphorylation which produces significant functional changes that include Ca$\sp{2+}$/calmodulin-independent activity and calmodulin trapping. Associations with other proteins localize CaM kinase to specific substrates and effectors which serves to optimize the efficiency and speed of signal transduction. In this thesis, we investigate the interactions that underlie the appropriate positioning of CaM kinase activity in cells. We demonstrate that the subcellular distribution of CaM kinase is dynamic in hippocampal slices exposed to anoxic/aglycemic insults and to high K$\sp{+}$-induced depolarization. We determine the localization of CaM kinase domains expressed in neurons and PC-12 cells and find that the C-terminal domain of the $\alpha$ subunit is necessary for localization to dendrites. Moreover, monomeric forms of the enzyme gain access to the nucleus. Attempts made to identify novel CaM kinase binding proteins using the yeast two-hybrid system resulted in the isolation of hundreds of positive clones. Those that have been sequenced are identical to CaM kinase isoforms. Finally, we report the discovery of specific regions within the C-terminal domain that are necessary and sufficient for subunit-subunit interactions. Differences between the $\alpha$ and $\beta$ isoforms were discovered that indicate unique structural requirements for oligomerization. A model for how CaM kinase subunits interact to form holoenzymes and how structural heterogeneity might influence CaM kinase function is presented. ^

Structural conservations and diversities of dsRNA viruses: Structural studies of the cytoplasmic polyhedrosis virus by electron cryomicroscopy and 3D reconstruction

The Reoviridae virus family is a group of economically and pathologically important viruses that have either single-, double-, or triple-shelled protein layers enclosing a segmented double stranded RNA genome. Each virus particle in this family has its own viral RNA dependent RNA polymerase and the enzymatic activities necessary for the mature RNA synthesis. Based on the structure of the inner most cores of the viruses, the Reoviridae viruses can be divided into two major groups. One group of viruses has a smooth surfaced inner core, surrounded by complete outer shells of one or two protein layers. The other group has an inner core decorated with turrets on the five-fold vertices, and could either completely lack or have incomplete outer protein layers. The structural difference is one of the determinant factors for their biological differences during the infection. ^ Cytoplasmic polyhedrosis virus (CPV) is a single-shelled, turreted virus and the structurally simplest member in Reoviridae. It causes specific chronic infections in the insect gut epithelial cells. Due to its wide range of insect hosts, CPV has been engineered as a potential insecticide for use in fruit and vegetable farming. Its unique structural simplicity, unparalleled capsid stability and ease of purification make CPV an ideal model system for studying the structural basis of dsRNA virus assembly at the highest possible resolution by electron cryomicroscopy (cryoEM) and three-dimensional (3D) reconstruction. ^ In this thesis work, I determined the first 3D structure of CPV capsids using 100 kV cryoEM. At an effective resolution of 17 Å, the full capsid reveals a 600-Å diameter, T = 1 icosahedral shell decorated with A and B spikes at the 5-fold vertices. The internal space of the empty CPV is unoccupied except for 12 mushroom-shaped densities that are attributed to the transcriptional enzyme complexes. The inside of the full capsid is packed with icosahedrally-ordered viral genomic RNA. The interactions of viral RNA with the transcriptional enzyme complexes and other capsid proteins suggest a mechanism for RNA transcription and subsequent release. ^ Second, the interactions between the turret proteins (TPs) and the major capsid shell protein (CSPs) have been identified through 3D structural comparisons of the intact CPV capsids with the spikeless CPV capsids, which were generated by chemical treatments. The differential effects of these chemical treatment experiments also indicated that CPV has a significantly stronger structural integrity than other dsRNA viruses, such as the orthoreovirus subcores, which are normally enclosed within outer protein shells. ^ Finally, we have reconstructed the intact CPV to an unprecendented 8 Å resolution from several thousand of 400kV cryoEM images. The 8 Å structure reveals interactions among the 120 molecules of each of the capsid shell protein (CSP), the large protrusion protein (LPP), and 60 molecules of the turret protein (TP). A total of 1980 α-helices and 720 β-sheets have been identified in these capsid proteins. The CSP structure is largely conserved, with the majority of the secondary structures homologous to those observed in the x-ray structures of corresponding proteins of other reoviruses, such as orthoreovirus and bluetongue virus. The three domains of TP are well positioned to play multifunctional roles during viral transcription. The completely non-equivalent interactions between LPP and CSP and those between the anchoring domain of TP and CSP account for the unparalleled stability of this structurally simplest member of the Reoviridae. ^

Impact of BCR -ABL on DNA repair

The BCR-ABL fusion gene is the molecular hallmark of Philadelphia-positive leukemias. Normal Bcr is a multifunctional protein, originally localized to the cytoplasm. It has serine kinase activity and has been implicated in cellular signal transduction. Recently, it has been reported that Bcr can interact with xeroderma pigmentosum group B (XPB/ERCC3)—a nuclear protein active in UV-induced DNA repair. Two major Bcr proteins (p160 Bcr and p130Bcr) have been characterized, and our preliminary results using metabolic labeling and immunoblotting demonstrated that, while both the p160 and p130 forms of Bcr localized to the cytoplasm, the p130 form (and to a lesser extent p160) could also be found in the nucleus. Furthermore, electron microscopy confirmed the presence of Bcr in the nucleus and demonstrated that this protein associates with metaphase chromatin as well as condensed interphase heterochromatin. Since serine kinases that associate with condensed DNA are often cell cycle regulatory, these observations suggested a novel role for nuclear Bcr in cell cycle regulation and/or DNA repair. However, cell cycle synchronization analysis did not demonstrate changes in levels of Bcr throughout the cell cycle. Therefore we hypothesized that BCR serves as a DNA repair gene, and its function is altered by formation of BCR-ABL. This hypothesis was investigated using cell lines stably transfected with the BCR-ABL gene, and their parental counterparts (MBA-1 vs. M07E and Bcr-AblT1 vs. 4A2+pZAP), and several DNA repair assays: the Comet assay, a radioinimunoassay for UV-induced cyclobutane pyrimidine dimers (CPDs), and clonogenic assays. Comet assays demonstrated that, after exposure to either ultraviolet (UV)-C (0.5 to 10.0 joules m −2) or to gamma radiation (200–1000 rads) there was greater efficiency of DNA repair in the BCR-ABL-transfected cells compared to their parental controls. Furthermore, after UVC-irradiation, there was less production of CPDs, and a more rapid disappearance of these adducts in BCR-ABL-bearing cells. UV survival, as reflected by clonogenic assays, was also greater in the BCR-ABL-transfected cells. Taken together, these results indicate that, in our systems, BCR-ABL confers resistance to UVC-induced damage in cells, and increases DNA repair efficiency in response to both UVC- and gamma-irradiation. ^

Collection of data on plant height along the plant diversity gradient in the Jena Experiment (Main Experiment, time series since 2002)

This data set contains a time series of plant height measurements (vegetative and reproductive) from the main experiment plots of a large grassland biodiversity experiment (the Jena Experiment; see further details below). In addition, data on species specific plant heights for the main experiment are available from 2002. In the main experiment, 82 grassland plots of 20 x 20 m were established from a pool of 60 species belonging to four functional groups (grasses, legumes, tall and small herbs). In May 2002, varying numbers of plant species from this species pool were sown into the plots to create a gradient of plant species richness (1, 2, 4, 8, 16 and 60 species) and functional richness (1, 2, 3, 4 functional groups). Plots were maintained by bi-annual weeding and mowing. 1. Plant height was recorded, generally, twice a year just before biomass harvest (during peak standing biomass in late May and in late August). Methodologies of measuring height have varied somewhat over the years. In earlier year the streched plant height was measured, while in later years the standing height without streching the plant was measured. Vegetative height was measured either as the height of the highest leaf or as the length of the main axis of non-flowering plants. Regenerating height was measured either as the height of the highest flower on a plant or as the height of the main axis of flowering. Sampled plants were either randomly selected in the core area of plots or along transects in defined distances. For details refer to the description of individual years. Starting in 2006, also the plots of the management experiment, that altered mowing frequency and fertilized subplots (see further details in the general description of the Jena Experiment) were sampled. 2. Species specific plant height was recorded two times in 2002: in late July (vegetative height) and just before biomass harvest during peak standing biomass in late August (vegetative and regenerative height). For each plot and each sown species in the species pool, 3 plant individuals (if present) from the central area of the plots were randomly selected and used to measure vegetative height (non-flowering indviduals) and regenerative height (flowering individuals) as stretched height. Provided are the means over the three measuremnts per plant species per plot.

El contrato territorial en España : Régimen jurídico y grado de implementación

El contrato territorial es una figura jurídica de aparición reciente destinada a promover el desarrollo sostenible del medio rural. Mediante él, se busca concretar el potencial de una explotación agropecuaria para proveer ciertas funciones sociales y ambientales (no tradicionales) tendientes a un desarrollo rural sostenible. A cambio de estas externalidades positivas, la explotación agropecuaria es compensada. En España, el contrato territorial ha surgido a la luz de dos ámbitos programáticos diferentes: al amparo de la legislación comunitaria europea y de las normas dictadas en su consecuencia a nivel nacional por un lado, y al abrigo de normas estrictamente de origen nacional, por el otro. Por el momento, sólo seis Comunidades Autónomas se han animado a implementarlo; sin embargo, de cara al nuevo Real Decreto 1336/2011 que regula las bases comunes del contrato territorial se espera mayor coherencia, eficiencia y utilización en el uso de esta herramienta

Proceso de institucionalización de la agricultura familiar y nuevo referencial para el desarrollo rural en la Argentina

Este artículo pone en relieve las ideas y los intereses políticos y económicos que condujeron a la reciente institucionalización de la agricultura familiar en la Argentina. Este sector, que fue motor de la primera agriculturización de la Argentina, había perdido toda representación a partir de la centralidad que tuvo el desarrollo tecnológico desde mediados de los años '70. Se analiza el proceso que relegitimó esta categoría socio-política y productiva y se presentan los dispositivos que muestran la consecuente transformación de la acción pública. El paso de una política social y paliativa hacia una políticade sectorización de la agricultura familiar refleja la transformación del referencial de desarrollo rural y las nuevas representaciones del sector agrícola en la Argentina y más ampliamente en el Cono Sur. Así, se reconstituye una representación de la agricultura familiar que identifica y reconoce la dimensión multifuncional de su actividad productiva. Esto conduce a una fragmentación del sector agrícola en dos referenciales: uno centrado en la agricultura familiar y otro centrado en la agricultura empresarial.

Proceso de institucionalización de la agricultura familiar y nuevo referencial para el desarrollo rural en la Argentina

Este artículo pone en relieve las ideas y los intereses políticos y económicos que condujeron a la reciente institucionalización de la agricultura familiar en la Argentina. Este sector, que fue motor de la primera agriculturización de la Argentina, había perdido toda representación a partir de la centralidad que tuvo el desarrollo tecnológico desde mediados de los años '70. Se analiza el proceso que relegitimó esta categoría socio-política y productiva y se presentan los dispositivos que muestran la consecuente transformación de la acción pública. El paso de una política social y paliativa hacia una políticade sectorización de la agricultura familiar refleja la transformación del referencial de desarrollo rural y las nuevas representaciones del sector agrícola en la Argentina y más ampliamente en el Cono Sur. Así, se reconstituye una representación de la agricultura familiar que identifica y reconoce la dimensión multifuncional de su actividad productiva. Esto conduce a una fragmentación del sector agrícola en dos referenciales: uno centrado en la agricultura familiar y otro centrado en la agricultura empresarial.

El contrato territorial en España : Régimen jurídico y grado de implementación

El contrato territorial es una figura jurídica de aparición reciente destinada a promover el desarrollo sostenible del medio rural. Mediante él, se busca concretar el potencial de una explotación agropecuaria para proveer ciertas funciones sociales y ambientales (no tradicionales) tendientes a un desarrollo rural sostenible. A cambio de estas externalidades positivas, la explotación agropecuaria es compensada. En España, el contrato territorial ha surgido a la luz de dos ámbitos programáticos diferentes: al amparo de la legislación comunitaria europea y de las normas dictadas en su consecuencia a nivel nacional por un lado, y al abrigo de normas estrictamente de origen nacional, por el otro. Por el momento, sólo seis Comunidades Autónomas se han animado a implementarlo; sin embargo, de cara al nuevo Real Decreto 1336/2011 que regula las bases comunes del contrato territorial se espera mayor coherencia, eficiencia y utilización en el uso de esta herramienta

Proceso de institucionalización de la agricultura familiar y nuevo referencial para el desarrollo rural en la Argentina

Este artículo pone en relieve las ideas y los intereses políticos y económicos que condujeron a la reciente institucionalización de la agricultura familiar en la Argentina. Este sector, que fue motor de la primera agriculturización de la Argentina, había perdido toda representación a partir de la centralidad que tuvo el desarrollo tecnológico desde mediados de los años '70. Se analiza el proceso que relegitimó esta categoría socio-política y productiva y se presentan los dispositivos que muestran la consecuente transformación de la acción pública. El paso de una política social y paliativa hacia una políticade sectorización de la agricultura familiar refleja la transformación del referencial de desarrollo rural y las nuevas representaciones del sector agrícola en la Argentina y más ampliamente en el Cono Sur. Así, se reconstituye una representación de la agricultura familiar que identifica y reconoce la dimensión multifuncional de su actividad productiva. Esto conduce a una fragmentación del sector agrícola en dos referenciales: uno centrado en la agricultura familiar y otro centrado en la agricultura empresarial.

El contrato territorial en España : Régimen jurídico y grado de implementación

El contrato territorial es una figura jurídica de aparición reciente destinada a promover el desarrollo sostenible del medio rural. Mediante él, se busca concretar el potencial de una explotación agropecuaria para proveer ciertas funciones sociales y ambientales (no tradicionales) tendientes a un desarrollo rural sostenible. A cambio de estas externalidades positivas, la explotación agropecuaria es compensada. En España, el contrato territorial ha surgido a la luz de dos ámbitos programáticos diferentes: al amparo de la legislación comunitaria europea y de las normas dictadas en su consecuencia a nivel nacional por un lado, y al abrigo de normas estrictamente de origen nacional, por el otro. Por el momento, sólo seis Comunidades Autónomas se han animado a implementarlo; sin embargo, de cara al nuevo Real Decreto 1336/2011 que regula las bases comunes del contrato territorial se espera mayor coherencia, eficiencia y utilización en el uso de esta herramienta

Proceso de institucionalización de la agricultura familiar y nuevo referencial para el desarrollo rural en la Argentina

Este artículo pone en relieve las ideas y los intereses políticos y económicos que condujeron a la reciente institucionalización de la agricultura familiar en la Argentina. Este sector, que fue motor de la primera agriculturización de la Argentina, había perdido toda representación a partir de la centralidad que tuvo el desarrollo tecnológico desde mediados de los años '70. Se analiza el proceso que relegitimó esta categoría socio-política y productiva y se presentan los dispositivos que muestran la consecuente transformación de la acción pública. El paso de una política social y paliativa hacia una políticade sectorización de la agricultura familiar refleja la transformación del referencial de desarrollo rural y las nuevas representaciones del sector agrícola en la Argentina y más ampliamente en el Cono Sur. Así, se reconstituye una representación de la agricultura familiar que identifica y reconoce la dimensión multifuncional de su actividad productiva. Esto conduce a una fragmentación del sector agrícola en dos referenciales: uno centrado en la agricultura familiar y otro centrado en la agricultura empresarial.

Spatial compartmentalization of free radical formation and mitochondrial heterogeneity in bivalve gills revealed by live-imaging techniques, supplementary material

Live-imaging techniques (LIT) utilize target-specific fluorescent dyes to visualize biochemical processes using confocal and multiphoton scanning microscopy, which are increasingly employed as non-invasive approach to physiological in-vivo and ex-vivo studies. Here we report application of LIT to bivalve gills for ex-vivo analysis of gill physiology and mapping of reactive oxygen (ROS) and nitrogen (RNS) species formation in the living tissue. Our results indicate that H2O2, HOO. and ONOO- radicals (assessed through C-H2DFFDA staining) are mainly formed within the blood sinus of the filaments and are likely to be produced by hemocytes as defense against invading pathogens. The oxidative damage in these areas is controlled by enhanced CAT (catalase) activities recorded within the filaments. The outermost areas of the ciliated epithelial cells composing the filaments, concentrated the highest mitochondrial densities (MTK Deep Red 633 staining) and the most acidic pH values (as observed with ageladine-a). These mitochondria have low (depolarized) membrane potentials (D psi m) (JC-1 staining), suggesting that the high amounts of ATP required for ciliary beating may be in part produced by non-mitochondrial mechanisms, such as the enzymatic activity of an ATP-regenerating kinase. Nitric oxide (NO, DAF-2DA staining) produced in the region of the peripheral mitochondria may have an effect on mitochondrial electron transport and possibly cause the low membrane potential. High DAF-2DA staining was moreover observed in the muscle cells composing the wall of the blood vessels where NO may be involved in regulating blood vessel diameter. On the ventral bend of the gills, subepithelial mucus glands (SMG) contain large mucous vacuoles showing higher fluorescence intensities for O2.- (DHE staining) than the rest of the tissue. Given the antimicrobial properties of superoxide, release of O2.- into the mucus may help to avoid the development of microbial biofilms on the gill surface. However, cells of the ventral bends are paying a price for this antimicrobial protection, since they show significantly higher oxidative damage, according to the antioxidant enzyme activities and the carbonyl levels, than the rest of the gill tissue. This study provides the first evidence that one single epithelial cell may contain mitochondria with significantly different membrane potentials. Furthermore, we provide new insight into ROS and RNS formation in ex-vivo gill tissues which opens new perspectives for unraveling the different ecophysiological roles of ROS and RNS in multifunctional organs such as gills.