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. ^

The determinants of offshore production by Multinational Corporations (MNCs) : a comparison of Japanese and US MNCs

This study employs confidential affiliate-level panel data to improve measures of foreign affiliate activities of Japanese firms in manufacturing sectors. Combining existing data on U.S. MNCs with the Japanese data, we illustrate the pattern and determinant of their foreign affiliate sales by destination market across countries and industries for the period 1989-2005. Among our results, Japanese and U.S. MNCs are similar in the substantial growth of their foreign affiliate sales and the importance of sales to local markets. However, Japanese MNCs are distinctive from U.S. MNCs in that Japanese affiliate sales in Asia were prominently higher in host markets with lower educational attainment.

Dimensions and determinants of upward mobility : a study based on longitudinal data from Delhi slums

This study based on two primary surveys of the same households in two different years (2007/08 and 2012) assesses the extent of inter-temporal change in income of the individual workers and makes an attempt to identify the factors which explain upward mobility in alternate econometric framework, envisaging endogeneity problem. It also encompasses a host of indicators of wellbeing and constructs the transition matrix to capture the extent of change over time at the household level. The findings are indicative of a rise in the income of workers across a sizeable percentage of households though many of them remained below the poverty line notwithstanding this increase. In fact, there is a wide spread deterioration in the wellbeing index constructed at the household level. Among several determinants of income rise two important policy prescriptions can be elicited. Inadequate education reduces the probability of upward mobility while education above a threshold level raises it. Savings are crucial for upward mobility impinging on the importance of asset creation. Views that entail neighbourhood spill-over effects also received validation. Besides, investment in housing and basic amenities turns out to be crucial for improvement in wellbeing levels.

Flexibility, innovation and sustainability in business models as determinants of firm performance heterogeneity.

La comprensión actual de la heterogeneidad de las empresas académico rendimiento en el entorno entra industria necesita un mayor desarrollo. Gestión estratégica y el discurso sobre la literatura empresarial necesita mayor explicación de por qué los modelos de negocio aparentemente similares en el mismo sector actúan de forma diferente. También qué factores del entorno sectorial y operativo determina el surgimiento y funcionamiento de los modelos de negocio sostenibles e innovadoras permanecen sin explorar. Un marco se conceptualiza acompañado de estudios de caso sobre la compañía aérea y las industrias de energía renovable. El estudio lleva a una visión basada en los recursos de los modelos de negocio que las empresas alcancen posiciones heterogéneas de recursos. Una explicación para la heterogeneidad firme desempeño que se busca por intermediación conocimiento que genera valor a partir de la utilización eficaz de los recursos de conocimientos adquiridos a entornos entra y inter-empresa. Un marco para la aparición de nuevos modelos de negocios verdes se conceptualiza y deducciones se obtienen mediante un estudio de caso sobre la base de la industria de biocombustibles de energías renovables para explicar la dinámica de los mercados verdes y cómo se puede crear valor sostenible y capturó e innovadora de los modelos de negocios verdes. El marco desarrollado proporciona una visión cíclica de la flexibilidad del modelo de negocio en la que se propaga la acumulación de recursos basada en el conocimiento del modelo de negocio a través de los ambientes dentro y inter-empresa. Estrategias de conocimiento de corretaje del resultado ambientes inter e dentro-firma en un mejor desempeño del modelo de negocio. La flexibilidad del modelo de negocio que adquiere está determinada por la eficiencia con la acumulación de recursos está alineado con su ambiente externo. Las características de la que el modelo de negocio alcanza ventajas competitivas, como la innovación y la flexibilidad se atribuyen a la heterogeneidad de los recursos. La investigación se extiende a la literatura orientación de servicio al conceptualizar y medir la orientación a servicios como un requisito clave para la innovación del modelo de negocio, mientras que aboga por la necesidad de identificar correctamente las competencias básicas de la empresa, especialmente relevante en el contexto de la empresa orientada a los servicios, donde la creación de valor requiere recursos y la prestación eficiente de los servicios. La investigación trata de llegar a una descripción de los modelos de negocio sostenibles verdes y argumenta que la innovación, la flexibilidad y la sostenibilidad son los tres habilitadores básicos de los cuales el concepto de modelo de negocio verde puede evolucionar la explotación de nuevos mecanismos de mercado y los mercados para crear y capturar valor en el mantenimiento de su innovadora ambiente externo. La investigación integra efectivamente los conceptos de corretaje de conocimientos y modelos de negocio a partir de un recurso basado en la acumulación de vista y al mismo tiempo llega a la heterogeneidad rendimiento de los modelos de negocio aparentemente similares dentro de la misma industria. La investigación indica cómo se producen perturbaciones del mercado en una industria como resultado de modelos de negocio innovador y flexible, y cómo los nuevos modelos de negocio evolucionan en base a estos trastornos. Avanza la comprensión de cómo la estrategia de núcleo competencia y la innovación del modelo de negocio construcciones se comportan en el esfuerzo de la empresa de servicios para obtener una ventaja competitiva sostenible. Los resultados tienen implicaciones en el rendimiento de las empresas que empiezan sin recursos distintos de los suyos, o que utilizan un modelo de negocio imitado, para lograr un mejor rendimiento a través de la evolución del modelo de negocio alineado con las exitosas estrategias de conocimiento de corretaje. Dicho marco puede permitir a las empresas a evaluar y elegir un modelo de negocio basado en la innovación, la flexibilidad, la sostenibilidad y las opciones para el cambio. La investigación se suma a la literatura acumulación de recursos, explicando cómo los recursos pueden ser efectivamente adquirida para crear valor. La investigación también se suma a la literatura empresarial verde, explicando cómo las empresas crear y capturar valor en los nuevos mecanismos dinámicos de mercado.

Ecological and genetic determinants of Pepino mosaic virus emergence

Virus emergence is a complex phenomenon, which generally involves spread to a new host from a wild host, followed by adaptation to the new host. Although viruses account for the largest fraction of emerging crop pathogens, knowledge about their emergence is incomplete. We address here the question of whether Pepino mosaic virus (PepMV) emergence as a major tomato pathogen worldwide could have involved spread from wild to cultivated plant species and host adaptation. For this, we surveyed natural populations of wild tomatoes in southern Peru for PepMV infection. PepMV incidence, genetic variation, population structure, and accumulation in various hosts were analyzed. PepMV incidence in wild tomatoes was high, and a strain not yet reported in domestic tomato was characterized. This strain had a wide host range within the Solanaceae, multiplying efficiently in most assayed Solanum species and being adapted to wild tomato hosts. Conversely, PepMV isolates from tomato crops showed evidence of adaptation to domestic tomato, possibly traded against adaptation to wild tomatoes. Phylogenetic reconstructions indicated that the most probable ancestral sequence came from a wild Solanum species. A high incidence of PepMV in wild tomato relatives would favor virus spread to crops and its efficient multiplication in different Solanum species, including tomato, allowing its establishment as an epidemic pathogen. Later, adaptation to tomato, traded off against adaptation to other Solanum species, would isolate tomato populations from those in other hosts.

Genetic and Metabolic Determinants of Human Epigenetic Variation

Peer reviewed

Molecular determinants of coordinated proton and zinc inhibition of N-methyl-d-aspartate NR1/NR2A receptors

Modulation of the N-methyl-d-aspartate (NMDA)-selective glutamate receptors by extracellular protons and Zn2+ may play important roles during ischemia in the brain and during seizures. Recombinant NR1/NR2A receptors exhibit a much higher apparent affinity for voltage-independent Zn2+ inhibition than receptors with other subunit combinations. Here, we show that the mechanism of this apparent high-affinity, voltage-independent Zn2+ inhibition for NR2A-containing receptors results from the enhancement of proton inhibition. We also show that the N-terminal leucine/isoleucine/valine binding protein (LIVBP)-like domain of the NR2A subunit contains critical determinants of the apparent high-affinity, voltage-independent Zn2+ inhibition. Mutations H42A, H44G, or H128A greatly increase the Zn2+ IC50 (by up to ≈700-fold) with no effect on the potencies of glutamate and glycine or on voltage-dependent block by Mg2+. Furthermore, the amino acid residue substitution H128A, which mediates the largest effect on the apparent high-affinity Zn2+ inhibition among all histidine substitutions we tested, is also critical to the pH-dependency of Zn2+ inhibition. Our data revealed a unique interaction between two important extracellular modulators of NMDA receptors.

Demographic and epidemiological determinants of healthcare costs in Netherlands: cost of illness study

Objectives: To determine the demands on healthcare resources caused by different types of illnesses and variation with age and sex.

Determinants of RNA polymerase alpha subunit for interaction with beta, beta', and sigma subunits: hydroxyl-radical protein footprinting.

Escherichia coli RNA polymerase (RNAP) alpha subunit serves as the initiator for RNAP assembly, which proceeds according to the pathway 2 alpha-->alpha 2-->alpha 2 beta-->alpha 2 beta beta'-->alpha 2 beta beta' sigma. In this work, we have used hydroxyl-radical protein footprinting to define determinants of alpha for interaction with beta, beta', and sigma. Our results indicate that amino acids 30-75 of alpha are protected from hydroxyl-radical-mediated proteolysis upon interaction with beta (i.e., in alpha 2 beta, alpha 2 beta beta', and alpha 2 beta beta' sigma), and amino acids 175-210 of alpha are protected from hydroxyl-radical-mediated proteolysis upon interaction with beta' (i.e., in alpha 2 beta beta' and alpha 2 beta beta' sigma). The protected regions are conserved in the alpha homologs of prokaryotic, eukaryotic, archaeal, and chloroplast RNAPs and contain sites of substitutions that affect RNAP assembly. We conclude that the protected regions define determinants of alpha for direct functional interaction with beta and beta'. The observed maximal magnitude of protection upon interaction with beta and the observed maximal magnitude of protection upon interaction with beta' both correspond to the expected value for complete protection of one of the two alpha protomers of RNAP (i.e., 50% protection). We propose that only one of the two alpha protomers of RNAP interacts with beta and that only one of the two alpha protomers of RNAP interacts with beta'.

Common molecular determinants of local anesthetic, antiarrhythmic, and anticonvulsant block of voltage-gated Na+ channels.

Voltage-gated Na+ channels are the molecular targets of local anesthetics, class I antiarrhythmic drugs, and some anticonvulsants. These chemically diverse drugs inhibit Na+ channels with complex voltage- and frequency-dependent properties that reflect preferential drug binding to open and inactivated channel states. The site-directed mutations F1764A and Y1771A in transmembrane segment IVS6 of type IIA Na+ channel alpha subunits dramatically reduce the affinity of inactivated channels for the local anesthetic etidocaine. In this study, we show that these mutations also greatly reduce the sensitivity of Na+ channels to state-dependent block by the class Ib antiarrhythmic drug lidocaine and the anticonvulsant phenytoin and, to a lesser extent, reduce the sensitivity to block by the class Ia and Ic antiarrhythmic drugs quinidine and flecainide. For lidocaine and phenytoin, which bind preferentially to inactivated Na+ channels, the mutation F1764A reduced the affinity for binding to the inactivated state 24.5-fold and 8.3-fold, respectively, while Y1771A had smaller effects. For quinidine and flecainide, which bind preferentially to the open Na+ channels, the mutations F1764A and Y1771A reduced the affinity for binding to the open state 2- to 3-fold. Thus, F1764 and Y1771 are common molecular determinants of state-dependent binding of diverse drugs including lidocaine, phenytoin, flecainide, and quinidine, suggesting that these drugs interact with a common receptor site. However, the different magnitude of the effects of these mutations on binding of the individual drugs indicates that they interact in an overlapping, but nonidentical, manner with a common receptor site. These results further define the contributions of F1764 and Y1771 to a complex drug receptor site in the pore of Na+ channels.

Protein kinase C chimeras: catalytic domains of alpha and beta II protein kinase C contain determinants for isotype-specific function.

Protein kinase C (PKC) is involved in the proliferation and differentiation of many cell types. In human erythroleukemia (K-562) cells, the PKC isoforms alpha and beta II play distinct functional roles. alpha PKC is involved in phorbol 12-myristate 13-acetate-induced cytostasis and megakaryocytic differentiation, whereas beta II PKC is required for proliferation. To identify regions within alpha and beta II PKC that allow participation in these divergent pathways, we constructed chimeras in which the regulatory and catalytic domains of alpha and beta II PKC were exchanged. These PKC chimeras can be stably expressed, exhibit enzymatic properties similar to native alpha and beta II PKC in vitro, and participate in alpha and beta II PKC isotype-specific pathways in K-562 cells. Expression of the beta/alpha PKC chimera induces cytostasis in the same manner as overexpression of wild-type alpha PKC. In contrast, the alpha/beta II PKC chimera, like wild-type beta II PKC, selectively translocates to the nucleus and leads to increased phosphorylation of the nuclear envelope polypeptide lamin B in response to bryostatin-1. Therefore, the catalytic domains of alpha and beta II PKC contain determinants important for alpha and beta II PKC isotype function. These results suggest that the catalytic domain represents a potential target for modulating PKC isotype activity in vivo.

Determinants of the phagocytic signal mediated by the type IIIA Fc gamma receptor, Fc gamma RIIIA: sequence requirements and interaction with protein-tyrosine kinases.

The Fc gamma receptor-associated gamma and zeta subunits contain a conserved cytoplasmic motif, termed the immunoglobulin gene tyrosine activation motif, which contains a pair of YXXL sequences. The tyrosine residues within these YXXL sequences have been shown to be required for transduction of a phagocytic signal. We have previously reported that the gamma subunit of the type IIIA Fc gamma receptor (Fc gamma RIIIA) is approximately 6 times more efficient in mediating phagocytosis than the zeta subunit of Fc gamma RIIIA. By exchanging regions of the cytoplasmic domains of the homologous gamma and zeta chains, we observed that the cytoplasmic area of the gamma chain bearing a pair of the conserved YXXL sequences is important in phagocytic signaling. Further specificity of phagocytic signaling is largely determined by the two internal XX amino acids in the YXXL sequences. In contrast, the flanking amino acids of the YXXL sequences including the seven intervening amino acids between the two YXXL sequences do not significantly affect the phagocytic signal. Furthermore, the protein-tyrosine kinase Syk, but not the related kinase ZAP-70, stimulated Fc gamma RIIIA-mediated phagocytosis. ZAP-70, however, increased phagocytosis when coexpressed with the Src family kinase Fyn. These data demonstrate the importance of the two specific amino acids within the gamma subunit YXXL cytoplasmic sequences in phagocytic signaling and explain the difference in phagocytic efficiency of the gamma and zeta chains. These results indicate the importance of Syk in Fc gamma RIIIA-mediated phagocytosis and demonstrate that ZAP-70 and syk differ in their requirement for a Src-related kinase in signal transduction.

Genetic and redox determinants of nitric oxide cytotoxicity in a Salmonella typhimurium model.

Paradoxically, nitric oxide (NO) has been found to exhibit cytotoxic, antiproliferative, or cytoprotective activity under different conditions. We have utilized Salmonella mutants deficient in antioxidant defenses or peptide transport to gain insights into NO actions. Comparison of three NO donor compounds reveals distinct and independent cellular responses associated with specific redox forms of NO. The peroxynitrite (OONO-) generator 3-morpholinosydnonimine hydrochloride mediates oxygen-dependent Salmonella killing, whereas S-nitrosoglutathione (GSNO) causes oxygen-independent cytostasis, and the NO. donor diethylenetriamine-nitric oxide adduct has no antibacterial activity. GSNO has the greatest activity for stationary cells, a characteristic relevant to latent or intracellular pathogens. Moreover, the cytostatic activity of GSNO may best correlate with antiproliferative or antimicrobial effects of NO, which are unassociated with overt cell injury. dpp mutants defective in active dipeptide transport are resistant to GSNO, implicating heterolytic NO+ transfer rather than homolytic NO. release in the mechanism of cytostasis. This transport system may provide a specific pathway for GSNO-mediated signaling in biological systems. The redox state and associated carrier molecules are critical determinants of NO activity.