Effects of deletions in the central helix of calmodulin on enzyme activation and peptide binding.

Journal ArticleUsing site-directed mutagenesis we have expressed in Escherichia coli three engineered calmodulins (CaM) containing deletions in the solvent-exposed region of the central helix. These are CaM delta 84, Glu-84 removed; CaM delta 83-84, Glu-83 and Glu-84 removed; and CaM delta 81-84, Ser-81 through Glu-84 removed. The abilities of these proteins to activate skeletal muscle myosin light chain kinase, plant NAD kinase, and bovine brain calcineurin activities were determined, as were their abilities to bind a synthetic peptide based on the calmodulin-binding domain of skeletal muscle myosin light chain kinase. Similar results were obtained with all three deletion proteins. Vm values for enzymes activated by the deletion proteins are all within 10-20% of those values obtained with bacterial control calmodulin. Relative to bacterial control values, changes in Kact or Kd values associated with the deletions are all less than an order of magnitude: Kact values for NAD kinase and myosin light chain kinase are increased 5-7-fold, Kd values for binding of the synthetic peptide are increased 4-7-fold, and Kact values for calcineurin are increased only 1-3-fold. In assays of NAD kinase and myosin light chain kinase activation some differences between bovine calmodulin and bacterial control calmodulin were observed. With NAD kinase, Kact values for the bacterial control protein are increased 4-fold relative to values for bovine calmodulin, and Vm values are increased by 50%; with myosin light chain kinase, Kact values are increased 2-fold and Vm values are decreased 10-15% relative to those values obtained with bovine calmodulin. These differences between bacterial control and bovine calmodulins probably can be attributed to known differences in postranslational processing of calmodulin in bacterial and eucaryotic cells. No differences between bovine and control calmodulins were observed in assays of calcineurin activation or peptide binding. Our observations indicate that contacts with the deleted residues, Ser-81 through Glu-84, are not critical in the calmodulin-target complexes we have evaluated. Formation of these calmodulin-target complexes also does not appear to be greatly affected by the global alterations in the structure of calmodulin that are associated with the deletions. In models in which the central helix is maintained in the altered calmodulins, each deleted residue causes the two lobes of calmodulin to be twisted 100 degrees relative to one another and brought 1.5 A closer together.(ABSTRACT TRUNCATED AT 400 WORDS

Modulation of CD4(+) T Cell-Dependent Specific Cytotoxic CD8(+) T Cells Differentiation and Proliferation by the Timing of Increase in the Pathogen Load

Background. Following infection with viruses, bacteria or protozoan parasites, naive antigen-specific CD8(+) T cells undergo a process of differentiation and proliferation to generate effector cells. Recent evidences suggest that the timing of generation of specific effector CD8(+) T cells varies widely according to different pathogens. We hypothesized that the timing of increase in the pathogen load could be a critical parameter governing this process. Methodology/Principal Findings. Using increasing doses of the protozoan parasite Trypanosoma cruzi to infect C57BL/6 mice, we observed a significant acceleration in the timing of parasitemia without an increase in mouse susceptibility. in contrast, in CD8 deficient mice, we observed an inverse relationship between the parasite inoculum and the timing of death. These results suggest that in normal mice CD8(+) T cells became protective earlier, following the accelerated development of parasitemia. the evaluation of specific cytotoxic responses in vivo to three distinct epitopes revealed that increasing the parasite inoculum hastened the expansion of specific CD8(+) cytotoxic T cells following infection. the differentiation and expansion of T. cruzi-specific CD8(+) cytotoxic T cells is in fact dependent on parasite multiplication, as radiation-attenuated parasites were unable to activate these cells. We also observed that, in contrast to most pathogens, the activation process of T. cruzi-specific CD8(+) cytotoxic T cells was dependent on MHC class II restricted CD4(+) T cells. Conclusions/Significance. Our results are compatible with our initial hypothesis that the timing of increase in the pathogen load can be a critical parameter governing the kinetics of CD4(+) T cell-dependent expansion of pathogen-specific CD8(+) cytotoxic T cells.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Millennium Institute for Vaccine Development and TechnologyConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo, Escola Paulista Med, CINTERGEN, São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilUniv Fed Rio de Janeiro, Inst Biofis Carlos Chagas Filho, Ilha Fundao, Ctr Ciencias Saude, BR-21941 Rio de Janeiro, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, CINTERGEN, São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilMillennium Institute for Vaccine Development and Technology: CNPq - 420067/2005-1Web of Scienc

Diffractive Optical Elements with a Large Angle of Operation Recorded in Acrylamide Based Photopolymer on Flexible Substrates

A holographic device characterised by a large angular range of operation is under development. The aim of this study is to increase the angular working range of the diffractive lens by stacking three layers of high efficiency optical elements on top of each other so that light is collected (and focussed) from a broader range of angles. The angular range of each individual lens element is important, and work has already been done in an acrylamide-based photosensitive polymer to broaden the angular range of individual elements using holographic recording at a low spatial frequency.This paper reports new results on the angular selectivity of stacked diffractive lenses. A working range of 12∘ is achieved. The diffractive focussing elements were recorded holographically with a central spatial frequency of 300 l/mm using exposure energy of 60 mJ/cm2 at a range of recording angles. At this spatial frequency with layers of thickness 50 ± 5 �m, a diffraction efficiency of 80% and 50% was achieved in the single lens element and combined device, respectively. The optical recording process and the properties of the multilayer structure are described and discussed. Holographic recording of a single lens element is also successfully demonstrated on a flexible glass substrate (Corning(R) Willow(R) Glass) for the first time

Fluorescence quenching studies of structure and dynamics in calmodulin-eNOS complexes

This is the peer reviewed version of the following article: Arnett David C.,Persechini Anthony,Tran Quang-Kim,Black D.J. and Johnson Carey K.(2015), Fluorescence quenching studies of structure and dynamics in calmodulin–eNOS complexes, FEBS Letters, 589, doi: 10.1016/j.febslet.2015.03.035, which has been published in final form at http://doi.org/10.1016/j.febslet.2015.03.035. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.Activation of endothelial nitric oxide synthase (eNOS) by calmodulin (CaM) facilitates formation of a sequence of conformational states that is not well understood. Fluorescence decays of fluorescently labeled CaM bound to eNOS reveal four distinct conformational states and single-molecule fluorescence trajectories show multiple fluorescence states with transitions between states occurring on time scales of milliseconds to seconds. A model is proposed relating fluorescence quenching states to enzyme conformations. Specifically, we propose that the most highly quenched state corresponds to CaM docked to an oxygenase domain of the enzyme. In single-molecule trajectories, this state occurs with time lags consistent with the oxygenase activity of the enzyme

Fluorescence quenching studies of structure and dynamics in calmodulin-eNOS complexes

This is the peer reviewed version of the following article: Arnett David C.,Persechini Anthony,Tran Quang-Kim,Black D.J. and Johnson Carey K.(2015), Fluorescence quenching studies of structure and dynamics in calmodulin–eNOS complexes, FEBS Letters, 589, doi: 10.1016/j.febslet.2015.03.035, which has been published in final form at http://doi.org/10.1016/j.febslet.2015.03.035. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.Activation of endothelial nitric oxide synthase (eNOS) by calmodulin (CaM) facilitates formation of a sequence of conformational states that is not well understood. Fluorescence decays of fluorescently labeled CaM bound to eNOS reveal four distinct conformational states and single-molecule fluorescence trajectories show multiple fluorescence states with transitions between states occurring on time scales of milliseconds to seconds. A model is proposed relating fluorescence quenching states to enzyme conformations. Specifically, we propose that the most highly quenched state corresponds to CaM docked to an oxygenase domain of the enzyme. In single-molecule trajectories, this state occurs with time lags consistent with the oxygenase activity of the enzyme

Museus de Ciência e a popularização do conhecimento no Brasil

We analyzed the role and importance of the popularization of science in science museums, and its perspectives in Brazil, from the experience we have developed over the years in the Espaço Ciência Viva; and our participation, observations and studies related to the popularization of science in other science museums and science centers in Brazil and abroad. From an analysis of the historical environment that started in the early 1980’s, to a new era of popularization of science in Brazil and abroad, we briefly evaluate its current state in Brazil and make some considerations on popularization of science, which we need to support for the future economic and social development of our country.Nous avons analysé le rôle, l'importance et l'avenir de la popularisation de la science à travers les musées des sciences au Brésil, en nous basant sur notre expérience acquise au fil des années au sein de Espaço Ciência Viva, ainsi que sur notre participation, nos observations et nos études sur la popularisation de la science dans d'autres musées ou centres dédiés, au Brésil et ailleurs. En partant du contexte historique au début des années 1980 jusqu'à sa situation actuelle, nous évaluons brièvement la popularisation de la science au Brésil, et commentons ce phénomène qu'il est nécessaire de soutenir pour le développement économique et social de notre pays.Analizamos el papel y la importancia de la popularización de la ciencia en los museos científicos, y sus expectativas en Brasil, basados en la experiencia que hemos obtenido a lo largo de los años en el Espaço Ciência Viva, y en nuestra participación, observaciones y estudios relacionados con la popularización de la ciencia en otros museos científicos y centros científicos en Brasil y en el extranjero. Desde un análisis del entorno histórico que empezó a principios de los años 80, a una nueva era de popularización de la ciencia en Brasil y en el mundo, evaluamos brevemente su situación actual en Brasil y hacemos algunas consideraciones sobre la popularización de la ciencia, que debemos apoyar para el futuro económico y el desarrollo social de nuestro país.Fazemos uma análise do papel e da importância da divulgação científica através dos museus e de suas perspectivas no Brasil, a partir da experiência que temos desenvolvido ao longo dos anos no Espaço Ciência Viva  e de nossas participações, observações e estudos relacionados à divulgação científica em outros centros e museus de Ciência no Brasil e no exterior. Partindo de uma análise do ambiente histórico que deu início, a partir da década de 1980, a uma nova era na divulgação científica no Brasil e no mundo, discutimos brevemente seu estado hoje no Brasil e fazemos algumas considerações visando refletir sobre a divulgação científica que precisamos para apoiar o processo de desenvolvimento econômico e social que se anuncia para o Brasil num futuro próximo

Science Museums and the Popularization of Science in Brazil

We analyzed the role and importance of the popularization of science in science museums, and its perspectives in Brazil, from the experience we have developed over the years in the Espaço Ciência Viva ; and our participation, observations and studies related to the popularization of science in other science museums and science centers in Brazil and abroad. From an analysis of the historical environment that started in the early 1980’s, to a new era of popularization of science in Brazil and abroad, we briefly evaluate its current state in Brazil and make some considerations on popularization of science, which we need to support for the future economic and social development of our country.Nous avons analysé le rôle, l'importance et l'avenir de la popularisation de la science à travers les musées des sciences au Brésil, en nous basant sur notre expérience acquise au fil des années au sein de Espaço Ciência Viva, ainsi que sur notre participation, nos observations et nos études sur la popularisation de la science dans d'autres musées ou centres dédiés, au Brésil et ailleurs. En partant du contexte historique au début des années 1980 jusqu'à sa situation actuelle, nous évaluons brièvement la popularisation de la science au Brésil, et commentons ce phénomène qu'il est nécessaire de soutenir pour le développement économique et social de notre pays.Analizamos el papel y la importancia de la popularización de la ciencia en los museos científicos, y sus expectativas en Brasil, basados en la experiencia que hemos obtenido a lo largo de los años en el Espaço Ciência Viva, y en nuestra participación, observaciones y estudios relacionados con la popularización de la ciencia en otros museos científicos y centros científicos en Brasil y en el extranjero. Desde un análisis del entorno histórico que empezó a principios de los años 80, a una nueva era de popularización de la ciencia en Brasil y en el mundo, evaluamos brevemente su situación actual en Brasil y hacemos algunas consideraciones sobre la popularización de la ciencia, que debemos apoyar para el futuro económico y el desarrollo social de nuestro país.Fazemos uma análise do papel e da importância da divulgação científica através dos museus e de suas perspectivas no Brasil, a partir da experiência que temos desenvolvido ao longo dos anos no Espaço Ciência Viva  e de nossas participações, observações e estudos relacionados à divulgação científica em outros centros e museus de Ciência no Brasil e no exterior. Partindo de uma análise do ambiente histórico que deu início, a partir da década de 1980, a uma nova era na divulgação científica no Brasil e no mundo, discutimos brevemente seu estado hoje no Brasil e fazemos algumas considerações visando refletir sobre a divulgação científica que precisamos para apoiar o processo de desenvolvimento econômico e social que se anuncia para o Brasil num futuro próximo

Activation and inactivation of neuronal nitric oxide synthase: characterization of Ca2+-dependent [125I]Calmodulin binding

Constitutive isoforms of nitric oxide synthase (NOS) are activated by transient binding of Ca(2+)/Calmodulin. Here, we characterize the binding of Calmodulin to purified neuronal NOS (nNOS). [125I]Calmodulin bound to a single class of non-interacting and high affinity sites on nNOS. [125I]Calmodulin binding achieved rapid saturation, was linear with nNOS concentration, and exhibited a strict dependence on [Ca(2+)]. Neither affinity nor extent of [125I]Calmodulin binding was affected by L-arginine, NADPH or Tetrahydrobiopterin. Native Calmodulin and engineered Calmodulin homologs [i.e., duplicated N-terminal (CaMNN)] potently displaced [125I]Calmodulin. CaMNN supported nNOS catalysis, but required approximately five-fold more Ca(2+) for comparable activity with native Calmodulin. Taken with results from kinetic analyses of [125I]Calmodulin association and dissociation, our findings suggest four sequential steps in activation of nNOS by Calmodulin: (1) Ca(2+) binds to Calmodulin's C-lobe, (2) the C-lobe of Calmodulin binds NOS, (3) Ca(2+) binds to the N-lobe of Calmodulin, and (4) the N-lobe binds to nNOS. Activation of nNOS only occurs after completion of step (4), with the displacement of nNOS's autoinhibitory insert. Upon intracellular Ca(2+) sequestration, deactivation of nNOS would proceed in reverse order

ATP activates a Reactive Oxygen Species-dependent oxidative stress response and secretion of pro-inflammatory cytokines in macrophages

Secretion of the proinflammatory cytokines, interleukin (IL)-1β and IL-18, usually requires two signals. The first, due to microbial products such as lipopolysaccharide, initiates transcription of the cytokine genes and accumulation of the precursor proteins. Cleavage and secretion of the cytokines is mediated by caspase-1, in association with an inflammasome containing Nalp3, which can be activated by binding of extracellular ATP to purinergic receptors. We show that treatment of macrophages with ATP results in production of reactive oxygen species (ROS), which stimulate the phosphatidylinositol 3-kinase (PI3K) pathway and subsequent Akt and ERK1/2 activation. ROS exerts its effect through glutathionylation of PTEN (phosphatase and tensin homologue deleted from chromosome 10), whose inactivation would shift the equilibrium in favor of PI3K. ATP-dependent ROS production and PI3K activation also stimulate transcription of genes required for an oxidative stress response. In parallel, ATP-mediated ROS-dependent PI3K is required for activation of caspase-1 and secretion of IL-1β and IL-18. Thus, an increase in ROS levels in ATP-treated macrophages results in activation of a single pathway that promotes both adaptation to subsequent exposure to oxidants or inflammation, and processing and secretion of proinflammatory cytokines