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

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

A Dynamic Model of Interactions of Ca^(2+), Calmodulin, and Catalytic Subunits of Ca^(2+)/Calmodulin-Dependent Protein Kinase II

During the acquisition of memories, influx of Ca^(2+) into the postsynaptic spine through the pores of activated N-methyl-D-aspartate-type glutamate receptors triggers processes that change the strength of excitatory synapses. The pattern of Ca^(2+) influx during the first few seconds of activity is interpreted within the Ca^(2+)-dependent signaling network such that synaptic strength is eventually either potentiated or depressed. Many of the critical signaling enzymes that control synaptic plasticity, including Ca^(2+)/calmodulin-dependent protein kinase II (CaMKII), are regulated by calmodulin, a small protein that can bind up to 4 Ca^(2+) ions. As a first step toward clarifying how the Ca^(2+)-signaling network decides between potentiation or depression, we have created a kinetic model of the interactions of Ca^(2+), calmodulin, and CaMKII that represents our best understanding of the dynamics of these interactions under conditions that resemble those in a postsynaptic spine. We constrained parameters of the model from data in the literature, or from our own measurements, and then predicted time courses of activation and autophosphorylation of CaMKII under a variety of conditions. Simulations showed that species of calmodulin with fewer than four bound Ca^(2+) play a significant role in activation of CaMKII in the physiological regime, supporting the notion that processing ofCa^(2+) signals in a spine involves competition among target enzymes for binding to unsaturated species of CaM in an environment in which the concentration of Ca^(2+) is fluctuating rapidly. Indeed, we showed that dependence of activation on the frequency of Ca^(2+) transients arises from the kinetics of interaction of fluctuating Ca^(2+) with calmodulin/CaMKII complexes. We used parameter sensitivity analysis to identify which parameters will be most beneficial to measure more carefully to improve the accuracy of predictions. This model provides a quantitative base from which to build more complex dynamic models of postsynaptic signal transduction during learning

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

Structures of smooth muscle myosin and heavy meromyosin in the folded, shutdown state

Remodelling of the contractile apparatus within smooth muscle cells is an essential process that allows effective contractile activity over a wide range of cell lengths. The thick filaments may be redistributed via depolymerisation into inactive myosin monomers that have been detected in vitro, in which the long tail has a folded conformation. The structure of this folded molecule has been controversial. Using negative stain electron microscopy of individual folded molecules from turkey gizzard we show they are more compact than previously described, with heads and the three segments of the folded tail closely packed. Smooth muscle heavy meromyosin (HMM), which lacks two-thirds of the tail, closely resembles the equivalent parts of whole myosin. Image processing reveals a characteristic head region morphology for both HMM and myosin whose features are identifiable by comparison with less compact molecules. The two heads associate asymmetrically: the tip of one motor domain touches the base of the other, resembling the blocked and free heads of this HMM when it forms 2-D crystals on lipid. The tail of HMM lies between the heads, contacting the blocked motor domain, unlike in the 2-D crystal. The tail of the intact myosin is bent sharply and consistently at two positions close to residues 1175 and 1535. The first bend position correlates with a skip in the coiled coil sequence, the second does not. The first segment runs between the heads from the head-tail junction. Unexpectedly, the other segments associate only with the blocked head rather than both heads, such that the second bend lies at a specific position near the C-lobe of the blocked head regulatory light chain. Quantitative analysis of tail flexibility shows that the single coiled coil of HMM has an apparent Young’s modulus of about 0.5 GPa. The folded tail of the intact molecule is less flexible indicating interactions between the segments. The folded tail does not modify the compact head arrangement but stabilises it, indicating a structural mechanism for the very low ATPase activity of the folded molecule

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

Fast-Response Calmodulin-Based Fluorescent Indicators Reveal Rapid Intracellular Calcium Dynamics

Faithful reporting of temporal patterns of intracellular Ca 2 + dynamics requires the working range of indicators to match the signals. Current genetically encoded calmodulin-based fluorescent indicators are likely to distort fast Ca 2 + signals by apparent saturation and integration due to their limiting fluorescence rise and decay kinetics. A series of probes was engineered with a range of Ca 2 + affinities and accelerated kinetics by weakening the Ca 2 + -calmodulin-peptide interactions. At 37 °C, the GCaMP3-derived probe termed GCaMP3 fast is 40-fold faster than GCaMP3 with Ca 2 + decay and rise times, t 1/2 , of 3.3 ms and 0.9 ms, respectively, making it the fastest to-date. GCaMP3 fast revealed discreet transients with significantly faster Ca 2 + dynamics in neonatal cardiac myocytes than GCaMP6f. With 5-fold increased two-photon fluorescence cross-section for Ca 2 + at 940 nm, GCaMP3 fast is suitable for deep tissue studies. The green fluorescent protein serves as a reporter providing important novel insights into the kinetic mechanism of target recognition by calmodulin. Our strategy to match the probe to the signal by tuning the affinity and hence the Ca 2 + kinetics of the indicator is applicable to the emerging new generations of calmodulin-based probe

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

Design and mechanistic insight into ultrafast calcium indicators for monitoring intracellular calcium dynamics.

Calmodulin-based genetically encoded fluorescent calcium indicators (GCaMP-s) are powerful tools of imaging calcium dynamics from cells to freely moving animals. High affinity indicators with slow kinetics however distort the temporal profile of calcium transients. Here we report the development of reduced affinity ultrafast variants of GCaMP6s and GCaMP6f. We hypothesized that GCaMP-s have a common kinetic mechanism with a rate-limiting process in the interaction of the RS20 peptide and calcium-calmodulin. Therefore we targeted specific residues in the binding interface by rational design generating improved indicators with GCaMP6fu displaying fluorescence rise and decay times (t1/2) of 1 and 3 ms (37 °C) in vitro, 9 and 22-fold faster than GCaMP6f respectively. In HEK293T cells, GCaMP6fu revealed a 4-fold faster decay of ATP-evoked intracellular calcium transients than GCaMP6f. Stimulation of hippocampal CA1 pyramidal neurons with five action potentials fired at 100 Hz resulted in a single dendritic calcium transient with a 2-fold faster rise and 7-fold faster decay time (t1/2 of 40 ms) than GCaMP6f, indicating that tracking high frequency action potentials may be limited by calcium dynamics. We propose that the design strategy used for generating GCaMP6fu is applicable for the acceleration of the response kinetics of GCaMP-type calcium indicators

Arquivo Público e informação: acesso à informação nos arquivos públicos estaduais do Brasil

Access conditions to information kept in public archival institutions are analyzed In this research through the study of Brazilian state public archives. The theoretical framework of this research is centered in a real and common concern in Brazil related to the contradiction between the rights one has to get information and one's real access to information. The research method adopted was the survey and the data v^s collected through a questionnaire sent by mail and answered by fifteen outof the twenty five existing Brazilian state public archives. After the study of federal and some state archival legislation, data analysis was carried out focusing on aspects winich affect significantly the access to the archives, such as: the location of the archival institution within the administrative structure of the state, the existence ofrecords management and transfer programs, the ways of organizing, storing and describing holdings, the existence of finding aids and, finally, the availability of human and technological resources. It was concluded that the access to the archiveskept in Brazilian state public archives is limited, to a great extent, althoughguaranteed by law. Either federal and some state legislation analyzed ensure that any citizen has the nght of accessing the archives. Nevertheless, it has been found several obstacles which prevent archival institutions of making the documents available to Brazilian citizens, either for research purposes or as evidence of rights.As condições de acesso à informação nos arquivos públicos são analisadas nesta pesquisa, através de estudo realizado nos Arquivos Públicos Estaduais brasileiros. O eixo teórico da pesquisa foi o questionamento ocorrente hoje no Brasil, que consiste na contradição existente entre o direito à informação e de acesso pleno aos documentos produzidos e acumulados pela administraçãopública e a realidade das instituições arquivístivas brasileiras, com dificuldade em tornar essas informações acessíveis para o público. A pesquisa de campo, utilizando o questionário, como instrumento de coleta de dados, analisou 15. instituições arquivísticas estaduais, dentre as 25 existentes no país. Após o estudo da legislação arquivística federal e de algumas leis estaduais, enviadas pelas instituições pesquisadas, procedeu-se à análise dos fatores consideradosimportantes no que tange à organização e as condições de acesso aos arquivos. Entre eles. a inserção dos Arquivos dentro da estrutura administrativa do Estado, a existência de programas de gestão de documentos e de recolhimento, as formas de organização, descrição e consen/ação do acervo, assim como a infra-estrutura dos arquivos, a existência de instrumentos de pesquisa e as possibilidades de recursos humanos e tecnológicos. Os resultados obtidos indicam que o acesso aos conjuntos de documentos, hoje, sob custódia dos. Arquivos Públicos Estaduais do Brasil encontra-se bastante limitado, apesar degarantido por lei. A legislação federal e as diversas leis estaduais estudadas, são suficientes para garantir ao cidadão o acesso aos arquivos. No entanto, são inúmeras as dificuldades encontradas pelas instituições arquivísticas para manter seus acervos disponíveis para consulta e para atender ao cidadão que procura os Arquivos, seja com o objetivo de realizar pesquisa cientifica ou de buscar a comprovação de direitos