An approach to the study of G-protein-coupled receptor kinases: an in vitro-purified membrane assay reveals differential receptor specificity and regulation by G beta gamma subunits.

Phosphorylation of GTP-binding-regulatory (G)-protein-coupled receptors by specific G-protein-coupled receptor kinases (GRKs) is a major mechanism responsible for agonist-mediated desensitization of signal transduction processes. However, to date, studies of the specificity of these enzymes have been hampered by the difficulty of preparing the purified and reconstituted receptor preparations required as substrates. Here we describe an approach that obviates this problem by utilizing highly purified membrane preparations from Sf9 and 293 cells overexpressing G-protein-coupled receptors. We use this technique to demonstrate specificity of several GRKs with respect to both receptor substrates and the enhancing effects of G-protein beta gamma subunits on phosphorylation. Enriched membrane preparations of the beta 2- and alpha 2-C2-adrenergic receptors (ARs, where alpha 2-C2-AR refers to the AR whose gene is located on human chromosome 2) prepared by sucrose density gradient centrifugation from Sf9 or 293 cells contain the receptor at 100-300 pmol/mg of protein and serve as efficient substrates for agonist-dependent phosphorylation by beta-AR kinase 1 (GRK2), beta-AR kinase 2 (GRK3), or GRK5. Stoichiometries of agonist-mediated phosphorylation of the receptors by GRK2 (beta-AR kinase 1), in the absence and presence of G beta gamma, are 1 and 3 mol/mol, respectively. The rate of phosphorylation of the membrane receptors is 3 times faster than that of purified and reconstituted receptors. While phosphorylation of the beta 2-AR by GRK2, -3, and -5 is similar, the activity of GRK2 and -3 is enhanced by G beta gamma whereas that of GRK5 is not. In contrast, whereas GRK2 and -3 efficiently phosphorylate alpha 2-C2-AR, GRK5 is quite weak. The availability of a simple direct phosphorylation assay applicable to any cloned G-protein-coupled receptor should greatly facilitate elucidation of the mechanisms of regulation of these receptors by the expanding family of GRKs.

Differential Apaf-1 levels allow cytochrome c to induce apoptosis in brain tumors but not in normal neural tissues.

Brain tumors are typically resistant to conventional chemotherapeutics, most of which initiate apoptosis upstream of mitochondrial cytochrome c release. In this study, we demonstrate that directly activating apoptosis downstream of the mitochondria, with cytosolic cytochrome c, kills brain tumor cells but not normal brain tissue. Specifically, cytosolic cytochrome c is sufficient to induce apoptosis in glioblastoma and medulloblastoma cell lines. In contrast, primary neurons from the cerebellum and cortex are remarkably resistant to cytosolic cytochrome c. Importantly, tumor tissue from mouse models of both high-grade astrocytoma and medulloblastoma display hypersensitivity to cytochrome c when compared with surrounding brain tissue. This differential sensitivity to cytochrome c is attributed to high Apaf-1 levels in the tumor tissue compared with low Apaf-1 levels in the adjacent brain tissue. These differences in Apaf-1 abundance correlate with differences in the levels of E2F1, a previously identified activator of Apaf-1 transcription. ChIP assays reveal that E2F1 binds the Apaf-1 promoter specifically in tumor tissue, suggesting that E2F1 contributes to the expression of Apaf-1 in brain tumors. Together, these results demonstrate an unexpected sensitivity of brain tumors to postmitochondrial induction of apoptosis. Moreover, they raise the possibility that this phenomenon could be exploited therapeutically to selectively kill brain cancer cells while sparing the surrounding brain parenchyma.

Convergent differential regulation of parvalbumin in the brains of vocal learners.

Spoken language and learned song are complex communication behaviors found in only a few species, including humans and three groups of distantly related birds--songbirds, parrots, and hummingbirds. Despite their large phylogenetic distances, these vocal learners show convergent behaviors and associated brain pathways for vocal communication. However, it is not clear whether this behavioral and anatomical convergence is associated with molecular convergence. Here we used oligo microarrays to screen for genes differentially regulated in brain nuclei necessary for producing learned vocalizations relative to adjacent brain areas that control other behaviors in avian vocal learners versus vocal non-learners. A top candidate gene in our screen was a calcium-binding protein, parvalbumin (PV). In situ hybridization verification revealed that PV was expressed significantly higher throughout the song motor pathway, including brainstem vocal motor neurons relative to the surrounding brain regions of all distantly related avian vocal learners. This differential expression was specific to PV and vocal learners, as it was not found in avian vocal non-learners nor for control genes in learners and non-learners. Similar to the vocal learning birds, higher PV up-regulation was found in the brainstem tongue motor neurons used for speech production in humans relative to a non-human primate, macaques. These results suggest repeated convergent evolution of differential PV up-regulation in the brains of vocal learners separated by more than 65-300 million years from a common ancestor and that the specialized behaviors of learned song and speech may require extra calcium buffering and signaling.

Differential predictability of four dimensions of affect intensity.

Individual differences in affect intensity are typically assessed with the Affect Intensity Measure (AIM). Previous factor analyses suggest that the AIM is comprised of four weakly correlated factors: Positive Affectivity, Negative Reactivity, Negative Intensity and Positive Intensity or Serenity. However, little data exist to show whether its four factors relate to other measures differently enough to preclude use of the total scale score. The present study replicated the four-factor solution and found that subscales derived from the four factors correlated differently with criterion variables that assess personality domains, affective dispositions, and cognitive patterns that are associated with emotional reactions. The results show that use of the total AIM score can obscure relationships between specific features of affect intensity and other variables and suggest that researchers should examine the individual AIM subscales.

Differential developmental trajectories of magnetic susceptibility in human brain gray and white matter over the lifespan

As indicated by several recent studies, magnetic susceptibility of the brain is influenced mainly by myelin in the white matter and by iron deposits in the deep nuclei. Myelination and iron deposition in the brain evolve both spatially and temporally. This evolution reflects an important characteristic of normal brain development and ageing. In this study, we assessed the changes of regional susceptibility in the human brain in vivo by examining the developmental and ageing process from 1 to 83 years of age. The evolution of magnetic susceptibility over this lifespan was found to display differential trajectories between the gray and the white matter. In both cortical and subcortical white matter, an initial decrease followed by a subsequent increase in magnetic susceptibility was observed, which could be fitted by a Poisson curve. In the gray matter, including the cortical gray matter and the iron-rich deep nuclei, magnetic susceptibility displayed a monotonic increase that can be described by an exponential growth. The rate of change varied according to functional and anatomical regions of the brain. For the brain nuclei, the age-related changes of susceptibility were in good agreement with the findings from R2* measurement. Our results suggest that magnetic susceptibility may provide valuable information regarding the spatial and temporal patterns of brain myelination and iron deposition during brain maturation and ageing. © 2013 Wiley Periodicals, Inc.

Differential antibody responses to Pneumococcal and Tetanus vaccines in asymptomatic HIV-infected patients :a serological and cellular study

info:eu-repo/semantics/nonPublished

Differential scanning calorimetry in life science: thermodynamics, stability, molecular recognition and application in drug design

All biological phenomena depend on molecular recognition, which is either intermolecular like in ligand binding to a macromolecule or intramolecular like in protein folding. As a result, understanding the relationship between the structure of proteins and the energetics of their stability and binding with others (bio)molecules is a very interesting point in biochemistry and biotechnology. It is essential to the engineering of stable proteins and to the structure-based design of pharmaceutical ligands. The parameter generally used to characterize the stability of a system (the folded and unfolded state of the protein for example) is the equilibrium constant (K) or the free energy (deltaG(o)), which is the sum of enthalpic (deltaH(o)) and entropic (deltaS(o)) terms. These parameters are temperature dependent through the heat capacity change (deltaCp). The thermodynamic parameters deltaH(o) and deltaCp can be derived from spectroscopic experiments, using the van't Hoff method, or measured directly using calorimetry. Along with isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC) is a powerful method, less described than ITC, for measuring directly the thermodynamic parameters which characterize biomolecules. In this article, we summarize the principal thermodynamics parameters, describe the DSC approach and review some systems to which it has been applied. DSC is much used for the study of the stability and the folding of biomolecules, but it can also be applied in order to understand biomolecular interactions and can thus be an interesting technique in the process of drug design.

Differential T and B cell responses against Mycobacterium tuberculosis heparin-binding hemagglutinin adhesin in infected healthy individuals and patients with tuberculosis.

Because only 10% of individuals infected with Mycobacterium tuberculosis will eventually develop disease, antigens that are recognized differently by the immune systems of infected healthy and diseased subjects may constitute potential vaccine candidates. Here, the heparin-binding hemagglutinin adhesin (HBHA) is identified as such an antigen. Lymphocytes from 60% of healthy infected individuals (n=25) produced interferon (IFN)-gamma after stimulation with HBHA, compared with only 4% of patients with active tuberculosis (n=24). In the responders, both CD4(+) and CD8(+) cells secreted HBHA-specific IFN-gamma, and the antigen was presented by both major histocompatibility complex class I and II molecules. In contrast to the reduced ability of patients with tuberculosis to produce HBHA-specific IFN-gamma, most of them (82%) produced anti-HBHA antibodies, compared with 36% of the infected healthy subjects. These observations indicate that HBHA is recognized differently by the immune systems of patients with tuberculosis and infected healthy individuals and might provide a marker for protection against tuberculosis.

Let´s teach geometry

In this work we present an activity for High School students in which various mathematical concepts of plane and spatial geometry are involved. The final objective of the proposed tasks is constructing a particular polyhedron, the cube, by using a modality of origami called modular origami.

Conceptualización de área del rectángulo con la mediación del programa Cabri Geometry

En esta charla se presentará el trabajo realizado durante el año 2010 por el grupo Nuevas Tecnologías de EDUMAT-UIS coordinado por el Dr. Martín Eduardo Acosta Gempeler. El grupo viene realizando un trabajo de capacitación a profesores de varios colegios del área metropolitana de Bucaramanga en cuanto a la implementación de software de geometría dinámica en la enseñanza de diferentes conceptos geométricos en secundaria.

Ethnomathematical research and drama in education techniques: developing a dialogue in a geometry class of 10th grade students

Ethnomathematical research, together with digital technologies (WebQuest) and Drama-in- Education (DiE) techniques, can create a fruitful learning environment in a mathematics classroom—a hybrid/third space—enabling increased student participation and higher levels of cognitive engagement. This article examines how ethnomathematical ideas processed within the experiential environment established by the Drama-in-Education techniques challenged students‘ conceptions of the nature of mathematics, the ways in which students engaged with mathematics learning using mind and body, and the ̳dialogue‘ that was developed between the Discourse situated in a particular practice and the classroom Discourse of mathematics teaching. The analysis focuses on an interdisciplinary project based on an ethnomathematical study of a designing tradition carried out by the researchers themselves, involving a search for informal mathematics and the connections with context and culture; 10th grade students in a public school in Athens were introduced to the mathematics content via an original WebQuest based on this previous ethnomathematical study; Geometry content was further introduced and mediated using the Drama-in-Education (DiE) techniques. Students contributed in an unfolding dialogue between formal and informal knowledge, renegotiating both mathematical concepts and their perception of mathematics as a discipline.

Material properties, geometry and their effect on the fatigue life of two flip-chip models

This paper describes how modeling technology has been used in providing fatigue life time data of two flip-chip models. Full-scale three-dimensional modeling of flip-chips under cyclic thermal loading has been combined with solder joint stand-off height prediction to analyze the stress and strain conditions in the two models. The Coffin-Manson empirical relationship is employed to predict the fatigue life times of the solder interconnects. In order to help designers in selecting the underfill material and the printed circuit board, the Young's modulus and the coefficient of thermal expansion of the underfill, as well as the thickness of the printed circuit boards are treated as variable parameters. Fatigue life times are therefore calculated over a range of these material and geometry parameters. In this paper we will also describe how the use of micro-via technology may affect fatigue life

Moment decay rates of solutions of stochastic differential equations

The objective of this paper is to investigate the p-ίh moment asymptotic stability decay rates for certain finite-dimensional Itό stochastic differential equations. Motivated by some practical examples, the point of our analysis is a special consideration of general decay speeds, which contain as a special case the usual exponential or polynomial type one, to meet various situations. Sufficient conditions for stochastic differential equations (with variable delays or not) are obtained to ensure their asymptotic properties. Several examples are studied to illustrate our theory.