ABSTRACT
We have previously shown that the heterodimeric cytokine interleukin-12, and the homodimer of its larger subunit p40, both bind to heparin and heparan sulfate with relatively high affinity. In the present study we characterised these interactions using a series of chemically modified heparins as competitive inhibitors. Human interleukin-12 and p40 homodimer show indistinguishable binding profiles with a panel of heparin derivatives, but that of murine interleukin-12 is distinct. Heparin markedly protects the human and murine p40 subunits, but not the p35 subunits, from cleavage by the bacterial endoprotease LysC, further implicating the larger subunit as the location of the heparin binding site. Moreover the essential role of the carboxyterminal D3 domain in heparin binding is established by the failure of a truncated construct of the p40 subunit lacking this domain to bind. Predictive docking calculations indicate that a cluster of basic residues at the tip of the exposed C'D' loop within D3 is important in heparin binding. However since the human and murine C'D' loops differ considerably in length, the mode and three dimensional orientation of heparin binding are likely to differ substantially between the human and murine p40s. Thus overall the binding of IL-12 via its p40 subunit to heparin-related polysaccharides of the extracellular matrix appears to be functionally important since it has been conserved across mammalian species despite this structural divergence.
Subject(s)
Heparin/metabolism , Interleukin-12 Subunit p40/metabolism , Animals , Binding Sites/physiology , Dimerization , Extracellular Matrix/metabolism , Humans , Interleukin-12 Subunit p35/metabolism , Mice , Protein Binding/physiology , Signal Transduction/physiologyABSTRACT
Rotaviruses are prototypical double-stranded RNA viruses whose triple-layered icosahedral capsid constitutes transcriptional machinery activated by the release of the external layer. To understand the molecular basis of this activation, we studied the structural interplay between the three capsid layers by electron cryo-microscopy and digital image processing. Two viral particles and four virus-like particles containing various combinations of inner (VP2)-, middle (VP6)-, and outer (VP7)-layer proteins were studied. We observed that the absence of the VP2 layer increases the particle diameter and changes the type of quasi-equivalent icosahedral symmetry, as described by the shift in triangulation number (T) of the VP6 layer (from T = 13 to T = 19 or more). By fitting X-ray models of VP6 into each reconstruction, we determined the quasi-atomic structures of the middle layers. These models showed that the VP6 lattices, i.e., curvature and trimer contacts, are characteristic of the particle composition. The different functional states of VP6 thus appear as being characterized by trimers having similar conformations but establishing different intertrimeric contacts. Remarkably, the external protein VP7 reorients the VP6 trimers located around the fivefold axes of the icosahedral capsid, thereby shrinking the channel through which mRNA exits the transcribing rotavirus particle. We conclude that the constraints arising from the different geometries imposed by the external and internal layers of the rotavirus capsid constitute a potential switch regulating the transcription activity of the viral particles.
Subject(s)
Base Pair Mismatch , Rotavirus/physiology , Transcription, Genetic , Virion/physiology , Animals , Antigens, Viral/genetics , Capsid Proteins/genetics , Cell Line , Cryoelectron Microscopy , Polymorphism, Genetic , Rotavirus/genetics , Spodoptera , Virion/geneticsABSTRACT
We show, using a murine NK cell line which responds quantitatively to rmIL-12, that treatment with ChABCase, but not other GAGases, results in substantial reductions in the secretion of gamma-IFN. Likewise, treatment of the cells with a beta-D-xyloside inhibitor of proteoglycan biosynthesis inhibits this cytokine response. In both treatments, the addition of soluble, exogenous GAGs does not relieve the inhibition of gamma-IFN secretion. We also demonstrate by ELISA that rmIL-12 binds to CS B. Overall, our studies on this in vitro cellular model of the initiation of Th1 immune responses indicate a major role for cell-surface, iduronate-rich, CS proteoglycan in the biological activity of IL-12.
Subject(s)
Dermatan Sulfate/physiology , Interferon-gamma/metabolism , Interleukin-12/pharmacology , Killer Cells, Natural/drug effects , Animals , Cell Differentiation/drug effects , Enzyme-Linked Immunosorbent Assay , In Vitro Techniques , Killer Cells, Natural/metabolism , MiceABSTRACT
Os objetos esportivos destinados à criança são parte da cultura material infantil. Colocam em jogo o corpo da criança, com suas reais características físicas, psicológicas, intelectuais e sociais, e as representações de criança pensadas no momento de concepção e produção dos objetos que convêm a cada idade. O objetivo deste artigo foi proceder, através de uma pesquisa qualitativa, à análise dos objetos esportivos, considerando a sua materialidade e influência sobre os corpos das crianças, bem como desvendar as formas de significar a infância e o esporte.
Sports objects intended for children are part of childrens material culture. They put into play the childs body with its actual physical, psychological, intellectual and social characteristics, and representations on the child at the time of the design and production of objects most suitable to each age. The aim of this article was to conduct, through qualitative research, an assessment of sports objects, considering their materiality and influence on childrens bodies, and to unveil ways to signify childhood and sports.
Los objetos deportivos destinados a los niños forman parte de la cultura material infantil. Ponen en juego el cuerpo del niño, con sus reales características físicas, psicológicas, intelectuales y sociales, y las representaciones del niño pensadas en el momento de la concepción y producción de los objetos adecuados para cada edad. Este artículo presenta, a través de una investigación cualitativa, el análisis de los objetos deportivos, considerando su materialidad e influencia sobre los cuerpos de los niños, y busca desvendar las formas de significar la infancia y el deporte.