Mutational Analysis of the Active Site and Antibody Epitopes of the Complement-inhibitory Glycoprotein, CD59

The Ly-6 superfamily of cell surface molecules includes CD59, a potent regulator of the complement system that protects host cells from the cytolytic action of the membrane attack complex (MAC). Although its mechanism of action is not well understood, CD59 is thought to prevent assembly of the MAC by binding to the C8 and/or C9 proteins of the nascent complex. Here a systematic, structure-based mutational approach has been used to determine the region(s) of CD59 required for its protective activity. Analysis of 16 CD59 mutants with single, highly nonconservative substitutions suggests that CD59 has a single active site that includes Trp-40, Arg-53, and Glu-56 of the glycosylated, membrane-distal face of the disk-like extracellular domain and, possibly, Asp-24 positioned at the edge of the domain. The putative active site includes residues conserved across species, consistent with the lack of strict homologous restriction previously observed in studies of CD59 function. Competition and mutational analyses of the epitopes of eight CD59-blocking and non-blocking monoclonal antibodies confirmed the location of the active site. Additional experiments showed that the expression and function of CD59 are both glycosylation independent

Mapping the Active Site of CD59

CD59 is a widely distributed membrane-bound inhibitor of the cytolytic membrane attack complex (MAC) of complement. This small (77 amino acid) glycoprotein is a member of the Ly6 superfamily of proteins and is important in protecting host cells from the lytic and proinflammatory activity of the MAC. CD59 functions by binding to C8 and/or C9 in the nascent MAC and interfering with C9 membrane insertion and polymerization. We present data obtained from a combination of molecular modeling and mutagenesis techniques, which together indicate that the active site of CD59 is located in the vicinity of a hydrophobic groove on the face of the molecule opposite to a “hydrophobic strip” suggested earlier. In addition, removal of the single N-linked glycosylation site at Asn18 of CD59 resulted in an enhancement of complement inhibitory activity

Interplay between REST and nucleolin transcription factors: a key mechanism in the overexpression of genes upon increased phosphorylation

Non-malignant cells can be transformed via the activation of kinases that control degradation of neural-restrictive silencer factor (REST). Here, we identify a mechanism that contributes to the activation of genes, expression of which is controlled by responsive elements containing overlapping binding sites for REST and nucleolin. We demonstrate that both phosphorylated and non-phosphorylated nucleolin-bound DNA; however, only phosphorylated nucleolin successfully competed with either full-length REST or a REST-derived DNA-binding peptide, REST68, for binding to the overlapping binding sites. We show that this interplay between the two transcription factors regulates the activation of cell survival and immunomodulatory genes in tumors and non-malignant cells with activated protein kinase C, which is accompanied with alterations in cell proliferation and apoptosis. We propose a model for the regulation of these genes, which brings a new insight into the molecular mechanisms that control cellular transformation driven by activation of protein kinases

Androgen receptor-binding regions of androgen-responsive genes

SIGLEAvailable from British Library Document Supply Centre-DSC:DX192017 / BLDSC - British Library Document Supply CentreGBUnited Kingdo

Attention: mr J W Pienaar

Re: St Peter's Church site, South End. Application for the upliftment of the moratorium in relation to the St Peter's Church sit

‘Placing’ Caring Relationships in Education: Addressing Abstraction and Domination

The purpose of this paper is twofold: to underscore the possible dangers of abstraction, objectification and reification as roots of human domination over other human and non-human beings; and to suggest that, as one possibility, place-based education, can counter these dominating patterns by pulling up their roots through fostering relational ontologies based on care and emotional/sensuous experience. The author will foreground the work of Henri Lefebvre, Neil Evernden, and R. D. Laing in the abstraction discussion, while the discussion of place-based education will draw largely on the writings of David Gruenewald

Fermenting the free folk school: tending a culture of place-based ecological learning and living

This research examines an example of a place-based learning initiative focused on sharing folk/self-sufficiency skills (the Free Folk School) at the neighbourhood level in helping participants put their socio-ecological values and concerns into action in order to live better in place. The work is inspired by place-based education and folk schooling literature. It mixes the approaches of Grounded Theory and Participatory Action Research, and what the author calls, “place-based marginal praxis.” Discussion of results focuses on ingredients that help in tending a culture where it is more appealing for people to act more in line with their socio-ecological values. The ingredients include: unlearning alienation, reskilling in both “hard” and “soft” skills, reclaiming “self-sufficiency” to mean “community sufficiency” or “self-enoughness,” sharing as a useful tool for adapting to change, searching for living/integrated knowledge, and reconnecting with ancient traditions or intergenerational interactions