Astroglial expression of the P-glycoprotein is controlled by intracellular CNTF

Background - The P-glycoprotein (P-gp), an ATP binding cassette transmembrane transporter, is expressed by astrocytes in the adult brain, and is positively modulated during astrogliosis. In a search for factors involved in this modulation, P-gp overexpression was studied in long-term in vitro astroglial cultures. Results - Surprisingly, most factors that are known to induce astroglial activation in astroglial cultures failed to increase P-gp expression. The only effective proteins were IFNγ and those belonging to the IL-6 family of cytokines (IL-6, LIF, CT-1 and CNTF). As well as P-gp expression, the IL-6 type cytokines - but not IFNγ - stimulated the expression of endogenous CNTF in astrocytes. In order to see whether an increased intracellular level of CNTF was necessary for induction of P-gp overexpression by IL-6 type cytokines, by the same cytokines analysis was carried out on astrocytes obtained from CNTF knockout mice. In these conditions, IFNγ produced increased P-gp expression, but no overexpression of P-gp was observed with either IL-6, LIF or CT-1, pointing to a role of CNTF in the intracellular signalling pathway leading to P-gp overexpression. In agreement with this suggestion, application of exogenous CNTF -which is internalised with its receptor - produced an overexpression of P-gp in CNTF-deficient astrocytes. Conclusions - These results reveal two different pathways regulating P-gp expression and activity in reactive astrocytes, one of which depends upon the intracellular concentration of CNTF. This regulation of P-gp may be one of the long searched for physiological roles of CNTF.

Leadership and the negotiation of order in small groups

This thesis is focussed on the role differentiationhypothesis as it relates to small groups (Bales, 1958). The hypothesis is systematically examined, both conceptually and empirically, in the light of the Equilibrium Hypothesis (Bales, 1953) and the Negotiated Order Theory of leadership (e.g. Hosking, 1988). Chapter 1 sketches in a context for the research,which was stimulated by attempts during the 60s and 70s to organise small groups without leaders (the leaderless group, based on isocratic principles). Chapter 2 gives a conceptual and developmental overview of Bales' work, concentrating on the Equilibrium Hypothesis. It is argued that Bales' conceptual approach, if developed, can potentially integrate the disparate small groups and leadership literatures. Chapters 3 and 4 examine the concepts `group', `leader' and `leadership' in terms of the Negotiated Order perspective. In chapter 3 it is argued that two aspects of the concept group need to be taken separately into account; physical attributes and social psychological aspects (the metaphysical glue). It is further argued that a collection of people becomes a group only when they begin to establish a shared sense of social order. In chapter 4 it is argued that leadership is best viewed as a process of negotiation between those who influence and those who are influenced, in the context of shared values about means and ends. It is further argued that leadership is the process by which a shared sense of social order is established and maintained, thus linking the concepts `leadership' and `group' in a single formulation. The correspondences with Bales' approach are discussed at the end of the chapter. Chapters 5 to 8 present a detailed critical description and evaluation of the empirical work which claims to show role differentiation or test the hypothesis, both Bales original work and subsequent studies. It is argued here, that the measurement and analytical procedures adopted by Bales and others, in particular the use of simple means as summaries of group structures, are fundamentally flawed, and that role differentiation in relation to particular identifiable groups has not been demonstrated clearly anywhere in the literature. Chapters 9 to 13 present the empirical work conducted for the thesis. 18 small groups are examined systematically for evidence of role differentiation using an approach based on early sociometry (Moreno, 1934). The results suggest that role differentiation, as described by Bales, does not occur as often as is implied in the literature, and not equivocally in any case. In particular structures derived from Liking are typically distributed or weak. This suggests that one of Bales' principal findings, that Liking varies independently of his other main dimensions, is the product of statistical artifact. Chapter 14 presents a general summary of results and presents some considerations about future research.

The role of the extracellular loops of the CGRP receptor, a family B GPCR

The CGRP (calcitonin gene-related peptide) receptor is a family B GPCR (G-protein-coupled receptor). It consists of a GPCR, CLR (calcitonin receptor-like receptor) and an accessory protein, RAMP1 (receptor activity-modifying protein 1). RAMP1 is needed for CGRP binding and also cell-surface expression of CLR. There have been few systematic studies of the ECLs (extracellular loops) of family B GPCRs. However, they are likely to be especially important for the interaction of the N-termini of the peptide agonists that are the natural agonists for these receptors. We have carried out alanine scans on all three ECLs of CLR, as well as their associated juxtamembrane regions. Residues within all three loops influence CGRP binding and receptor activation. Mutation of Ala203 and Ala206 on ECL1 to leucine increased the affinity of CGRP. Residues at the top of TM (transmembrane) helices 2 and 3 influenced CGRP binding and receptor activation. L351A and E357A in TM6/ECL3 reduced receptor expression and may be needed for CLR association with RAMP1. ECL2 seems especially important for CLR function; of the 16 residues so far examined in this loop, eight residues reduce the potency of CGRP at stimulating cAMP production when mutated to alanine.