Revisiting the 'Missing Middle' in English Sub-National Governance

In the light of the new Coalition Government’s proposed ‘rescaling’ of sub-national governance away from the regional level, it is an opportune time to re-consider the strength and weaknesses of the city or sub-regional approach to economic development and to search, once more, for the ‘missing middle’ in English Governance. In this context, the article initially assesses the case for city or sub regions as tiers of economic governance, before examining the lessons to be learnt from the experiences of the existing city regions in the North East of England. It argues that while contemporary plans to develop Local Enterprise Partnerships (LEPs) can be usefully considered within the context of the emerging city regional developments under the previous Labour Governments, a number of important challenges remain, particularly in relation to ensuring accountable structures of governance, a range of appropriate functions, adequate funding, and comprehensive coverage across a variety of sub-regional contexts. While the proposals of the new Government create the necessary ‘space’ to develop sub-regional bodies and offer genuine opportunities for both city and county LEPs, the scale of the sub-regional challenge should not be underestimated, particularly given the context of economic recession and major reductions in the public sector

Ligand-Receptor Interactions

The formation and dissociation of specific noncovalent interactions between a variety of macromolecules play a crucial role in the function of biological systems. During the last few years, three main lines of research led to a dramatic improvement of our understanding of these important phenomena. First, combination of genetic engineering and X ray cristallography made available a simultaneous knowledg of the precise structure and affinity of series or related ligand-receptor systems differing by a few well-defined atoms. Second, improvement of computer power and simulation techniques allowed extended exploration of the interaction of realistic macromolecules. Third, simultaneous development of a variety of techniques based on atomic force microscopy, hydrodynamic flow, biomembrane probes, optical tweezers, magnetic fields or flexible transducers yielded direct experimental information of the behavior of single ligand receptor bonds. At the same time, investigation of well defined cellular models raised the interest of biologists to the kinetic and mechanical properties of cell membrane receptors. The aim of this review is to give a description of these advances that benefitted from a largely multidisciplinar approach

Force Measurements of TCR/pMHC Recognition at T Cell Surface

The rupture forces and adhesion frequencies of single recognition complexes between an affinity selected peptide/MHC complex and a TCR at a murine hybridoma surface were measured using Atomic Force Microscopy. When the CD8 coreceptor is absent, the adhesion frequency depends on the nature of the peptide but the rupture force does not. When CD8 is present, no effect of the nature of the peptide is observed. CD8 is proposed to act as a time and distance lock, enabling the shorter TCR molecule to bridge the pMHC and have time to finely read the peptide. Ultimately, such experiments could help the dissection of the sequential steps by which the TCR reads the peptide/MHC complex in order to control T cell activation

Kinetics and locus of failure of receptor-ligand-mediated adhesion between latex spheres. I. Protein-carbohydrate bond.

We previously described the use of a counter-rotating cone and plate rheoscope to measure the time and force dependence of break-up of doublets of sphered, swollen, and fixed red cells (SSRC) cross-linked by monoclonal IgM antibody. It has been shown that doublet break-up can occur by extraction of receptors from the membrane, rather than by antibody-antigen bond break-up, and is a stochastic process. We therefore prepared 4.62-microns carboxyl modified latex spheres with a covalently coupled synthetic blood group B antigen trisaccharide. Using a two-step carbodiimide process, ethylene diamine was covalently linked to the carboxyl modified latex spheres, and the trisaccharide, having an eight carbon spacer modified to bear a terminal carboxyl group, was linked to the ethylene diamine. Using these antigen spheres we carried out studies in Couette flow, in a transparent cone and plate rheoscope, of the shear-induced break-up of doublets cross-linked by monoclonal IgM anti-B antibody in 19% and 15% Dextran 40. As previously found with SSRC, over a range of normal force from 55 to 175 pN, there was a distribution in times to break-up. However, the fraction of antigen sphere doublets broken up, which increased from 0.08 to 0.43 at 75 pM IgM, and from 0.06 to 0.20 at 150 pM IgM, was significantly lower than that for the SSRC, where the fraction broken up at 150 pM IgM increased from 0.10 to 0.47. Thus, significantly higher forces were required to achieve the same degree of break-up for doublets of antigen-linked spheres than for SSRC. Computer simulation using a stochastic model of break-up showed that the differences between antigen sphere and SSRC doublet break-up were due to a change in bond character (the range and depth of the bond energy minimum) rather than to an increase in the number of bonds linking antigen-sphere doublets. This supports the notion that antibody-antigen bonds are ruptured in the case of antigen spheres, whereas antigen is able to be extracted from the membrane of SSRC, although changes of receptor substrate from cell to latex and the possibility of latex strand extraction from the microspheres are potential complicating factors

Interaction forces between red cells agglutinated by antibody. IV. Time and force dependence of break-up.

We report on an extension of a previously described method to measure the hydrodynamic force to separate doublets of fixed, sphered and swollen red cells cross-linked by antibody (S. P. Tha, J. Shuster, and H. L. Goldsmith. 1986. Biophys. J. 50:1117-1126). With a traveling microtube apparatus, doublets are tracked and videotaped in a slowly accelerating Poiseuille flow in 150-microns-diameter tubes, and the hydrodynamic normal force at break-up, Fn, is computed from the measured doublet velocity and radial position. Previous results showed a large range of Fn, the mean of which increased with [antiserum], and an absence of clustering at discrete values of Fn. Since it was assumed that the cells separate the instant a critical force to break all crossbridges was reached, lack of clustering could have been due to the use of a polyclonal antiserum. We therefore studied the effect of monoclonal IgM or IgA antibody on the distribution of Fn. The results showed that the data are as scattered as ever, with Fn varying from 2 to 200 pN, and exhibit no evidence of clustering. However, the scatter in Fn could be due to the stochastic nature of intercellular bonds (E. Evans, D. Berk, and A. Leung. 1991a. Biophys. J. 59:838-848). We therefore studied the force dependence of the time to break-up under constant shear stress (Fn from 30 to 200 pN), both in Poiseuille and Couette flow, the latter by using a counter-rotating cone and plate rheoscope. When 280 doublets were rapidly accelerated in the traveling microtube and then allowed to coast in steady flow for up to 180 s, 91% survived into the constant force region; 16% of these broke up after time intervals, tP, of 2-30s. Of 340 doublets immediately exposed to constant shear in the rheoscope, 37% broke after time intervals, tc, from < 1 to 10 s. Thus, doublets do indeed break up under a constant shear stress, if given time. The average time to break-up decreased significantly with increasing force, while the fraction of doublets broken up increased. At a given Fn, the fraction of break-ups decreased with increasing [IgM], suggesting that the average number of bonds had also increased. Using a stochastic model of break-up (G. I. Bell. 1978. Science (Washington DC). 200:618-627; E. Evans, D. Berk,and A. Leung. 1991. Biophys. J. 59:838-848) and a Poisson distribution for the number of bonds, Nb, break-up in slowly accelerating Poiseuille flow and in immediate shear application in Couette flow was simulated. In Poiseuille flow, the observed range and scatter in Fn could be reproduced assuming (Nb) > 5. In the rheoscope, the time intervals and number of rotations to break-up, tc, were quite well reproduced assuming (Nb) = 4. The similarity of (Fn) for monoclonal IgM and IgA for doublet break-up under constant slow acceleration is compatible with the conclusion of Evans et al. (1991 a) for normal red cells and Xia et al. (manuscript submitted for publication) for sphered and swollen red cells, that the applied force extracts the antigen from the cell membrane

Probabilistic modeling of shear-induced formation and breakage of doublets cross-linked by receptor-ligand bonds.

A model was constructed to describe previously published experiments of shear-induced formation and breakage of doublets of red cells and of latexes cross-linked by receptor-ligand bonds (. Biophys. J. 65:1318-1334; Tees and Goldsmith. 1996. Biophys. J. 71:1102-1114;. Biophys. J. 71:1115-1122). The model, based on McQuarrie's master equations (1963. J. Phys. Chem. 38:433-436), provides unifying treatments for three distinctive time periods in the experiments of particles in a Couette flow in which a doublet undergoes 1) formation upon two-body collision between singlets; 2) evolution of bonds at low shear rate; and 3) break-up at high shear rate. Neglecting the applied force at low shear rate, the probability of forming a doublet per collision as well as the evolution of probability distribution of bonds in a preformed doublet were solved analytically and found to be in quite good agreement with measurements. At high shear rate with significant force acting to accelerate bond dissociation, the predictions for break-up of doublets were obtained numerically and compared well with data in both individual and population studies. These comparisons enabled bond kinetic parameters for three types of particles cross-linked by two receptor-ligand systems to be calculated, which agreed well with those computed from Monte Carlo simulations. This work can be extended to analyze kinetics of receptor-ligand binding in cell aggregates, such as those of neutrophils and platelets in the circulation