Structural features of human immunoglobulin G that determine isotype-specific differences in complement activation.

Although very similar in sequence, the four subclasses of human immunoglobulin G (IgG) differ markedly in their ability to activate complement. Glu318-Lys320-Lys322 has been identified as a key binding motif for the first component of complement, C1q, and is present in all isotypes of Ig capable of activating complement. This motif, however, is present in all subclasses of human IgG, including those that show little (IgG2) or even no (IgG4) complement activity. Using point mutants of chimeric antibodies, we have identified specific residues responsible for the differing ability of the IgG subclasses to fix complement. In particular, we show that Ser at position 331 in gamma 4 is critical for determining the inability of that isotype to bind C1q and activate complement. Additionally, we provide further evidence that levels of C1q binding do not necessarily correlate with levels of complement activity, and that C1q binding alone is not sufficient for complement activation

Short-scale break-up in unsteady interactive layers: Local development of normal pressure gradients and vortex wind-up

Following the finite-time collapse of an unsteady interacting boundary layer (step 1), shortened length and time scales are examined here in the near-wall dynamics of transitional-turbulent boundary layers or during dynamic stall. The next two steps are described, in which (step 2) normal pressure gradients come into operation along with a continuing nonlinear critical-layer jump and then (step 3) vortex formation is induced typically. Normal pressure gradients enter in at least two ways, depending on the internal or external flow configuration. This yields for certain internal flows an extended KdV equation with an extra nonlinear integral contribution multiplied by a coefficient which is proportional to the normal rate of change of curvature of the velocity profile locally and whose sign turns out to be crucial. Positive values of the coefficient lead to a further finite-time singularity, while negative values produce a rapid secondary instability phenomenon. Zero values in contrast allow an interplay between solitary waves and wave packets to emerge at large scaled times, this interplay eventually returning the flow to its original, longer, interactive, boundary-layer scales but now coupled with multiple shorter-scale Euler regions. In external or quasi-external flows more generally an extended Benjamin–Ono equation holds instead, leading to a reversal in the roles of positive and negative values of the coefficient. The next step, 3, typically involves the strong wind-up of a local vortex, leading on to explosion or implosion of the vortex. Further discussion is also presented, including the three-dimensional setting, the computational implications, and experimental links

A novel approach to the site-selective dual labelling of a protein via chemoselective cysteine modification

Local protein microenvironment is used to control the outcome of reaction between cysteine residues and 2,5-dibromohexanediamide. The differential reactivity is exploited to introduce two orthogonal reactive handles onto the surface of a double cysteine mutant of superfolder green fluorescent protein in a regioselective manner. Subsequent elaboration with commonly used thiol and alkyne containing reagents affects site-selective protein dual labelling

Overexpression of CD44 in acquired tamoxifen-resistant breast cancer cells augments their migratory response to heregulin beta 1

Background Acquired endocrine resistance in breast cancer cells is accompanied by altered growth factor receptor signalling [1] and a highly migratory cell phenotype [2]. Interestingly, in tamoxifen-resistant (TamR) MCF7 cells, our microarray analysis has demonstrated elevated levels of CD44, a transmembrane glycoprotein known to interact with, and modulate the function of, growth factor receptors [3]. Here we have explored the role of CD44 as a modulator of heregulin beta-1-induced migratory signalling in TamR cells. Methods Expression of CD44 (standard and v3 isoforms) were confirmed by RT-PCR and western blotting and their association with erbB family members determined by both immunofluorescence microscopy and immunoprecipitation. Activation of intracellular signalling following heregulin beta 1 treatment (10 ng/ml) in the presence or absence of CD44 (using siRNA-mediated inhibition) was determined by western blotting using phosphospecific antibodies. Cellular migration was determined by seeding cells (control and CD44 siRNA-treated) into fibronectin-coated transwell chambers (8.0 μm pore size) in the presence or absence of heregulin beta 1. After 24 hours, migratory cells were fixed, stained with crystal violet and counted. Results Both standard and v3 isoforms of CD44 were overexpressed in TamR cells at both gene and protein levels (mean fold increase in CD44s protein (TamR versus MCF7): 4.26 ± 1.2, P < 0.05). Moreover, CD44s and v3 colocalised with Her2 and Her3 receptors at the cell surface and were also detectable in Her2/Her3 cellular immunoprecipitates. Treatment of TamR cells with heregulin resulted in phosphorylation of erbB receptors together with a number of downstream signalling intermediates, including Akt, Src and FAK, and resulted in enhanced cellular migration. Significantly, heregulin-induced intracellular signalling was dramatically reduced in cells in which the expression of CD44 was suppressed (via siRNA), with a corresponding loss of heregulin-induced migratory behaviour (mean fold change in cell migration versus untreated control: 6.7 ± 1.1, P < 0.05 (heregulin beta 1); 1.8 ± 0.9 (CD44 siRNA); 1.47 ± 0.6, P < 0.05 (heregulin beta 1 + CD44 siRNA)). Conclusion These data demonstrate a role for CD44 as a modulator of erbB receptor function in endocrine-resistant breast cancer cells, where it augments heregulin beta 1 migratory signalling

Flow and geometry induced scattering of high frequency acoustic duct modes

Cut-on cut-off transition of acoustic modes in hard-walled ducts with irrotational mean flow is well understood for Helmholtz numbers of order unity. Previous finite-element simulations of this phenomenon, however, appear to indicate the possibility of energy scattering into neighbouring modes at moderately large Helmholtz numbers. In this paper, such scattering phenomena are explained and predicted in slowly varying aeroengine ducts using a multiple-scales approach. It is found that, for sufficiently high frequencies, two mechanisms exist whereby energy can be scattered into neighbouring modes by an incident propagating mode. One mechanism occurs only when there is a mean flow inside the duct and induces scattering at significantly lower frequencies than the other mechanism which remains present without mean flow. A coupled system of ordinary differential equations is derived and then solved numerically for a number of example cases to obtain the corresponding transmitted and reflected amplitudes of the scattered modes as well as the overall acoustic pressure field. The theory appears to demonstrate that some exchange of energy between the acoustic and mean flow fields occurs during scattering

Investigating the central executive in adult dyslexics: Evidence from phonological and visuospatial working memory performance

There is long-standing evidence for verbal working memory impairments in both children and adults with dyslexia. By contrast, spatial memory appears largely to be unimpaired. In an attempt to distinguish between phonological and central executive accounts of the impairments in working memory, a set of phonological and spatial working memory tasks was designed to investigate the key issues in working memory, task type, task demands (static, dynamic, and updating), and task complexity. Significant differences emerged between the dyslexic and nondyslexic participants on the verbal working memory tasks employed in Experiment 1, thereby providing further evidence for continuing dyslexic impairments of working memory into adulthood. The nature of the deficits suggested a problem with the phonological loop, with there being little evidence to implicate an impairment of the central executive. Due to the difficulties associated with separating verbal working memory and phonological processing, however, performance was investigated in Experiment 2 using visuospatial measures of working memory. The results of the visuospatial tasks indicated no between-group differences in static spatial memory, which requires the short-term storage of simultaneously presented information. In almost all conditions there were no between-group differences in dynamic spatial memory that demands the recall of both locationand order of stimuli presented sequentially. However, a significant impairment occurred on the dynamic task under high memory updating load, on which dyslexic adults showed nonphonological working memory deficits. In the absence of an explanation involving verbal recoding, this finding is interpreted in terms of a central executive or automaticity impairment in dyslexia