Domain swapping in the human histamine H-1 receptor

G-protein-coupled receptors (GPCRs) represent the largest family of receptors involved in transmembrane signaling. Although these receptors were generally believed to be monomeric entities, accumulating evidence supports the presence of GPCRs in multimeric forms. Here, using immunoprecipitation as well as time-resolved fluorescence resonance energy transfer to assess protein-protein interactions in living cells, we unambiguously demonstrate the occurrence of dimerization of the human histamine H-1 receptor. We also show the presence of domain-swapped H-1 receptor dimers in which there is the reciprocal exchange of transmembrane domain TM domains 6 and 7 between the receptors present in the dimer. Mutation of aspartate(107) in transmembrane (TM) 3 or phenylalanine(432) in TM6 to alanine results in two radioligand-binding-deficient mutant H-1 receptors. Coexpression of H-1 D(107)A and H-1 F(432)A, however, results in a reconstituted radioligand binding site that exhibits a pharmacological profile that corresponds to the wildtype H-1 receptor. Interestingly, the H-1 receptor radioligands [H-3] mepyramine and [H-3]-(-)- trans-1-phenyl-3-N, N-dimethylamino-1,2,3,4-tetrahydronaphthalene show differential saturation binding values (B-max) for wild-type H-1 receptors but not for the radioligand binding site that is formed upon coexpression of H-1 D(107)A and H-1 F(432)A receptors, suggesting the presence of different H-1 receptor populations.

Isolation of recombinant antibodies against EspA and intimin of Escherichia coli O157:H7

Intimin, Tir, and EspA proteins are expressed by attaching-effacing Escherichia coli, which include enteropathogenic and enterohemorrhagic E. coli pathotypes. EspA proteins are part of the type three secretion system needle complex that delivers Tir to the host epithelial cell, while surface arrayed intimin docks the bacterium to the translocated Tir. This intimate attachment leads to attaching and effacing lesions. Recombinant forms of these effector proteins from enterohemorrhagic E. coli O157:H7 were produced by using E. coli expression vectors. Binding of intimin and Tir fragments in enzyme-linked immunosorbent assay (ELISAs) demonstrated the interaction of intimin fragments containing the C-terminal 282 or 188 amino acids to a Tir fragment containing amino acid residues 258 to 361. Recombinant intimin and EspA proteins were used to elicit immune responses in rabbits and immune phage-display antibody libraries were produced. Screening of these immune libraries by conventional phage-antibody panning and colony filter screening produced a panel of antibodies with specificity for EspA or intimin. Antibodies recognizing different C-terminal epitopes on intimin bound specifically to the gamma intimin of O157:H7 and not to other classes of intimin. Antibodies recognizing EspA from E. coli O157 also recognized the protein from the eae-deficient O157 mutant DM3 and from E. coli O111. Anti-intimin antibodies were also produced as fusion proteins coupled to the reporter molecule alkaline phosphatase, allowing the one-step detection of gamma intimin. The isolated recombinant monoclonal antibodies were functional in a range of assay formats, including ELISA, Western blotting, and dot blots, thus demonstrating their diagnostic potential.

Recombinant anti-EspA antibodies block Escherichia coli O157:H7-induced attaching and effacing lesions in vitro

Intimin and EspA proteins are virulence factors expressed by attaching and effacing Escherichia coli (AEEC) such as enteropathogenic and enterohaemorrhagic E. coli. The EspA protein makes up a filament structure forming part of the type III secretion system (TTSS) that delivers effector proteins to the host epithelial cell. Bacterial surface displayed intimin interacts with translocated intimin receptor in the host cell membrane leading to intimate attachment of the bacterium and subsequent attaching and effacing lesions. Here, we have assessed the use of recombinant monoclonal antibodies against E. coli O157:147 EspA and intimin for the disruption of AEEC interaction with the host cell. Anti-gamma intimin antibodies did not reduce either adhesion of E. coli O157:H7 to host cell mono-layers or subsequent host cell actin rearrangement. Anti-EspA antibodies similarly had no effect on bacterial adhesion however they had a marked effect upon E. coli O157:H7-induced host cell actin rearrangement, where both monoclonal and polyclonal antibodies completely blocked cytoskeletal changes within the host cell. Furthermore, these anti-EspA antibodies were shown to reduce actin rearrangement induced by some but not all other AEEC serotypes tested. Both polyclonal and monoclonal antibodies could be used to label E. coli O157 EspA filaments and these immunoreagents did not inhibit the formation of such filaments. This is the first report of monoclonal antibodies to EspA capable of disrupting the TTSS function of E. coli O157:H7. (c) 2005 Elsevier SAS. All rights reserved.

Type specimens of G.E. Post in Beirut and The Natural History Museum, London

The Flora of Syria, Palestine and Sinai, a pioneer Flora of the region, was published in 1896 by George Edward Post (1838–1909). Lesser known are his series of Diagnoses plantarum novarum orientalium, published in the Journal of the Linnean Society Botany, and 10 papers, Plantae Postianae, which appeared in Swiss journals from 1890 to 1900. A greatly expanded second edition of the Flora was prepared by John Edward Dinsmore and published in Beirut in 1932 and 1933. Post's plant collection is part of the Post Herbarium (BEI), with about 63 000 specimens, that has been well maintained, despite civil war and inadequate staffing. This work involves the identification of around 150 types in BEI and BM, and improvement of the accessibility of the specimens. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 159, 315–321.

Capturing UK real estate volitility

Volatility, or the variability of the underlying asset, is one of the key fundamental components of property derivative pricing and in the application of real option models in development analysis. There has been relatively little work on volatility in real terms of its application to property derivatives and the real options analysis. Most research on volatility stems from investment performance (Nathakumaran & Newell (1995), Brown & Matysiak 2000, Booth & Matysiak 2001). Historic standard deviation is often used as a proxy for volatility and there has been a reliance on indices, which are subject to valuation smoothing effects. Transaction prices are considered to be more volatile than the traditional standard deviations of appraisal based indices. This could lead, arguably, to inefficiencies and mis-pricing, particularly if it is also accepted that changes evolve randomly over time and where future volatility and not an ex-post measure is the key (Sing 1998). If history does not repeat, or provides an unreliable measure, then estimating model based (implied) volatility is an alternative approach (Patel & Sing 2000). This paper is the first of two that employ alternative approaches to calculating and capturing volatility in UK real estate for the purposes of applying the measure to derivative pricing and real option models. It draws on a uniquely constructed IPD/Gerald Eve transactions database, containing over 21,000 properties over the period 1983-2005. In this first paper the magnitude of historic amplification associated with asset returns by sector and geographic spread is looked at. In the subsequent paper the focus will be upon model based (implied) volatility.