Human immunoglobulin A (IgA)-specific ligands from combinatorial engineering of protein A.

Affinity reagents capable of selective recognition of the different human immunoglobulin isotypes are important detection and purification tools in biotechnology. Here we describe the development and characterization of affinity proteins (affibodies) showing selective binding to human IgA. From protein libraries constructed by combinatorial mutagenesis of a 58-amino-acid, three-helix bundle domain derived from the IgG-binding staphylococcal protein A, variants showing IgA binding were selected by using phage display technology and IgA monoclonal antibodies (myeloma) as target molecules. Characterization of selected clones by biosensor technology showed that five out of eight investigated affibody variants were capable of IgA binding, with dissociation constants (K(d)) in the range between 0.5 and 3 microm. One variant (Z(IgA1)) showing the strongest binding affinity was further analyzed, and showed that human IgA subclasses (IgA(1) and IgA(2)) as well as secretory IgA were recognized with similar efficiencies. No detectable cross-reactivity towards human IgG, IgM, IgD or IgE was observed. The potential use of the Z(IgA1) affibody as a ligand in affinity chromatography applications was first demonstrated by selective recovery of IgA protein from a spiked Escherichia coli total cell lysate, using an affinity column containing a divalent head-to-tail Z(IgA1) affibody dimer construct as a ligand. In addition, efficient affinity recovery of IgA from unconditioned human plasma was also demonstrated.     

An interaction map of endoplasmic reticulum chaperones and foldases

Chaperones and foldases in the endoplasmic reticulum (ER) ensure correct protein folding. Extensive protein-protein interaction maps have defined the organization and function of many cellular complexes, but ER complexes are under-represented. Consequently, chaperone and foldase networks in the ER are largely uncharacterized. Using complementary ER-specific methods, we have mapped interactions between ER-lumenal chaperones and foldases and describe their organization in multiprotein complexes. We identify new functional chaperone modules, including interactions between protein-disulfide isomerases and peptidyl-prolyl cis-trans-isomerases. We have examined in detail a novel ERp72-cyclophilin B complex that enhances the rate of folding of immunoglobulin G. Deletion analysis and NMR reveal a conserved surface of cyclophilin B that interacts with polyacidic stretches of ERp72 and GRp94. Mutagenesis within this highly charged surface region abrogates interactions with its chaperone partners and reveals a new mechanism of ER protein-protein interaction. This ability of cyclophilin B to interact with different partners using the same molecular surface suggests that ER-chaperone/foldase partnerships may switch depending on the needs of different substrates, illustrating the flexibility of multichaperone complexes of the ER folding machinery.     

Computer simulation of surface-induced aggregation of ferritin.

Models are presented describing the transient mass-transport limited adsorption and cluster growth of ferritin at a solid surface. Computer simulations are carried out on a hexagonal lattice using a computer model that can be characterized as a two-dimensional stochastic cellular automaton allowing different rules regarding association, lateral interaction and dissociation to be incorporated in the model. The fractal dimensions of individual clusters were extracted from simulated aggregates and for similar rules found to be consistent with literature values on reversible diffusion-limited aggregation in two dimensions. The distribution of clusters versus free surface were shown to be affected by neighbor-dependent association probability. Low fractal dimension clusters were generated by a combination of strong lateral cohesion and neighbor-dependent dissociation to the bulk. By comparing computer simulated aggregation to experimental electron micrographs of adsorbed ferritin layers it is suggested that neighbor-dependent association, neighbor-dependent dissociation and lateral interactions are important factors in the complex dynamics of adsorbed protein layers.     

Reduced natriuretic response to acute sodium loading in COMT Gene deleted mice

Background  

The intrarenal natriuretic hormone dopamine (DA) is metabolised by catechol-O-methyltransferase (COMT) and monoamine oxidase (MAO). Inhibition of COMT, as opposed to MAO, results in a potent natriuretic response in the rat. The present study in anaesthetized homozygous and heterozygous COMT gene deleted mice attempted to further elucidate the importance of COMT in renal DA and sodium handling. After acute intravenous isotonic sodium loading, renal function was followed.     

The MIPS mammalian protein-protein interaction database

SUMMARY: The MIPS mammalian protein-protein interaction database (MPPI) is a new resource of high-quality experimental protein interaction data in mammals. The content is based on published experimental evidence that has been processed by human expert curators. We provide the full dataset for download and a flexible and powerful web interface for users with various requirements.     

Genetic diversity of JC virus in the Saami and the Finns: implications for their population history

The JC virus (JCV) genotyping method was used to gain insights into the population history of the Saami and the Finns, both speaking Finno-Ugric languages and living in close geographic proximity. Urine samples from Saami and Finns, collected in northern and southern Finland, respectively, were used to amplify a 610-bp JCV-DNA region containing abundant type-specific mutations. Based on restriction site polymorphisms in the amplified fragments, we classified JCV isolates into one of the three superclusters of JCV, type A, B, or C. All 15 Saami isolates analyzed and 41 of 43 Finnish isolates analyzed were classified as type A, the European type, and two samples from Finns were classified as type B, the African/Asian type. We then amplified and sequenced a 583-bp JCV-DNA region from the type A isolates of Saami and Finns. According to type-determining nucleotides within the region, we classified type A isolates into EU-a1, -a2, or -b. Most type A isolates from Saami were classified as EU-a1, while type A isolates from Finns were distributed among EU-a1, EU-a2, and EU-b. This trend in the JCV-genotype distribution was statistically significant. On a phylogenetic tree based on complete sequences, most of the type A isolates from Saami were clustered in a single clade within EU-a1, while those from Finns were distributed throughout EU-a1, EU-a2, and EU-b. These findings are discussed in the context of the population history of the Saami and the Finns. This study provides new complete JCV DNA sequences derived from populations of anthropological interest.     

A molecular phylogeny of the north‐east Atlantic species of the genus Idotea (Isopoda) with focus on the Baltic Sea

Today, the marine isopods of the genus Idotea Fabricius, 1798 consist of 26 accepted species. Most species can be found in the Northern Hemisphere. While some species have a cosmopolitan distribution, others are endemic to a few coastlines. In the Baltic Sea, Idotea species have a key role as important meso‐grazers. Only three species can be found in this brackish environment, I. balthica, I. chelipes and I. granulosa, while nine species are described from the whole European coast. The goal of the present study was to reconstruct the phylogeny of the genus Idotea and to test whether the Baltic low‐salinity tolerant species represent a single lineage within the genus. The phylogeny of north‐east Atlantic Idotea species was investigated based on a fragment of the nuclear gene 28S and the mitochondrial gene COI for eight Idotea species. The phylogenetic reconstructions showed that the genus Idotea might not be monophyletic. Neither I. linearis nor I. urotoma did belong to the separated and well‐supported Idotea clade of I. balthica, I. chelipes, I. emarginata, I. granulosa, I. metallica and I. pelagica. The three Idotea species found in the Baltic belonged to different lineages, with estimated COI‐based divergence time older than 7 Myr. This suggests that the tolerance to low salinity has likely evolved in the genus Idotea more than once.     

Fluorescence-microscopy-based image analysis for analyte-dependent particle doublet detection in a single-step immunoagglutination assay

A novel fluorescence-microscopy-based image analysis method for classification of singlet and doublet latex particles is demonstrated and applied to a particle-based immunoagglutination assay for quantification of biomolecules in microliter-volume bulk samples. The image analysis method, verified by flow cytometric agglutination analysis, is based on a pattern recognition algorithm employing Gaussian-base-function fitting which allows robust identification and counting of singlets, doublets, and higher agglomerates of fluorescent microparticles. The immunoagglutination assay is experimentally modeled by a biotin-streptavidin interaction, with the goal of both theoretically and experimentally investigating the performance of a general immunoagglutination-based assay. For this purpose a theoretical model of the initial agglutination kinetics, based on particle diffusion combined with a steric factor determined by the level of specific and nonspecific agglutination, was developed. The theoretical model combined with the experimental data can be used to optimize an agglutination-based assay with regard to sensitivity and dynamic range and to estimate the affinity, receptor surface density, molecular and binding site sizes, and level of nonspecific binding that is present in the assay. The experimental results are in good agreement with the theoretical model, indicating the usefulness of the model for immunoagglutination assay optimization.