Functions of LIM proteins in cell polarity and chemotactic motility

LimC and LimD are two novel LIM proteins of Dictyostelium, which are comprised of double and single LIM domains, respectively. Green fluorescent protein-fused LimC and LimD proteins preferentially accumulate at areas of the cell cortex where they co-localize with actin and associate transiently with cytoskeleton-dependent dynamic structures like phagosomes, macropinosomes and pseudopods. Furthermore, both LimC and LimD interact directly with F-actin in vitro. Mutant cells that lack either LimC or LimD, or both, exhibit normal growth. They are, however, significantly impaired in growth under stress conditions and are highly sensitive to osmotic shock, suggesting that LimC and LimD contribute towards the maintenance of cortical strength. Moreover, we noted an altered morphology and F-actin distribution in LimD(–) and LimC(–)/D(–) mutants, and changes in chemotactic motility associated with an increased pseudopod formation. Our results reveal both unique and overlapping roles for LimC and LimD, and suggest that both act directly on the actin cytoskeleton and provide rigidity to the cortex

Multiplex Assay for Protein Profiling and Potency Measurement of German Cockroach Allergen Extracts

<div><p>Background</p><p>German cockroach (GCr) allergens induce IgE responses and may cause asthma. Commercial GCr allergen extracts are variable and existing assays may not be appropriate for determining extract composition and potency.</p><p>Objective</p><p>Our aim was to develop a multiplex antibody/bead-based assay for assessment of GCr allergen extracts.</p><p>Methods</p><p>Single chain fragment variable (scFv) antibodies against GCr were obtained by screening libraries derived from naïve human lymphocytes and hyperimmunized chicken splenocytes and bone marrow. Selected clones were sequenced and characterized by immunoblotting. Eighteen scFv antibodies (17 chicken, 1 human) coupled to polystyrene beads were used in this suspension assay; binding of targeted GCr allergens to antibody-coated beads was detected using rabbit antisera against GCr, and against specific allergens rBla g 1, rBla g 2, and rBla g 4. The assay was tested for specificity, accuracy, and precision. Extracts were also compared by IgE competition ELISA.</p><p>Results</p><p>Chicken scFv’s generated eight different binding patterns to GCr proteins from 14 to 150 kDa molecular weight. Human scFv’s recognized a 100 kDa GCr protein. The multiplex assay was found to be specific and reproducible with intra-assay coefficient of variation (CV) of 2.64% and inter-assay CV of 10.0%. Overall potencies of various GCr extracts were calculated using mean logEC50s for eight selected scFvs. Overall potency measures were also analyzed by assessing the contributions to potency of each target.</p><p>Conclusions</p><p>An scFv antibody-based multiplex assay has been developed capable of simultaneously measuring different proteins in a complex mixture, and to determine the potencies and compositions of allergen extracts.</p></div

Western blots of E6Cg, rBla g 1, rBla g 2, and rBla g 4.

<p>Purified soluble human (A) and chicken (B) scFvs were used as primary antibodies (1.0 μg/mL) and horseradish peroxidase-conjugated anti-hemagglutinin (1:1000) was used for detection using a chemiluminescent substrate. Antibody designations are listed on the top of each panel, and the separated protein sources are indicated at the bottom. Brackets indicate eight different banding patterns.</p

Comparison of laboratory-scale GCr extracts.

<p>All scFv-coupled beads for 8 antibodies (see text and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140225#pone.0140225.s006" target="_blank">S1 Table</a>) were mixed in PBS containing 1% BSA and dispensed in wells containing diluted extract (50 μL/well). Overall mean logEC₅₀ values are indicated on y-axis. Each point represents the signal from one scFv-coupled bead; horizontal bar is the mean value. **, p<0.05; ***, p<0.001.</p

Comparison of GCr extracts from three different manufacturers and E6Cg by competition ELISA using E2Cg as the reference extract.

<p>Antibody sources are rabbit anti-E6Cg sera, human serum pool S1Cr, and mix of 8 scFvs. Relative potencies are determined by comparing x intercepts of parallel inhibition curves compared to E2Cg.</p

Comparison of GCr extracts from three different manufacturers.

<p>(A) SDS-PAGE analysis of GCr extracts was performed under reducing conditions. (B) Overall mean log EC₅₀ values are indicated on y-axis. Each point represents the signal from one scFv-coupled bead; horizontal bar is the mean value. **, p<0.05.</p

Depletion of E6Cg and detection of specific scFv target proteins.

<p>(A) E6Cg depleted of Bla g 1 and mock-depleted analyzed using scFv αBg 1 (C) E6Cg depleted of Bla g 3 and mock-depleted analyzed using scFv 2A1, (E) E6Cg depleted of Bla g 7 and mock-depleted analyzed with scFv 6A2. E6Cg depleted and mock-depleted were also analyzed using non-related scFv antibodies during each run, shown in panels B (scFv 2A1 for detection of Bla g 3), D (scFv 6A2 for detection of Bla g 7), and F (scFv 6A3 for detection of vitellogenin).</p

MAEPA specificity.

<p>Serially diluted E6Cg (A), cat pelt (B), short ragweed pollen (C) and Alternaria alternata (D) extracts were added to a mix of all 18 scFv-coupled bead sets in 96-well filter bottom plates. Mixture of rabbit anti-E6Cg, anti Bla g 1, anti Bla g 2, and anti Bla g 4 sera (1:500) were used as primary antibody followed by biotinylated anti-rabbit (1:1000) and streptavidin-RPE (1:500). MFI detected for each scFv is on y-axis; extract dilution (log scale) is on x-axis.</p

Role of Anthrax Toxins in Dissemination, Disease Progression, and Induction of Protective Adaptive Immunity in the Mouse Aerosol Challenge Model▿ †

Anthrax toxins significantly contribute to anthrax disease pathogenesis, and mechanisms by which the toxins affect host cellular responses have been identified with purified toxins. However, the contribution of anthrax toxin proteins to dissemination, disease progression, and subsequent immunity after aerosol infection with spores has not been clearly elucidated. To better understand the role of anthrax toxins in pathogenesis in vivo and to investigate the contribution of antibody to toxin proteins in protection, we completed a series of in vivo experiments using a murine aerosol challenge model and a collection of in-frame deletion mutants lacking toxin components. Our data show that after aerosol exposure to Bacillus anthracis spores, anthrax lethal toxin was required for outgrowth of bacilli in the draining lymph nodes and subsequent progression of infection beyond the lymph nodes to establish disseminated disease. After pulmonary exposure to anthrax spores, toxin expression was required for the development of protective immunity to a subsequent lethal challenge. However, immunoglobulin (immunoglobulin G) titers to toxin proteins, prior to secondary challenge, did not correlate with the protection observed upon secondary challenge with wild-type spores. A correlation was observed between survival after secondary challenge and rapid anamnestic responses directed against toxin proteins. Taken together, these studies indicate that anthrax toxins are required for dissemination of bacteria beyond the draining lymphoid tissue, leading to full virulence in the mouse aerosol challenge model, and that primary and anamnestic immune responses to toxin proteins provide protection against subsequent lethal challenge. These results provide support for the utility of the mouse aerosol challenge model for the study of inhalational anthrax

Cross-reaction between Formosan termite (Coptotermes formosanus) proteins and cockroach allergens.

Cockroach allergens can lead to serious allergy and asthma symptoms. Termites are evolutionarily related to cockroaches, cohabitate in human dwellings, and represent an increasing pest problem in the United States. The Formosan subterranean termite (Coptotermes formosanus) is one of the most common species in the southern United States. Several assays were used to determine if C. formosanus termite proteins cross-react with cockroach allergens. Expressed sequence tag and genomic sequencing results were searched for homology to cockroach allergens using BLAST 2.2.21 software. Whole termite extracts were analyzed by mass-spectrometry, immunoassay with IgG and scFv antibodies to cockroach allergens, and human IgE from serum samples of cockroach allergic patients. Expressed sequence tag and genomic sequencing results indicate greater than 60% similarity between predicted termite proteins and German and American cockroach allergens, including Bla g 2/Per a 2, Bla g 3/Per a 3, Bla g 5, Bla g 6/Per a 6, Bla g 7/Per a 7, Bla g 8, Per a 9, and Per a 10. Peptides from whole termite extract were matched to those of the tropomyosin (Bla g 7), arginine kinase (Per a 9), and myosin (Bla g 8) cockroach allergens by mass-spectrometry. Immunoblot and ELISA testing revealed cross-reaction between several proteins with IgG and IgE antibodies to cockroach allergens. Several termite proteins, including the hemocyanin and tropomyosin orthologs of Blag 3 and Bla g 7, were shown to crossreact with cockroach allergens. This work presents support for the hypothesis that termite proteins may act as allergens and the findings could be applied to future allergen characterization, epitope analysis, and clinical studies