The level of α7, β2 or β4 nAChR subunits in the brain sections of experimental mice studied by immunohistochemistry.

<p>Confocal microscopy images of the hippocampus CA1 and striatum of non-treated (Ctrl), α7(1–208)-immunized or LPS-injected mice stained with biotinylated α7-, β2- or β4-specific antibodies and developed with Extravidin-Cy3 (<i>red</i>). Cell nuclei are stained with DAPI (<i>blue</i>). Bar corresponds to 50μm, actual for each fragment of the panel.</p

The level of nAChR-specific antibodies in the blood and of nAChR subtypes in the brain of experimental mice studied by ELISA (A) or Sandwich ELISA (B).

<p><b>A</b>—7(1–208)-specific antibodies in the blood sera (1:50) of mice immunized with 7(1–208) (n = 8) or injected with LPS (n = 5) for 5 months compared to non-treated (Ctrl, n = 9) and adjuvant-“immunized” animals (CFA, n = 5). <b>B</b>—3, 4, 7, β2 and β4 nAChR subunits in the brain detergent lysates of the same groups of mice (4 mice from each group). <b>C</b>—Pearson coefficients (R) of correlation between the levels of nAChR subunits in the brain and those of 7(1–208)-specific antibodies in the blood. The columns correspond to M±SE, *—p<0.05; **—p<0.005; ***—p<0.0005 compared to Ctrl.</p

Biochemical and biophysical analysis of i-α1-ECD.

<p><b>A</b>) <b>Gel filtration analysis</b>. The right y axis shows the absorbance at 280 nm (A₂₈₀) and the left y axis the bound ¹²⁵I-α-BTX (Δcpm) in the filter assay, using 20 μL of each gel filtration fraction and 3,000 cpm of ¹²⁵I-α-BTX. The values are the mean and standard deviation (±S.D.). Arrows indicate the peaks of eluted protein markers of known molecular mass. <b>B</b>) <b>Far-UV CD analysis</b>. Deconvolution of the presented spectrum with CDPro software showed 8% α-helical content, 40% β-sheet structure, 22% β-turns and 30% unordered portion. NRMSD (normalized root mean square deviation) value was 0.05, reflecting the accurate analysis of secondary structure. <b>C</b>) <b>DLS analysis</b>. The size distribution by volume of the gel-filtration isolated i-α1-ECD molecules is shown. The calculated hydrodynamic diameter of i-α1-ECD is 4 nm with a polydispersity value of 29%. Protein sample was derived from gel filtration analysis and its concentration was 0.3 mg/mL. SDS-PAGE analysis followed by D) Coomassie Brilliant Blue staining and <b>E</b>) Western blot analysis using the anti-α1 nAChR mAb 198. Lane 1: Positions of protein Mr standards; Lane 2: y-α1-ECD; Lane 3: i-α1-ECD. Protein samples were derived from gel filtration chromatography.</p

Episodic memory of experimental mice studied in the “Novel Object Recognition” test.

<p>Discrimination indexes calculated for mice immunized with 7(1–208) (n = 8) or injected with LPS (n = 5) compared to non-treated animals (n = 9) or those “immunized” with complete Freund’s adjuvant (CFA, n = 5). ***—p<0.0005 compared to non-treated mice (Ctrl).</p

Visualization of biotinylated antibody within the brain at different periods after injection.

<p>Confocal microscopy images of the striatum of mice injected with α7(1–208)-specific antibody (A-B) or non-specific IgG (C) in 15 min (A) or 3h (B-C) after injection. Antibodies were developed with Extravidin-Cy3 (<i>red</i>). Bar corresponds to 100μm, actual for each fragment of the panel. D—Antibody-specific ELISA signal of the primary brain supernatants and detergent lysates of mice either pre-treated or not with LPS; the brains were removed in 15 min or 3 h after the antibody injection. Each column corresponds to mean±SE of three repeats in ELISA; *—p<0.05; **—p<0.005 compared to the data of LPS non-treated mice.</p

The GFAP-positive astrocytes (A) and the number of nucleated cells (B) in the brain sections of experimental mice studied by immunohistochemistry.

<p><b>A</b>—Confocal microscopy images of the hippocampus CA1 (Hip), motor/somatosensory cortex (Crtx) or striatum (Str) of non-treated (Ctrl), α7(1–208)-immunized or LPS-injected mice stained with rabbit GFAP-specific antibody and developed with anti-rabbit Alexa 488 (<i>green</i>). Cell nuclei are stained with DAPI (<i>blue</i>). Bar corresponds to 50μm, actual for each fragment of the panel. <b>B</b>—The number of nucleated cells (DAPI-positive) in corresponding brain regions studied in all available sections (12 to 16 for each treatment for each region); *—p<0.05; **—p< 0.005.</p

The levels of different Aβ isoforms in the brain detergent lysates of experimental mice studied by Sandwich ELISA.

<p><b>A</b>—total Aβ₄₀ and Aβ₄₂, <b>B</b>—Aβ₄₀ and Aβ₄₂ bound to α7 nAChR in mice immunized with 7(1–208) (n = 8) or injected with LPS (n = 5) compared to non-treated animals (Ctrl, n = 9) and to those “immunized” with complete Freund’s adjuvant (CFA, n = 5). The columns correspond to M±SE (n = 5); *—p<0.05; **—p<0.005; ***—p<0.0005 compared to Ctrl.</p

The level of Aβ₄₀ and Aβ₄₂ in the brain sections of experimental mice studied by immunohistochemistry.

<p>Confocal microscopy images of the hippocampus CA1 or motor/somatosensory cortex of non-treated (Ctrl), α7(1–208)-immunized or LPS-injected mice stained with biotinylated Aβ₄₀- or Aβ₄₂-specific antibodies and developed with Extravidin-Cy3 (<i>red</i>) and with α7-specific antibody developed with anti-rabbit Alexa 488 (<i>green</i>). Bar corresponds to 50μm, actual for each fragment of the panel.</p

SPR recordings of Cro interaction with AChBP from <i>L</i>. <i>stanalis</i>.

<p>An arrow indicates injection of the analyte. Line 1 corresponds to injection of buffer solution. Curve 2–5 correspond to injections of solutions with Cro at concentrations of 5.5, 11.5, 23 and 46 μg/ml, respectively.</p

Interaction of non-venom proteins with <i>T</i>.<i>californica</i> nAChR.

<p>Inhibition of the initial rate of specific [¹²⁵I]-α-Bgt binding to <i>T</i>.<i>californica</i> nAChR by RNAse (squares) and cytochrome C (circles).</p