Journal: BioMed Research International

Article Title: Analysis of the Glycosylation Profile of Disease-Associated Water-Soluble Prion Protein Using Lectins

doi: 10.1155/2019/1053282

Figure Lengend Snippet: Analysis of the glycosylation profile of ws-PrP and classical PrP and effect of treatment with GdnHCl . ((a)-(c)) Glycosylation profile of PrP Sc from high-speed supernatant (S HS ) and plasma is similar: ((a) and (b)) lectin-binding profile to single glycans in PrP Sc captured by 3F4 and 6H4, respectively, from S HS and plasma; (c) glycans total content in PrP Sc captured by 3F4 and 6H4 from S HS and plasma. (d) Comparison between signals of PrP Sc captured by specific anti-PrP polyclonal antibody from 263K-infected hamster plasma and signals of immunoglobulin (IgG) prepared from 263K-infected hamster plasma, before and after treatment with guanidine hydrochloride (GdnHCl) in ELISA. Signals were detected by 6H4 antibody/secondary antibody or only with secondary antibody. (e) Representative silver-stained 12% SDS-PAGE gel of hamster plasma total proteins showing effect of GdnHCl on high-abundance proteins removal from blood. ((f)-(m)) Glycosylation profile of ws-PrP differs from that of classical PrP: ((f) and (g)) lectins binding profile to single glycans in PrP Sc captured by 3F4 and 6H4, respectively, from S HS and high-speed pellet (P HS ); (h) glycans total content in PrP Sc captured by 3F4 and 6H4 from S HS and P HS ; (i) percentage of glycans total content in S HS PrP Sc with respect to that in P HS PrP Sc ; (j) lectins binding profile to glycans in PrP Sc captured by 3F4 from noninfected S HS , plasma and P HS ; ((k) and (l)) lectins binding profile to single glycans in PrP Sc captured by 3F4 from P HS (k) and S HS (l) before and after treatment with GdnHCl; (m) glycans total content in PrP Sc captured by 3F4 from S HS and P HS before and after treatment with GdnHCl. Total proteins from plasma, S HS and P HS were loaded into 96-well ELISA plates that were precoated with the 3F4 or 6H4 antibodies, and the lectin-binding profile was determined by staining with ConA, Concanavalin A; WGA, wheatgerm agglutinin; RCA, Ricinus communis agglutinin; DBA, Dolichos biflorus agglutinin; PNA, peanut agglutinin; SBA, soybean agglutinin; and UEA-1, Ulex europaeus agglutinin I. The sizes of molecular mass markers in kilodaltons are indicated on the left. Data are means ± SD and are representative of at least three independent assays and three different preparations, performed in duplicate. ∗ P<0.05, ∗∗ P<0.01, ∗∗∗ P<0.001, ∗∗∗∗ P<0.0001.

Article Snippet: Signals were developed by a sequential incubation with biotinylated lectins (10 μ g/mL of ConA, Concanavalin A; WGA, wheat germ agglutinin; RCA, Ricinus communis agglutinin; DBA, Dolichos biflorus agglutinin; PNA, peanut agglutinin; SBA, soybean agglutinin; and UEA-1, Ulex europaeus agglutinin I) (Vector Laboratories) for 2 h at room temperature, with a streptavidin horseradish peroxidase (HRP)-conjugated (2.5 μ g/mL) (Vector Laboratories) for 1 h at room temperature, and with an ABTS Substrate (Vector Laboratories) for 20 min in the dark at room temperature.

Techniques: Binding Assay, Infection, Enzyme-linked Immunosorbent Assay, Staining, SDS Page

Journal: BioMed Research International

Article Title: Analysis of the Glycosylation Profile of Disease-Associated Water-Soluble Prion Protein Using Lectins

doi: 10.1155/2019/1053282

Figure Lengend Snippet: Glycosylation profile of hamster plasma total proteins is essential for their stability in blood . ((a)-(c)) Binding profile of ConA, Concanavalin A; WGA, wheatgerm agglutinin; RCA, Ricinus communis agglutinin; DBA, Dolichos biflorus agglutinin; PNA, peanut agglutinin; SBA, soybean agglutinin; and UEA-1, Ulex europaeus agglutinin I to equal amounts of total proteins from noninfected and terminally scrapie-infected hamster plasma ((a) and (b)) and from scrapie-infected hamster plasma collected at different time points after infection (c). ((a) and (c)) Lectin-ELISA analysis, (b) lectin-blot analysis (10% SDS-PAGE gel). The figures show that lectins binding profile to plasma total proteins is not due to the infection or disease duration but rather to potential relevance of the glycans recognized by ConA and RCA for maintaining the stability of plasma total proteins including PrP against certain insults. (d) Comparison between the regression lines of the binding profile of ConA, WGA, RCA, DBA, PNA, SBA, and UEA-1 toward plasma total proteins throughout disease period. The basal immunoreactivity of ConA and RCA is the highest and this trend is maintained also throughout disease period; only immunoreactivity of ConA (increase) and DBA (decrease) correlates with disease progression (r = 0.90); lectins basal immunoreactivity values are potential predictors of the expected effect by the relative sugars on proteins stability state. (e) The increase in the immunoreactivity of ConA is proportional to the sum of reduction in the immunoreactivity of DBA, SBA, and UEA-I. The left side of the figure shows that the two effects (decrease and increase of immunoreactivity) are inversely correlated between each other during disease progression, while the right side of the figure shows that the two effects (decrease and increase of immunoreactivity) are mathematically very close but opposite. The increase and decrease in the lectins reactivity were calculated by normalization of the absorbance values against the basal reactivity values for each lectin. Lectins basal reactivity is indicated by a circle shown on . The sizes of molecular mass markers in kilodaltons are indicated on the left. Data are means ± SD and are representative of at least three independent assays and three different preparations, performed in duplicate. ∗ P<0.05, ∗∗ P<0.01.

Article Snippet: Signals were developed by a sequential incubation with biotinylated lectins (10 μ g/mL of ConA, Concanavalin A; WGA, wheat germ agglutinin; RCA, Ricinus communis agglutinin; DBA, Dolichos biflorus agglutinin; PNA, peanut agglutinin; SBA, soybean agglutinin; and UEA-1, Ulex europaeus agglutinin I) (Vector Laboratories) for 2 h at room temperature, with a streptavidin horseradish peroxidase (HRP)-conjugated (2.5 μ g/mL) (Vector Laboratories) for 1 h at room temperature, and with an ABTS Substrate (Vector Laboratories) for 20 min in the dark at room temperature.

Techniques: Binding Assay, Infection, Enzyme-linked Immunosorbent Assay, SDS Page

Journal: PLoS ONE

Article Title: Blood Group Substances as Potential Therapeutic Agents for the Prevention and Treatment of Infection with Noroviruses Proving Novel Binding Patterns in Human Tissues

doi: 10.1371/journal.pone.0089071

Figure Lengend Snippet: (A) Sections were incubated with anti-A (a,b), anti-B (c,d) and biotinylated Ulex lectin (e,f). (B) Sections were incubated with PBS in place of anti-A, anti-B and Ulex . (C) Sections were incubated with GI.1 VLP and then with anti-GI.1 antibody. (D) Sections were incubated with GII.2 VLP and then with anti-GII.2 antibody. (E) Sections were incubated with GII.6 VLP and then with anti-GII.6 antibody. (F) Sections were incubated with GII.6 after incubation with the PGM-(A + H + ), the flying squid liver (B + H + ) and PGM (A − H + ) preparations, respectively, and then with anti-GII.6 antibody. (G) Section from A blood type were incubated with (a) and without (b) GI.1 VLP and then treated with the PGM-(A + H + ) followed by incubation with anti-GI.1 antibody. The section from B blood type was incubated with (c) and without (d) the VLP from GI.1 and then treated with the flying squid liver (B + H + ) followed by incubation with anti-GI.1 antibody. The section from O blood type was incubated with (e) and without (f) the GII.2 VLP and then treated with the PGM (A − H + ) followed by incubation with anti-GII.2 antibody. Immunostaining of all the sections from (A) to (G) was followed by an ABC detection system. See the details in the Text. Magnifications: (a), (c) and (e) in (A) to (F) and (a) to (f) in (G), ×100; (b), (d) and (f) in (A) to (F), ×400.

Article Snippet: Anti-A, anti-B, anti-Le a and anti-Le b mouse monoclonal antibodies were obtained from Ortho Clinical Diagnosis (Rochester, NY) and biotinylated Ulex lectin was from Seikagaku (Tokyo, Japan).

Techniques: Incubation, Immunostaining

Journal: mBio

Article Title: Burkholderia pseudomallei Penetrates the Brain via Destruction of the Olfactory and Trigeminal Nerves: Implications for the Pathogenesis of Neurological Melioidosis

doi: 10.1128/mBio.00025-14

Figure Lengend Snippet: B. pseudomallei penetrates degraded olfactory and intact respiratory epithelium (RE). All sections were immunolabeled with anti- B . pseudomallei antibodies (green) and DAPI (blue); some were also labeled with anti-OMP antibodies and UEA1 lectin as indicated. (A) A coronal section of the nasal cavity (NC) shows that one side has extensive infection (arrow) while the other side has little evidence of infection. Boxed areas are shown in panels F to H as indicated. (B) A higher-magnification view of uninfected olfactory epithelium (OE) shows a uniform structure. (C) OE in an inoculated mouse shows an extensive presence of B. pseudomallei (arrow; green) in the NC at 24 h. The OE is crenellated (arrow with tail); the thin respiratory epithelium (RE) in the ventral NC was not visually affected. The asterisk indicates nonspecific autofluorescence. (D) Bacteria (green) occasionally penetrated relatively intact epithelium, but only in patches where neurons (immunolabeled with OMP; red) were absent (arrow). (E) In the olfactory epithelium, bacteria (green; arrow) were not associated with Bowman’s glands (labeled with UEA1 lectin; white; arrow with tail); olfactory neurons (red) are labeled with anti-OMP antibodies. (F to H) Higher-magnification views of the boxed areas indicated in panel A. (F) B. pseudomallei (arrow) was present on the surface of the OE, but no morphological reaction was apparent. (G) Ulceration of the OE (dashed line) was seen, although the presence of bacteria was limited (arrow). (H) The OE showed extensive destruction and loss of integrity, and bacteria were present (arrows) within the epithelium. Bacteria were not detected in the lamina propria (LP) underlying the OE (G and H). (I) In patches of respiratory epithelium, there was widespread infection with B. pseudomallei (arrow), but bacteria did not penetrate the deeper layers. (J and K) OMP immunolabeling (red) demonstrates that healthy epithelium was not penetrated by bacteria (arrow) despite their presence in the adjacent nasal cavity, but that epithelium was penetrated as the neurons partially degraded; panel K shows the same section as that in panel J but with the red channel (OMP) turned off. (L to O) OMP immunolabeling became patchy with some areas showing low levels of OMP reactivity (arrow with tail). Bacteria penetrated the outer layers and were present in nerve bundles in the lamina propria (arrows in panels N and O); panels M and O show the same sections as those in panels L and N, respectively, but with the red channel (OMP) turned off. (P and Q) Complete loss of the neuronal layer led to colonization of the remaining layer by bacteria (arrows); arrows with tails point to neurons in the nasal cavity and remaining epithelium. (R to T) Schematics summarizing the infection of the epithelium. (R) Sagittal view of the nasal cavity, olfactory bulbs (OB), and cortex (Cx). (S) In uninfected mice, the olfactory epithelium is uniform and neurons (red) are distributed throughout the epithelium. (T) When B. pseudomallei (green) is present, the majority of epithelium becomes crenellated but neurons remain within the epithelium and bacteria cannot penetrate. In some regions, the neurons are lost (arrow) and bacteria penetrate the remaining layers. Bar sizes are in μm.

Article Snippet: The biotinylated lectin UEA1 (1:100; Dako) was incubated similarly to the antibodies, followed by streptavidin-Alexa Fluor 647 (1:400; Invitrogen).

Techniques: Immunolabeling, Labeling, Infection, Bacteria