Journal: PLoS ONE

Article Title: Correlation Index-Based Responsible-Enzyme Gene Screening (CIRES), a Novel DNA Microarray-Based Method for Enzyme Gene Involved in Glycan Biosynthesis

doi: 10.1371/journal.pone.0001232

Figure Lengend Snippet: The expression patterns of about 1000 glycan-related genes were profiled in a set of six different cell lines (A–F) by comparing the microarray binding of cellular cDNA and reference polyA(+) RNA and calculating the relative expression values . The polygons in the left web graphs represent the relative gene expression profiles of eight glycan-related genes selected as examples. In these graphs, the difference in relative gene expression is expressed on a log scale, where the edge of the polygon corresponds to the strongest expression in each cell line (A–F). The same set of six cell lines were examined for cell-surface glycan expression using fluorescently labeled plant lectins and flow cytometry; the strength of the glycan expression is plotted as relative values among the six lines, where the edge of the polygon represents the strongest expression (web graph on top right). The glycan expression profiles were analyzed for correlations with the glycan-related gene expression profiles. Similarities and dissimilarities between the profiles were assessed using Pearson's correlation coefficient, which has values ranging from -1 (no correlation) to 1 (perfect correlation). A complete list of the genes found to be positively or negatively correlated with plant lectin staining patterns is presented in . Genes known to affect the biosynthesis of an epitope were selected from among the correlated genes (shown for each lectin in the tables on the right in – ). A correlated gene identified by CIRES was confirmed as the gene responsible for regulating the biosynthesis of a particular glycan by transferring the gene into another cell line of the set, via gene transfer techniques such as retrovirus-mediated overexpression, and looking for a related change in epitope expression.

Article Snippet: Two commercially available sets of biotin-conjugated plant lectins (Plant Lectin Set I and II) were obtained from Seikagaku (Tokyo, Japan).

Techniques: Expressing, Glycoproteomics, Microarray, Binding Assay, Gene Expression, Labeling, Flow Cytometry, Staining, Transferring, Over Expression

Journal: PLoS ONE

Article Title: Correlation Index-Based Responsible-Enzyme Gene Screening (CIRES), a Novel DNA Microarray-Based Method for Enzyme Gene Involved in Glycan Biosynthesis

doi: 10.1371/journal.pone.0001232

Figure Lengend Snippet: (A, C, D) Expected glycan structures for lectin recognition (left), web graphs of the lectin staining profiles (depicted as polygons) obtained using a set of six B-cell lines (middle), and the correlation indexes (CI, Pearson's correlation coefficient for profile matching) of the relevant genes that correlated with the plant lectin staining profiles and the P values of the correlations (right). The correlation orders of the glycan-related genes selected from the complete list of correlated genes are indicated as numbers in parentheses in the box for each gene, with a smaller number indicating a stronger correlation between gene expression and glycan expression profiles. Genes with a negative correlation are indicated by an N before the order number. The lectins used were (A) PHA-L4, (C) SSA, and (D) PNA. Lectin epitopes shown in the figures are taken from the literature unless otherwise specified , . (B) Namalwa cells were infected with MSCV harboring MGAT5-IRES-EGFP . Control cells were infected with empty vector ( IRES-EGFP ) or the same vector encoding B3GNT2 or MGAT3 . Flow cytometry results for PHA-L4 staining were compared between EGFP-positive cells (solid line) and EGFP-negative cells (dashed line).

Article Snippet: Two commercially available sets of biotin-conjugated plant lectins (Plant Lectin Set I and II) were obtained from Seikagaku (Tokyo, Japan).

Techniques: Glycoproteomics, Staining, Gene Expression, Expressing, Infection, Control, Plasmid Preparation, Flow Cytometry

Journal: PLoS ONE

Article Title: Correlation Index-Based Responsible-Enzyme Gene Screening (CIRES), a Novel DNA Microarray-Based Method for Enzyme Gene Involved in Glycan Biosynthesis

doi: 10.1371/journal.pone.0001232

Figure Lengend Snippet: Presentation is the same as in except that the plant lectins used were (A) LCA, (B) UEA-I, and (C) RCA 120. N.D. in the gene order list indicates that no gene was determined to have a correlation with the lectin staining.

Article Snippet: Two commercially available sets of biotin-conjugated plant lectins (Plant Lectin Set I and II) were obtained from Seikagaku (Tokyo, Japan).

Techniques: Staining

Journal: PLoS ONE

Article Title: Correlation Index-Based Responsible-Enzyme Gene Screening (CIRES), a Novel DNA Microarray-Based Method for Enzyme Gene Involved in Glycan Biosynthesis

doi: 10.1371/journal.pone.0001232

Figure Lengend Snippet: Presentation is the same as in except that the plant lectins used were (A, B) DSA and (C–E) PHA-E4. N.D. in the gene order list indicates that no significant correlation was detected. (B, D) Flow cytometric staining patterns for EGFP-positive Namalwa cells are shown in bold lines, and those for EGFP-negative (control) cells are shown in gray dashed lines. The overexpression of MGAT5 resulted in a subtle (60%) increase in DSA staining. The overexpression of MGAT3 resulted in a 2-fold increase in PHA-E4 staining. (E) PHA-E4 lectin blotting was performed using the membrane fraction of the same set of cell lines. A plot of the quantified signals reveals differences in the PHA-E4 staining profile among the six cell lines (C, E), as discussed in the text.

Article Snippet: Two commercially available sets of biotin-conjugated plant lectins (Plant Lectin Set I and II) were obtained from Seikagaku (Tokyo, Japan).

Techniques: Staining, Negative Control, Over Expression, Membrane

Journal: PLoS ONE

Article Title: Correlation Index-Based Responsible-Enzyme Gene Screening (CIRES), a Novel DNA Microarray-Based Method for Enzyme Gene Involved in Glycan Biosynthesis

doi: 10.1371/journal.pone.0001232

Figure Lengend Snippet: Presentation is the same as in except that the plant lectins used were (A–B) MAM, (C) WGA, and (D) Con-A. The epitopes of the two different lectins of MAM, MAL and MAH, are illustrated separately. WGA essentially recognizes a cluster of N-acetyl groups, as indicated, and thus required Neu5Ac as a Sia species. Con-A recognizes mannose-containing glycans with varying affinities. High-mannose-type glycans (upper diagram in (D)) bind best to this lectin. (B) Namalwa cells were infected with retroviruses encoding various GlcNAc transferases. The MAM staining patterns of the EGFP-positive populations of each infectant are shown. MGAT5 overexpression did not shift the staining pattern in comparison with the vector control (data not shown).

Article Snippet: Two commercially available sets of biotin-conjugated plant lectins (Plant Lectin Set I and II) were obtained from Seikagaku (Tokyo, Japan).

Techniques: Infection, Staining, Over Expression, Comparison, Plasmid Preparation, Control

Journal: Addiction Biology

Article Title: Social threat exposure in juvenile mice promotes cocaine‐seeking by altering blood clotting and brain vasculature

doi: 10.1111/adb.12373

Figure Lengend Snippet: Increased blood coagulation and impairments in brain microvasculature in S‐S cocaine‐abstinent mice. (a) Decreased prothrombin time (PT) and (b) increased vWF mRNA expression were observed in cocaine‐abstinent S‐S mice (PT, N : NS‐S = 3, S‐S = 4; vWF, N: NS‐S = 6, S‐S = 5). (c) Representative images of NeuN immunostaining (left column) and blood vessels stained with L. esculentum (tomato) lectin of the dorsal striatum (STR; top), dentate gyrus (DG; middle) and nucleus accumbens (NAC; bottom) of S‐S and NS‐S mice ( N : NS‐S = 4, S‐S = 4) during withdrawal. (d) Quantitative analysis of blood vessels showing that S‐S mice had fewer capillary branch points and (e) lower signal intensity in the STR, DG and NAC. (f) Cumulative plots of distribution of capillary diameters in the STR and (g) NAC in NS‐S (filled circle) and S‐S (open circle) mice revealing smaller diameter vessels in S‐S compared with NS‐S mice. P < 0.001; *** P < 0.001; ** P < 0.01; * P < 0.05

Article Snippet: For lectin staining (Thurston et al. ) brain sections containing NAc, striatum or hippocampus (dentate gyrus) were first incubated overnight at 4°C in PBS containing 0.3 percent Triton X‐100 with biotin‐conjugated Lycopersicon esculentum (tomato) lectin (1 µg/ml; BD Pharmingen, San Diego, CA) and rabbit anti‐NeuN (1:500; Millipore, Billerica, Massachusetts, USA).

Techniques: Coagulation, Expressing, Immunostaining, Staining