186,211 research outputs found
Activation of Silent gal Genes in the lac-gal Regulon of Streptococcus thermophilus
No full textStreptococcus thermophilus strain CNRZ 302 is unable to ferment galactose, neither that generated intracellularly by lactose hydrolysis nor the free sugar. Nevertheless, sequence analysis and complementation studies with Escherichia coli demonstrated that strain CNRZ 302 contained structurally intact genes for the Leloir pathway enzymes. These were organized into an operon in the order galKTE, which was preceded by a divergently transcribed regulator gene, galR, and followed by a galM gene and the lactose operon lacSZ. Results of Northern blot analysis showed that the structural gal genes were transcribed weakly, and only in medium containing lactose, by strain CNRZ 302. However, in a spontaneous galactose-fermenting mutant, designated NZ302G, the galKTE genes were well expressed in cells grown on lactose or galactose. In both CNRZ 302 and the Gal+ mutant NZ302G, the transcription of the galR gene was induced by growth on lactose. Disruption of galR indicated that it functioned as a transcriptional activator of both the gal and lac operons while negatively regulating its own expression. Sequence analysis of the gal promoter regions of NZ302G and nine other independently isolated Gal+ mutants of CNRZ 302 revealed mutations at three positions in the galK promoter region, which included substitutions at positions -9 and -15 as well as a single-base-pair insertion at position -37 with respect to the main transcription initiation point. Galactokinase activity measurements and analysis of gusA reporter gene fusions in strains containing the mutated promoters suggested that they were gal promoter-up mutations. We propose that poor expression of the gal genes in the galactose-negative S. thermophilus CNRZ 302 is caused by naturally occurring mutations in the galK promoter.
Anti-gal-9 mAbs efficiently neutralize gal-9-induced apoptosis in primary T cells.
No full textCD3+ T-cells were isolated from healthy donors, activated by a combination of CD3/CD28 antibodies and treated or not with gal-9 (gal-9S; 40 nM) alone or in combination with lactose (5 mM), control isotype mAbs (ctrl IgG1) or anti-gal-9 mAbs (Gal-Nab1 and Gal-Nab2) at 67 nM (i.e. 10 μg/mL). After 36 h, they were subjected to annexin-V/PI staining and flow cytometry analysis. A. Examples of flow cytometry plots for purified CD3+ cells from one donor. B. Synthesis of data from 3 similar experiments made with CD3+ cells from 3 donors. C and D. Dose-response curves for apoptotic cell death (annexin-V+ PI+) (C) or PS translocation (annexin-V+ PI-) (D) in activated CD3+ cells treated for 36h with gal-9 combined with increasing concentrations of Gal-Nab1 and Gal-Nab2 (0.3 to 100 nM). Empty squares indicate the percentages obtained in conditions without gal-9. Black crosses indicate the percentages obtained with isotype control IgG1 mAbs used at maximal concentration (100 nM). Data are presented as means ± SEM of three independent experiments made with three distinct donors.</p
Gal-3 expression in microglia cells.
No full textImmunohistochemical staining of Gal-3 (red) expressing microglia. All microglia were detected using the microglia marker Iba1 (green). At 0 DIV no Gal-3-expressing cells were found (A, B). In controls, at 3, 4 and 7 DIV Iba1/Gal-3 co-expressing cells were found and only in the GCL (C, D, G, H, K, and L). LPS-treated retinas displayed larger numbers of Iba1/Gal-3 co-expressing cells that were located in the GCL, INL and OPL at 3, 4 and 7 DIV. Scale bar: 200 μm.</p
H-gal-GP is flexible.
No full textThe three main ways in which H-gal-GP appears to move, as determined by principal component analysis of multi-body refinement, are illustrated in each row A-C.</p
Subunit fitting to H-gal-GP EM density.
No full textA EM density of one-winged H-gal-GP (4.5 Å map) fitted with models of aspartyl protease PEP 1 (red), MEP3 (dark blue) and cysteine protease (cyan). A cropped view of A, through the centre of the H-gal-GP complex (B) and cropped to just show two of the MEP3 domains (C). D H-gal-GP map viewed from the base showing the fitting of the MEP subunits. Fitted subunits of the H-gal-GP complex viewed from the side (E) and base (F) and colored red (PEP1), cyan (cysteine protease) and orange, blue, green and grey for the four MEPs.</p
Gal-9 binds and recruits to <i>Mtb</i>.
No full text(A) Experimental design for Mtb pull-down mass spectrometry identification of Mtb-binding proteins. (B) Domain organization of Gal-9. CRD, carbohydrate recognition domain. (C) Immunoblot of in vitro binding reactions between indicated pathogens and Gal-9-FLAG THP-1 lysate, probed with anti-FLAG antibody; IN, input; Lm, Listeria monocytogenes; Stm, Salmonella enteria serovar Typhimurium; Cn, Cryptococcus neoformans; Mtb, Mycobacterium tuberculosis. (D) Confocal microscopy of WT BMMs infected with WT or ΔeccC Mtb-GFP (MOI = 2) 8 hours post-infection and immunostained for endogenous Gal-9 and Gal-3. (E) Quantification of Mtb-GFP colocalization with Gal-9 or Gal-3 at indicated time points. Figures represent two independent experiments (D, E). An average of 882 cells were analyzed per technical replicate (D, E). Error bars represent SD from 3 technical replicates. The schematic was created with BioRender.com.</p
X-gal-negative Newly Formed Cardiomyocytes Increase Following Cardiac Injury.
No full text(A) X-gal staining and immunofluorescent images of sections comparing sham and myocardial infarction (MI) CreLacZ mice: left panel, sham; middle panel, MI remote area; and right panel, MI area. The upper panels show X-gal-stained images. The bottom panels show the corresponding immunofluorescent images (SA-actinin, green; laminin, red). Asterisks show X-gal-negative cardiomyocytes. Scale bar, 20 μm. The X-gal-negative cardiomyocytes are further enlarged in the inset. Scale bar, 10 μm. (B) Number of X-gal-negative cardiomyocytes per area 3 months after MI: sham (n = 5) and MI (n = 5). *p < 0.05. (C) Comparison of the number of X-gal-negative cardiomyocytes in the MI remote and MI area 3 months after MI (n = 5 per area). *p < 0.05. (D) Left: comparison of the cross-sectional area between X-gal-negative (MI2w neg) and -positive (MI2w pos) cardiomyocytes at 2 weeks after MI. Right: comparison of the cross-sectional area of X-gal-negative cardiomyocytes at 2 weeks (MI2w neg) and 6 months (MI6m neg) after MI. n = 50–66 cardiomyocytes pooled from two MI mice per group. *p < 0.05. The Mann–Whitney U-test was used for statistical analysis.</p
Anti-gal-9 mAbs inhibit the “Th1-like” phenotype shifting induced by gal-9 in resting or stimulated PBMCs.
No full textA-C. PBMCs from healthy donors were stimulated or not with anti-CD3/CD28 antibodies and treated for one week with human recombinant gal-9 (Gal-9S; 40 nM) with or without combination with lactose (5 mM), control isotype antibody (ctrl IgG1), Gal-Nab1 or Gal-Nab2 (67 nM i.e. 10 μg/mL). Intracellular cytokine expression was assessed by flow cytometry as explained under “Materials and Methods”. At least 50% of the cells were alive. Dead cells were gated out. A. Examples of flow cytometry plots obtained with stimulated PBMCs for one donor after gating on the CD3+ T cell population: IFN-γ (upper plots) and IL-2 (lower plots) expression were analyzed. B-C. Percentages of CD3+ IFNγ+ (upper histogram) and CD3+ IL-2+ (lower histogram) cells were normalized with the basal percentages obtained in untreated cells using either unstimulated (B) or stimulated (C) PBMCs. Data are represented as means ± SEM of four independent experiments with different donors. All statistical differences displayed are compared with gal-9 treatment; ***p<0.001; **p<0.01; *p<0.05; ns: not significant (one-way ANOVA/Dunnet post-test).</p
Gal-9 is shed by neutrophil mediated DP of CD44 upon activation.
No full text(A) Percent colocalization of CD44 and Gal-9 on neutrophils quantified using an Amnis ImageStream. (B) Cumulative percent colocalization of Gal-9 and CD44 on the surface of neutrophils. (C) Representative plot of neutrophils with a high and low delta centroid XY. (D) Representative images of capped and dispersed CD44 on neutrophils. (E) Representative plot of LPS-treated neutrophil radial delta centroid. (F) Cumulative data of CD44 capping (delta centroid >1) in unstimulated and LPS-treated neutrophils. (G) Representative plot showing changing Gal-9 and CD32 expression on neutrophils untreated or stimulated with LPS. (H) Cumulative results showing the percent expression of surface Gal-9 on unstimulated and LPS stimulated neutrophils. (I) Cumulative results showing the MFI of surface Gal-9 on unstimulated and LPS stimulated neutrophils. (J) Cumulative data showing correlation between % Gal-9 expression on neutrophils and CD44 capping. (K) Surface expression of Gal-9 on neutrophils untreated or treated with MBCD. (L) Surface expression of Gal-9 on neutrophils untreated or treated with LPS in the presence or absence of a RAP-1 inhibitor, (M) ezrin inhibitor, and a (N) DP.I. (O) Cumulative data of Gal-9 mRNA expression and (P) shed Gal-9 in culture supernatants of LPS-activated neutrophils in neutrophils from HCs once stimulated with LPS for 3 hours in the presence or absence of DP.I. The underlying data can be found in S1 Data. DP, depalmitoylation; DP.I, depalmitoylation inhibitor; Gal-9, Galectin-9; HC, healthy control; LPS, lipopolysaccharide; MBCD, methyl-beta-cyclodextran; MFI, median fluorescence intensity.</p
Gal-9 shedding by activated neutrophils is regulated by ROS and CaMKII.
No full text(A) Visual model showing the mechanistic action of phloretin on the GLUT1 transporter. (B) Representative plots showing the effect of phloretin on Gal-9 shedding. (C) Cumulative data showing surface Gal-9 expression in untreated or LPS-treated neutrophil in the presence or absence of phloretin. (D) Cumulative data of Gal-9 mRNA expression and (E) shed Gal-9 in culture supernatants of LPS-activated neutrophils in neutrophils from HCs once stimulated with LPS for 3 hours in the presence or absence of phloretin. (F) Cumulative data showing percent CD44 capping in the presence or absence of phloretin in untreated or LPS-treated neutrophils. (G) Representative plots and (H) cumulative data representing the MFI of ROS in untreated and LPS-treated neutrophils. (I) Representative plots and (J) cumulative data showing the change in MFI of ROS expression in the presence or absence of LPS and phloretin. (K) Representative histogram and (L) cumulative data showing percent Gal-9 expression in the absence and presence of LPS and LPS + Apoc. (M) Cumulative data of Gal-9 mRNA expression and (N) shed Gal-9 in culture supernatants of LPS-activated neutrophils in neutrophils from HCs once stimulated with LPS for 3 hours in the presence or absence of Apoc. (O) Cumulative data showing percent Gal-9 expression and (P) CD44 capping in the absence or presence of LPS and a CaMKII inhibitor. (Q) Cumulative data of Gal-9 mRNA expression and (R) shed Gal-9 in culture supernatants of LPS-activated neutrophils in neutrophils from HCs once stimulated with LPS for 3 hours in the presence or absence of CAMKII inhibitor. The underlying data can be found in S1 Data. Apoc, Apocynin; CaMKII, calcium/calmodulin-dependent protein kinase II; Gal-9, Galectin-9; HC, healthy control; LPS, lipopolysaccharide; MFI, median fluorescence intensity; ROS, reactive oxygen species.</p
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