Journal:

Article Title: Cooperative regulation of light-harvesting complex II phosphorylation via the plastoquinol and ferredoxin-thioredoxin system in chloroplasts

doi:

Figure Lengend Snippet: Modulation of PSII protein phosphorylation by electron transfer inhibitors, reducing agents, and quantity of light. (A and B) Thylakoids isolated from dark-adapted leaves were phosphorylated in vitro for 15 or 30 min in the presence of the following reagents: 20 μM 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) or 10 μM 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) (A) or 2 mM trans-4,5-dihydroxy-1,2-dithiane (DTTox), 2 mM DTT, or 10 mM ascorbate (B). Immunoblots present the phosphorylation level of the D1 and LHCII proteins. (C) Steady-state phosphorylation level of PSII proteins in vivo. Leaf discs were incubated in darkness (D) or illuminated for 2 h at a photon flux density of 30 (LL), 200 (GL), or 1000 (HL) μmol·m−2·s−1. Phosphorylated PSII proteins were detected by immunoblotting with phosphothreonine antibody. P-CP43, P-D2, P-D1, and P-LHCII, phosphorylated forms of CP43, D2, D1, and LHCII proteins, respectively.

Article Snippet: Before phosphorylation assays, isolated thylakoid membranes were preincubated at 23°C for 5–10 min either in darkness or in light, in the presence of the following chemicals: l -ascorbic acid (Sigma); 3-(3,4-dichlorophenyl)-1,1-dimethylurea (Sigma); 2,5-dibromo-3-methyl-6-isopropyl- p -benzoquinone (TCI America, Portland, OR); DTT (Roche Molecular Biochemicals); trans -4,5-dihydroxy-1,2-dithiane (Sigma); reduced and oxidized glutathione (Sigma); N -ethylmaleimide (NEM) (Sigma); or chloroplast thioredoxin f and m of pea.

Techniques: Isolation, In Vitro, Western Blot, In Vivo, Incubation

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Disruption to tRNA Modification by Queuine Contributes to Inflammatory Bowel Disease

doi: 10.1016/j.jcmgh.2023.02.006

Figure Lengend Snippet: Alterations of metabolites related to QTRT1 in human IBD patients. In tRNA containing a GUN anticodon (where N = any base), guanine at position 34 is exchanged with queuine to obtain queuosine-modified tRNA, a reaction carried out by the eTGT enzyme, which is composed of subunits of QTRT1 and QTRT2 subunits. The abundance of guanine was compared between IBD patients and control subjects by revisiting metabolite data from Metabolomics Workbench. Alteration of guanine in ( A ) UC and ( B ) CD patients. Two datasets were included in the analysis with project numbers PR000639 (UC: n = 145; CD: n = 266; control: n = 134) and PR000677 (UC: n = 76; CD: n = 88; control: n = 56). The data are shown as mean ± SD, Welch’s t test. ( C ) The abundance of the 4 tRNA synthetases that are modified with queuine (asparaginyl, aspartyl, histidyl, and tyrosyl) was compared in UC patients (n = 14) and control subjects (n = 5) sourced from GEO datasets with accession number GSE9452 . ( D ) Four synthetases of tRNAs (asparaginyl, aspartyl, histidyl, tyrosyl) were compared between CD patients (n = 36) and control subjects (n = 32) sourced from ArrayExpress with accession number E-MTAB-5783. The data are shown as mean ± SD, Welch’s t test.

Article Snippet: After 24 hours of culture, queuine hydrochloride (Toronto Research Chemicals, Toronto, Canada) was added to each well at a final concentration of 5 μM for 24, 48, and 72 hours.

Techniques: Modification, Control

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Disruption to tRNA Modification by Queuine Contributes to Inflammatory Bowel Disease

doi: 10.1016/j.jcmgh.2023.02.006

Figure Lengend Snippet: Queuine treatment regulates cell proliferation and permeability. ( A ) An MTT assay showing the proliferation of control and Caco-2 BBE cells treated with queuine hydrochloride was conducted at 24, 48, and 72 hours after treatment. Data are expressed as mean ± SD, generalized linear mixed models. n = 6 per group. ( B ) The mRNA expression level of PCNA in Caco-2 BBE cells treated with queuine for 72 hours were measured by real-time PCR. Data are expressed as mean ± SD, Welch’s t test. n = 3 (one-repeat well) per group. ( C ) The protein expression of PCNA in Caco-2 BBE cells treated with queuine for 72 hours was measured by Western blotting. Data are expressed as mean ± SD, Welch’s t test. n = 4 per group. ( D ) Immunofluorescence staining of the cell proliferation marker Ki67 was performed in Caco-2 BBE cells after 72 hours of queuine treatment. The Ki67 index was calculated with the formula: Ki67-positive cells/total cells. The relative fluorescence intensity was quantified using ImageJ by counting 3 images for each sample. Data are expressed as mean ± SD, Welch’s t test. n = 3 per group. ( E ) The TEER value was measured and monitored on HT-29 cells at 24, 48, and 72 hours after treatment with queuine. Data were expressed as mean ± SD, two-way analysis of variance. n = 6 per group. ( F ) The mRNA expression levels of QTRT1, β-catenin, and claudin-5 in Caco-2 BBE cells treated with queuine for 72 hours were measured by real-time PCR. Data are expressed as mean ± SD, Welch’s t test. n = 3 (one-repeat well) per group. ( G ) The protein expression of QTRT1, β-catenin, and claudin-5 in Caco-2 BBE cells treated with queuine for 72 hours was measured by Western blotting. Data are expressed as mean ± SD, Welch’s t test. n = 3 per group. ( H ) IF staining of the cell proliferation markers QTRT1, β-catenin, and claudin-5 was performed in Caco-2 BBE cells after 72 hours of queuine treatment. The relative fluorescence intensity was quantified using ImageJ by counting 3 images from each sample. Data are expressed as mean ± SD, Welch’s t test. n = 3 per group.

Article Snippet: After 24 hours of culture, queuine hydrochloride (Toronto Research Chemicals, Toronto, Canada) was added to each well at a final concentration of 5 μM for 24, 48, and 72 hours.

Techniques: Permeability, MTT Assay, Control, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Immunofluorescence, Staining, Marker, Fluorescence

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Disruption to tRNA Modification by Queuine Contributes to Inflammatory Bowel Disease

doi: 10.1016/j.jcmgh.2023.02.006

Figure Lengend Snippet: Queuine treatment in human colonoids. ( A ) Human colon organoids from healthy subjects were treated with queuine hydrochloride and monitored for 96 hours. The volume of the organoids was calculated with the formula: volume (μm 3 ) = [(4 × π × (dimeter/2) 2 ]/3. The scale bar is 750 μm. Data are expressed as mean ± SD, two-way analysis of variance. n = 10 per group. ( B ) Mouse organoids were treated with DSS for 3 hours. Data are expressed as mean ± SD, one-way analysis of variance. n = 4 per group. ( C ) The working model of this study. Queuine base, and its corresponding nucleoside (queuosine), are produced by bacteria and salvaged by human body via the intestinal epithelium. Queuine replaces guanine at the wobble position of tRNAs and promotes efficient mRNA translation. Gut dysbiosis, one of the most common symptoms of the IBD patients, could critically impact on the functions of intestinal epithelium and queuine-to-guanine transportation, and mRNA translation. The protein expression of QTRT1, the catalytic subunit of the queuine-insertase complex, is suppressed in IBD patients. Reduced QTRT1 may lead to increased permeability and dysfunction of intestinal epithelial cells through modulating claudin-5 and β-catenin expression.

Article Snippet: After 24 hours of culture, queuine hydrochloride (Toronto Research Chemicals, Toronto, Canada) was added to each well at a final concentration of 5 μM for 24, 48, and 72 hours.

Techniques: Produced, Bacteria, Expressing, Permeability