Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Proline Absorption and SGK1 Expression are Inhibited in Intestinal Tis7 Transgenic Mice.

doi: 10.1159/000443094

Figure Lengend Snippet: Fig. 6. Reduced intestinal apical membrane expression of the proline-specific transporter SLC6A20 in tis7tg mice. (A) Immunoblot analysis of total cellular expression of the intestinal brush border protein sucra se-isomaltase (SI), and transporters for Glc (SGLT1), Pro (SLC6A20 and SLC36A1) and Leu (SLC6A19) in the supernatant of jejunum mucosa. (B) Expression in the apical membrane-bound fraction isolated through cell-surface biotinylation and analyzed by immunoblot. β-actin and SGLT1 served as the respective loading controls. Expression was quantified as the normalized densities of protein bands, as in (C) and (D), respecti vely. The inserted chart in (C) is a scale-adjusted quantification of SI, SGLT1, SLC6A19 and SLC6A20 protein expression. * p ≤ 0.05 WT vs. Tis7tg jejunum ; error bars represent the SEM.

Article Snippet: Anti-pThr256-SGK (sc16744R), anti-β-actin (sc47778), anti-SGLT1 (sc98974), anti-sucrase-isomaltase (SI) (sc27603), anti-SLC6A20 (sc134762), antiSLC6A19 (sc160812), anti-SLC36A1 (sc368553) antibody and donkey anti-goat IgG-HRP (sc-2020) were from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Membrane, Expressing, Western Blot, Isolation

Journal: Bioorganic & medicinal chemistry

Article Title: A novel assay revealed that ribonucleotide reductase is functionally important for interstrand DNA crosslink repair

doi: 10.1016/j.bmc.2015.09.045

Figure Lengend Snippet: RNR inhibitors inhibit both replication-dependent and -independent ICLR. A) The reporter plasmid (pGL-ICL) containing no LgT-SV40ori to specifically measure replication-independent ICLR activity. ICLR activity in GM04312 (XPA (−)) cells (B) and in GM15876 (XPA (+)) cells (C), measured using pGL(LgT-SV40ori)-ICL (Figure 1A)/ pRL(LgT-SV40ori) (Figure 1B) or pGL-ICL (Figure 3A)/ pGL4.75 (identical to pRL containing no LgT-SV40ori, control for inhibition of reporter generation by the CMV promoter, see Methods) in the absence or presence of gemcitabine (1 μM). Absence of XPA prevents replication-independent ICLR. Accordingly, no ICLR activity was observed in GM04312 for pGL-ICL. Both gemcitabine (D) and 3-AP (E) showed ICLR inhibition equipotent for pGL(LgT-SV40ori)-ICL and pGL-ICL in HT1080 cells. Error bars represent standard deviation (n=3).

Article Snippet: GM04312 ( 8 ), GM15876, and GM08437 (XP2YO[SV] ( 9 )) cells were obtained from Coriell Institute Biorepository (Camden, NJ).

Techniques: Plasmid Preparation, Activity Assay, Control, Inhibition, Standard Deviation

Journal: Human mutation

Article Title: ERCC4 Variants Identified in a Cohort of Patients with Segmental Progeroid Syndromes

doi: 10.1002/humu.23367

Figure Lengend Snippet: ERCC4 variants in two unrelated segmental progeroid syndrome probands.

Article Snippet: Cases of CS without any features of XP are quite rare among ERCC4 mutants ( Kashiyama, et al., 2013 ).

Techniques:

Journal: Human mutation

Article Title: ERCC4 Variants Identified in a Cohort of Patients with Segmental Progeroid Syndromes

doi: 10.1002/humu.23367

Figure Lengend Snippet: Characteristics of the four ERCC4 variants identified in the proband

Article Snippet: Cases of CS without any features of XP are quite rare among ERCC4 mutants ( Kashiyama, et al., 2013 ).

Techniques: Variant Assay

Journal: Human mutation

Article Title: ERCC4 Variants Identified in a Cohort of Patients with Segmental Progeroid Syndromes

doi: 10.1002/humu.23367

Figure Lengend Snippet: Mutation analyses of ERCC4 mRNA in the two probands

Article Snippet: Cases of CS without any features of XP are quite rare among ERCC4 mutants ( Kashiyama, et al., 2013 ).

Techniques: Mutagenesis

Journal: Human mutation

Article Title: ERCC4 Variants Identified in a Cohort of Patients with Segmental Progeroid Syndromes

doi: 10.1002/humu.23367

Figure Lengend Snippet: Expression analysis of XPF-ERCC1 complex. (A) Levels of full length mRNAs are shown for ERCC4/XPF and ERCC1 as measured by qRT-PCR in CALIF1010 fibroblasts. C5R0 is used as a normal control and set to 1. XP51RO is from a patient with XFE progeroid syndrome caused by a mutation in ERCC4. (B) For MME1010 LCL, GM03798 was used as a normal control and set to 1.0. LB313 cells are from an XP-F patient with low but detectable NER capacity. The bar graphs represent the mean values ± SD of the 6 reactions carried out for each gene. Statistical analysis (One-way ANOVA post hoc test) showed no significant differences in ERCC4 or ERCC1 mRNA levels among patients and control cell lines. (C) Western blot analysis of nuclear fractions of cells of patients’ LCL and fibroblast cells showing levels of ERCC4, LMNA and β-actin. LMNA used was used as a marker for nuclei and β-actin was utilized as an additional loading control. Control 1 and control 2 correspond to extracts from control LCLs and fibroblasts, respectively. (D) Immunodetection of ERCC4 and ERCC1 in healthy donor fibroblasts (C5RO) and cells from an ERCC4 progeroid patient (XP51RO) and the new CALIF1010 patient. GAPDH was used as the loading control. Both ERCC4 and ERCC1 protein levels are substantially lower in both progeroid patients compared to controls.

Article Snippet: Cases of CS without any features of XP are quite rare among ERCC4 mutants ( Kashiyama, et al., 2013 ).

Techniques: Expressing, Quantitative RT-PCR, Control, Mutagenesis, Western Blot, Marker, Immunodetection

Journal: Human mutation

Article Title: ERCC4 Variants Identified in a Cohort of Patients with Segmental Progeroid Syndromes

doi: 10.1002/humu.23367

Figure Lengend Snippet: Distribution of the reported ERCC4 variants and conservation of the Gln496, Ala860, and Arg799 amino acids throughout evolution. (A) The structural domains in ERCC4 and the locations of amino acid alterations in previously reported patients. XP pathogenic variants are grouped above the functional domains. Pathogenic variants reported in other conditions are shown above the XP variants along with the corresponding diagnosis. ERCC4 includes an N-terminal disrupted helicase domain and a nuclease domain. The C-terminal domain, including the nuclease domain and helix-hairpin-helix (HhH) motifs, shows similarity to that of ERCC1. Double-lined squares show the variants found in MME1010 and CALIF1010. (B) Conservation of the altered amino acids. Amino acids colored in gray indicate conservation between groups of highly similar properties. (C) A proposed model for the impact of the MME1010 patient’s variant residing in the HhH domain of ERCC4. This C-terminal part of the protein is critical for interaction with ERCC1 and for heteroduplex stability. ERCC4/XPF in green. ERCC1 is represented in blue. The red spheres represent the mutant residue Asp860. The overlapping stick representation in orange represents the wildtype residue Ala860 superimposed, illustrating little structural deformity caused by the variant. The model was generated using Mutagenesis wizard of Pymol visualization software. The model was calculated on HADDOCK web server for electrostatics, desolvation and van der Waals energy refinement (van Dijk, et al., 2012; van Zundert, et al., 2016)

Article Snippet: Cases of CS without any features of XP are quite rare among ERCC4 mutants ( Kashiyama, et al., 2013 ).

Techniques: Functional Assay, Biomarker Discovery, Variant Assay, Mutagenesis, Residue, Generated, Software