Journal: Human mutation

Article Title: Cellular pathophysiological consequences of BCS1L mutations in mitochondrial complex III enzyme deficiency.

doi: 10.1002/humu.21294

Figure Lengend Snippet: Figure 1. A: Schematic representation of the BCS1L cDNA (GenBank reference sequence NM_004328.4), slightly modified from Fernandez- Vizarra et al. [2007]. Amino acid or nucleotide changes found in the patients (P1–P6) are shown with arrows. Noncoding exons are indicated as 1 and 2. Coding exons representative of the BCS1L open-reading frame are indicated as 1 to 7. The numbered bar indicates the amino acid position along the protein sequence. The ATG 11 translation initiation site is indicated in gray with an arrow. The BCS1L functional domains, deduced by homology with yeast bcs1, are indicated as follows: TMD, transmembrane domain; MTS, mitochondrial targeting sequence; IAS, import auxiliary sequence; BCS1L, activity and stability domain; AAA-ATPase, AAA-protein family specific ATPase domain. B: Effect of the BCS1L mutations on cell growth. Cells were plated on P10 plates at 5 105 cells per plate in glucose-containing media and counted on a daily basis for 4 days. Each data point represents the mean7SD of values obtained from each cell line, tested in triplicate. C1–C2, control fibroblasts. P1–P6, patients’ fibroblasts.

Article Snippet: Western blot analyses were performed with antibodies raised against the following proteins: polyclonal BCS1L (ProteinTech Group, Chicago, IL); monoclonal BCS1L (Abnova, Taipei City, Taiwan); mitochondrial complex I NDUFA9 and NDUFS3 subunits, complex II SDHA subunit, complex III RISP, and core 2 protein, and complex IV COX2 and COX5A subunits (Molecular Probes, Eugene, OR); DRP1 and OPA1 (BD Biosciences, Enzyme activities measured in our laboratory are expressed as nmol min 1 mg 1 protein.

Techniques: Sequencing, Modification, Functional Assay, Activity Assay, Control

Journal: Human mutation

Article Title: Cellular pathophysiological consequences of BCS1L mutations in mitochondrial complex III enzyme deficiency.

doi: 10.1002/humu.21294

Figure Lengend Snippet: Figure 2. A: Western blot analysis of the BCS1L protein and mitochondrial respiratory chain subunits. Samples corresponding to 30 mg protein from whole fibroblast lysates were run through 10% SDS–PAGE gels and subsequently blotted. The levels of the BCS1L protein and the different respiratory chain subunits were detected using antibodies against the indicated proteins. The signal corresponding to b-actin was used for normalization. B: Subcellular fractionations were performed in control and patients’ fibroblasts, and 15 mg protein from cytosolic fractions were subject to Western blot analysis. C: To evaluate the expression and assembly of the intramitochondrial BCS1L protein, BN–PAGE analysis was performed with mitochondrial-enriched fractions from control and BCS1L-mutated fibroblasts, followed by Western blot analysis using an antibody against BCS1L. The approximate molecular masses of three detected BCS1L complexes are indicated. D: Assembly of the mitochondrial respiratory chain complexes was analyzed with 40 mg of digitonin-isolated mitochondria on 4–15% blue native gels. A complex I in-gel activity (IGA) assay is shown in the upper panel. Duplicate gels were subject to Western blot analysis using antibodies against the following OXPHOS subunits: complex I (CI) NDUFS3, complex III (CIII) Rieske Iron-Sulfur protein (RISP), complex IV (CIV) subunit COX5A, and complex II (CII) SDHA subunit. C1–C3, controls. P1–P6, patients.

Article Snippet: Western blot analyses were performed with antibodies raised against the following proteins: polyclonal BCS1L (ProteinTech Group, Chicago, IL); monoclonal BCS1L (Abnova, Taipei City, Taiwan); mitochondrial complex I NDUFA9 and NDUFS3 subunits, complex II SDHA subunit, complex III RISP, and core 2 protein, and complex IV COX2 and COX5A subunits (Molecular Probes, Eugene, OR); DRP1 and OPA1 (BD Biosciences, Enzyme activities measured in our laboratory are expressed as nmol min 1 mg 1 protein.

Techniques: Western Blot, SDS Page, Control, Expressing, Isolation, Activity Assay, IgA Assay

Journal: Human mutation

Article Title: Cellular pathophysiological consequences of BCS1L mutations in mitochondrial complex III enzyme deficiency.

doi: 10.1002/humu.21294

Figure Lengend Snippet: Figure 3. Mitochondrial morphology in control and BCS1L-mutated fibroblasts grown in normal glucose media. A: The mitochondrial morphology of fixed cells was analyzed by immunofluorescence using an anticomplex V a-subunit antibody. C1–C2, controls. P1–P6, BCS1L- mutated fibroblasts. Scale bar 5 20 mm. B: Different shapes of the mitochondrial network. (a, b) Depict controls with the classic filamentous mitochondrial network. The most common shapes found among patients’ fibroblasts included dense entangled perinuclear mitochondrial networks (c, d), intermediate shapes with elongated and short mitochondrial tubules (e, f), circular structures (g), small tubules with short branches (h, i, j), vesicles (k), and long fragmented mitochondria (l). C: The proportions of cells with essentially elongated filamentous mitochondria (% elongated), with intermediate mitochondrial morphology (% intermediate), or with only punctuate mitochondria (% punctuate) were determined. Data are presented as mean7SD of the analysis of 4400 cells from each patient and five controls. D: Mitochondrial diameter in mutant fibroblasts grown in normal glucose medium. The results shown are the means7SD of at least 200 determinations from each cell line. One-way ANOVA analysis: a significant main effect (Po0.001) was found. Scheffe´ test post hoc: Po0.01, significantly different from controls.

Article Snippet: Western blot analyses were performed with antibodies raised against the following proteins: polyclonal BCS1L (ProteinTech Group, Chicago, IL); monoclonal BCS1L (Abnova, Taipei City, Taiwan); mitochondrial complex I NDUFA9 and NDUFS3 subunits, complex II SDHA subunit, complex III RISP, and core 2 protein, and complex IV COX2 and COX5A subunits (Molecular Probes, Eugene, OR); DRP1 and OPA1 (BD Biosciences, Enzyme activities measured in our laboratory are expressed as nmol min 1 mg 1 protein.

Techniques: Control, Immunofluorescence, Mutagenesis

Journal: Human mutation

Article Title: Cellular pathophysiological consequences of BCS1L mutations in mitochondrial complex III enzyme deficiency.

doi: 10.1002/humu.21294

Figure Lengend Snippet: Figure 5. Increased ROS production and altered expression of the cellular antioxidant defenses in the BCS1L-mutated fibroblasts. Hydrogen peroxide levels were analyzed in live cells (A) by confocal microscopy or (B) by flow cytometry, using DCF-DA as a probe. C, Control without DCF-DA incubation; C, control incubated with DCF-DA; P1–P6, patients’ fibroblasts incubated with DCF-DA. C: Unbalanced expression of the cellular antioxidant defenses. 30 mg of total protein from cell lysates were run through 10% SDS–PAGE gels and immunoblotted using the indicated antibodies. MnSOD, mitochondrial superoxide dismutase. CAT, catalase. GPx, gluthatione peroxidase. GR, gluthatione reductase. C1, C2, controls. P1–P6, patients. D: The signals from three independent blots were quantified and the densitometric values were normalized with the anti-b-actin antibody. (a.u.), arbitrary units. Data are presented as mean7SD of the analysis of individual patients’ fibroblasts and five different controls.

Article Snippet: Western blot analyses were performed with antibodies raised against the following proteins: polyclonal BCS1L (ProteinTech Group, Chicago, IL); monoclonal BCS1L (Abnova, Taipei City, Taiwan); mitochondrial complex I NDUFA9 and NDUFS3 subunits, complex II SDHA subunit, complex III RISP, and core 2 protein, and complex IV COX2 and COX5A subunits (Molecular Probes, Eugene, OR); DRP1 and OPA1 (BD Biosciences, Enzyme activities measured in our laboratory are expressed as nmol min 1 mg 1 protein.

Techniques: Expressing, Confocal Microscopy, Flow Cytometry, Control, Incubation, SDS Page