Two-dimensional Blue Native Gel Electrophoresis Research Articles

Nicotinic Receptor Subunit α5 Modifies Assembly, Up-regulation, and Response to Pro-inflammatory Cytokines

In the mammalian brain high affinity nicotine-binding sites are composed of at least the alpha4 and beta2 subunits. Additional nicotinic acetylcholine receptor subunits that are often co-expressed with alpha4+beta2 include alpha5. The introduction of alpha5 into 293 cells expressing alpha4+beta2 strongly favors assembly of alpha4+alpha5+beta2 receptors, increases constitutive ligand binding density as measured using [(3)H]epibatidine, but reduces the magnitude of up-regulation in response to chronic nicotine. In contrast, when beta4 is substituted for beta2, alpha5 interferes with the assembly of these receptors, demonstrating an important role for the beta subunit in this process. When cells co-express alpha4+alpha5+beta2+beta4, over 50% of the subunit associations include all four subunits, but they fail to be detected using [(3)H]epibatidine binding. However, complexes of alpha4+alpha5+beta2 do preferentially emerge from these subunit mixtures, and these mixtures bind ligand. In previous studies of alpha4+beta2+beta4 co-expression by 293 cells, the inflammatory cytokines IL-1beta and TNFalpha influenced the outcome of receptor assembly (Gahring, L. C., Days, E. L., Kaasch, T., González de Mendoza, M., Owen, L., Persiyanov, K., and Rogers, S. W. (2005) J. Neuroimmunol. 166, 88-101). When alpha5 is included in this subunit mixture, and cells are exposed to either inflammatory cytokine, subunit association is no longer altered. These findings suggest that alpha5 is an influential modulator of alpha4+beta2 nicotinic acetylcholine receptor assembly and stabilizes their expression in response to fluctuations in external conditions.

Exploring Rat Plasmatic Proteomes: What Triggered the Liver Regeneration? (Supplementary materials)

To further clarify the priming mechanism of liver regeneration, proteins and protein complexes from rat plasma (normal group, partial hepatectomy (PHx) group and sham-operation group) were comparatively studied by two-dimensional gel electrophoresis and two-dimensional blue native gel electrophoresis. Our results suggested that Kupffer cell--NF-kappaB/ROS might trigger the liver regeneration.

Two-dimensional Blue Native/SDS Gel Electrophoresis of Multiprotein Complexes from Helicobacter pylori

The study of protein interactions constitutes an important domain to understand the physiology and pathogenesis of microorganisms. The two-dimensional blue native/SDS-PAGE was initially reported to analyze membrane protein complexes. In this study, both cytoplasmic and membrane complexes of a bacterium, the strain J99 of the gastric pathogen Helicobacter pylori, were analyzed by this method. It was possible to identify 34 different proteins grouped in 13 multiprotein complexes, 11 from the cytoplasm and two from the membrane, either previously reported partially or totally in the literature. Besides complexes involved in H. pylori physiology, this method allowed the description of interactions involving known pathogenic factors such as (i) urease with the heat shock protein GroEL or with the putative ketol-acid reductoisomerase IlvC and (ii) the cag pathogenicity island CagA protein with the DNA gyrase GyrA as well as insight on the partners of TsaA, a peroxide reductase/stress-dependent molecular chaperone. The two-dimensional blue native/SDS-PAGE combined with mass spectrometry is a potential tool to study the differences in complexes isolated in various situations and also to study the interactions between bacterial and eucaryotic cell proteins.

X‐linked NDUFA1 gene mutations associated with mitochondrial encephalomyopathy

Mitochondrial complex I deficiency is the commonest diagnosed respiratory chain defect, being genetically heterogeneous. The male preponderance of previous patient cohorts suggested an X-linked underlying genetic defect. We investigated mutations in the X-chromosomal complex I structural genes, NDUFA1 and NDUFB11, as a novel cause of mitochondrial encephalomyopathy. We sequenced 12 nuclear genes and the mitochondrial DNA-encoded complex I genes in 26 patients with respiratory chain complex I defect. Novel mutations were confirmed by polymerase chain reaction restriction length polymorphism. Assembly/stability studies in fibroblasts were performed using two-dimensional blue native gel electrophoresis. Two novel p.Gly8Arg and p.Arg37Ser hemizygous mutations in NDUFA1 were identified in two unrelated male patients presenting with Leigh's syndrome and with myoclonic epilepsy and developmental delay, respectively. Two-dimensional blue native gel electrophoresis showed decreased levels of intact complex I with no accumulation of lower molecular weight subcomplexes, indicating that assembly, stability, or both are compromised. Mutations in the X-linked NDUFA1 gene result in complex I defect and encephalomyopathy. Assembly/stability analysis might give an explanation for the different clinical phenotypes and become useful for future diagnostic purposes.

SURFEIT-1 Gene Analysis and Two-Dimensional Blue Native Gel Electrophoresis in Cytochrome c Oxidase Deficiency

Leigh syndrome, a progressive, often fatal, neurodegenerative disorder, is frequently associated with a deficiency in the activity of cytochrome c oxidase (COX), the last enzyme of the mitochondrial respiratory chain. In contrast to NADH:ubiquinone oxidoreductase and succinate dehydrogenase deficiencies, no mutations in nuclear genes encoding COX subunits have been identified thus far. Very recently, however, a Leigh syndrome complementation group has been identified which showed mutations in the SURFEIT-1 (SURF-1) gene. The results of a mutational detection study in 16 new randomly selected COX-deficient patients revealed a new mutation (C688T) in 2 patients and the earlier reported 845delCT mutation in 2 additional patients. In addition, we evaluated the diagnostic value of two-dimensional blue native gel electrophoresis. We show that this technique reveals distinct patterns of both fully and partially assembled COX complexes and is thereby capable of discrimination between COX-deficient SURF-1 and non-SURF-1-mutated patients.