Quantitative phenotyping of inflammatory bowel disease in the IL-10-deficient mouse by use of noninvasive magnetic resonance imaging.

BACKGROUND: Although magnetic resonance imaging (MRI) is an increasingly used diagnostic tool in the assessment of inflammatory bowel disease (IBD) in humans, diagnosis and quantitation of intestinal inflammation in animal models of IBD still depends on ex vivo techniques. The aim of this study was to evaluate whether high-field MRI is suitable for the quantitative phenotyping of gut inflammation in a dextran sulfate sodium (DSS)-triggered interleukin (IL)10-deficient (IL-10(-/-)) mouse model of IBD, especially in longitudinal studies. METHODS: Using colitis-susceptible and -resistant backgrounds, MRI and ex vivo analyses were applied to characterize this specific model, differentiating disease severity and time-dependent alterations. Colon wall thickness, cecum wall tissue intensity, spleen, and mesenteric lymph node (MLN) volumes were evaluated 1, 2, 4, and 12 weeks after disease onset by T2-weighted MRI. Ex vivo parameters included histology, spleen, and MLN weight and analysis of cytokine expression. RESULTS: MRI and ex vivo determined parameters correlated well, revealing a mouse strain-specific colitis development over time with characteristics typical for the DSS model in the initial and for the IL-10(-/-) model in the chronic phase. To evaluate the use of high-field MRI for monitoring therapeutic studies, mice with a profound colitis were treated with IL-10-producing Saccharomyces boulardii and monitored by MRI. CONCLUSIONS: MRI can be utilized to quantify colitis development in the IL-10(-/-) model of IBD. Therefore, this noninvasive technique might be highly advantageous for an individual follow-up of colitis development in chronic models of IBD, facilitating the reduction of animal numbers in this kind of research.

OXPHOS supercomplexes as a hallmark of the mitochondrial phenotype of adipogenic differentiated human MSCs.

Mitochondria are essential organelles with multiple functions, especially in energy metabolism. Recently, an increasing number of data has highlighted the role of mitochondria for cellular differentiation processes. Metabolic differences between stem cells and mature derivatives require an adaptation of mitochondrial function during differentiation. In this study we investigated alterations of the mitochondrial phenotype of human mesenchymal stem cells undergoing adipogenic differentiation. Maturation of adipocytes is accompanied by mitochondrial biogenesis and an increase of oxidative metabolism. Adaptation of the mt phenotype during differentiation is reflected by changes in the distribution of the mitochondrial network as well as marked alterations of gene expression and organization of the oxidative phosphorylation system (OXPHOS). Distinct differences in the supramolecular organization forms of cytochrome c oxidase (COX) were detected using 2D blue native (BN)-PAGE analysis. Most remarkably we observed a significant increase in the abundance of OXPHOS supercomplexes in mitochondria, emphasizing the change of the mitochondrial phenotype during adipogenic differentiation.

Saccharomyces cerevisiae porin pore forms complexes with mitochondrial outer membrane proteins Om14p and Om45p.

Numerous transport processes occur between the two mitochondrial (mt) membranes due to the diverse functions and metabolic processes of the mt organelle. The metabolite and ion transport through the mt outer membrane (OM) is widely assumed to be mediated by the porin pore, whereas in the mt inner membrane (IM) specific carriers are responsible for transport processes. Here, we provide evidence by means of Blue Native (BN)-PAGE analysis, co-immunoprecipitation, and tandem affinity purification that the two mt OM proteins Om14p and Om45p associate with the porin pore. Porin molecules seem to assemble independently to build the core unit. A subpopulation of these core units interacts with Om14p and Om45p. With preparative tandem affinity purification followed by MS analysis, we could identify interaction partners of this OM complex, which are mainly localized within the mt IM and function as carriers for diverse molecules. We propose a model for the role of the two OM proteins in addressing the porin pore to bind to specific channels in the mt IM to facilitate transport of metabolites.

Characterizing cytochrome c states--TERS studies of whole mitochondria.

Protein structures (cytochrome c) were visualized by TERS measurements on whole mitochondria referring to specific spectral features describing the electronic state of the heme moiety.

HCC1, the Arabidopsis homologue of the yeast mitochondrial copper chaperone SCO1, is essential for embryonic development.

The Arabidopsis HCC1 gene is a homologue of the copper chaperone SCO1 from the yeast Saccharomyces cerevisiae. SCO1 (synthesis of cytochrome c oxidase 1) encodes a mitochondrial protein that is essential for the correct assembly of complex IV in the respiratory chain. GUS analyses showed HCC1 promoter activity in vascular tissue, guard cells, hydathodes, trichome support cells, and embryos. HCC1 function was studied in two hcc1 T-DNA insertion lines, hcc1-1 and hcc1-2. Gametophyte development was not affected by the disruption of HCC1, but homozygous hcc1-1 and hcc1-2 embryos became arrested at various developmental stages, mostly at the heart stage. Both the wild-type HCC1 gene and the modified gene coding for the C-terminally SNAP-tagged HCC1 were able to complement the embryo-lethal phenotype of the hcc1-1 line. Localization of the SNAP-tagged HCC1 in transgenic lines identified HCC1 as a mitochondrial protein. To determine if HCC1 is a functional homologue to Sco1p, the respiratory-deficient yeast sco1 mutant was transformed with chimeric constructs containing different combinations of HCC1 and SCO1 sequences. One of the resulting chimeric proteins restored respiration in the yeast mutant. This protein had the N-terminal mitochondrial targeting signal and the single transmembrane domain derived from Sco1p and the C-terminal half (including the copper-binding motif) derived from HCC1. Growth of the complemented yeast mutant was enhanced by the addition of copper to the medium. The data demonstrate that HCC1 is essential for embryo development in Arabidopsis, possibly due to its role in cytochrome c oxidase assembly.

Interaction and interrelation of P2X7 and P2X4 receptor complexes in mouse lung epithelial cells.

P2X4 and P2X7 receptors are ATP-gated ion channels that are co-expressed in alveolar epithelial type I cells. Both receptors are localized to the plasma membrane and partly associated with lipid rafts. Here we report on our study in an alveolar epithelial cell line of the molecular organization of P2X7R and P2X4R receptors and the effect of their knockdown. Native gel electrophoresis reveals three P2X7R complexes of approximately 430, approximately 580 and approximately 760 kDa. The latter two correspond exactly in size to signals of Cav-1, the structural protein of caveolae. Interestingly knockdown of P2rx7 affects protein levels, the intracellular distribution and the supramolecular organization of Cav-1 as well as of P2X4R, which is mainly detected in a complex of approximately 430 kDa. Our data suggest upregulation of P2X4R as a compensatory mechanism of P2X7R depletion.

Ubiquinone biosynthesis in Saccharomyces cerevisiae: the molecular organization of O-methylase Coq3p depends on Abc1p/Coq8p.

Coenzyme Q is a redox-active lipid that functions as an electron carrier in the mitochondrial respiratory chain. Q-biosynthesis in Saccharomyces cerevisiae requires at least nine proteins (Coq1p-Coq9p). The molecular function of Coq8p is still unknown; however, lack of Q and the concomitant accumulation of the intermediate 3-hexaprenyl-4-hydroxybenzoic acid in the absence of Coq8p suggest an essential role in Q-biosynthesis. Localization studies identify Coq8p as a soluble mitochondrial protein, with characteristics of a protein of the matrix or associated with the inner mitochondrial membrane. Coq8p forms homomeric structure(s) as revealed by two-hybrid analysis and tandem affinity purification. Two-dimensional (2D)-Blue Native/sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis suggests that Coq8p - together with Coq2p and Coq10p - is predominantly associated with a complex of about 500 kDa, whereas Coq3p, Coq5p and Coq9p are mainly organized in a 1.3 MDa Q-biosynthesis complex that is not associated with the complex III and IV supracomplexes of the respiratory chain. Loss of Coq8p is accompanied by destabilization of Coq3p, but not of Coq9p from the 1.3 MDa Q-biosynthesis complex. This effect cannot be reversed by Q(6) supplementation. The detection of Coq3p isoforms by 2D-isoelectric focusing is in line with the proposed function of Coq8p as a kinase, with Coq3p as a target.

Yeast pyruvate dehydrogenase complex is regulated by a concerted activity of two kinases and two phosphatases.

The activity of yeast pyruvate dehydrogenase complex is regulated by reversible phosphorylation. Recently we identified two enzymes that are involved in the phosphorylation (Pkp1p) and dephosphorylation (Ppp1p) of Pda1p, the alpha-subunit of the pyruvate dehydrogenase complex. Here we provide evidence that two additional mitochondrial proteins, Pkp2p (Ygl059wp) and Ppp2p (Ycr079wp), are engaged in the regulation of this complex by affecting the phosphorylation state of Pda1p. Our data indicate complementary activities of the kinases and a redundant function for the phosphatases. Both proteins are associated with the complex. We propose a model for the role of the regulatory enzymes and the phosphorylation state of Pda1p in the assembly process of the pyruvate dehydrogenase complex.

YIL042c and YOR090c encode the kinase and phosphatase of the Saccharomyces cerevisiae pyruvate dehydrogenase complex.

In Saccharomyces cerevisiae the pyruvate dehydrogenase (PDH) complex is regulated by reversible phosphorylation of its Pda1p subunit. We here provide evidence that Pda1p is phosphorylated by the mitochondrial kinase Yil042cp. Deletion of YOR090c, encoding a putative mitochondrial phosphatase, results in a decreased PDH activity, indicating that Yor090cp acts as the corresponding PDH phosphatase. We demonstrate by means of blue native gel electrophoresis and tandem affinity purification that both enzymes are associated with the PDH complex.

Saccharomyces cerevisiae translational activator Cbs1p is associated with translationally active mitochondrial ribosomes.

In the yeast Saccharomyces cerevisiae, mitochondrial translation of most, if not all, mitochondrially encoded genes is regulated by an individual set of gene-specific activators. Translation of the COB mRNA encoding cytochrome b requires the function of two nuclearly encoded proteins, Cbs1p and Cbs2p. Genetic data revealed that the 5'-untranslated region of COB mRNA is the target of both proteins. Recently, we provided evidence for an interaction of Cbs2p with mitochondrial ribosomes. We demonstrate here by means of blue native gel electrophoresis, density gradient centrifugation and tandem affinity purification that a portion of Cbs1p is also associated with mitochondrial ribosomes. In addition, we demonstrate that the amount of ribosome-associated Cbs1p is elevated in the presence of chloramphenicol, which is known to stall ribosomes on mRNAs. In the presence of puromycin, which strips off the mRNA and nascent protein chains from ribosomes, Cbs1p is no longer associated with ribosomes. Our data indicate that the observed interaction is mediated by ribosome-bound mRNA, thus restricting the association to ribosomes actively translating cytochrome b.

Saccharomyces cerevisiae translational activator Cbs2p is associated with mitochondrial ribosomes.

A characteristic feature of the mitochondrial expression system in Saccharomyces cerevisiae is the requirement for gene-specific translational activator proteins. Translation of mitochondrial apocytochrome b mRNA requires the nucleus-encoded proteins Cbs1p and Cbs2p. These proteins are thought to tether cytochrome b mRNA to the mitochondrial inner membrane via binding to the 5' untranslated mRNA leader. Here, we demonstrate by the use of affinity chromatography and coimmunoprecipitation that Cbs2p interacts with the mitoribosomes. We further provide evidence that the C-terminus of Cbs2p is important for ribosome association, while the N-terminal portion is essential for the formation of homomeric structures.