After the introduction of mitochondria with a mixture of mutant and wild-type mitochondrial DNA (mtDNA) into a human rho degree cell line (143B.206), Yoneda et al. [Yoneda, M., Chomyn, A., Martinuzzi, A., Hurko, O. & Attardi, G. (1992) Proc. Natl. Acad. Sci. USA 89, 11164-11168] observed a shift in the proportion of the two mitochondrial genotypes in a number of cybrid clones. In every case where a shift was observed, there was an increase in the proportion of mutant mtDNA. By using the same cell line (143B.206 rho degree), we also generated cybrids that were either stable in their mitochondrial genotype or showed an increase in the proportion of mutant mtDNA. However, temporal analysis of the same mutant mtDNA type in another rho degree cell line revealed a quite distinct outcome. Those clones that showed a change shifted toward higher levels of wild-type rather than mutant mtDNA. These results indicate that the nuclear genetic background of the recipient (rho degree) cell can influence the segregation of mutant and wild-type mitochondrial genomes in cell cybrids.
Cardiovascular disease (CVD) remains the major cause of excess mortality in patients with non-alcoholic fatty liver disease (NAFLD). The aim of this study was to investigate the individual contribution of NAFLD to CVD risk factors in the absence of pathogenic influences from other comorbidities often found in NAFLD patients, by using an established in-vitro model of hepatic steatosis.Histopathological events in non-alcoholic fatty liver disease were recapitulated by focused metabolic nutrient overload of hepatoblastoma C3A cells, using oleate-treated-cells and untreated controls for comparison. Microarray and proteomic data from cell culture experiments were integrated into a custom-built systems biology database and proteogenomics analysis performed. Candidate genes with significant dysregulation and concomitant changes in protein abundance were identified and STRING association and enrichment analysis performed to identify putative pathogenic pathways.The search strategy yielded 3 candidate genes that were specifically and significantly up-regulated in nutrient-overloaded cells compared to untreated controls: fibrinogen alpha chain (2.2 fold), fibrinogen beta chain (2.3 fold) and fibrinogen gamma chain (2.1 fold) (all rank products pfp <0.05). Fibrinogen alpha and gamma chain also demonstrated significant concomitant increases in protein abundance (3.8-fold and 2.0-fold, respectively, p <0.05).In-vitro modelling of NAFLD and reactive oxygen species formation in nutrient overloaded C3A cells, in the absence of pathogenic influences from other comorbidities, suggests that NAFLD is an isolated determinant of CVD. Nutrient overload-induced up-regulation of all three fibrinogen component subunits of the coagulation cascade provides a possible mechanism to explain the excess CVD mortality observed in NAFLD patients.
Oxidative stress is the central to molecular events leading to the progression of simple steatosis to steatohepatitis in nonalcoholic fatty liver disease (NAFLD). We have previously shown that an in vitro cellular steatosis model using C3A cells treated with energy substrates; lactate (L), pyruvate (P), octanoate (O) and ammonia (N), recapitulates the sequence of events in dietary-induced NAFLD; namely enhanced acute respiration and reactive oxygen species (ROS) formation leading to mitochondrial impairment. In contrast, treatment with oleate results in similar triglyceride accumulation but with relatively low ROS. Using a combined microarray, proteomic and metabolomic approach, we aimed to explore how triglyceride accumulation and enhanced ROS affect glutathione metabolism in our in vitro cell model.
Methods
C3A cells were treated with either LPON or oleate for 72 hours. Microarray RNA expression was measured using Illumina® Whole Human Genome BeadChip H12 Microarray. For proteomics, peptides were analysed by liquid chromatography (LC) coupled mass spectrometry (MS) (Agilent HPLC/OrbitrapXL). Data were quantified label-free using Progenesis LC-MS and MASCOT. For metabolomics, LC separation was performed using hydrophilic interaction chromatography with a ZIC–HILIC. MS was performed using Orbitrap Exactive with HESI 2 probe. Raw LC/MS data were processed with XCMS Centwave and mzMatch.
Results
LPON led to 2-fold downregulation of GCLC (encodes glutamate-cysteine ligase catalytic subunit, the rate limiting enzyme for glutathione synthesis) and upregulation of GPX1 and TXNDC12. Expression of GCLC and TXNDC12 was unchanged with oleate. Metabolomics confirmed that oxidised glutathione, glutathione disulfide, was higher in LPON- than oleate-treated cells. Among glutathione S-transferase genes, GSTA1 was unchanged with oleate but was upregulated by LPON (2.4-fold). Similarly, GSTT1, GSTK1 and GSTO1 were significantly increased by LPON. In contrast, MGST2 expression was higher in oleate than LPON-treated cells. Finally, proteomics showed that microsomal glutathione S-transferase 2 was downregulated by 2.5-fold by LPON.
Conclusion
Our data show that increased ROS formation rather than triglyceride accumulation alters glutathione metabolism. Such alterations may influence susceptibility to further insults, particularly those accelerating glutathione depletion, for example, paracetamol overdose.
Wiki technology has become a ubiquitous mechanism for dissemination of information, and places strong emphasis on collaboration. We aimed to leverage wiki technology to allow small groups of researchers to collaborate around a specific domain, for example a biological pathway. Automatically gathered seed data could be modified by the group and enriched with domain specific information. We describe a software system, BioKb, implemented as a plugin for the TWiki engine, and designed to facilitate construction of a field-specific wiki containing collaborative and automatically generated content. Features of this system include: query of publicly available resources such as KEGG, iHOP and MeSH, to generate 'seed' content for topics; simple definition of structure for topics of different types via an administration page; and interactive incorporation of relevant PubMed references. An exemplar is shown for the use of this system, in the creation of the RAASWiki knowledgebase on the renin-angiotensin-aldosterone system (RAAS). RAASWiki has been seeded with data by use of BioKb, and will be the subject of ongoing development into an extensive knowledgebase on the RAAS. The BioKb system is available from http://www.bioinf.mvm.ed.ac.uk/twiki/bin/view/TWiki/BioKbPlugin as a plugin for the TWiki engine.
We have studied the dynamics of mitochondrial DNA maintenance and segregation in human cells using serial cybrid transfer of partially duplicated mitochondrial DNA, from a mitochondrial myopathy patient, to two distinct recipient cell types. The results indicate two radically different outcomes dependent upon nuclear background. In one case (lung carcinoma) there is systematic loss of the partial duplication by an implied recombinational mechanism. In another nuclear background (osteosarcoma) the duplicated molecules can survive, having only a marginal effect on mitochondrial respiratory function. Moreover, in the osteosarcoma nuclear background further disturbances of mtDNA maintenance frequently follow from cybrid transfer. These are progressive, catastrophic loss of mtDNA and further rearrangement to generate partially triplicated molecules. The results imply differential expression of nuclear genes regulating mtDNA copy number, replication and recombination in different human cell types.
Type 2 diabetes ultimately results from pancreatic β-cell failure. Abnormally elevated intracellular regeneration of glucocorticoids by the enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) in fat or liver may underlie pathophysiological aspects of the metabolic syndrome. Elevated 11β-HSD1 is also found in pancreatic islets of obese/diabetic rodents and is hypothesized to suppress insulin secretion and promote diabetes. To define the direct impact of elevated pancreatic β-cell 11β-HSD1 on insulin secretion, we generated β-cell-specific, 11β-HSD1-overexpressing (MIP-HSD1) mice on a strain background prone to β-cell failure. Unexpectedly, MIP-HSD1(tg/+) mice exhibited a reversal of high fat-induced β-cell failure through augmentation of the number and intrinsic function of small islets in association with induction of heat shock, protein kinase A, and extracellular signal-related kinase and p21 signaling pathways. 11β-HSD1(-/-) mice showed mild β-cell impairment that was offset by improved glucose tolerance. The benefit of higher β-cell 11β-HSD1 exhibited a threshold because homozygous MIP-HSD1(tg/tg) mice and diabetic Lep(db/db) mice with markedly elevated β-cell 11β-HSD1 levels had impaired basal β-cell function. Optimal elevation of β-cell 11β-HSD1 represents a novel biological mechanism supporting compensatory insulin hypersecretion rather than exacerbating metabolic disease. These findings have immediate significance for current therapeutic strategies for type 2 diabetes.
Understanding fundamental molecular mechanisms that govern the transmigration and interstitial migration of leukocytes to sites of tissue damage and infection is of potential significance in identifying novel therapeutic targets for the management of chronic inflammatory disorders. CD31 is a mammalian cell adhesion molecule that regulates the recruitment of leukocytes from the circulation. Our recent unpublished work has suggested that homophilic ligation of CD31 can negatively regulate the ether-à-go-go-related gene (ERG) current within leukocytes to regulate cell-cell adhesion. To validate and probe the functional significance of ERG in leukocytes, we developed an infected wound model of inflammation in zebrafish and assessed the efficacy of two ERG-specific inhibitors, dofetilide and E4031, as well as an ERG-specific antisense RNA morpholino on neutrophil recruitment. Our data confirm a hitherto undescribed role for ERG in leukocytes, where inhibition or translational knockdown of ERG resulted in significant attenuation of the inflammatory response to an infectious stimulus. Inhibition of ERG was verified independently by a decrease in the ventricular heart rate, where ERG also functions in the repolarization of the cardiac action potential. Our results suggest that ERG-specific Class III antiarrhythmic drugs can modulate inflammatory responses to infection.
EXCESS’ MODEL OF NAFLD IN HUMAN HEPATOCYTES 1Leonard J Nelson , 1Philipp Treskes, 2Donald R Dunbar, 2Jonathan R Manning, 1Ann Lockman, 1Steve Morley, 1,3Peter C Hayes, 1,3John N Plevris. 1University of Edinburgh Hepatology Laboratory, Chancellor’s Building; 2Bioinformatics Team, University/BHF Centre for Cardiovascular Science, University of Edinburgh; & 3The Royal Infirmary of Edinburgh. EDINBURGH, SCOTLAND, UK
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