Liver mtDNA content increases during development: a comparison of methods and the importance of age- and tissue-specific controls for the diagnosis of mtDNA depletion

BACKGROUND: The quantitative loss of mitochondrial DNA (mtDNA) known as mtDNA depletion, often gives rise to liver disease. The diagnosis of mtDNA depletion syndrome is frequently imprecise, both for technical reasons and because of the lack of established age-adjusted normal ranges. We aimed to refine quantitative methods for diagnosing the hepatic type of mtDNA depletion syndrome, firstly by establishing an age-matched reference range for mitochondrial to nuclear DNA ratio (henceforth "mtDNA content") and secondly by investigating mtDNA in fibroblasts. METHODS: By comparing realtime PCR with an established method for quantifying mtDNA content we established a reference range for young children using biopsy and post-mortem material from patients <15 years. In addition, we investigated the arrangement of mtDNA in nucleoids from fibroblasts using fluorescence microscopy. RESULTS: Both methods showed that the mtDNA content of liver increases rapidly over the perinatal period. In a patient whose liver mtDNA content fell, but remained within the reference range, early investigation and age-matched controls were essential, as we found a progressive increase in muscle mtDNA copy number, respiratory chain activity and muscle power with age. In three further patients, fluorescence microscopy of the fibroblasts proved diagnostic. In one case a movement disorder was an important pointer. CONCLUSIONS: These cases highlight the (i) need for comparing mtDNA copy number data generated from patients to DNA isolated from an age-matched normal range from the tissue of interest and (ii) the utility of mtDNA staining with PicoGreen as a method to detect aberrant nucleoid morphology in mtDNA depletion patient fibroblast lines when affected tissues are not available for measuring mtDNA copy number.     

Resistance Gene Analog Polymorphism (RGAP) Markers Co-Localize with Disease Resistance Genes and QTL in Common Bean

Resistance (R) genes containing nucleotide-binding site (NBS)-leucine rich repeats (LRR) are the most prevalent types of R gene in plants. The objective of this study was to develop PCR-based R-gene analog polymorphism (RGAP) markers for common bean (Phaseolus vulgaris L). Twenty degenerate primers were designed from the conserved kinase-1a (GVGKTT) and hydrophobic domains (GLPLAL) of known NBS-LRR type R-genes and from EST databases. Sixty-six of the 100 primer combinations tested yielded polymorphism. Thirty-two RGAP markers were mapped in the BAT 93/Jalo EEP558 core mapping population for common bean. The markers mapped to 10 of 11 linkage groups with a strong tendency for clustering. In addition, the RGAP markers co-located, on six linkage groups, with 15 resistance gene analogs (RGAs) that were previously mapped in other populations of common bean. The distance between the priming sites in NBS-LRR type R-genes is around 500 bp. Of the 32 RGAP markers, 19 had sizes larger and 13 less than 500 bp. RGAP markers mapped close to known R-genes on B11, and to QTLs for resistance on B1, B2, B6, B7, B8, B10, and B11. RGAP appears to provide a useful marker technique for tagging and mapping R-genes in segregating common bean populations, discovery of candidate genes underlying resistance QTL, and future cloning of R-genes in common bean.     

Colonization with Heligmosomoides polygyrus suppresses mucosal IL-17 production

Helminth exposure appears to protect hosts from inappropriate inflammatory responses, such as those causing inflammatory bowel disease. A recently identified, strongly proinflammatory limb of the immune response is characterized by T cell IL-17 production. Many autoimmune type inflammatory diseases are associated with IL-17 release. Because helminths protect from these diseases, we examined IL-17 production in helminth-colonized mice. We colonized mice with Heligmosomoides polygyrus, an intestinal helminth, and analyzed IL-17 production by lamina propria mononuclear cells (LPMC) and mesenteric lymph node (MLN) cells. Colonization with H. polygyrus reduces IL-17A mRNA by MLN cells and inhibits IL-17 production by cultured LPMC and MLN cells. Helminth exposure augments IL-4 and IL-10 production. Blocking both IL-4 and IL-10, but not IL-10 alone, restores IL-17 production in vitro. Colonization of colitic IL-10-deficient mice with H. polygyrus suppresses LPMC IL-17 production and improves colitis. Ab-mediated blockade of IL-17 improves colitis in IL-10-deficient mice. Thus, helminth-associated inhibition of IL-17 production is most likely an important mechanism mediating protection from inappropriate intestinal inflammation.