A Fluorescence Detection and Selection Device for Dna-Molecules Bound to Microspheres

Abstract

A device for the detection and selection of DNA-molecules bound to microspheres has been realized. It is based on a quartz chip with a capillary flowsystem, and confocal fluorescence detection of DNA. The device is to be used to identify and isolate single DNA-molecules for sequencing experiments.     

Cardiac oxidative stress in a mouse model of neutral lipid storage disease

Cardiac oxidative stress has been implicated in the pathogenesis of hypertrophy, cardiomyopathy and heart failure. Systemic deletion of the gene encoding adipose triglyceride lipase (ATGL), the enzyme that catalyzes the rate-limiting step of triglyceride lipolysis, results in a phenotype characterized by severe steatotic cardiac dysfunction. The objective of the present study was to investigate a potential role of oxidative stress in cardiac ATGL deficiency. Hearts of mice with global ATGL knockout were compared to those of mice with cardiomyocyte-restricted overexpression of ATGL and to those of wildtype littermates. Our results demonstrate that oxidative stress, measured as lucigenin chemiluminescence, was increased ~ 6-fold in ATGL-deficient hearts. In parallel, cytosolic NADPH oxidase subunits p67phox and p47phox were upregulated 4–5-fold at the protein level. Moreover, a prominent upregulation of different inflammatory markers (tumor necrosis factor α, monocyte chemotactant protein-1, interleukin 6, and galectin-3) was observed in those hearts. Both the oxidative and inflammatory responses were abolished upon cardiomyocyte-restricted overexpression of ATGL. Investigating the effect of oxidative and inflammatory stress on nitric oxide/cGMP signal transduction we observed a ~ 2.5-fold upregulation of soluble guanylate cyclase activity and a ~ 2-fold increase in cardiac tetrahydrobiopterin levels. Systemic treatment of ATGL-deficient mice with the superoxide dismutase mimetic Mn(III)tetrakis (4-benzoic acid) porphyrin did not ameliorate but rather aggravated cardiac oxidative stress. Our data suggest that oxidative and inflammatory stress seems involved in lipotoxic heart disease. Upregulation of soluble guanylate cyclase and cardiac tetrahydrobiopterin might be regarded as counterregulatory mechanisms in cardiac ATGL deficiency.     

Molecular Characterization of Parabasalian Symbionts Coronympha clevelandii and Trichonympha subquasilla from the Hawaiian Lowland Tree Termite Incisitermes immigrans

An important and undervalued challenge in characterizing symbiotic protists is the accurate identification of their host species. Here, we use DNA barcoding to resolve one confusing case involving parabasalian symbionts in the hindgut of the Hawaiian lowland tree termite, Incisitermes immigrans, which is host to several parabasalians, including the type species for the genus Coronympha, C. clevelandii. We collected I. immigrans from its type locality (Hawaii), confirmed its identity by DNA barcoding, and characterized the phylogenetic position of two symbionts, C. clevelandii and Trichonympha subquasilla. These data show that previous molecular surveys of “I. immigrans” are, in fact, mainly derived from the Caribbean termite I. schwarzi, and perhaps also another related species. These results emphasize the need for host barcoding, clarify the relationship between morphologically distinct Coronympha species, and also suggest some interesting distribution patterns of nonendemic termite species and their symbionts.     

Evaluation of new immunological targets in neuromyelitis optica

The detection of reactivity against autoantigens plays a crucial role in the diagnosis of autoimmune diseases. However, only a few autoantibodies are known in each disease, and their precise targets are often not precisely defined. In neuromyelitis optica (NMO), an autoimmune disease of the central nervous system, anti‐aquaporin 4 antibodies are currently the only available immunological markers, although they are not detected in 10–50% of patients. Using enzyme‐linked immunosorbent assays, we evaluated the reactivity against 19 structurally defined peptides in 26 NMO sera compared with 21 healthy subjects. We observed increased levels of IgG against myelin basic protein sequence MBP(156–175), pyruvate dehydrogenase sequence PDH(167–186) and CSF114(Glc), the last of these having a possible correlation with onset of inflammatory relapse. These preliminary results may suggest that the aquaporin 4 is not the unique target in NMO and that the study of reactivity against these peptides would be helpful for the diagnosis and follow‐up of the disease. Complementary studies are however warranted to confirm these results. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

A New Approach to Harness Probiotics Against Common Bacterial Skin Pathogens: Towards Living Antimicrobials

Probiotics and Antimicrobial Proteins - In this study, the potential of certain lactic acid bacteria—classified as probiotics and known to be antimicrobially active against pathogens or...     

Camptothecin induces p53-dependent and -independent apoptogenic signaling in melanoma cells

Various DNA-targeting agents may initiate p53-dependent as well as p53-independent response and subsequent apoptosis via alternative cellular systems which include for instance p73, caspase-2 or Bcl-2 family proteins. The scope of involvement of individual molecules in this process and the mechanisms governing their potential interplay are still not entirely understood, in particular in highly aggressive cancers such as in malignant melanoma. In this work we investigated the role and involvement of both p53-dependent and -independent mechanisms in selected melanoma cell lines with differing status of p53 using a model DNA topoisomerase I inhibitor camptothecin (CPT). Here we report that CPT induced in Bowes melanoma cells apoptosis which is essentially p53 and mitochondria-dependent but with some involvement of caspase-2 and p73. Conversely, in mutant p53 melanoma cells overall levels of CPT-induced apoptosis are significantly lower, with p73 and caspase-2 signaling playing important roles. In addition, in these cells the expression of micro RNAs family 34 (miR-34) were low compared to wild-type p53 cells. The ectopic expression of wild type p53 than restored apoptotic response of cells to CPT despite the fact that the expression of miR-34 and miR-155 were not influenced. These results suggest that CPT induces multivariate cellular stress responses including activation of DNA-damage response-p53 pathway as well as p53-independent signaling and their mutual crosstalk play the decisive role in the efficient triggering of apoptosis in melanoma cells.