Dmitri B Papkovsky

Publications (91) View all

• Article: Mitochondrial pyrimidine nucleotide carrier (PNC1) regulates mitochondrial biogenesis and the invasive phenotype of cancer cells.

  C Favre, A Zhdanov, M Leahy, D Papkovsky, R O'Connor
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  ABSTRACT: The insulin-like growth factor (IGF-I) signalling pathway is essential for metabolism, cell growth and survival. It induces expression of the mitochondrial pyrimidine nucleotide carrier 1 (PNC1) in transformed cells, but the consequences of this for cell phenotype are unknown. Here we show that PNC1 is necessary to maintain mitochondrial function by controlling mitochondrial DNA replication and the ratio of transcription of mitochondrial genes relative to nuclear genes. PNC1 suppression causes reduced oxidative phosphorylation and leakage of reactive oxygen species (ROS), which activates the AMPK-PGC1alpha signalling pathway and promotes mitochondrial biogenesis. Overexpression of PNC1 suppresses mitochondrial biogenesis. Suppression of PNC1 causes a profound ROS-dependent epithelial-mesenchymal transition (EMT), whereas overexpression of PNC1 suppresses both basal EMT and induction of EMT by TGF-beta. Overall, our findings indicate that PNC1 is essential for mitochondria maintenance and suggest that its induction by IGF-I facilitates cell growth whereas protecting cells from an ROS-promoted differentiation programme that arises from mitochondrial dysfunction.
  Oncogene 05/2010; 29(27):3964-76. · 6.37 Impact Factor
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  Article: Comparative bioenergetic assessment of transformed cells using a cell energy budget platform.

  A V Zhdanov, C Favre, L O'Flaherty, J Adam, R O'Connor, P J Pollard, D B Papkovsky
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  ABSTRACT: The aberrant expression and functional activity of proteins involved in ATP production pathways may cause a crisis in energy generation for cells and compromise their survival under stressful conditions such as excitation, starvation, pharmacological treatment or disease states. Under resting conditions such defects are often compensated for, and therefore masked by, alternative pathways which have significant spare capacity. Here we present a multiplexed 'cell energy budget' platform which facilitates metabolic assessment and cross-comparison of different cells and the identification of genes directly or indirectly involved in ATP production. Long-decay emitting O(2) and pH sensitive probes and time-resolved fluorometry are used to measure changes in cellular O(2) consumption, glycolytic and total extracellular acidification (ECA), along with the measurement of total ATP and protein content in multiple samples. To assess the extent of spare capacity in the main energy pathways, the cells are also analysed following double-treatment with carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone and oligomycin. The four-parametric platform operating in a high throughput format has been validated with two panels of transformed cells: mouse embryonic fibroblasts (MEFs) lacking the Krebs cycle enzyme fumarate hydratase (Fh1) and HeLa cells with reduced expression of pyrimidine nucleotide carrier 1. In both cases, a marked reduction in both respiration and spare respiratory capacity was observed, accompanied by a compensatory activation of glycolysis and consequent maintenance of total ATP levels. At the same time, in Fh1-deficient MEFs the contribution of non-glycolytic pathways to the ECA did not change.
  Integrative Biology 11/2011; 3(11):1135-42. · 4.51 Impact Factor
• Article: Analysis of total aerobic viable counts in raw fish by high-throughput optical oxygen respirometry.

  A Hempel, N Borchert, H Walsh, K Roy Choudhury, J P Kerry, D B Papkovsky
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  ABSTRACT: A simple, miniaturized, and automated screening assay for the determination of total aerobic viable counts in fish samples is presented here. Fish tissue homogenates were prepared in peptone buffered water medium, according to standard method, and aliquots were dispensed into wells of a 96-well plate with the phosphorescent, oxygen-sensing probe GreenLight. Sample wells were covered with mineral oil (barrier for ambient oxygen), and the plate was monitored on a standard fluorescent reader at 30°C. The samples produced characteristic profiles, with a sharp increase in fluorescence above the baseline level at a certain threshold time, which could be correlated with initial microbial load. Five different fish species were analyzed: salmon, cod, plaice, mackerel, and whiting. Using a conventional agar plating method, the relationship between the threshold time and total aerobic viable counts load (in CFU per gram) was established, calibration curve generated, and the test was validated with 169 unknown fish samples. It showed a dynamic range of 10(4) to 10(7) CFU/g, accuracy of ± 1 log(CFU/g), assay time of 2 to 12 h (depending on the level of contamination), ruggedness with respect to the key assay parameters, simplicity (three pipetting steps, no serial dilutions), real-time data output, high sample throughput, and automation. With this test, quality of fish samples, CFU-per-gram levels, and their respective time profiles were determined.
  Journal of food protection 05/2011; 74(5):776-82. · 1.94 Impact Factor
• Article: Analysis of close proximity quenching of phosphorescent metalloporphyrin labels in oligonucleotide structures.

  M Burke, P J O'Sullivan, G V Ponomarev, D V Yashunsky, D B Papkovsky
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  ABSTRACT: Quenching of phosphorescent platinum(II) and palladium(II) coproporphyrin (MeCP) labelled oligonucleotides was investigated. Strong hybridization-specific quenching was observed in duplex DNA structures with a variety of quenchers and with two identical porphyrin labels when in close proximity. Classical resonance energy transfer mechanism was ruled out, since quenching did not correlate with spectral overlaps and lifetime changes were insignificant. Quenching of MeCP by the free quenchers in solution revealed that porphyrin-porphyrin quenching is predominantly static while other dyes quench dynamically. The results suggest that the quenching in DNA duplex proceeds via direct contact.
  Analytica chimica acta 03/2007; 585(1):139-46. · 4.31 Impact Factor
• Article: Comparative Stability Assessment of Laccases from the Basidiomycetes Coriolus hirsutus and Coriolus zonatus in the Presence of Effectors

  E. V. Stepanova, O. V. Koroleva, V. P. Gavrilova, E. O. Landesman, A. Makower, D. B. Papkovsky
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  ABSTRACT: Stability characteristics of the laccases of the basidiomycetes Coriolus hirsutus and Coriolus zonatus were measured comparatively at temperatures of 25 and 40C in the presence of various effectors (proteins, salts, polyalcohols, polyacids, and polyelectrolytes). Stabilization effects of cations on the laccases from C. hirsutus and C. zonatus decreased in the descending series Cu2+ > Mg2+ > Ca2+ and Ca2+ > Mg2+ > Mn2+, respectively. Tween 20 caused insignificant stabilization of the two enzymes. The C. zonatus laccase was also insignificantly stabilized as a result of treatment with bovine serum albumin. The enzymatic activity of the laccase preparations from C. hirsutus and C. zonatus was conserved virtually completely after vacuum drying (84 and 93%, respectively). The most effective stabilizer of the C. hirsutus laccase was found to be dextran (17 kDa). Dry preparations treated with this agent conserved up to 95% of the enzymatic activity. The most effective stabilizer of the C. zonatus laccase was polyacrylic acid (102% of the initial activity).
  Applied Biochemistry and Microbiology 01/2003; 39(5):482-487. · 0.56 Impact Factor