RNA polymerase II large subunit degradation induced by ecteinascidin 743: Molecular characterization and subsequent rational investigation of antitumor mechanisms in cancers with clinical response

Ecteinascidin 743 (Et-743), which is a novel DNA minor groove alkylator with a unique spectrum of antitumor activity, is currently being evaluated in phase II/III clinical trials. Although the precise molecular mechanisms responsible for the observed antitumor activity are poorly understood, recent data suggests that post-translational modifications of RNA polymerase II Large Subunit (RNAPII LS) may play a central role in the cellular response to this promising anticancer agent. The stalling of an actively transcribing RNAPII LS at Et-743-DNA adducts is the initial cellular signal for transcription-coupled nucleotide excision repair (TC-NER). In this manner, Et-743 poisons TC-NER and produces DNA single strand breaks. Et-743 also inhibits the transcription and RNAPII LS-mediated expression of selected genes. Because the poisoning of TC-NER and transcription inhibition are critical components of the molecular response to Et-743 treatment, we have investigated if changes in RNAPII LS contribute to the disruption of these two cellular pathways. In addition, we have studied changes in RNAPII LS in two tumors for which clinical responses were reported in phase I/II clinical trials: renal cell carcinoma and Ewing's sarcoma. Our results demonstrate that Et-743 induces degradation of the RNAPII LS that is dependent on active transcription, a functional 26S proteasome, and requires functional TC-NER, but not global genome repair. Additionally, we have provided the first experimental data indicating that degradation of RNAPII LS might lead to the inhibition of activated gene transcription. A set of studies performed in isogenic renal carcinoma cells deficient in von Hippel-Lindau protein, which is a ubiquitin-E3-ligase for RNAPII LS, confirmed the central role of RNAPII LS degradation in the sensitivity to Et-743. Finally, we have shown that RNAPII LS is also degraded in Ewing's sarcoma tumors following Et-743 treatment and provide data to suggest that this event plays a role in decreased expression of the Ewing's sarcoma oncoprotein, EWS-Fli1. Altogether, these data implicate degradation of RNAPII LS as a critical event following Et-743 exposure and suggest that the clinical activity observed in renal carcinoma and Ewing's sarcoma may be mediated by disruption of molecular pathways requiring a fully functional RNAPII LS. ^

PRODUCTION, DEGRADATION AND ACCUMULATION OF ORGANICS BY WASTE STABILIZATION POND MICROORGANISMS

The objectives of this research were: to determine the contribution of algae to commonly run water quality variables, to evaluate waste pond micoorganisms' capacity to degrade and accumulate ten EPA priority pollutants, and to determine the environmental fate of those compounds in a laboratory

Regulation of Protein Degradation in the Heart by AMP-Activated Protein Kinase

The degradation of proteins by the ubiquitin proteasome system is essential for cellular homeostasis in the heart. An important regulator of metabolic homeostasis is AMP-activated protein kinase (AMPK). During nutrient deprivation, AMPK is activated and intracellular proteolysis is enhanced through the ubiquitin proteasome system (UPS). Whether AMPK plays a role in protein degradation through the UPS in the heart is not known. Here I present data in support of the hypothesis that AMPK transcriptionally regulates key players in the UPS, which, under extreme conditions can be detrimental to the heart. The ubiquitin ligases MAFbx /Atrogin-1 and MuRF1, key regulators of protein degradation, and AMPK activity are increased during nutrient deprivation. Pharmacologic and genetic activation of AMPK is sufficient for the induction of MAFbx/Atrogin-1 and MuRF1 in cardiomyocytes and in the heart in vivo. Comprehensive experiments demonstrate that the molecular mechanism by which AMPK regulates MuRF1 expression is through the transcription factor myocyte enhancer factor 2 (MEF2), which is involved in stress response and cardiomyocyte remodeling. MuRF1 is required for AMPK-mediated protein degradation through the UPS in cardiomyocytes. Consequently, the absence of MuRF1 during chronic fasting preserves cardiac function, possibly by limiting degradation of critical metabolic enzymes. Furthermore, during cardiac hypertrophy, chronic activation of AMPK also leads to cardiac dysfunction, possibly through enhanced protein degradation and metabolic dysregulation. Collectively, my findings demonstrate that AMPK regulates expression of ubiquitin ligases which are required for UPS-mediated protein degradation in the heart. Based on these results, I propose that specific metabolic signals may serve as modulators of intracellular protein degradation in the heart.

Regulation of Protein Degradation in the Heart by AMP-Activated Protein Kinase

The degradation of proteins by the ubiquitin proteasome system is essential for cellular homeostasis in the heart. An important regulator of metabolic homeostasis is AMP-activated protein kinase (AMPK). During nutrient deprivation, AMPK is activated and intracellular proteolysis is enhanced through the ubiquitin proteasome system (UPS). Whether AMPK plays a role in protein degradation through the UPS in the heart is not known. Here I present data in support of the hypothesis that AMPK transcriptionally regulates key players in the UPS, which, under extreme conditions can be detrimental to the heart. The ubiquitin ligases MAFbx /Atrogin-1 and MuRF1, key regulators of protein degradation, and AMPK activity are increased during nutrient deprivation. Pharmacologic and genetic activation of AMPK is sufficient for the induction of MAFbx/Atrogin-1 and MuRF1 in cardiomyocytes and in the heart in vivo. Comprehensive experiments demonstrate that the molecular mechanism by which AMPK regulates MuRF1 expression is through the transcription factor myocyte enhancer factor 2 (MEF2), which is involved in stress response and cardiomyocyte remodeling. MuRF1 is required for AMPK-mediated protein degradation through the UPS in cardiomyocytes. Consequently, the absence of MuRF1 during chronic fasting preserves cardiac function, possibly by limiting degradation of critical metabolic enzymes. Furthermore, during cardiac hypertrophy, chronic activation of AMPK also leads to cardiac dysfunction, possibly through enhanced protein degradation and metabolic dysregulation. Collectively, my findings demonstrate that AMPK regulates expression of ubiquitin ligases which are required for UPS-mediated protein degradation in the heart. Based on these results, I propose that specific metabolic signals may serve as modulators of intracellular protein degradation in the heart.

Measurements of formation, degradation, and sinking velocity of diatom aggregates from laboratory studies performed at 15 °C and 4 °C

Most deep ocean carbon flux profiles show low and almost constant fluxes of particulate organic carbon (POC) in the deep ocean. However, the reason for the non-changing POC fluxes at depths is unknown. This study presents direct measurements of formation, degradation, and sinking velocity of diatom aggregates from laboratory studies performed at 15 °C and 4 °C during a three-week experiment. The average carbon-specific respiration rate during the experiment was 0.12 ± 0.03 at 15 °C, and decreased 3.5-fold when the temperature was lowered to 4 °C. No direct influence of temperature on aggregate sinking speed was observed. Using the remineralisation rate measured at 4 °C and an average particle sinking speed of 150 m d**-1, calculated carbon fluxes were similar to those collected in deep ocean sediment traps from a global data set, indicating that temperature plays a major role for deep ocean fluxes of POC.

Copepod abundance and fecal pellet production, sinking rate, and degradation in samples obtained during Leg 7 of Galathea 3 cruise 2006

The vertical distribution of copepods, fecal pellets and the fecal pellet production of copepods were measured at seven stations across the Southern Indian Ocean from productive areas off South Africa to oligotrophic waters off Northern Australia during October/November 2006. We quantified export of copepod fecal pellet from surface waters and how much was retained. Furthermore, the potential impact of Oncaea spp. and harpacticoid copepods on fecal pellets degradation was evaluated and found to be regional substantial. The highest copepod abundance and fecal pellet production was found in the western nutrient-rich stations close to South Africa and the lowest at the central oligotrophic stations. The in situ copepod fecal pellet production varied between 1 and 1,000 µg C/m**3/day. At all stations, the retention of fecal pellets in the upper 400 m of the water column was more than 99% and the vertical export of fecal pellets was low (<0.02 mg/m**2/day).

Impact of changing carbonate chemistry, temperature, and salinity on growth and test degradation of the benthic foraminifer Ammonia aomoriensis

The present study investigated the combined effects of ocean acidification, temperature, and salinity on growth and test degradation of Ammonia aomoriensis. This species is one of the dominant benthic foraminifera in near-coastal habitats of the southwestern Baltic Sea that can be particularly sensitive to changes in seawater carbonate chemistry. To assess potential responses to ocean acidification and climate change, we performed a fully crossed experiment involving three temperatures (8, 13, and 18°C), three salinities (15, 20, and 25) and four pCO2 levels (566, 1195, 2108, and 3843 µatm) for six weeks. Our results highlight a sensitive response of A. aomoriensis to undersaturated seawater with respect to calcite. The specimens continued to grow and increase their test diameter in treatments with pCO2 <1200 µatm, when Omega calc >1. Growth rates declined when pCO2 exceeded 1200 µatm (Omega calc <1). A significant reduction in test diameter and number of tests due to dissolution was observed below a critical Omega calc of 0.5. Elevated temperature (18°C) led to increased Omega calc, larger test diameter, and lower test degradation. Maximal growth was observed at 18°C. No significant relationship was observed between salinity and test growth. Lowered and undersaturated Omega calc, which results from increasing pCO2 in bottom waters, may cause a significant future decline of the population density of A. aomoriensis in its natural environment. At the same time, this effect might be partially compensated by temperature rise due to global warming.

Optimization of InGaAsN(Sb)/GaAs quantum dots for optical emission at 1.55 µm with low optical degradation

Low optical degradation in GaInAsN(Sb)/GaAs quantum dots (QDs) p–i–n structures emitting up to 1.55 μm is presented in this paper. We obtain emission at different energies by means of varying N content from 1 to 4%. The samples show a low photoluminescence (PL) intensity degradation of only 1 order of magnitude when they are compared with pure InGaAs QD structures, even for an emission wavelength as large as 1.55 μm. The optimization studies of these structures for emission at 1.55 μm are reported in this work. High surface density and homogeneity in the QD layers are achieved for 50% In content by rapid decrease in the growth temperature after the formation of the nanostructures. Besides, the effect of N and Sb incorporation in the redshift and PL intensity of the samples is studied by post-growth rapid thermal annealing treatments. As a general conclusion, we observe that the addition of Sb to QD with low N mole fraction is more efficient to reach 1.55 μm and high PL intensity than using high N incorporation in the QD. Also, the growth temperature is determined to be an important parameter to obtain good emission characteristics. Finally, we report room temperature PL emission of InGaAsN(Sb)/GaAs at 1.4 μm.