Light-independent degradation of stromal proteins in intact chloroplasts isolated from Pisum sativum L. leaves: requirement for divalent cations

Intact chloroplasts were isolated from mature pea (Pisum sativum L.) leaves in order to study the degradation of several stromal proteins in organello. Changes in the abundances of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39), phosphoribulokinase (EC 2.7.1.19), glutamine synthetase (EC 6.3.1.2) and ferredoxin-dependent glutamine:α-ketoglutarate aminotransferase (glutamate synthase; EC 1.4.7.1) were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Coomassie-staining of the gels or immunoblotting using specific antibodies for the different enzymes. Degradation of several stromal proteins was strongly stimulated when intact chloroplasts were incubated in the light in the presence of dithiothreitol. Since free radicals may artificially accumulate in the chloroplast under such conditions and interfere with the stability of stromal proteins, the general relevance of these processes remains questionable. In the absence of light, proteolysis proceeded slowly in isolated chloroplasts and was not stimulated by dithiothreitol. Inhibition by ethylenediaminetetraacetic acid (EDTA), 1,10-phenanthroline or excess zinc ions as well as the requirement for divalent cations suggested that a zinc-containing metalloprotease participated in this process. Furthermore, light-independent degradation of ribulose-1,5-bisphosphate carboxylase/oxygenase and phosphoribulokinase was enhanced in chloroplasts isolated from leaves in which senescence was accelerated by nitrogen starvation. Our results indicate that light-independent stromal protein degradation in intact chloroplasts may be analogous to proteolysis that occurs in intact leaves during senescence.

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.

Proceedings of the Twenty-First Annual Biochemical Engineering Symposium

The 21st Annual Biochemical Engineering Symposium was held at Colorado State University on April 20, 1991. The primary goals of this symposium series are to provide an opportunity for students to present and publish their research work and to promote informal discussions on biochemical engineering research. Contents High Density Fed-Batch Cultivation and Energy Metabolism of Bacillus thuringtensis; W.-M. Liu, V. Bihari, M. Starzak, and R.K. Bajpai Influences of Medium Composition and Cultivation Conditions on Recombinant Protein Production by Bacillus subtilis; K. Park, P.M. Linzmaier, and K.F. Reardon Characterization of a Foreign Gene Expression in a Recombinant T7 Expression System Infected with λ Phages; F. Miao and D.S. Kompala Simulation of an Enzymatic Membrane System with Forced Periodic Supply of Substrate; N. Nakaiwa, M. Yashima, L.T. Fan, and T. Ohmori Batch Extraction of Dilut Acids in a Hollow Fiber Module; D.G. O'Brien and C.E. Glatz Evaluation of a New Electrophoretic Device for Protein Purification; M.-J. Juang and R.G. Harrison Crossflow Microfiltration and Membrane Fouling for Yeast Cell Suspension; S. Redkar and R. Davis Interaction of MBP-β-Galactosidase Fusion Protein with Starch; L. Taladriz and Z. Nikolov Predicting the Solubility of Recombinant Proteins in Escherichia coli; D.L. Wilkinson and R.G. Harrison Evolution of a Phase-Separated, Gravity-Independent Bioractor; P.E. Villeneuve and E.H. Dunlop A Strategy for the Decontamination of Soils Containing Elevated Levels of PCP; S. Ghoshal, S. K. Banelji, and RK. Bajpai Practical Considerations for Implementation of a Field Scale In-Situ Bioremediation Project; J.P. McDonald, CA Baldwin, and L.E. Erickson Parametric Sensitivity Studies of Rhizopus oligosporus Solid Substrate Fermentation; J. Sargantanis, M.N. Karim, and V.G. Murphy, and RP. Tengerdy Production of Acetyl-Xylan Esterase from Aspergillus niger; M.R Samara and J.C. Linden Biological and Latex Particle Partitioning in Aqueous Two-Phase Systems; D.T.L. Hawker, RH. Davis, P.W. Todd, and R Lawson Novel Bioreactor /Separator for Microbial Desulfurization of Coal; H. Gecol, RH. Davis, and J .R Mattoon Effect of Plants and Trees on the Fate, Transport and Biodegradation of Contaminants in the Soil and Ground Water; W. Huang, E. Lee, J.F. Shimp, L.C. Davis, L.E. Erickson, and J.C. Tracy Sound Production by Interfacial Effects in Airlift Reactors; J. Hua, T.-Y. Yiin, LA Glasgow, and L.E. Erickson Soy Yogurt Fermentation of Rapid Hydration Hydrothermal Cooked Soy Milk; P. Tuitemwong, L.E. Erickson, and D.Y.C. Fung Influence of Carbon Source on Pentachlorophenol Degradation by Phanerochaete chrysosportum in Soil; C.-Y.M. Hsieh, RK. Bajpai, and S.K. Banerji Cellular Responses of Insect Cells Spodopiera frugiperda -9 to Hydrodynamic Stresses; P.L.-H. Yeh and RK. Bajpa1 A Mathematical Model for Ripening of Cheddar Cheese; J. Kim, M. Starzak, G.W. Preckshoi, and R.K. Bajpai

(Table 1) Characteristics of South Pole personnel according to forced expiratory flow (FEF25-75) response to altitude

The impact of acute altitude exposure on pulmonary function is variable. A large inter-individual variability in the changes in forced expiratory flows (FEFs) is reported with acute exposure to altitude, which is suggested to represent an interaction between several factors influencing bronchial tone such as changes in gas density, catecholamine stimulation, and mild interstitial edema. This study examined the association between FEF variability, acute mountain sickness (AMS) and various blood markers affecting bronchial tone (endothelin-1, vascular endothelial growth factor (VEGF), catecholamines, angiotensin II) in 102 individuals rapidly transported to the South Pole (2835 m). The mean FEF between 25 and 75% (FEF25-75) and blood markers were recorded at sea level and after the second night at altitude. AMS was assessed using Lake Louise questionnaires. FEF25-75 increased by an average of 12% with changes ranging from -26 to +59% from sea level to altitude. On the second day, AMS incidence was 36% and was higher in individuals with increases in FEF25-75 (41 vs. 22%, P = 0.05). Ascent to altitude induced an increase in endothelin-1 levels, with greater levels observed in individuals with decreased FEF25-75. Epinephrine levels increased with ascent to altitude and the response was six times larger in individuals with decreased FEF25-75. Greater levels of endothelin-1 in individuals with decreased FEF25-75 suggest a response consistent with pulmonary hypertension and/or mild interstitial edema, while epinephrine may be upregulated in these individuals to clear lung fluid through stimulation of beta2-adrenergic receptors.

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).

Simulated Bromoform emission and concentration using the ocean biogeochemistry model MPIOM/HAMOCC forced by 6-hourly NCEP data

Bromoform (CHBr3) is one important precursor of atmospheric reactive bromine species that are involved in ozone depletion in the troposphere and stratosphere. In the open ocean bromoform production is linked to phytoplankton that contains the enzyme bromoperoxidase. Coastal sources of bromoform are higher than open ocean sources. However, open ocean emissions are important because the transfer of tracers into higher altitude in the air, i.e. into the ozone layer, strongly depends on the location of emissions. For example, emissions in the tropics are more rapidly transported into the upper atmosphere than emissions from higher latitudes. Global spatio-temporal features of bromoform emissions are poorly constrained. Here, a global three-dimensional ocean biogeochemistry model (MPIOM-HAMOCC) is used to simulate bromoform cycling in the ocean and emissions into the atmosphere using recently published data of global atmospheric concentrations (Ziska et al., 2013) as upper boundary conditions. Our simulated surface concentrations of CHBr3 match the observations well. Simulated global annual emissions based on monthly mean model output are lower than previous estimates, including the estimate by Ziska et al. (2013), because the gas exchange reverses when less bromoform is produced in non-blooming seasons. This is the case for higher latitudes, i.e. the polar regions and northern North Atlantic. Further model experiments show that future model studies may need to distinguish different bromoform-producing phytoplankton species and reveal that the transport of CHBr3 from the coast considerably alters open ocean bromoform concentrations, in particular in the northern sub-polar and polar regions.