Helix-coil transition of the self-complementary dG-dG-dA-dA-dT-dT-dC-dC duplex.

The helix-coil transition of the octanucleotide self-complementary duplex dG-dG-dA-dA-dT-dT-dC-dC has been monitored at the Watson-Crick protons, the base and sugar nonexchangeable protons and the backbone phosphates by high-resolution nuclear magnetic resonance (NMR) spectroscopy. The melting transition of the octanucleotide monitored by ultraviolet absorbance spectroscopy is characterized by the thermodynamic parameters delta H degree = -216.7 kJ/mol and delta S degree (25 degrees C) = -0.632 KJ mol-1 K-1 in 0.1 M NaCl, 10 mM phosphate solution. Correlation of the transition midpoint values monitored by the ultraviolet absorbance studies at strand concentrations below 0.2 mM and by NMR studies at 5.3 mM suggest that both methods are monitoring the octanucleotide duplex-to-strand transition. The NMR spectra of the Watson-Crick ring NH protons of the octanucleotide duplex have been followed as a function of temperature. The resonance from the terminal dG.dC base pairs broadens out at room temperature while the resonances from the other base pairs broaden simultaneously with the onset of the melting transition. The nonexchangeable base and sugar H-1' protons are resolved in the duplex and strand states and shift as average peaks through the melting transition. The experimental shifts on duplex formation have been compared with calculated values based on ring-current and atomic diamagnetic anisotropy contributions for a B-DNA base-pair-overlap geometry in solution. Several nonexchangeable proton resonances broaden in the fast-exchange region during the duplex-to-strand transition and the excess widths yield a duplex dissociation rate constant for the octanucleotide of 1.9 x 10(3) s-1 at 32 degrees C (fraction of duplex = 0.86) in 0.1 M NaCl, 10 mM phosphate buffer. The 31P resonances of the seven internucleotide phosphates are distributed over 0.6 ppm in the duplex state, shift downfield during the duplex-to-strand transition and undergo additional downfield shifts during the stacked-to-unstacked strand transition with increasing temperature.     

Inactivation of sortilin (a novel lysosomal sorting receptor) by dominant negative competition and RNA interference

To assess the role of sortilin in the sorting and trafficking of sphingolipid activator proteins (SAPs) the function of sortilin was abolished by a dominant-negative mutant and by the use of RNAi. Mutant sortilin lacking the carboxyl-terminal region that contains the sorting signal abolished the trafficking of SAPs to the lysosomes. Both sortilin and SAPs were retained in the Golgi apparatus. The use of chemically synthesized siRNA effectively blocked the trafficking of SAPs to the lysosomes as well. Additionally, we created a stable COS-7 cell line transfected with the pSilencer 3.1 H1 neo vector containing a selected siRNA template oligonucleotide (small hairpin interference RNA) where the levels of sortilin were greatly suppressed. The elimination of sortilin by this method will permit to determine whether or not sortilin is involved in a general mechanism of lysosomal sorting that involves the trafficking of various soluble lysosomal proteins other than SAPs.     

AP-1 and retromer play opposite roles in the trafficking of sortilin between the Golgi apparatus and the lysosomes

Sortilin has been implicated in the sorting of one soluble hydrolase and two sphingolipid activator proteins to the lysosomes. While the GGA adaptor proteins have been demonstrated to play a role in the targeting of sortilin to the endosomes, the recycling of sortilin has not yet been elucidated. Here we examine the role of two adaptor protein complexes, AP-1 and retromer. Our results demonstrate that AP-1 is required for the transport of sortilin to the endosomes and retromer for the recycling of sortilin to the Golgi apparatus. While inhibition of AP-1 causes accumulation of sortilin in the Golgi apparatus, RNAi depletion of retromer results in retention of sortilin in the lysosomes. We also demonstrate that the interaction of sortilin with retromer occurs through a YXXΦ site in its cytosolic tail. In conclusion, our observations indicate that retromer and AP-1 play opposite roles in the trafficking of sortilin.     

Annexin A2 Reduces PCSK9 Protein Levels via a Translational Mechanism and Interacts with the M1 and M2 Domains of PCSK9

Annexin A2 (AnxA2) was reported to be an extracellular endogenous inhibitor of proprotein convertase subtilisin kexin type 9 (PCSK9) activity on cell-surface LDL receptor degradation. In this study, we investigated the effect of silencing the expression of AnxA2 and PCSK9 in HepG2 and Huh7 cells to better define the role of AnxA2 in PCSK9 regulation. AnxA2 knockdown in Huh7 cells significantly increased PCSK9 protein levels as opposed to AnxA2 knockdown in HepG2 cells. However, HepG2 cells overexpressing AnxA2 had lower levels of PCSK9 protein. Overall, our data revealed a plausible new role of AnxA2 in the reduction of PCSK9 protein levels via a translational mechanism. Moreover, the C-terminal Cys/His-rich domain of PCSK9 is crucial in the regulation of PCSK9 activity, and we demonstrated by far-Western blot assay that the M1 and M2 domains are necessary for the specific interaction of PCSK9''s C-terminal Cys/His-rich domain and AnxA2. Finally, we produced and purified recombinant PCSK9 from humans and mice, which was characterized and used to perform 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate LDL cell-based assays on the stable knockdown HepG2 and Huh7 cells. We also demonstrated for the first time the equipotency of human and mouse PCSK9 R218S on human cells.     

Assignment of the imidazole ring nitrogen protons of histidine 48 in the proton NMR spectrum of ribonuclease A in water solution.

Several exchangeable resonances, designated a, b, c and d are observed in the 11-14 ppm (from 2,2-dimethyl-2-silapentane-5-sulfonate) region of the proton spectrum of ribonuclease A in water solution. We describe a number of lines of evidence suggesting the assignment of peaks b and c to the N1 and N3 protons of His 48, which occupies an interior position in the protein remote from the active site. This evidence includes the observation that the binding of Cu(II) and 3'-CMP (cytidine 3'-monophosphate) has no effect on these resonances. Further evidence includes pH titration data showing a pKa of approx. 2 for these protons, solvent exchange rates in the native state and with disulfide bridges IV-V and III-VIII cleaved, the observation of the carboxymethylated enzymes CM-His12-RNAase A and CM-His119-RNAase A, and of the modified enzymes Des(1-21)-RNAase A (S-protein) and Des(119-124)-RNAase A.     

Photomineralization in a boreal hydroelectric reservoir: a comparison with natural aquatic ecosystems

In order to evaluate the role of photochemistry in the carbon dioxide (CO₂) generation from a 10-year-old boreal reservoir, the photomineralization of dissolved organic matter (DOM) was assessed and compared to a boreal river as well as to boreal and temperate lakes during July and August, 2003. Sterile water samples were irradiated by sunlight over the whole photoperiod and subsequently analyzed for CO₂. Mean energy-normalized apparent photochemical yield of CO₂ (an index of DOM photoreactivity normalized for the energy absorbed by samples) was significantly higher in the reservoir (27.7 ± 13.0 mg CO₂·m⁻³·kJ⁻¹) and the boreal river (35.8 ± 2.3 mg CO₂·m⁻³·kJ⁻¹) than in the boreal lakes (15.5 ± 5.1 mg CO₂·m⁻³·kJ⁻¹). The DOM photoreactivity of the temperate lakes (20.9 ± 8.1 mg CO₂·m⁻³·kJ⁻¹) was not statistically different from any type of boreal water bodies. There was no significant difference in either the integrated photoproduction of CO₂ (273–433 mg CO₂·m⁻²·d⁻¹) or the potential photochemical contribution to CO₂ diffusive fluxes (56–92%) among these water bodies. DOM photoreactivity was significantly affected by the cumulative hydrological residence time (CHRT) when considering the whole data set. However, when considering only the boreal water bodies, iron (Fe) and manganese (Mn) also intervened. The fact that DOM photoreactivity was related to CHRT as well as to Fe and Mn concentrations, which are respectively permanent and long-lasting features of the reservoir, suggests that the photoproduction of CO₂ is not likely to decrease over time. This process may therefore play a substantial role in the long-term CO₂ emissions from boreal reservoirs during the summer, its potential contribution to CO₂ diffusive fluxes being estimated at 56 ± 29 %.     

Development of Composite Indices to Measure the Adoption of Pro-Environmental Behaviours across Canadian Provinces

Objective

The adoption of pro-environmental behaviours reduces anthropogenic environmental impacts and subsequent human health effects. This study developed composite indices measuring adoption of pro-environmental behaviours at the household level in Canada.

Methods

The 2007 Households and the Environment Survey conducted by Statistics Canada collected data on Canadian environmental behaviours at households'' level. A subset of 55 retained questions from this survey was analyzed by Multiple Correspondence Analysis (MCA) to develop the index. Weights attributed by MCA were used to compute scores for each Canadian province as well as for socio-demographic strata. Scores were classified into four categories reflecting different levels of adoption of pro-environmental behaviours.

Results

Two indices were finally created: one based on 23 questions related to behaviours done inside the dwelling and a second based on 16 questions measuring behaviours done outside of the dwelling. British Columbia, Quebec, Prince-Edward-Island and Nova-Scotia appeared in one of the two top categories of adoption of pro-environmental behaviours for both indices. Alberta, Saskatchewan, Manitoba and Newfoundland-and-Labrador were classified in one of the two last categories of pro-environmental behaviours adoption for both indices. Households with a higher income, educational attainment, or greater number of persons adopted more indoor pro-environmental behaviours, while on the outdoor index, they adopted fewer such behaviours. Households with low-income fared better on the adoption of outdoors pro-environmental behaviours.

Conclusion

MCA was successfully applied in creating Indoor and Outdoor composite Indices of pro-environmental behaviours. The Indices cover a good range of environmental themes and the analysis could be applied to similar surveys worldwide (as baseline weights) enabling temporal trend comparison for recurring themes. Much more than voluntary measures, the study shows that existing regulations, dwelling type, households composition and income as well as climate are the major factors determining pro-environmental behaviours.     

The biogeochemistry of toluene in coastal seawater: radiotracer experiments in controlled ecosystems

The fate of toluene in coastal seawater was investigated in controlled ecosystems using¹⁴C- and³H-toluene as tracers. Under winter-like conditions, 80% of the toluene volatilized from the water column in 2 months. Microbial degradation was less important than volatilization and sorption onto particulate matter with resultant loss to the sediments was minor. During summer most of the toluene was degraded by microbes. Nearly 80% of the toluene was converted to CO₂ within 1 week and the label remained in the water column as dissolved CO₂. The experimental results were applied to estimate the removal rates and the residence time of toluene in adjacent Narragansett Bay, Rhode Island. In winter volatilization would dominate the loss of toluene and a residence time of 6 d would be predicted. However, rapid biodegradation in summer would result in a residence time of < 1 d.     

The sorting and trafficking of lysosomal proteins

For a long time lysosomes were considered terminal organelles involved in the degradation of different substrates. However, this view is rapidly changing by evidence demonstrating that these organelles and their content display specialized functions in addition to the degradation of substances. Many lysosomal proteins have been implicated in specialized cellular functions and disorders such as antigen processing, targeting of surfactant proteins, and most lysosomal storage disorders. To date, about fifty lysosomal hydrolases have been identified, and the majority of them are targeted to the lysosomes via the mannose-6-phosphate receptor (M6P-Rc). However, recent studies on the intracellular trafficking of the non-enzymic lysosomal proteins prosaposin and GM2 activator (GM2AP) demonstrated that they use an alternative receptor termed "sortilin". Existing evidence suggests that some hydrolases traffic to the lysosomes in a mannose 6-phophate-indepentend manner. The possibility that sortilin is implicated in the targeting of some soluble hydrolases, as well as the consequences of this process, is addressed in the present review.     

The carbon cycle of Quebec boreal reservoirs investigated by elemental compositions and isotopic values

Dissolved and particulate organic matter (POM) of three Quebec boreal reservoirs of different ages (Laforge-1, 7 years; Robert-Bourassa, 25 years and Cabonga, 70 years at the time of sampling) and sets of lakes from the same watersheds was analyzed using organic carbon concentrations, C/N and C/P elemental composition, δ¹³C and δ¹⁵N isotopic values. The reservoirs are characterized by lower dissolved organic carbon concentrations with lower C/N ratios and by lower δ¹³C and higher δ¹⁵N in POM. They contain more autochthonous dissolved organic matter and less terrigenous organic matter than the lakes. Some of those characteristics are more pronounced in the younger than in the older reservoirs. The differences can be attributed to two causes: (1) more extended degradation of terrigenous organic matter, caused by an increase in residence time; and (2) differences in food web structure resulting from the phenomenon known as trophic upsurge, in newly flooded reservoirs. The results indicate that some effects of reservoir creation on the carbon cycle are short term perturbations, others however long term features of those reservoirs. The implications of these findings for CO₂ emissions from reservoirs are discussed.