Anti-apoptotic activity of low levels of wild-type p53.

Induction of apoptosis is a function of both an external stimulus and the physiology of the cell, which includes the expression of multiple oncogenes and tumor suppressors. Here we have studied the apoptotic response of immortalized mouse fibroblasts to serum withdrawal. We show that, in addition to the p53-independent apoptosis observed in p53- cells, overexpression of wild-type p53 tumor suppressor results in a high rate of programmed cell death. However, physiological range, low levels of the p53 protein protect fibroblasts from induction of apoptosis. Our results indicate that, as a function of its dose, the wild-type p53 can either protect from death or promote apoptosis. This new, anti-apoptotic, activity of p53 may have implications for the understanding of the role played by p53 in embryonic development as well as in initial stages of oncogenesis.     

Experimental evidence for lack of homodimerization of the G protein-coupled human N-formyl peptide receptor

A large number of G protein-coupled receptors have been shown to form homodimers based on a number of different techniques such as receptor coimmunoprecipitation, cross-linking, and fluorescence resonance energy transfer. In addition, functional assays of cells coexpressing a mutant receptor with a wild-type receptor have shown receptor phenotypes that can best be explained through dimerization. We asked whether the human neutrophil N-formyl peptide receptor (FPR) forms dimers in Chinese hamster ovary cells by coexpressing wild-type FPR with one of two mutants: D71A, which is uncoupled from G protein, and N297A, which has a defect in receptor phosphorylation and endocytosis. Experiments measuring chemotaxis, ligand-induced release of intracellular calcium, and p42/44 mitogen-activated protein kinase activation did not show an inhibitory effect of the coexpressed FPR D71A mutant. Coexpressed wild-type receptor was efficiently internalized, but failed to correct the endocytosis defects of the D71A and the N297A mutants. To explore the possibility that the mutations themselves prevented dimerization, we examined the coimmunoprecipitation of differentially epitope-tagged FPR. Immunoprecipitation of hemagglutinin-tagged FPR failed to coimmunoprecipitate coexpressed c-myc-tagged FPR and vice versa. Together, these data suggest that, unlike many other G protein-coupled receptors, FPR does not form homodimers.     

Detection and quantification of apoptosis in transiently transfected adherent cells.

Apoptosis is a highly conserved form of cell death present in all eukaryotic cell types and controlled by multiple genes. Several methods have been developed to quantify apoptosis, but none is adapted for all cell types. It is particularly difficult to reliably assay apoptosis of adherent cells. We describe a new, rapid and reliable flow cytometric method which can be used for quantifiying apoptosis in a sub-population of transiently transfected adherent cells. This technique is based on the detection of transfected cells and the apoptotic sub-population by immunofluorescence and Annexin-V labelling, respectively.     

Toxigenic saprophytic fungi in marine shellfish farming areas

Toxigenic saprophytic fungi were isolated from samples of shellfish, sediment and seawater obtained from marine shellfish farming areas. The 456 strains identified included 12 different genera, with a clear predominance (68%) of Penicillium, Aspergillus, Trichoderma and Cladosporium. To assess the risk of poisoning due to the presence of these fungi in shellfish farming areas, the strains were cultured in liquid medium, filtered, and tested on larvae of Artemia salina, a small crustacean highly sensitive to mycotoxins. Thirty-five point five percent of the strains proved active with this test. This study confirms the existence of fungi in shellfish farming areas, as suggested by our earlier work showing that filter-feeding shellfish accumulate toxic metabolites of fungal origin. The presence of fungi in the marine environment represents a real risk of poisoning through the consumption of contaminated shellfish.This revised version was published online in October 2005 with corrections to the Cover Date.     

Different endocytosis pathways of the C5a receptor and the N-formyl peptide receptor

Two chemoattractant receptors, C5aR (the complement fragment C5a receptor) and FPR (the N-formyl peptide receptor), are involved in neutrophil activation at sites of inflammation. In this study, we found major differences in the intracellular trafficking of the receptors in transfected Chinese hamster ovary (CHO) cells. Western blot analysis showed that FPR was stable during a 3 h stimulation with ligand, but C5aR was reduced in quantity by 50%. Not all C5aR was targeted directly for degradation however; a small, but visible fraction of the receptor became re-phosphorylated upon subsequent addition of ligand, suggesting that some of the receptor had cycled to the cell surface. Light membrane fractions isolated from activated cells showed C5aR distribution at the bottom of a glycerol gradient, colocalizing with the main distribution of the late endosomal/lysosomal marker LAMP2, whereas FPR was found at the bottom of the gradient as well as in the middle of the gradient, where it cofractionated with the early/sorting endosomal marker Rab5. Using fluorescence microscopy, we observed ligand-dependent redistribution of C5aR-EGFP from the plasma membrane to LAMP2-positive compartments, whereas FPR-EGFP showed significant colocalization with the early/sorting endosomes. Analysis of endogenous C5aR and FPR in neutrophils revealed a pattern similar to the CHO transfectants: C5aR underwent degradation after prolonged ligand stimulation, while FPR did not. Finally, we confirmed the down-regulation of C5aR in a functional assay by showing reduced chemotaxis toward C5a in both CHO transfectants and neutrophils after preincubation with C5a. A similar decrease in FPR-mediated chemotaxis was not observed.     

Variations des zymogrammes estérasiques chez les larves de Palaemon serratus soumises à différentes durées de choc thermique

Palaemon serratus at larval stage 2, acclimatized at 16°, were subjected to a temperature increase of 15° for periods of 5, 20 or 40 min with or without return to the initial temperature for 12 h. The soluble protein concentration of larvae subjected to a thermal shock of 20 min duration is lower than in control larvae. Total esterase-2C activity per mg of soluble protein after a thermal shock of 20 min duration is less than in control larvae but, in contrast with larvae subjected to a shock of 40 min duration, the initial activity is restored after 12 hours. Esterase-2C activity zymograms, after polyacrylamide gradient gel electrophoresis, show twelve isozymes. The activity of each isozyme examined varies according to the duration of the thermal shock.     

Glucose transport in isolated brush-border and lateral-basal plasma-membrane vesicles from intestinal epithelila cells

Free-flow electrophoresis was used to separate microvilli from the lateral basal plasma membrane of the epithelial cells from rat small intestine. The activities of the marker enzyme for the microvillus membrane, i.e. alkaline phosphatase (EC 3.1.31), was clearly separated from the marker for the lateral-basal plasma membrane, i.e. the (Na⁺, K⁺)-ATPase (EC 3.6.1.3). A microvillus membrane fraction was obtained with a high specific activity of alkaline phosphatase (an 8-fold enrichement over the starting homogenate). The lateral-basal plasma membrane fraction contained (Na⁺, K⁺)-ATPase (5-fold over homogenate) with some alkaline phosphatase (2-fold over homogenate).Glucose transport was studied in both membrane fractions. The uptake of d-glucose was much faster than that of l-glucose in either plasma membrane, d-Glucose uptake could be accounted for completely by its transport into an osmotically active space. Interestingly, the characteristics of the glucose transport of the microvillus membrane were different from those of the lateral-basal plasma membrane. In particular: Na⁺ stimulated the d-glucose transport by the microvillus membrane, but not by the lateral-basal plasma membrane. In addition, the glucose transport of the microvillus membrane was much more sensitive to phlorizin inhibition than that of the lateral-basal plasma membrane.These experiments thus provide evidence not only for an asymmetrical distribution of the enzymes, but also for differences in the transport properties with respect to glucose between the two types of plasma membrane of the intestinal epithelial cell.     

Life cycle assessment and process simulation of prospective battery-grade cobalt sulfate production from Co-Au ores in Finland

Purpose

The soaring demand for cobalt for lithium-ion batteries has increased interest in the utilization of non-conventional cobalt sources. Such raw materials include complex ores containing minerals such as cobaltite and skutterudite, which, while rare, occur around the world, including in Finland, Canada, and the USA. The goal of this study was to evaluate the cradle-to-gate impacts of cobalt sulfate recovery from unutilized cobalt- and gold-bearing ores with the use of process simulation.

Methods

A literature analysis was conducted to establish the state-of-the-art processing methods for complex cobalt ores containing significant amounts of gold. The drafted process was simulated using HSC Sim software to obtain a mass and energy balance, which was compiled into a life cycle inventory (LCI). The environmental impact categories (global warming, acidification, eutrophication, ozone depletion, photochemical smog creation, water use) were calculated in GaBi software. Uncertainty regarding the possible future raw material composition was studied, and the simulation was used to investigate process performance and to evaluate the effect of variation in the process parameters on the environmental impact indicators.

Results and discussion

The results indicated that the main cobalt mineral type (cobaltite, linnaeite) had only minor effects on the evaluated impact categories. With cobaltite-dominated ores (High As case), the global warming potential (GWP) was estimated to be 20.9 kg CO₂-eq, of which 12.7 kg CO₂-eq was attributed to the hydrometallurgical process. With linnaeite-dominated ores, the equivalent values were 20.4 kg CO₂-eq and 11.0 kg CO₂-eq. The production of a high grade concentrate was observed to greatly decrease the impacts of the hydrometallurgical process, but the cobalt losses in the beneficiation stage and the mineral processing impacts would likely increase. The simulation showed that there is still potential to improve the cobalt recovery (to approximately 96%), which would also affect the indicator values.

Conclusions

The impacts were estimated prior to intensive metallurgical testing to determine the possible high impact areas in the process. Based on this, it is suggested that, during hydrometallurgical processing, improved treatment of cobalt-containing wash waters and the optimization of oxygen utilization efficiency in pressure leaching are the most significant ways to decrease the environmental impacts. Optimal solutions for the concentrate could be found when experimental data on the minerals processing steps becomes available.