Fractionation at pilot-plant scale of an haemoglobin hydrolysate by strong anionic exchange chromatography Application to the preparation of an amphiphilic peptide

The development and the scale-up of high performance anion chromatography to obtain 1 milligram to 1 gram yields of a peptide fraction from a complex peptic haemoglobin hydrolysate is described here. The chromatographic conditions were developed using a 1 cm³ Mono Q analytical column and progressively scaled-up to a 6 dm³ Q Sepharose Fast Flow column. For easy recovery of peptide and easy adjustment of conditions for final purification, a volatile buffer, ethanolamine/HCl buffer 20 mmol dm⁻³, pH 10·5, was employed; desalting was carried out by a pilot-plant scale electrodialysis which permitted the elimination of 99% NaCl without important loss of peptide (less than 15%). A combination of these techniques with reverse phase HPLC proved a useful strategy for fractionation of a complex peptide mixture and enabled pure peptides to be obtained in sufficient quantities for further analyses and biological tests. The example of preparation and purification of an amphiphilic peptide is described. Its ability to solubilize an insoluble photosensitizer, protoporphyrin IX, was determined in order to study its utilization as a carrier for photochemotherapy. © 1998 SCI.     

Investigations on the synthesis of polyimides with coronand structure via tandem Diels-Alder addition polymerization

The tandem Diels-Alder polymerization of 11 bismaleimides with 4,4'-dimethyl-6,6'-(octamethylene)di-2-pyrone was investigated. Polymers with coronand structure were formed only in case of bismaleimides possessing flexible spacer groups; rigid spacer groups led to network formation.     

Kinetics of PCr to ATP and β-ATP to β-ADP phosphoryl conversion are modified in working rat skeletal muscle after training

Kinetics of phosphoryl transfers from PCr to γ-ATP and from β-ATP to β-ADP were measured by magnetization transfer in an in vivo³¹P NMR experiment in working rat skeletal hind leg muscles. Two groups were examined. One group was submitted to a 6-week training program of treadmill running. The other group was composed of sedentary animals. Metabolic oxidative capacity and mechanical performance were improved greatly by training as shown previously. Phosphoryl transfer of PCr→γ-ATP or β-ATP→β-ADP total fluxes were identical in resting trained and untrained muscles. Under stimulation, the flux of creatine kinase transfer was significantly inhibited by 23% compared with resting level in untrained muscles; by contrast, it was not inhibited and maintained at the high resting level in trained muscles. Thus physiological changes probably linked to a decrease of the production of anions, which could inhibit creatine kinase, were able to maintain creatine kinase flux. The flux of β-ATP to β-ADP transfer were enhanced largely in working muscles from 1.4±0.8 and 2±0.8 at rest to 4±1.6 and 6.6±2.7 mM s⁻¹ for untrained and trained muscles respectively; the effect was more pronounced in trained than in untrained muscles. These results showed an acceleration of phosphoryl turnover in working muscles after training, which could contribute to improve oxidative and mechanical performances. Such kinetic measurements of phosphoryl conversion may provide information on ATP turnover in pathophysiologic situations where ADP accumulates because of impaired ATP synthesis (mitochondrial myopathies, lower perfusion level).     

Physicochemical characteristics and bronchial epithelial cell cytotoxicity of Folpan 80 WG® and Myco 500®, two commercial forms of folpet

Background

Pesticides, in particular folpet, have been found in rural and urban air in France in the past few years. Folpet is a contact fungicide and has been widely used for the past 50 years in vineyards in France. Slightly water-soluble and mostly present as particles in the environment, it has been measured at average concentration of 40.1 μg/m³ during its spraying, 0.16–1.2 μg/m³ in rural air and around 0.01 μg/m³ in urban air, potentially exposing both the workers and the general population. However, no study on its penetration by inhalation and on its respiratory toxicity has been published. The objective of this study was to determine the physicochemical characteristics of folpet particles (morphology, granulometry, stability) in its commercial forms under their typical application conditions. Moreover, the cytotoxic effect of these particles and the generation of reactive oxygen species were assessed in vitro on respiratory cells.

Results

Granulometry of two commercial forms of folpet (Folpan 80WG^® and Myco 500^®) under their typical application conditions showed that the majority of the particles (>75%) had a size under 5 μm, and therefore could be inhaled by humans. These particles were relatively stable over time: more than 75% of folpet remained in the particle suspension after 30 days under the typical application conditions. The inhibitory concentration (IC₅₀) on human bronchial epithelial cells (16HBE14o-) was found to be between 2.89 and 5.11 μg/cm² for folpet commercial products after 24 h of exposure. Folpet degradation products and vehicles of Folpan 80 WG^® did not show any cytotoxicity at tested concentrations. At non-cytotoxic and subtoxic concentrations, Folpan 80 WG^® was found to increase DCFH-DA fluorescence.

Conclusion

These results show that the particles of commercial forms of folpet are relatively stable over time. Particles could be easily inhaled by humans, could reach the conducting airways and are cytotoxic to respiratory cells in vitro. Folpet particles may mediate its toxicity directly or indirectly through ROS-mediated alterations. These data constitute the first step towards the risk assessment of folpet particles by inhalation for human health. This work confirms the need for further studies on the effect of environmental pesticides on the respiratory system.     

Species richness, structural diversity and species composition in meadows created by visitors of a botanical garden in Switzerland

Biodiversity is an increasingly important criterion in designing ecological compensation areas and urban green areas. However, little is known about how important biodiversity is to the general public. This paper presents the results of two complementary studies investigating the meadow preferences of visitors to a botanical garden in Switzerland. In the first study, 152 people were asked to create their own favourite meadow patch by selecting 25 out of 779 local wild plants (54 species) that were displayed in flowerpots. In the second study, 143 people were asked with the help of a written questionnaire to imagine a bare piece of land the size of 100 m × 100 m close to their place of living and mentally create the meadow of their dreams. In both cases, study participants assembled meadows that were species-rich and rich in structural diversity (expressed by plant height and leaf forms). Moreover, they explicitly stated diversity as their main assemblage criterion. Although study participants favoured plants with large or colourful flowers, only a third of all plants in the meadows created with real plants were flowering and in people's imagined meadows grasses were frequently included, indicating that people like diverse meadows consisting of a green matrix with some colourful flowers.     

Permeability of low molecular weight organics through nanofiltration membranes

The removal of natural organic matter (NOM) using nanofiltration (NF) is increasingly becoming an option for drinking water treatment. Low molecular weight (LMW) organic compounds are nevertheless only partially retained by such membranes. Bacterial regrowth and biofilm formation in the drinking water distribution system is favoured by the presence of such compounds, which in this context are considered as the assimilable organic carbon (AOC). In this study, the question of whether NF produces microbiologically stable water was addressed. Two NF membranes (cut-off of about 300 Da) were tested with different natural and synthetic water samples in a cross-flow filtration unit. NOM was characterised by liquid chromatography with organic carbon detection (LC-OCD) using a size-exclusion column in addition to specific organic acid measurements, while AOC was measured in a batch growth bioassay.Similarly to high molecular weight organic compounds like polysaccharides or humic substances that have a permeability lower than 1%, charged LMW organic compounds were efficiently retained by the NF membranes tested and showed a permeability lower than 3%. However, LMW neutrals and hydrophobic organic compounds permeate to a higher extent through the membranes and have a permeability of up to 6% and 12%, respectively. Furthermore, AOC was poorly retained by NF and the apparent AOC concentration measured in the permeated water was above the proposed limit for microbiologically stable water. This indicates that the drinking water produced by NF might be biologically unstable in the distribution system. Nevertheless, in comparison with the raw water, NF significantly reduced the AOC concentration.     

Optimization of PCR-based methods for rapid detection of Campylobacter jejuni, Campylobacter coli and Yersinia enterocolitica serovar 0:3 in wastewater samples

PCR-based methods were evaluated for their adequacy to assess the removal of pathogens from wastewater samples. For the development and optimization of the methods, samples were taken at two different sites from two different constructed wetlands. Campylobacter jejuni/coli and Yersinia enterocolitica serogroup 0:3 were selected as model pathogens and Enterococcus faecalis as a standard microbiological indicator. The chosen PCR protocols were optimized for wastewater DNA extracts in order to obtain high sensitivity and reproducibility independently of the background flora. All PCR protocols were successfully performed and reproducible with a background of up to 10(10) nontarget cells per reaction. Five cells of Y. enterocolitica, 50 cells of C. jejuni/coli, and 500 cells of E. faecalis per 100ml treated water could be detected. The method detection limit in the settled wastewater was higher: 200 cells per 100ml for Y. enterocolitica, 2000 cells per 100ml for C. jejuni/coli, and 20,000 cells per 100ml for E. faecalis. C. jejuni/coli and Y. enterocolitica PCRs were adapted to municipal wastewater, with higher loads of potential PCR inhibitors. Sensitivity was lower for this type of wastewater: 200 cells of Y. enterocolitica and 2000 cells of C. jejuni/coli were detected per 100ml treated wastewater, 2500 cells of Y. enterocolitica and 25,000 cells of C. jejuni/coli per 100ml settled wastewater. The developed PCR methods enable the detection of C. jejuni/coli, Y. enterocolitica serogroup 0:3 and E. faecalis within 12h. They show specificity, reproducibility and low detection limits for the investigated pathogens.