Characterisation of dissolved organic matter in Parisian urban aquatic systems: predominance of hydrophilic and proteinaceous structures

Understanding the nature of organic matter is a necessary first step in assessing contaminant bioavailability and allowing water supply managers to optimise the treatment train in the aim of providing safe and inexpensive drinking water. This study provides further insight into the composition, structure and functional groups of dissolved organic matter (DOM) (both hydrophobic and hydrophilic) from urban aquatic systems by means of various analytical techniques (DAX-8/XAD-4 fractionation, elemental analysis, UV and FTIR spectroscopies, ¹³C and ¹⁵N isotopic analysis, size exclusion chromatography and Pyrolysis-GC-MS). The analytical range chosen for this study constitutes a powerful tool in the characterisation of DOM in urban water. The inclusion of information from one technique to the next might not only serve as a support to each one, but also as a complement. The DOM fraction from treated effluent and, more generally, DOM from urban water (i.e. receiving treated effluent) display a strong hydrophilic characteristic [i.e. low humic substance (HS) content, low SUVA], along with a high distribution in molecular weights observed by SEC and low average molecular weight. Due to the origin of this DOM, proteinaceous structures constitute the main compounds, as observed by FTIR and Py-GC-MS. Such characteristics (i.e. heterogeneity, low average molecular weight and diverse functional groups, which make up a total of N) could explain that DOM from treated effluent displayed a strong reactive potential metals pollutants as previously demonstrated.     

Activation of potassium channels by hypoxia and reoxygenation in the human lung adenocarcinoma cell line A549

Active oxygen species are generated in cells during pathophysiologic conditions such as illflammation and postischemic reperfusion. If oxygen radical scavengers are added before reperfusion, then the magnitude of injury is reduced. We inves-tigated whether free radicals generated following exposure to hypoxia and reoxygenation activate voltage-dependent K⁺ ion channels in tumor cells in vitro. Using the technique of whole cell voltage clamping, we recorded currents from two families of potassium (K⁺) channels that were activated following reoxygenation. One of these groups possessed the electrophysical characteristics of a tetraethylammonium (TEA)-sensitive delayed rectifier channel and the other possessed characteristics of a Tea-insensitive slow inactivating channel. We present evidence which suggests that K⁺ channels are activated following reoxygenation but not during the hypoxia phase. The K⁺ currents decayed with time following reoxygenation. The decay characteristics of the K⁺ currents depended on the duration and level of hypoxia to which the cells were exposed. To determine whether activation of K⁺ channels by reoxygenation was initiated by free radicals, we pretreated cells with N-Acetyl L-Cysteine (NAC), a free radical scavenger, and found that this pretreatment abolished the currents induced by reoxygenation. We also present evidence that free radicals do not directly act on the channel itself, but activate a protein kinase which, in turn, activates the K⁺ channels. Taken together, these results indicate that one of the early responses to oxidative stress is the activation of K⁺ currents. © 1993 Wiley-Liss, Inc.     

Monoclonal antibody recognizes a conformational epitope in a random coil protein

The antigenic determinants for two monoclonal antibodies directed against horse apo-cytochrome c, a protein of disordered structure, as judged by spectroscopic and hydrodynamic criteria, have been studied by a combination of methods: antigen competition in solution by radio immunoassay and enzyme-linked immunoassay, and differential acetylation of free and antibody-bound antigen. In the latter method the accessibility of lysine residues of the antigen in the antigen-antibody complex is compared to the accessibility in the free antigen. The two antibodies against the heme-free protein do not recognize intact native cytochrome c, but they crossreact with the heme-containing peptides 1-38 and 1-65 of cytochrome c. The antigenic determinant recognized by monoclonal antibody SJL 2-4 is conformational and discontiguous, it is composed of residues close to the N-terminus and around position 25. The other monoclonal antibody, Cyt-1-59, seems to recognize a contiguous epitope close to the N-terminus. The present results show that even a seemingly disordered protein which is conventionally classified as a random coil may feature subtle spatial regularities. The presence of ordered conformational elements in apocytochrome c may be important for the enzyme-catalyzed covalent attachment of the heme and the import of cytochrome c into mitochondria. A discontiguous determinant for SJL 2-4 is particularly interesting because this antibody inhibits the proliferation of a T-cell clone specific for apo-cytochrome c [Corradin & Engers (1984) Nature (Lond.) 308, 547-548].     

Assembly properties of two CNBr fragments of avian desmin that correspond to the headpiece domain and helix 1B

To study how different domains of the muscle-specific intermediate filament protein, desmin, contribute to its polymerization, two of its CNBr fragments were examined as to their oligomeric structure under assembly conditions. One of these, D88, covers residues 1-88 and represents almost the entire headpiece; the other, D109, covers residues 145-254, and includes the entire Helix 1B and part of linker L12 of the intact molecule. Chemical cross-linking followed by SDS-PAGE, and analytical gel filtration, revealed that in 10 mM Tris-HCl, pH 8.5, conditions that favor tetramerization of intact desmin D88 formed only dimers. D109, on the other hand, formed primarily a dimeric species but low levels of trimeric and tetrameric species were also detectable. These data are consistent with the proposal that, during assembly of intact protein molecules into IF, the headpiece and Helix 1 contribute to dimerization of two polypeptides into a parallel, in-register coiled-coil. However, additional interactions, including headpiece-to-rod binding and hydrophobic interaction along the entire rod domain, are required to stabilize the tetramers and full-size IF.     

Effects of seasonal changes of soil salinity and soil nitrogen on the N-metabolism of the halophyteArthrocnemum fruticosum (L.) Moq.

Summary The N-metabolism ofArthrocnemum fruticosum (L.) Moq., growing in a saline area north-east of the Dead Sea in Jordan, was studied over its vegetative growth period from March to September 1981. Plant and soil samples were taken at monthly intervals. Water content, Na⁺, K⁺, Cl⁻, NH ₄ ⁺ , NO ₂ ⁻ and NO ₃ ⁻ concentrations were determined in the soil extracts, and the same determinations plus ash weight, soluble carbohydrates, proline, proteins andin vivo nitrate reductase in the plant roots and shoots. Soil humidity decreased and salinity increased from March to August, with re-wetting occurring in late July. K⁺ and Cl⁻ were much lower in the soils than Na⁺. Plant relative dry weight increased during summer due to the absorption of Na⁺ in addition to increased organic dry weight. The uptake of Na⁺ was not balanced by a similar uptake of Cl⁻. Ammonium and nitrate decreased in soil and plants in parallel with increasing salinity. Nitrite was only found in the roots and always in very low quantities. Proline was found only in March. The total soluble carbohydrates in the roots showed a short increase in June when the sodium in the plants also increased. It was concluded that carbohydrates may be used to balance osmotic shocks, but that another compatible compounds is necessary to maintatin long-term osmotic equilibrium. The nitrate reductase activity, measuredin vivo, and the soluble protein changed roughly in parallel with the internal nitrate from May to August, suggesting that nitrogen uptake and reduction in the plant is inhibited during summer when the soil is dry and very saline. This could be a direct effect of drought and/or salinity on the plants, or an indirect onevia an inhibition of nitrifying bacteria.     

Endogenous stimulant of prostaglandin endoperoxide synthase activity in human amniotic fluid

In this study we describe the discovery and characterization of a substance in human amniotic fluid that stimulates prostaglandin biosynthesis by a microsome-enriched preparation of bovine seminal vesicles. The stimulatory activity is not retained substantially upon anisotropic ultrafiltration through a filter with a molecular weight exclusion limit of 500. Stimulation of prostaglandin biosynthesis by this substance is time- and concentration-dependent; maximal stimulation of approx. 200% being observed within 20 min of commencing incubation with 1 ml-equivalent of stimulant fraction. Stimulatory activity is demonstrable both in the presence of reduced glutathione (1.3 mM) and L-tryptophan (20 mM), either separately or combined, and in the presence of exogenous arachidonic acid (5-120 microM). In the absence of added cofactors, the stimulatory substance increases the rates of biosynthesis of prostaglandin E2 and prostaglandin F2 alpha to equal extents. The amount of stimulatory substance added to incubations is correlated positively with increased oxygen consumption during incubations. The stimulatory substance is stable to heating at 100 degrees C for 10 min but is inactivated substantially (to less than 20% of original activity) by treatment with pronase. It is concluded that human amniotic fluid contains a substance of relatively low molecular weight, which is proteinaceous in character, that stimulates prostaglandin endoperoxide synthase activity.     

Flow Cytometric Characterization of Bovine Blood Neutrophil Phagocytosis of Fluorescent Bacteria and Zymosan Particles

A Flow Cytometric method for the evaluation of the phagocytic capacity of bovine blood neutrophils is described. The neutrophils were isolated from bovine blood by a one step discontinuous gradient of Percoll. By this technique of isolation, 90 ± 2.8 % (mean ± s) of the granulocytes in the whole blood were recovered. Isolated neutrophils were incubated with FITC labeled S. aureus or zymosan particles in a ratio of 1:20 and 1:10, respectively, and a final serum concentration of 10 %. Phagocytosis was terminated after 15 min and the number of extracellular bacteria or zymosan particles and the percentage of phagocytic granulocytes were registered by Flow Cytometry (FCM). FCM and microscopic studies revealed that eosinophils play a minor role in the phagocytosis of bacteria. The neutrophils were the main population of the granulocytes which were actively phagocytic. Variation among cows in the ability of their blood neutrophils to phagocytize bacteria was evident.     

Synthesis of 2-acetamido-2-deoxy-4-O-β-d-galactopyranosyl-3-O-methyl-d-glucopyranose (N-acetyl-3-O-methyllactosamine) and its benzyl α-glycoside

Benzyl 2-acetamido-2-deoxy-3-O-methyl-α-d-glucopyranoside (3) was obtained by deacetalation of its 4,6-O-benzylidene derivative (2). Compound 2 was prepared by methylation of benzyl 2-acetamido-4,6-O-benzylidene-2-deoxy-α-d-glucopyranoside with methyl iodide-silver oxide in N,N-dimethylformamide. Diol 3 was selectively benzoylated and p-toluenesulfonylated, to give the 6-benzoic and 6-p-toluenesulfonic esters (4 and 5, respectively). Displacement of the sulfonyl group of 5 with sodium benzoxide in benzyl alcohol afforded the 6-O-benzyl derivative (6). Glycosylation of 4 with 2,3,4,6-tetra-O-acetyl-α-d-galactopyranosyl bromide (7) in dichloromethane, in the presence of 1,1,3,3-tetramethylurea, furnished the disaccharide derivative 8. Similar glycosylation of compound 6 with bromide 7 gave the disaccharide derivative 10. O-Deacetylation of 8 and 10 afforded disaccharides 9 and 11. The structure of compound 9 was established by ¹³C-n.m.r. spectroscopy. Hydrogenolysis of the benzyl groups of 11 furnished the disaccharide 2-acetamido-2-deoxy-4-O-β-d-galactopyranosyl-3-O-methyl-d-glucopyranose (N-acetyl-3-O-methyllactosamine).