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].     

Sensitivity of methicillin-resistant Staphylococcus aureus strains to some antibiotics, antiseptics and disinfectants

The effects of antibiotics, antiseptics and disinfectants against some methicillin resistant (MRSA) and methicillin-sensitive (MSSA) Staphylococcus aureus strains have been studied. The MRSA and MSSA strains were equally sensitive to phenols, esters of para(4)-hydroxybenzoit acid and chlorhexidine but MRSA strains were slightly more resistant to quaternary ammonium compounds and considerably more so to dibromopropamidine isothionate. Some MRSA strains were also resistant to phenylmercuric nitrate (but not another organomercurial, thiomersal), mercuric chloride and cadmium chloride. All MRSA strains produced β-lactamase. Strains from the Royal Free Hospital, London were highly resistant to β-lactam antibiotics, erythromycin, trimethoprim and tetracyctines but were sensitive to other antibiotics. One strain from the University Hospital of Wales, Cardiff was resistant to gentamicin but sensitive to tetracycline and trimethoprim.     

Purification and properties ofl-serine dehydratase fromLactobacillus fermentum ATCC 14931

l-Serine dehydratase fromLactobacillus fermentum was purified 100-fold. It was stabilized by the presence of 1 mM l-cysteine in 50 mM phosphate buffer. M_r=150,000 was determined by gel filtration. The enzyme consists of four apparently identical subunits (M_r=40,000) that were observed after treatment with sodium dodecyl sulfate. The apparent K_m forl-serine was 65 mM. Fe⁺⁺ was required for the enzymatic activity, and the apparent K_m value for this reaction was 0.55 mM. Maximum enzymatic activity was observed at 45°C and pH 8.0 in 50 mM phosphate buffer. At pH values different from the optimum, a positive cooperativity between substrate molecules was observed. The activation energy of the reaction was 11,400 and 22,800 cal × mol^–1 for temperature values more than and less than 35°C respectively. The purified enzyme showed a maximum absorption between 400 and 420 nm, indicating the presence of pyridoxal-5-phosphate (PLP) as a prosthetic group. The PLP concentration was 0.027 µmoles per milligram of protein. The data suggest that there is 1 mol of PLP for each protein subunit.     

Hepatic mitochondrial cytochrome P-450 system. Purification and characterization of two distinct forms of mitochondrial cytochrome P-450 from beta-naphthoflavone-induced rat liver

We have purified two distinct isoforms of mitochondrial cytochrome P-450 from beta-naphthoflavone (beta-NF)-induced rat liver to greater than 85% homogeneity and characterized their molecular and catalytic properties. One of these isoforms showing an apparent molecular mass of 52 kDa is termed P-450mt1 and the second isoform with 54-kDa molecular mass is termed P-450mt2. Cytochrome P-450mt2 comigrates with similarly induced microsomal P-450c (the major beta-NF-inducible form) on sodium dodecyl sulfate-polyacrylamide gels and cross-reacts with polyclonal antibody monospecific for cytochrome P-450c. Cytochrome P-450mt2, however, represents a distinct molecular species since it failed to react with a monoclonal antibody to P-450c and produced V8 protease fingerprints different from P-450c. Cytochrome P-450mt1, on the other hand, did not show any immunochemical homology with P-450c or P-450mt2 as well as partially purified P-450 from control mitochondria. Electrophoretic comparisons and Western blot analysis show that both P-450mt1 and P-450mt2 are induced forms not present in detectable levels in control liver mitochondria. A distinctive property of mitochondrial P-450mt1 and P-450mt2 was that their catalytic activities could be reconstituted with both NADPH-cytochrome P-450 reductase as well as mitochondrial specific ferredoxin and ferredoxin reductase electron transfer systems, while P-450c showed exclusive requirement for NADPH-cytochrome P-450 reductase. Cytochromes P-450mt1 and P-450mt2 were able to metabolize xenobiotics like benzo(a)pyrene and dimethyl benzanthracene at rates only one-tenth with cytochrome P-450c. Furthermore, P-450mt1, P-450mt2, as well as partially purified P-450 from control liver, but not P-450c, showed varying activities for 25- and 26-hydroxylation of cholesterol and 25-hydroxylation of vitamin D3. These results provide evidence for the presence of at least two distinct forms of beta-NF-inducible cytochrome P-450 in rat hepatic mitochondria.     

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.     

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.