Effect of arsenate As (V) on the biomarkers of Myriophyllum alterniflorum in oligotrophic and eutrophic conditions.

Alternate watermilfoil, Myriophyllum alterniflorum is an aquatic macrophyte found in the Limousin rivers (France) whose potential for biomonitoring of metal pollution has been demonstrated. The objective of the present study carried out in vitro was to identify biomarkers for an early detection of the pollution by a metalloid As (V) in eutrophic and oligotrophic conditions. A synthetic medium of similar composition to the waters of the River Vienne was prepared. The morphological development of watermilfoil was monitored for 30 days, with or without contamination by 100 µg L(-1) As (V). In addition, the mineralization of plants and the analysis of biomarkers (chlorophylls, photosynthetic and respiratory intensities …) were investigated after 21 days. Our results indicated that eutrophic medium, induced a decrease in chlorophyll pigments, in growth and an increase in H2O2 compared to the oligotrophic medium. While, the presence of As (V), led to a decrease in the osmotic potential, pigment content, photosynthesis and respiration rates and an inhibition of shoot branching of plants in both conditions. However, a significant increase in H2O2 content was noted in the eutrophic medium. Finally, As (V) was found to be more accumulated in roots than shoots in both conditions but was more accumulated in oligotrophic one. Therefore, we can conclude that the water trophic level modifies the response of M. alterniflorum in presence of arsenate. Thus, M. alterniflorum shows a great promise in water-quality biomonitoring.

Transfer of metal(loid)s in a small vineyard catchment: contribution of dissolved and particulate fractions in river for contrasted hydrological conditions.

The use of inorganic pesticides in viticulture leads to the accumulation of metal(loid)s in soils which can be transferred to the hydro-systems (groundwater and surface water) via several processes. This study reports on the occurrence and behavior of metal(loid)s (Li, Al, Cr, Ni, Cu, Zn, As, Sr, and Ba), with a particular focus on Cu, Zn, and As, in a small stream draining a vineyard catchment. Base flow and flood events were monitored in order to assess the spatiotemporal variability of metal(loid) concentrations and to evaluate the contribution of the particulate fraction to the transfer of metal(loid)s according to the hydrological conditions. Results show that very different patterns of metal(loid)s were observed in the Morcille River according to the hydrological conditions. In base flow conditions, Cu and As were mainly transported in dissolved phase, which contributed to more than 70 and 80%, respectively, of the total load during this period. On the contrary, during base flow, Zn was mainly transported as associated to particles (90%). During the two storm events monitored, the particulate fraction was dominant, as its represented around 74-80%, 97%, and 50-70% of the total Cu, Zn, and As load in the river, respectively. Thus, despite a weaker affinity for particles during floods (decrease of particulate content during floods), metal(loid)s were mainly brought as particles, given that high amounts of suspended particulate matter (up to 2031 mg/L) were mobilized. Finally, comprehensive fluxes estimations confirmed that floods were responsible for more than 90% of the total Cu, Zn and 75% for As load transiting in the Morcille River in August within a very short period of time (less than 17%).

Fibrinogen Bondy: a new case of dysfibrinogenemia. Isolation of the abnormal fibrinogen molecules.

In a 81 year old health woman, gross abnormalities of fibrin formation led to the discovery of an abnormal fibrinogen named fibrinogen Bondy. Clottability of purified fibrinogen Bondy was only 53% compared to 95-98% for normal fibrinogen. Functional studies revealed (i) delayed coagulation by thrombin and batroxobin (Reptilase), (ii) incomplete release of fibrino-peptides A and B, (iii) poor fibrin monomer aggregation, (iv) delayed fibrin proteolysis by plasmin. Electrophoretic mobility of fibrinogen Bondy, its three chains and the products of fibrin cross-linking, was normal. Fibrinogen NH2-terminal residues of fibrinogen Bondy were found to be normal. The presence of Ala, in addition to Gly and Tyr in the fibrin clot and its supernatant, showed that a part of fibrinogen molecules was not clotted, i.e. either copolymerised with fibrin or remaining in solutions. Gel filtration of the supernatant allowed the separation of both soluble complexes and fibrinogen. This fibrinogen population was shown to be unclottable by thrombin and to inhibit clotting of normal fibrinogen.

Assessing pesticide concentrations and fluxes in the stream of a small vineyard catchment--effect of sampling frequency.

This study reports on the occurrence and behaviour of six pesticides and one metabolite in a small stream draining a vineyard catchment. Base flow and flood events were monitored in order to assess the variability of pesticide concentrations according to the season and to evaluate the role of sampling frequency on the evaluation of fluxes estimates. Results showed that dissolved pesticide concentrations displayed a strong temporal and spatial variability. A large mobilisation of pesticides was observed during floods, with total dissolved pesticide fluxes per event ranging from 5.7x10(-3) g/Ha to 0.34 g/Ha. These results highlight the major role of floods in the transport of pesticides in this small stream which contributed to more than 89% of the total load of diuron during August 2007. The evaluation of pesticide loads using different sampling strategies and method calculation, showed that grab sampling largely underestimated pesticide concentrations and fluxes transiting through the stream.

Positive gadolinium anomalies in wastewater treatment plant effluents and aquatic environment in the Hérault watershed (South France).

Anthropogenic gadolinium (Gd), used as a contrast agent in magnetic resonance imaging, may enter rivers and groundwaters with the effluents of wastewater treatment plant (WWTP). Such contaminations, which are mainly found in densely populated areas with highly developed medical systems, induce positive gadolinium anomalies in waters. This study reports on the occurrence of positive Gd anomaly in wastewaters, surface and groundwaters in a slightly populated Mediterranean watershed. Water samples have been collected along the Hérault River, in its tributaries, in wells and springs supplying drinking water and in WWTP effluents during two sampling campaigns in February and July 2003. Systematically pronounced positive gadolinium anomalies (Gd/Gd( *)) were observed in WWTP effluents with values reaching 306. These observations have shown that Gd/Gd( *) can also be found in wastewater drained from rural communities, not equipped with MRI facilities. Positive gadolinium anomalies were detected in two tributaries of the Hérault River and in some wells supplying drinking water, corresponding to an excess of anthropogenic Gd in water up to 15.4pM. A monthly monitoring on one well has confirmed the persistence of gadolinium anomalies all along the year, suggesting a continual wastewater contamination on this site. A spatial monitoring on one tributary showed that wastewater contribution modifies completely the normalized REE pattern of river water, resulting in a decrease of REE amount correlated to the Gd anomaly appearance.

Thrombin-induced endothelial cell dysfunction.

Endothelium is a multifunctional organ which can directly influence circulating blood components as well as other cells within the vessel wall. The clotting enzyme thrombin, generated at the surface of damaged endothelium, induces blood coagulation but also exerts a variety of functional effects on the endothelium itself. Thrombin acts on endothelial cells to stimulate synthesis and release of various agents, such as inflammatory mediators, vasoactive substances and growth factors. It causes leukocyte adhesion to the endothelium by triggering expression of adhesion molecules on the cell surface and causes disruption of endothelial permeability properties. The majority of thrombin effects on endothelial cells are mediated by its receptor and require its lytic activity. Differences have been observed among the response to thrombin of endothelial cells of different origin. In general microvascular endothelial cells appear to be particularly sensitive to this enzyme. Thrombin induced microvascular dysfunction can have pathological consequences and contribute to organ reactions to inflammation and ischaemia.

Molecular defect of prothrombin Barcelona. Substitution of cysteine for arginine at residue 273.

Prothrombin Barcelona has been isolated from a patient with a normal prothrombin antigen level but low prothrombin coagulant activity. The activation of this protein is impaired by the absence of one of the two factor Xa-catalyzed cleavages that normally lead to the formation of thrombin. Prothrombin Barcelona and prothrombin were isolated from patient plasma and normal plasma, respectively, in a single-step, high-yield immunoaffinity purification using conformation-specific antibodies immobilized on Sepharose. After reduction and alkylation, the purified proteins were subjected to trypsin hydrolysis. The resulting peptides were separated by reverse-phase high performance liquid chromatography. Comparison of the peptide maps of prothrombin Barcelona and prothrombin demonstrated that a peptide, identified as fragment 274-287 in prothrombin by automated Edman degradation, was missing in the prothrombin Barcelona digest. In the chromatogram derived from prothrombin Barcelona, an additional peptide was observed. The amino acid sequence of this peptide was Ala-Ile-Glu-Gly-Cys-Thr-Ala-Thr-Ser-Glu-Tyr-Gln-Thr-Phe-Phe-Asn-Pro-Arg, corresponding to residues 269-287 in prothrombin except for the substitution of cysteine for arginine at residue 273. The substitution of cysteine for arginine was confirmed by tryptic digestion of 14C-carboxymethylated prothrombin Barcelona. Edman degradation of fragment 269-287 indicated the association of 14C with the cysteine at residue 273. The replacement of arginine by cysteine at residue 273, adjacent to the known factor Xa cleavage site, precludes normal activation of prothrombin Barcelona by factor Xa and the generation of thrombin.

The synthetic chemoattractant Trp-Lys-Tyr-Met-Val-DMet activates neutrophils preferentially through the lipoxin A(4) receptor.

A D-methionine-containing peptide, Trp-Lys-Tyr-Met-Val-D-Met-NH(2) (WKYMVm), featuring a unique receptor specificity was investigated with respect to its ability to activate neutrophil effector functions. The peptide was found to be more potent than the N-formylated peptide N-formyl-Met-Leu-Phe (fMLF) at inducing neutrophil chemotaxis, mobilization of neutrophil complement receptor 3 (CR3), and activation of the neutrophil NADPH-oxidase. The fact that binding of fML[(3)H]F was inhibited by both fMLF and WKYMVm suggests that N-formyl peptide receptor (FPR) is shared by these peptides. However, the neutrophil response induced by the WKYMVm peptide was insensitive to the fMLF antagonists, cyclosporin H, and Boc-FLFLF that specifically block the function of the FPR. These results suggest that even though WKYMVm may bind FPR the cells are activated preferentially through a receptor distinct from the FPR. Using transfected HL-60 cells expressing either the FPR or its neutrophil homologue FPRL1, also referred to as LXA(4)R because it has been shown to bind lipoxin A(4), we show that WKYMVm is about 300-fold more active at mobilizing intracellular calcium through FPRL1 than through FPR. The WKYMVm activates FPRL1-expressing cells in a cyclosporin H-independent manner with an EC(50 )of around 75 pmol/L, whereas it activates FPR-expressing cells with an EC(50 )of around 25 nmol/L. The observation that exudated cells are primed in their response to WKYMVm suggests that FPRL1/LXA(4)R like FPR is stored in mobilizable organelles. (Blood. 2000;95:1810-1818)

Human complement 5a (C5a) anaphylatoxin receptor (CD88) phosphorylation sites and their specific role in receptor phosphorylation and attenuation of G protein-mediated responses. Desensitization of C5a receptor controls superoxide production but not receptor sequestration in HL-60 cells.

Upon agonist binding, the anaphylatoxin human complement 5a receptor (C5aR) has previously been found to be phosphorylated on the six serine residues of its carboxyl-terminal tail (Giannini, E., Brouchon, L., and Boulay, F. (1995) J. Biol. Chem. 270, 19166-19172). To evaluate the precise roles that specific phosphorylation sites may play in receptor signaling, a series of mutants were expressed transiently in COS-7 cells and stably in the physiologically relevant myeloid HL-60 cells. Ser(334) was found to be a key residue that controls receptor phosphorylation. Phosphorylation of either of two serine pairs, namely Ser(332) and Ser(334) or Ser(334) and Ser(338), was critical for the phosphorylation of C5aR and its subsequent desensitization. Full phosphorylation and desensitization of C5aR were obtained when these serines were replaced by aspartic acid residues. The mutation S338A had no marked effect on the agonist-mediated phosphorylation of C5aR, but it allowed a sustained C5a-evoked calcium mobilization in HL-60 cells. These findings and the ability of the S314A/S317A/S327A/S332A mutant receptor to undergo desensitization indicate that the phosphorylation of Ser(334) and Ser(338) is critical and sufficient for C5aR desensitization. The lack of phosphorylation was found to result not only in a sustained calcium mobilization and extracellular signal-regulated kinase 2 activity but also in the enhancement of the C5a-mediated respiratory burst in neutrophil-like HL-60 cells. For instance, the nonphosphorylatable S332A/S334A mutant receptor triggered a 1.8-2-fold higher production of superoxide as compared with the wild-type receptor. Interestingly, although the desensitization of this mutant was defective, it was sequestered with the same time course and the same efficiency as the wild-type receptor. Thus, in myeloid HL-60 cells, desensitization and sequestration of C5aR appear to occur through divergent molecular mechanisms.

Identification of a monoclonal antibody against platelet GPIIb that interacts with a calcium-binding site and induces aggregation.

We have characterized a monoclonal antibody named D33C, specific for platelet glycoprotein (GP) IIb, which induces fibrinogen binding and platelet aggregation. D33C Fab fragments interact with an average of 44,000 +/- 20,000 sites on resting platelet with a Kd value of 0.8 microM. This value decreased to 0.17 microM in the presence of 1 mM EDTA suggesting that Ca2+ chelation increases the antibody affinity. Purified IgGs and Fab fragments exhibit a similar potency and induce binding of fibrinogen and aggregation at levels comparable to those obtained with ADP. D33C-induced platelet aggregation, however, was not inhibited by 1 microM PGE1 and was not associated with a significant [14C]serotonin release, suggesting differences with ADP in the mechanism of activation. Among a large series of synthetic peptides corresponding to potential antigenic sequences within the structure of GPIIb, one peptide with the sequence DIDDNGYPDLIV was found to inhibit D33C activity. This peptide corresponds to a putative calcium-binding site whose sequence is highly homologous to similar sequences present in the alpha subunits of the fibronectin and the vitronectin receptors. Despite this homology, D33C interacts only with platelet GPIIb suggesting that the identified epitope may be differently exposed at the surface of the cells. This antibody may prove to be a valuable tool to study the induction reaction on recombinant GPIIbIIIa expressed in cells that lack the appropriate signal transduction reactions.

Effect of propeptide mutations on post-translational processing of factor IX. Evidence that beta-hydroxylation and gamma-carboxylation are independent events.

Post-translational processing of Factor IX includes glycosylation, cleavage of the signal peptide and propeptide, vitamin K-dependent carboxylation of specific glutamic acid residues to form gamma-carboxyglutamic acid, and beta-hydroxylation of aspartic acid at residue 64 to form beta-hydroxyaspartic acid. The human Factor IX cDNA coding sequence was modified in the propeptide region (residue -18 to -1) using oligonucleotide-directed site-specific mutagenesis, and the altered Factor IX cDNA was expressed in Chinese hamster ovary cells. The effects of the mutations on proteolytic processing, gamma-carboxylation, and beta-hydroxylation were assessed by direct structural analysis. After purification, the molecular weight of each of the recombinant Factor IX species and its NH2-terminal amino acid sequence were shown to be identical to those of plasma Factor IX. gamma-Carboxyglutamic acid and beta-hydroxyaspartic acid analyses revealed that recombinant wild-type Factor IX contained 9.2 gamma-carboxyglutamic acid and 0.3 beta-hydroxyaspartic acid residues/molecule compared with 11.4 gamma-carboxyglutamic acid and 0.39 beta-hydroxyaspartic acid residues in plasma Factor IX. When the 18-residue propeptide was deleted or when the cells were grown in the presence of sodium warfarin, secreted Factor IX contained no detectable gamma-carboxyglutamic acid but 0.36 and 0.40 residues of beta-hydroxyaspartic acid, respectively. Point mutations leading to substitution of alanine for phenylalanine at residue -16 or glutamic acid for alanine at residue -10 contained 0.2 and 1.7 gamma-carboxyglutamic acid residues, respectively, and 0.2 residues of beta-hydroxyaspartic acid. These data confirm that the propeptide mutations made do not interfere with proteolytic processing and that the Factor IX propeptide contains a recognition site that designates the adjacent glutamic acid-rich domain for gamma-carboxylation. In contrast, beta-hydroxylation of aspartic acid 64 is an independent process which does not require vitamin K and is mediated through a hydroxylation recognition site in the mature Factor IX, not in the propeptide.

Thrombin Metz: characterization of the dysfunctional thrombin derived from a variant of human prothrombin.

Thrombin Metz and normal thrombin, resulting from activation of the respective prothrombins by factor Xa in the presence of calcium, phospholipid, and factor Va, were purified by chromatography on sulfopropyl Sephadex. By physicochemical criteria, thrombin Metz is identical to normal thrombin. Its functional properties were investigated in some reactions in which thrombin is classically involved. Thrombin Metz exhibits less than 4% of fibrinogen clotting activity. Both Km and Kcat, determined on S2238, are abnormal. Titration with the high-affinity competitive inhibitor of thrombin, DAPA, shows that fluorescence enhancement of the probe is only 34% in binding to thrombin Metz when compared to that observed in binding to normal thrombin. High-performance liquid chromatography has been used to measure the simultaneous rate of release of fibrinopeptides A and B. A decreased release rate for both fibrinopeptides, more marked for fibrinopeptide B, results in a slow fibrin polymerization, as followed by absorbance at 450 nm. Thrombin Metz is less than 5% as effective as normal thrombin in inducing platelet aggregation. Interaction with antithrombin III is slower than normal when followed by SDS gel electrophoresis and inhibition of the amidolytic activity of thrombin on S2238. This abnormality is not observed in the presence of heparin. However, thrombin Metz binds less tightly to a heparin-Sepharose column, and the direct inhibition of heparin on its activity on S2238 is weaker. From these results, we can predict that the defect in thrombin Metz affects the catalytic site or its vicinity and, jointly or consequently, the region of interaction of thrombin with antithrombin III and heparin.

Covalent structures of beta and gamma autolytic derivatives of human alpha-thrombin.

Nonclotting beta- and gamma-thrombins have been prepared by autolysis of human alpha-thrombin at pH 8.6 in the presence of 0.4 M NaCl and purified on BioRex 70. Reduced and carbamidomethylated A and B chains fragments were separated by gel filtration and reverse phase high performance liquid chromatography. Structural characterization of these fragments demonstrated that alpha to beta conversion results from two cleavages at Arg 62 and Arg 73 in the B chain, releasing an intact 11-residue peptide. beta to gamma conversion corresponds to the additional loss of a fragment of the B chain stretching from Ile 124 to Lys 154. Autolysis is not accompanied by cleavages in the A chain. Loss of clotting activity is therefore related solely to the excision of residues 63 to 73 in the B chain. With the exception of cleavage at Arg 73, these results differ from a proposed model for alpha to gamma conversion of bovine thrombin.

Molecular basis of hemophilia B: a defective enzyme due to an unprocessed propeptide is caused by a point mutation in the factor IX precursor.

A mutant factor IX, designated factor IXCambridge, was isolated from a patient with hemophilia B. This protein includes an 18-residue propeptide attached to the NH2 terminus of factor IX. A point mutation at residue -1, from an arginine to a serine, precludes cleavage of the propeptide by a processing protease and interferes with gamma-carboxylation of the factor IX, indicating the importance of the leader sequence in substrate recognition by the vitamin K-dependent carboxylase. This represents an example of an enzyme defect due to the presence of a point mutation in a precursor protein (preproenzyme) that is the cause of a human hereditary disease. This defect will serve as a prototype for understanding the molecular basis of some forms of hemophilia and other hereditary enzyme deficiencies.