Influence of organic matters on AsIII oxidation by the microflora of polluted soils.

The global AsIII-oxidizing activity of microorganisms in eight surface soils from polluted sites was quantified with and without addition of organic substrates. The organic substances provided differed by their nature: either yeast extract, commonly used in microbiological culture media, or a synthetic mixture of defined organic matters (SMOM) presenting some common features with natural soil organic matter. Correlations were sought between soil characteristics and both the AsIII-oxidizing rate constants and their evolution in accordance with inputs of organic substrates. In the absence of added substrate, the global AsIII oxidation rate constant correlated positively with the concentration of intrinsic organic matter in the soil, suggesting that AsIII-oxidizing activity was limited by organic substrate availability in nutrient-poor soils. This limitation was, however, removed by 0.08 g/L of added organic carbon. In most conditions, the AsIII oxidation rate constant decreased as organic carbon input increased from 0.08 to 0.4 g/L. Incubations of polluted soils in aerobic conditions, amended or not with SMOM, resulted in short-term As mobilization in the presence of SMOM and active microorganisms. In contrast, microbial AsIII oxidation seemed to stabilize As when no organic substrate was added. Results suggest that microbial speciation of arsenic driven by nature and concentration of organic matter exerts a major influence on the fate of this toxic element in surface soils.

Long-term fate of exogenous metals in a sandy Luvisol subjected to intensive irrigation with raw wastewater.

From 1899 to 2002, sandy Luvisol in the Paris region has been intensively irrigated with raw wastewater, resulting in major soil pollution by metallic trace elements (MTE). To identify the soil phases implicated in retaining these metals, sequential extractions were performed on a solum irrigated with untreated wastewater and another reference solum. The endogenous and exogenous fractions of MTE in the contaminated soil were discriminated using correlations between MTE and major elements defined from unpolluted soils of the area. In the contaminated soil no exogenous lead and chromium are present below the surface horizon, whereas exogenous zinc and copper are found down to the base of the solum. The endogenous MTE are mainly found in the residual fraction. Exogenous MTE appear to be associated with organic matter in the surface horizon, and exogenous zinc seems to be readsorbed on iron and manganese oxyhydroxides in the underlying horizons.

Response of rhizobial populations to moderate copper stress applied to an agricultural soil.

The use of pesticides in agricultural soils may affect the soil microbiota. The effect of repeated application of copper sulfate in soil on indigenous populations of rhizobia was assessed in a medium-term field experiment. Copper sulfate was applied over 8 years at two different rates, 12.5 and 50 kg of CuSO4 ha(-1) year(-1), in the field. The concentrations of total copper in soil varied between 14.0 (control plots that did not receive copper sulfate) and 91.0 mg kg(-1) (the most contaminated plots) at the time of sampling, 3 years after the end of the copper treatments. All the other physicochemical parameters were similar among the plots that also shared the same cropping history. The target rhizobia were monospecific populations of Rhizobium leguminosarum bv. viciae nodulating Vicia sativa and communities of rhizobial species nodulating Phaseolus vulgaris. The size of the vetch rhizobial populations was significantly reduced in the soils with the higher Cu content, whereas the size of the Phaseolus rhizobial populations was not significantly affected. However, the number of nodules formed on both vetches and common beans were reduced for the plants grown in the most contaminated soils, suggesting an additional toxic effect of copper on plant physiology. The diversity (Simpson's indices) of rhizobial genotypes, as characterized by polymerase chain reaction restriction fragment length polymorphism of 16S-23S rDNA intergenic spacer (IGS), was not influenced by copper application. Also, the genetic structure of the R. leguminosarum bv. viciae populations was not modified by copper treatments. By contrast, a shift was observed in the composition of the Phaseolus-nodulating communities in relation to soil copper content. The communities were composed of three 16S rDNA haplotypes: one corresponding to the R. leguminosarum (biovar phaseoli) species, the two others forming a new lineage of Phaseolus rhizobia based on 16S rDNA sequence analysis. The reduced frequency of the R. leguminosarum species in the Phaseolus-nodulating communities from the copper-treated soils was linked to its higher sensitivity to copper as compared to the higher tolerance of isolates belonging to the other rhizobial lineage. The new lineage was functionally efficient for symbiotic nitrogen fixation with P. vulgaris. Our results suggest that functional redundancy among species exhibiting variability for copper tolerance preserved the size of Phaseolus-nodulating communities. In contrast, the abundance of the vetch-nodulating rhizobia, which was a monospecific functional group mainly constituted by copper-sensitive genotypes, was adversely affected by repeated application of copper sulfate.

Enhancing spatial estimates of metal pollutants in raw wastewater irrigated fields using a topsoil organic carbon map predicted from aerial photography.

Various approaches have been used to estimate metal pollutant element (TE) contents at unsampled locations in a 15-ha contaminated site located in the plain of Pierrelaye-Bessancourt (about 24 km Northwest of Paris). 87 samples of soil plough layer were randomly sampled at each mesh of a regular square grid over the whole study area and the total contents of Cd, Cr, Cu, Ni, Pb, and Zn were measured. A first set of 50 measurements, randomly selected from the 87 samples, was used for the prediction and another set of 37 measurements was kept for the validation. Topsoil organic carbon contents (SOC) were measured at 75 sites with 50 measurements sharing the same locations as TE. An aerial photography of the study area showing bare soils was selected for relating brightness intensities and SOC. Mapping procedures used were ordinary kriging (OK), cokriging (COK), collocated cokriging (CC), and kriging with external drift (KED). SOC maps used as exhaustively sampled information in KED and CC of TE were obtained by KED and CC procedures, respectively, accounting for 75 SOC measurements and the brightness intensities of numerical counts provided by the visible bands of the aerial photograph bare soils. Consequently, for each TE, four maps were generated: two maps resulting from KED and CC procedures (KED-SOC75P, CC-SOC75P), another one provided by standard cokriging (COK-TE50SOC75) accounting for TE prediction set plus 75 SOC measurements, and the last one corresponding to that estimated by ordinary kriging from only prediction set measurements (OK50). Three indices: (1) the mean prediction error (ME) and the mean absolute prediction error (|ME|); (2) the root mean square error (RMSE); and (3) the relative improvement (RI) of accuracy, as well as residuals analysis, were computed from the validation set (observed data) and predicted values. On the 37 test data, the results showed that the more accurate predictions were systematically those obtained by kriging accounting for SOC map predicted by KED from 75 SOC measurements and brightness values of the aerial photo (KED-SOC75P) followed closely by CC-SOC75P procedure, except for Cu and Zn where CC-SOC75P appeared to be slightly more accurate than KED-SOC75P. In regard to the RI of accuracy between prediction methods, the results confirmed once for all the benefit of accounting for SOC data set plus the exhaustively sampled information provided by the aerial photography regardless of the considered TE. Nevertheless, for Cd, Pb, and Zn, the RI of accuracy was less than 20% between the two most accurate methods (KED-SOC75P and CC-SOC75P) and standard cokriging in which the information provided by the aerial photography is ignored when mapping. The sensitivity of KED-SOC75P and CC-SOC75P approaches to the sampling density of the target variables (TE) was assessed using 10 random subsets of different sizes (25 and 33 observations) drawn from a prediction set that includes 50 data. Results have shown that the TE estimates by KED-SOC75P and CC-SOC75P approaches using only 25 TE samples were much more accurate than the estimates performed by OK50 and COK-TE50SOC75 approaches that use the whole samples of the prediction set. Moreover, the RI of accuracy was reduced by less than 15% if the original sampling density was reduced by a third.

[Differentiation of hepatic and hematopoietic stem cells: study of liver regulation protein (LRP)]. / Différenciation des progéniteurs hépatocytaires et hématopoïétiques: étude de la Liver Regulating Protein" (LRP).

In the adult liver, the plasma membrane Liver Regulating Protein (LRP) has been found to participate in the recognition signals between hepatocytes and rat liver epithelial cells (RLECs) and to play a critical role in the functional stability of hepatocytes. Here, we report the involvement of LRP in hematopoiesis. First, LRP was evidenced in the hematopoietic fetal rat liver and in adult rat bone marrow. Then, the involvement of LRP in adult rat and human hematopoiesis was investigated in coculture of hematopoietic cells with RLECs. In the rat, RLECs sustain long-term production of hematopoietic cells and committed progenitors as well as bone marrow stroma cells. These effects can be specifically blocked by addition of anti-LRP antibodies to both culture systems. The supportive activity of RLECs on human hematopoiesis was assessed by comparison to the reference MS-5 cell line from both unifractioned and purified CD34(+)CD38(-) hematopoietic cells. Taken together, our results argue for the involvement of LRP in heterotypic interaction signals mediated by RLEC and bone marrow stromal cells which lead to hematopoietic primitive progenitors development. They might lead to interesting applications in fundamental research, pharmacology and therapeutics.

Pattern of cytokine expression by rat liver epithelial cells supporting long-term culture of human CD34(+)umbilical cord blood cells.

Fetal liver is the main site of haematopoiesis during mid-gestation. The adult liver still provides a favourable environment for extramedullary haematopoiesis. Nevertheless, regulation of liver haematopoiesis by cell-cell contacts or by cytokines remains poorly understood. Recently, we have shown that rat liver epithelial cells (RLECs) support long-term survival and multilineage differentiation of adult human CD34(+)and CD34(+)/CD38(-)haematopoietic cells obtained from granulocyte-colony stimulating factor mobilized peripheral blood and from bone marrow respectively. In addition, the importance of physical proximity between haematopoietic cells and RLECs was clearly demonstrated. Here, our findings give evidence that RLECs belonging to the epithelial but non-parenchymal liver compartment also sustain the long-term production of progenitors from human CD34(+)umbilical cord blood cells. Moreover, to better analyse the regulation of haematopoiesis in this RLEC coculture model, we have investigated the cytokine expression by RLECs alone and by RLECs coming from coculture with CD34(+)cells from umbilical cord blood. We demonstrated that a broad spectrum of cytokines acting at different stages of haematopoiesis is produced by RLECs. Interestingly, an upregulation of leukemia inhibitory factor expression by RLECs in presence of CD34(+)haematopoietic cells was observed. These data suggest an important role of cell-cell interactions in the regulation of haematopoiesis.

Hematopoiesis-promoting activity of rat liver biliary epithelial cells: involvement of a cell surface molecule, liver-regulating protein.

Stromal cell lines from bone marrow and other blood-forming organs including fetal liver have been found to support hematopoiesis. In this paper, we demonstrate that rat liver biliary epithelial cells (RLEC), most likely originating from primitive bile ductules, are able to support long-term hematopoietic cell growth as well as burst-forming unit-erythroid (BFU-E) and colony-forming unit-granulocyte/macrophage (CFU-GM) production. RLEC have previously been shown to express a cell surface molecule named liver-regulating protein (LRP), which is involved in the long-term maintenance of hepatocyte functions in a coculture system. In addition, LRP-like molecules have been found in spleen, thymus, lymph nodes, and peripheral blood cells. In the present study, we found that hematopoietic cells and several stromal cell types from bone marrow were LRP-positive, and immunoprecipitation revealed polypeptides similar to those found in RLEC. We then investigated the biological role of LRP on hematopoiesis using short-term RLEC and bone marrow stromal cell culture systems. Addition of specific anti-LRP antibody to both systems reduced hematopoietic cell proliferation and committed progenitor production, whereas it did not directly affect the clonal proliferation and maturation of these progenitors in methylcellulose assays. Moreover, using diffusible chamber cultures that suppress direct contacts with hematopoietic cells, we observed low cell growth and no effect of monoclonal antibody (mAb) L8 treatment. All these results strongly argue for a cell proximity signal mediated by RLEC and bone marrow stromal cells and for the involvement of LRP-like molecules in this signal in liver and bone marrow hematopoietic function.

Rat liver biliary epithelial cells support long-term production of haemopoietic progenitors from human CD34 cells.

In this study we report the supportive activity of rat liver epithelial cells (RLEC) on human haemopoiesis in the absence of exogeneously supplied growth factors. RLEC is a rat cell line derived from primitive biliary cells with epithelial characteristics which induce the long-term differentiation of hepatocytes through cell-cell contacts. We have established the ability of these cells to sustain long-term survival and multilineage differentiation of human haemopoietic progenitors from unfractionated bone marrow and growth-factor mobilized peripheral blood cells, and from human CD34+ and CD34+ CD38- haemopoietic cells, with a higher efficiency than the murine MS-5 stromal cell line: the numbers of committed progenitors recovered from RLEC cocultures after 8 weeks were 3-fold higher than from MS-5 cocultures, with an unusually high BFU-E production. Furthermore, using diffusible insert cultures, we demonstrated that, despite the lack of strong adhesive interaction between haemopoietic cells and RLEC, physical proximity was absolutely required for optimum stimulation of LTC-IC by RLEC. Taken together, these results show that biliary epithelial cells support human haemopoiesis and cause speculation that common mechanisms might be used by RLEC to regulate both the hepatocyte and the haemopoietic progenitors differentiation.

Specialization switch in differentiating embryonic rat liver progenitor cells in response to sodium butyrate.

Embryonic (E) 12 rat liver epithelial cells constitute a population of bipotential progenitor cells which can differentiate along the hepatocyte (Hep) or biliary epithelial cell (BEC) lineage in primary culture. In the present study, E12 cells were seeded on fibronectin-coated substratum and exposed to sodium butyrate (SB) for various exposure times, and the emergence of the Hep or BEC phenotype was monitored by following the variations in albumin production and assessing the appearance of the two surface-exposed markers HES6 and BDS7. Continuous exposure to SB resulted into a major reduction in albumin production and, at Day 9 postseeding, few cells coexpressed BDS7 and albumin. When cells were exposed to SB for 5 days and then cultured for an additional 5 days without SB, they massively express BDS7, but very little HES6. Moreover, the reverse sequence, i.e., 5 days without SB followed by 5 days with it, led to the appearance of many cells expressing both HES6 and BDS7. These results indicate that progenitors committed preferentially along the Hep lineage still have the option to switch to BECs, at a transitional stage that we refer to as a "differentiation window."