Temporal evolution of redox processes and free Cd dynamics in a metal-contaminated soil after rewetting.

Soil testing procedures to address metals bioavailability currently use air-dried soil rewetted almost until saturation. Such practices may influence the redox state of soil and the related dynamics of metals. To assess this potential impact, a metal-contaminated soil was air-dried and rewetted to 90% water holding capacity. We monitored over a 21-day incubation period the temporal changes of soil redox potential and solution Cd concentration (either total or free). Other physico-chemical parameters were followed notably pH, ionic strength (I) and the concentrations of NO(3)(-), Mn, Fe and SO(4)(2-) in solution. Soil redox potential showed the progressive establishment of strong reducing conditions in soil, in agreement with the temporal changes of NO(3)(-), Mn, Fe and SO(4)(2-) concentrations. It decreased by 13 pe units over the culture period leading to sulphate-reducing conditions (pe<-3) within only 21days. Solution Cd concentration increased transitorily over the first 100-150h of incubation (2-fold increase) in relation with the parallel increase in the concentration of competing cations for adsorption (Ca, Mg). It steeply decreased over the last 300h of incubation (30-fold decrease) as a result of Cd precipitation as Cd sulphides. This biphasic evolution of Cd dynamics was related to the temporal changes of Cd resupply from the solid phase. Using the technique of DGT we described the kinetics of Cd resupply over time and needed to invoke the existence of two pools of Cd.

Is the transfer factor a relevant tool to assess the soil-to-plant transfer of radionuclides under field conditions?

The radiological impact of radionuclides released to the terrestrial environment is usually predicted with mathematical models in which the transfer of radionuclides from soil to the plant is described with the transfer factor (TF). This paper questions the validity of the protocols proposed by the International Atomic Energy Agency to measure TF in the field and in greenhouses conditions. We grew maize (Zea mays L.) both in the field after a surface application of radionuclides ((54)Mn, (57)Co, (65)Zn, and (134)Cs) and in a greenhouse with the same soil that has received the same fertilization and that had been previously sieved and homogeneously labeled with the same radionuclides before being repacked in pots. The analysis of the displacement of radionuclides in the field soil profile showed a higher concentration of the surface-applied radionuclides in the preferential flow path (PFP) in comparison to the soil matrix indicating that they infiltrated heterogeneously in the soil profile due to the structure-induced non-uniform water flow. A significantly higher recovery of (57)Co and (134)Cs was observed in the plants grown in the field soil, whereas no differences in the recovery of (54)Mn and (65)Zn between the two experiments were detected. These results suggest that (i) under field conditions the soil-to-plant transfer of radionuclides that co-exist as stable elements present at low concentrations in the soil and in the plant is higher than that measured under greenhouse conditions and (ii) the implicit assumption made when calculating the TF (that radionuclides are homogeneously distributed in the soil profile) is not valid, thereby preventing the calculation of an average concentration to obtain the TF parameter.

Regulation of the three-dimensional organization of thyroid epithelial cells into follicle structures by the matricellular protein, thrombospondin-1.

Thyroid epithelial cells in primary culture have the capacity to organize into thyroid-specific three-dimensional structures, the follicles, in response to TSH. We studied whether thrombospondin 1 (TSP1), which represents, besides thyroglobulin, the main protein secreted by thyroid cells, could play a role in the process of folliculogenesis. TSH promoted follicle formation and inhibited TSP1 production. On the contrary, the phorbol ester, 12-O-tetradecanoyl-phorbol 13-acetate (1-100 nM) prevented TSH-induced follicle formation and strongly increased the synthesis of TSP1. Activation of TSP1 synthesis was dependent upon messenger RNA synthesis. Transforming growth factor-beta, like 12-O-tetradecanoyl-phorbol 13-acetate, increased TSP1 synthesis and prevented TSH-induced follicle formation. Thus, signaling molecules that depressed or conversely activated TSP1 production, respectively promoted or prevented thyroid folliculogenesis. TSP1, purified from platelets, was devoid of effect on cell substratum attachment, but exerted a concentration-dependent inhibition of the TSH-activated reconstitution of thyroid follicles (half-inhibition at 40 microg/ml). TSP1 exhibited the same effect when added to thyroid cell aggregates representing primitive follicle structures. Our data suggest that the control of thyroid follicle formation may operate at least in part through regulation of the production of the matricellular protein TSP1, which acts as a negative modulator of the cell-cell adhesion process involved in thyroid follicle morphogenesis.

Expression of laminin and its possible role in adrenal cortex homeostasis.

The adult mammalian adrenal cortex undergoes permanent regeneration. This process implies a cellular proliferation step restricted to the external zone of the tissue, and a subsequent centripetal cell migration during which phenotypic transition from glomerulosa into fasciculata and reticularis cells and elimination of senescent cells through apoptosis occur. As the molecular mechanisms implied in adrenocortical cell migration are still generally unknown, we addressed that question in the present study. Of several extracellular matrix proteins tested, laminin was the most potent chemotactic and haptotactic factor for bovine fasciculata adrenocortical cells. The maximal chemotactic effect (3-fold stimulation) was observed with 50-75 micrograms/ml laminin, whereas the haptotactic effect (3.5-fold stimulation) plateaued for laminin concentrations in the coating solution over 25 micrograms/ml. Using an anti-Engelbreth-Holm-Swarm laminin antibody, we could demonstrate that adrenocortical cells actively synthesize and secrete Engelbreth-Holm-Swarm-laminin, with the A chain produced in limiting quantities. ACTH treatment of adrenocortical cells specifically induced a 2.7- to 4.5-fold increase in A chain synthesis, resulting in a corresponding increase in the amount of secreted laminin. The distribution of laminin in the adrenal cortex tissue was then evaluated by standard immunohistochemistry. The protein appeared to be uniformly expressed in the three zones of the cortex. This observation does not favor the hypothesis that laminin acts as an attractant driving centripetal cell migration. Laminin, which is synthesized under the control of the systemic hormone ACTH, appears as a permissive factor that facilitates proper homeostasis of the adrenocortical tissue.

Opposite regulation of thrombospondin-1 and corticotropin-induced secreted protein/thrombospondin-2 expression by adrenocorticotropic hormone in adrenocortical cells.

Corticotropin-induced secreted protein (CISP) is a trimeric glycoprotein secreted by primary cultures of bovine adrenortical cells in response to adrenocorticotropic hormone (ACTH). This protein was recently purified in our laboratory, and its N-terminal amino-acid sequence revealed a significant similarity with thrombospondin-2 (TSP2). We report here the nucleotide sequence of a 386 bp RT-PCR fragment specific for CISP. The deduced protein sequence shares 84% identity with the N-terminal portion of mature human TSP2, suggesting that CISP is its bovine counterpart. Northern analysis of adrenocortical cell RNA using the above cDNA fragment as a probe revealed a 6.0 kb CISP/TSP2 mRNA whose abundance was increased nearly fivefold following a 24 h cell treatment with 10(-7) M ACTH. Under the same conditions, the expression of TSP1 mRNA was reduced by tenfold. The protein levels of TSP1 and CISP/TSP2 varied accordingly with their respective mRNA levels, as shown by immunoprecipitation and immunofluorescence experiments. Taken together, these data show that ACTH induces a dramatic shift in the pattern of adrenocortical cell thrombospondin expression from TSP1 to CISP/TSP2. This observation suggests that these two members of the thrombospondin family exert distinct biological functions in the adrenal cortex. This hypothesis is further supported by the observation that anti-CISP antibodies inhibit the maintenance of the morphological changes of bovine adrenocortical cells induced by ACTH, whereas anti-TSP1 antibodies do not.

Transforming growth factors-beta s: a multifunctional cytokine family. Implication in the regulation of adrenocortical cell endocrine functions.

Knowledge of the structure of the first recognized transforming growth factor-beta (TGF-beta 1) has led to the identification of more than two dozen structurally related peptides which appear of crucial importance in the regulation of cell proliferation, cell differentiation and embryogenesis. TGF-beta 1 and its close homologs (TGF-beta 2-5) are multifunctional peptides whose effects on cell functions are dependent upon the cell type, the environment and the presence of other growth factors. TGF-beta 1 is produced and secreted as a latent macromolecular complex. One of the major steps in the control of TGF-beta activity may thus be its release (activation) from its latent form upon the effect of local factors. Adrenocortical cells may be taken as an example in which autocrine production of TGF-beta may be a component of a negative regulatory loop in balance with the positive effect of a systemic hormone (ACTH) in controlling the expression of the cell steroidogenic differentiated functions. In this system, latent TGF-beta can be activated by an ACTH-induced secreted protein (CISP), a member of the thrombospondin family. This points to the importance of the functional interaction between TGF-beta s and extracellular matrix components in the local regulation of cell activities.

Inhibition of angiogenesis by thrombospondin-2.

To assess the ability of proteins of the thrombospondin family to inhibit angiogenesis, recombinant murine thrombospondin-2, bovine thrombospondin-2/CISP and thrombospondin-5/COMP were purified and tested for ability to block the migration of capillary endothelial cells towards a variety of inducers and to inhibit neovascularization induced in the rat cornea. Both preparations of thrombospondin-2 were active inhibitors in vitro and in vivo whereas thrombospondin-5/COMP was inactive. These results define thrombospondin-2 as a newly identified naturally occurring inhibitor of angiogenesis and suggest that the properdin-like type 1 modules that it shares with antiangiogenic thrombospondin-1 and are missing in thrombospondin-5/COMP could contribute to this activity.

Transforming growth factors beta stimulate both thrombospondin-1 and CISP/thrombospondin-2 synthesis by bovine adrenocortical cells.

We recently observed that adrenocortical cells secrete, under ACTH treatment, a large trimeric glycoprotein (CISP) presenting amino acid sequence similarity with thrombospondin-2. We also observed that the same cells synthesize and secrete thrombospondin-1 whereas under smaller amounts. The aim of this study was to investigate the regulation of these two secreted proteins by members of the TGF beta family of regulatory peptides. We developed an appropriate immunoprecipitation technique that allowed us to quantitate synthesis of thrombospondin-1 and CISP/thrombospondin-2 in a single assay. Using this assay, we observed that thrombospondin-1 and CISP/thrombospondin-2 syntheses were respectively stimulated threefold and twofold by a 24-h treatment with 2 ng/ml TGF beta 1. These inductions were dose-dependent (half-maximal effect: 0.2 ng/ml) and time-dependent (detectable after 5 h and plateauing between 15 and 25 h of treatment). They were not observed when transcription was blocked by RNA polymerase inhibitors such as 5,6-dichlorobenzimidazole riboside or actinomycin D. Among members of the TGF beta family, TGF beta 1 and TGF beta 2 and to a lesser extent activin could stimulate thrombospondin-1 and CISP/thrombospondin-2 synthesis, whereas inhibin and Müllerian inhibiting substance were inactive. Taken together, these data represent the first study on the regulation of both thrombospondin-1 and CISP/thrombospondin-2 by TGF betas. They further support the concept that TGF beta is a local regulator of adrenocortical functions.

Distinct effects of thrombospondin-1 and CISP/thrombospondin-2 on adrenocortical cell spreading.

Corticotropin-induced secreted protein (CISP) is a trimeric protein secreted by bovine adrenocortical cells in response to ACTH, that is likely to represent the bovine form of thrombospondin-2 (TSP2). This study was aimed at delineating the respective effects of CISP/TSP2 and TSP1 (thrombospondin-1) on adrenocortical cell attachment and spreading. TSP1 and CISP/TSP2 were found to slightly reduce the attachment of adrenocortical cells to plastic in the presence of serum but exhibited a pronounced differential effect on cell spreading. CISP/TSP2 inhibited adrenocortical cell spreading in a dose-dependent manner (maximal effect with 40 micrograms/ml) whereas TSP1 (up to 100 micrograms/ml) did not influence this process. The inhibition of spreading was observed whether plates were coated with CISP/TSP2 alone or with a mixture of CISP/TSP2 and fibronectin. We suggest that the inhibition of in vitro adrenocortical cell spreading by CISP/TSP2 is indicative of an implication of this protein in the migration of adrenocortical cells in vivo.

The molecular structure of corticotropin-induced secreted protein, a novel member of the thrombospondin family.

CISP (corticotropin-induced secreted protein) is a secreted protein recently purified in our laboratory from the conditioned medium of ACTH-treated bovine adrenocortical cells. Partial amino acid sequencing of CISP revealed homology with thrombospondins (TSPs), a family of adhesive proteins and in particular with TSP2. We report here the characterization of the molecular structure of CISP. Analysis of CISP by polyacrylamide gel electrophoresis in the absence or presence of SDS indicated an apparent molecular mass approximately equal to 600 kDa for the unreduced protein and an apparent molecular mass of 195 kDa after reduction by 2-mercaptoethanol. The sedimentation coefficient of CISP determined by ultracentrifugation on sucrose gradients was shifted from 9.7 S in the absence to 5.7 S in the presence of 2-mercaptoethanol. These data are consistent with a trimeric organization of the CISP molecule in which 195-kDa monomers would be linked together by disulfide bonds. The trimeric structure of CISP could be observed by rotary shadowing/electron microscopy, where CISP appeared to be composed of three equally electron-dense nodules and of a fourth nodule formed by the close association of three smaller fragments. The overall size of the molecule was 60 nm. We also observed that CISP is sulfated and glycosylated. Using glycosylation inhibitors, we could determine that CISP is synthesized as a 175-kDa core protein, is then matured into a 190-kDa high-mannose form and secreted as a 195-kDa mature protein. Inhibition of sulfation by chlorate did not prevent CISP secretion, whereas inhibition of glycosylation by tunicamycin blocked it. Taken together, these data indicate that the TSP2-related CISP molecule presents both structural and functional properties very similar to those of TSP1. CISP differs greatly, however, from TSP1 by the inducibility of its synthesis by cAMP.

Corticotropin-induced secreted protein, an ACTH-induced protein secreted by adrenocortical cells, is structurally related to thrombospondins.

The treatment of primary cultures of bovine adrenocortical cells with nanomolar concentrations of ACTH induces a 10-fold increase in the synthesis of a secreted protein of apparent molecular mass 195 kDa on reducing SDS-polyacrylamide gels. This corticotropin-induced secreted protein (CISP) appears to be an oligomeric calcium-binding protein. Its secretion under serum-free culture conditions is sustained over 4 days in the continuous presence of ACTH. Induction of CISP secretion by ACTH is mimicked by cAMP analogs and adenylate cyclase activators. We report here the purification of CISP to apparent homogeneity with an overall yield of 43% using a combination of heparin-agarose and Mono-Q chromatographies. The NH2-terminal amino acid sequence and the sequence of several tryptic peptides revealed that CISP is structurally related to the members of the thrombospondin (TSP) family. Among these members, bovine CISP appeared to be more homologous to mouse TSP2 (85% identity in the 29 amino acid long NH2-terminal sequence) than to TSP1 (18% identity in the same region). We also observed that CISP binds Ca2+ and is an adhesive protein for bovine adrenocortical cells. Thus, CISP possesses both structural and functional properties of thrombospondins. Whether CISP represents the bovine form of TSP2 or a novel member of the expanding thrombospondin family will need to be elucidated by cloning and sequencing of a larger portion of the molecule.