Water column distribution of zooplanktonic size classes derived from in-situ plankton profilers: Potential use to contextualize contaminant loads in plankton.

Pollution is one of the main anthropogenic threats to marine ecosystems. Studies analysing the accumulation and transfer of contaminants in planktonic food webs tend to rely on samples collected in discrete water bodies. Here, we assessed the representativeness of measurements at the chlorophyll-a maximum layer during the MERITE-HIPPOCAMPE cruise for the entire water column by investigating the vertical distribution of particles and plankton obtained by in-situ optical profilers at nine stations across the Mediterranean Sea. We identified specific conditions where the interpretation of results from contaminant analyses can be improved by detailing plankton size structure and vertical distributions. First, the presence of higher than usual plankton concentrations can result in sampling issues that will affect biomass estimation within each size class and therefore bias our understanding of the contaminant dynamics. Secondly, the presence of an unsampled water layer with high zooplankton biomass might imply non-resolved contaminant pathways along the trophic structure. This study lays the basis for optimizing sampling strategy in contaminant studies.

Giant eagle syndrome with pseudoarthrosis and peripheral facial palsy. / Síndrome de Eagle con pseudoartrosis y parálisis facial periférica.

TITLE: Síndrome de Eagle con pseudoartrosis y parálisis facial periférica.

Modeling of PCB trophic transfer in the Gulf of Lions; 3D coupled model application.

3D coupled modeling approach is used for the PCB dispersion assessment in the Gulf of Lion and its transfer to zooplankton via biogeochemical processes. PCB budgets and fluxes between the different species of PCB: dissolved, particulate, biosorbed on plankton, assimilated by zooplankton, which are governed by different processes: adsorption/desorption, bacteria and plankton mortality, zooplankton excretion, grazing, mineralization, volatilization have been estimated. Model outputs were compared with the available in situ data. It was found that the Rhone River outflows play an important role in the organism contamination in the coastal zone, whereas the atmospheric depositions are rather more important in the offshore zones. The transfer of the available contaminant to bacteria and phytoplankton species is mainly related to the biomass present in the water column. Absorption fluxes (grazing) to zooplankton are rather higher than the passive sorption fluxes, which are themselves also linked to the sorption coefficient.

Glucagon-like peptide-1 receptor agonist treatment reduces beta cell mass in normoglycaemic mice.

AIMS/HYPOTHESIS: Incretin-based therapies improve glycaemic control in patients with type 2 diabetes. In animal models of diabetes, glucagon-like peptide-1 receptor agonists (GLP-1RAs) increase beta cell mass. GLP-1RAs are also evaluated in non-diabetic individuals with obesity and cardiovascular disease. However, their effect on beta cell mass in normoglycaemic conditions is not clear. Here, we investigate the effects of the GLP-1RA liraglutide on beta cell mass and function in normoglycaemic mice. METHODS: C57BL/6J mice were treated with the GLP-1RA liraglutide or PBS and fed a control or high-fat diet (HFD) for 1 or 6 weeks. Glucose and insulin tolerance tests were performed after 6 weeks. BrdU was given to label proliferating cells 1 week before the animals were killed. The pancreas was taken for either histology or islet isolation followed by a glucose-induced insulin-secretion test. RESULTS: Treatment with liraglutide for 6 weeks led to increased insulin sensitivity and attenuation of HFD-induced insulin resistance. A reduction in beta cell mass was observed in liraglutide-treated control and HFD-fed mice at 6 weeks, and was associated with a lower beta cell proliferation rate after 1 week of treatment. A similar reduction in alpha cell mass occurred, resulting in an unchanged alpha to beta cell ratio. In contrast, acinar cell proliferation was increased. Finally, islets isolated from liraglutide-treated control mice had enhanced glucose-induced insulin secretion. CONCLUSIONS/INTERPRETATION: Our data show that GLP-1RA treatment in normoglycaemic mice leads to increases in insulin sensitivity and beta cell function that are associated with reduced beta cell mass to maintain normoglycaemia.

[Molecular (de)regulation and cancer: new therapeutic strategies]. / (Dé)régulation moléculaire et cancer: nouvelles stratégies thérapeutiques.

The considerable progress of molecular biology within the past twenty years has permitted a more and more detailed characterization of the molecular mechanisms regulating cell proliferation. The corollary to these discoveries has been the identification of different deregulations yielding to cell transformation and cancer. The goal of this review is to present new therapeutic tools that stemmed from the now well understood logic underlying cell transformation. These tools, based on the intimate understanding of signalization pathways, aim at restoring the molecular controls which had been abrogated during the process of cell transformation. We present a survey of these new proposed therapeutic strategies. These new approaches will probably allow the clinician, in the near future, to combine traditional therapies with more targeted ones, and thus to limit side effects often associated with classical cancer therapies, while improving the overall effect of the treatment.

Bcl-XL expression correlates with primary macrophage differentiation, activation of functional competence, and survival and results from synergistic transcriptional activation by Ets2 and PU.1.

Depriving primary bone marrow-derived macrophages of colony-stimulating factor-1 (CSF-1) induces programmed cell death by apoptosis. We show that cell death is accompanied by decreases in the expression of anti-apoptotic Bcl-x(L) protein and the Ets2 and PU.1 proteins of the Ets transcription factor family. Macrophages require both priming and triggering signals independent of CSF-1 to kill neoplastic cells or microorganisms, and this activation of macrophage competence is accompanied by increased expression of bcl-x(L), ets2, and PU.1. Furthermore, we show that only Ets2 and PU.1, but not Ets1, function in a synergistic manner to transactivate the bcl-x promoter. The synergy observed between PU.1 and Ets2 is dependent on the transactivation domains of both proteins. Although other transcription factors like Fos, c-Jun, Myc, STAT3, and STAT5a are implicated in the activation of macrophage competence or in CSF-1 signaling, no synergy was observed between Ets2 and these transcription factors on the bcl-x promoter. We demonstrate that the exogenous expression of both Ets2 and PU.1 in macrophages increases the number of viable cells upon CSF-1 depletion and that Ets2 and PU.1 can functionally replace Bcl-x(L) in inhibiting Bax-induced apoptosis. Together, these results demonstrate that PU.1 and Ets2 dramatically increase bcl-x activation, which is necessary for the cytocidal function and survival of macrophages.

Ras controls tumor necrosis factor receptor-associated factor (TRAF)6-dependent induction of nuclear factor-kappa b. Selective regulation through receptor signaling components.

In the present study, we show that Ras activity differentially controls interleukin (IL)-1 induced transcription factor activation by selective regulation of responses mediated by receptor complex components. Initial experiments revealed that stimulation with IL-1 caused a rapid, matrix-dependent activation of Ras. The effect was transient, peaking at 5 min and returning to base levels after 30 min. Activation correlated with pronounced changes in cell shape in EGFPH-Ras transfected cells. Transfection with the dominant negative mutant, Ras(Asn-17), inhibited IL-1 induced activation of the IL-8 promoter as well as of NF-kappa B and AP-1 synthetic promoters in transient transfection assays. Furthermore, overexpression of the IL-1 signaling proteins TRAF6 or MyD88 gave characteristic activation of IL-8, which was accentuated in the presence of IL-1. Co-transfection with Ras(Asn-17) gave a dose-dependent inhibition of TRAF6-induced responses in the presence and absence of IL-1, but had no effect on MyD88 mediated activity. Similarly, induction of NF-kappa B was abolished by Ras(Asn-17) only in TRAF6-transfected cells. In contrast, inhibiting Ras activity limited AP-1-mediated responses through both receptor complex proteins. Constitutively active Ras(Val-12) increased the TRAF6 induced activity of the NF-kappa B pathway similar to the effect induced by IL-1, while the Ras(Val-12) induced activity was not inhibited by co-transfection with a dominant negative TRAF6. Our data show that activation of the Ras GTPase is an early, matrix-dependent response in IL-1 signaling which participates in structural regulation of IL-1-induced genes. In addition, they show that the Ras induced effect selectively regulates TRAF6-mediated activation of the NF-kappa B pathway, suggesting that Ras GTPase represents a convergence point in structural and cytokine responses, with distinct effects on a subset of downstream signaling events.

Dynamic shuttling of nuclear factor kappa B between the nucleus and cytoplasm as a consequence of inhibitor dissociation.

Activation of the nuclear factor kappaB (NFkappaB) transcription factor is intimately associated with its translocation from the cytoplasm to the nucleus. Using the nuclear export inhibitor leptomycin B, we demonstrate shuttling of the RELA subunit of NFkappaB and the inhibitory subunit IkappaBalpha between these two compartments in unstimulated cells. Determination of the kinetics of nuclear entry shows marked differences for the two components; the entry of IkappaBalpha occurs more rapidly than RELA. The shuttling is suggested to be a consequence of the cytoplasmic dissociation of the NFkappaB.IkappaB complex rather than its direct nuclear import or degradation and resynthesis of IkappaBalpha. Using previously published kinetic data, this proposition is born out by the deduction that 17% of NFkappaB is not complexed to IkappaBalpha in a resting cell. A numerical model is presented to validate the proposed regulation of NFkappaB subcellular localization consequent in part on the nuclear export function and in part on the cytoplasmic retention function of IkappaBalpha. We suggest that the non-saturated interaction of NFkappaB with the inhibitor may enhance the specificity of action of IkappaB proteins on different NFkappaB dimers and allow additional modes of regulation of IkappaB function.

Activation of nuclear factor kappaB in single living cells. Dependence of nuclear translocation and anti-apoptotic function on EGFPRELA concentration.

We have studied the dynamics of nuclear translocation during nuclear factor kappaB activation by using a p65(RELA)-enhanced green fluorescent protein (EGFP) fusion construct. Quantitation of expression levels indicates that EGFPRELA can be detected at physiological concentrations of about 60,000 molecules per cell. Stimulation of transfected fibroblasts with interleukin (IL)-1beta caused nuclear translocation of EGFPRELA, typically resulting in a 30-fold increase in nuclear protein at maximum induction and a concomitant 20% decrease in cytoplasmic levels. The response of individual cells to IL-1beta was graded, and the kinetics of nuclear translocation were dependent on the dose of IL-1beta and the level of EGFPRELA expression. The rate of nuclear uptake was saturable, and the time lag for uptake increased at higher EGFPRELA expression levels. Furthermore, nuclear translocation was reduced at less than saturating doses of IL-1beta suggesting that the pathway is limited by incoming signals. The response to IL-1beta was biphasic, demonstrating a decline in nuclear import rate at expression levels above three to four times endogenous. This correlated with the anti-apoptotic function of EGFPRELA which was more prominent at low expression levels and demonstrated successively less protection at higher levels. In comparison, transfection of p50 had no effect on the level of apoptosis and demonstrated some toxicity in combination with EGFPRELA.

Recruitment of a heparan sulfate subunit to the interleukin-1 receptor complex. Regulation by fibronectin attachment.

In this study, we identified an adhesion-regulated subunit of the interleukin-1 (IL-1) receptor complex. Transfection of fibroblasts with an IL-1 receptor-EGFP construct showed that the fusion protein was located at focal adhesions in cells attaching to fibronectin. Fibronectin attachment caused enhancement in endogenous IL-1 type I receptor levels from on average 2500 to 4300 receptors/cell. In addition, matrix attachment resulted in a decrease in binding affinity (Ka) from 1.0 x 10(9) (M-1) to 5.6 x 10(8) (M-1), due to a 2-fold reduction in association rate constant. The adhesion-mediated effects were reversed by soluble heparin. Cross-linking experiments showed that in cells attached to fibronectin, 50-70% of the radiolabeled IL-1 was associated with a heparinase sensitive, high molecular mass component of about 300 kDa, with a core protein of 80-90 kDa. Formation of the complex was dependent on cell interaction with the heparin binding region in fibronectin and required IL-1/type I IL-1 receptor binding. This report demonstrates the recruitment of a heparan sulfate to the IL-1 receptor complex, following attachment to fibronectin, which correlates with alterations in receptor function. The data suggest that the heparan sulfate constitutes an attachment regulated component of the IL-1 receptor complex with the role of mediating matrix regulation of IL-1 responses.

Regulation of the CYP1A1 promoter in transgenic mice: an exquisitely sensitive on-off system for cell specific gene regulation.

Mammalian cytochrome P-450s in the CYP1A gene family catalyse the oxidation of a wide range of drugs and foreign compounds resulting in their excretion. These enzymes are highly inducible by a range of compounds, including polycyclic aromatic hydrocarbons such as 3-methylcholanthrene (3-MC) and dioxins. Analysis of the CYP1A1 promoter has identified dioxin responsive enhancer elements which mediate the induction response. In order to evaluate this promoter as an in vivo regulatable expression system and to gain further insights into the tissue specific regulation of this gene, an 8.5 kb genomic fragment of the rat CYP1A1 promoter was cloned upstream of the lacZ reporter gene. This construct was used to generate transgenic mice and three independent lines were expanded for further study. The regulation of beta-galactosidase expression was determined in mock and 3-MC-treated mice in an extensive range of tissues. In untreated animals no transgene expression was detectable over non-transgenic controls. Treatment with 3-MC caused a profound increase in transgene expression (> 1,000-fold) in many tissues including liver, adrenal, kidney and intestine. Inducible transgene expression was also detectable in many of the other tissues including the spleen, lung, pancreas and the reproductive organs. Although the absolute levels of induction varied, no significant differences in the pattern of transgene expression were observed between the three different transgenic mouse lines. In addition, the pattern of transgene expression correlated closely with the reported regulation of CYP1A1 protein. These results indicate that the CYP1A1 promoter can drive expression of heterologous genes in a truly on/off manner in a variety of tissues and cell types which will allow the expression of other proteins to be controlled in vivo. This reporter system also provides a model for establishing the environmental and hormonal factors regulating the expression of the CYP1A1 gene.

Functional studies of E7 proteins from different HPV types.

We have performed comparative studies on the E7 proteins from malignant and non-malignant Human Papillomavirus types HPV 1, 6, 11, 16, 18, 33). GST/E7 fusion proteins from all these HPV types associate with Rb1, p107 and the cyclin A/CDK2 complex. As has been shown for Rb1, the association with p107 and Cyclin A was weaker for the 'low risk' HPV6 and 11 E7 proteins as compared to 'high risk' HPV16, 18 and 33 E7 proteins. In contrast the E7 protein of the benign type HPV1 bound Rb1, p107 and cyclin A with the same affinity as the 'high risk' E7 proteins. The affinities of the E7/Rb1 interaction have been confirmed in vivo by the 'two hybrid' method in the yeast Saccharomyces cerevisiae. Although HPV1 E7 showed the same affinity in vitro and in vivo for Rb1 as the high risk HPV E7s, it did not have the ability to activate the E2F-1 transcription factor inhibited by Rb1, nor did it have any transforming activity when coexpressed with activated ras in primary rodent cells.

Trans-activation of the adenovirus E2 promoter by human papillomavirus type 16 E7 is mediated by retinoblastoma-dependent and -independent pathways.

In common with the adenovirus E1A and simian virus 40 large T oncoproteins, the E7 protein of human papillomavirus (HPV) type 16 interacts with the retinoblastoma (Rb) tumour suppressor protein (pRb). The functional importance of this interaction for HPV-16 E7 protein was investigated by analysis of the transactivating function of E7 at the adenovirus E2 promoter in a set of breast tumour cell lines. Trans-activation by HPV-16 E7 in two pRb-deficient cell lines demonstrated that pRb is not essential for E7-mediated trans-activation, but reconstitution of Rb expression indicated the existence of an Rb-mediated pathway of E7 trans-activation. This pathway results from suppression by E7 of a trans-repressing function encoded by the Rb gene. The E7 protein is shown to be capable of interacting in vivo with the Rb-related protein p107. Furthermore, analysis of a fusion construct between the amino terminus of Rb and the carboxy terminus of p107 suggests that, in common with pRb, the p107 protein trans-represses the adenovirus E2 early promoter. Therefore it is proposed that the pRb-independent pathway of E7 trans-activation is a consequence of the suppression of trans-repression by p107.

HPV16 E7 protein associates with the protein kinase p33CDK2 and cyclin A.

E7 is the major transforming protein of human papillomavirus type 16 (HPV16). It has been found to associate with the retinoblastoma protein Rb1. We investigated whether HPV16 E7 protein was associated with other cellular proteins, in particular with those involved in cell cycle control. Immunoprecipitates from CaSki cell extracts with an anti E7 monoclonal antibody contained a histone H1 kinase. Recombinant E7, synthesized in yeast, when mixed with protein extracts from epithelial cells bound histone H1 kinase activity in vitro. The in vivo and the in vitro-formed E7-kinase complex had the same periodicity of activity during the cell cycle, being most active in S and G2/M. Immunoblotting of E7 immunoprecipitates with an antibody raised against the p33CDK2, revealed a 33 kDa protein band not detected by an anti-p34cdc2 antibody, suggesting that the E7-associated kinase activity is due to the p33CDK2. The interaction appears to be via cyclin A, since probing of similar immunoblots showed a 50 kDa band corresponding to cyclin A. The association of E7 with cyclin A appeared to be direct, not involving Rb 1 or other proteins.

Structural analysis of wild-type and mutant human immunodeficiency virus type 1 Tat proteins.

We expressed the human immunodeficiency virus type 1 transactivator protein, Tat, in the wheat germ cell-free translation system and found it to exist as a monomer. The first coding exon (residues 1 to 72) of wheat germ-expressed Tat was resistant to trypsin digestion, indicating that it is a highly folded, independently structured protein domain. Several mutant Tat proteins were dramatically more sensitive to trypsin than the wild type was, suggesting that their reduced transactivation activities are the result of destabilized structures. Mutant proteins with single-amino-acid substitutions were also identified that had reduced transactivation activities but wild-type structures in the trypsin assay. These mutants clustered in two regions of Tat, at acidic residues 2 and 5 in the amino terminus and between residues 18 and 32. These mutants, wild type in structure but reduced in activity, identify residues in the wild-type protein that may directly contact other molecules during Tat function.

Mutational analysis of the conserved cysteine-rich region of the human immunodeficiency virus type 1 Tat protein.

The Tat transactivator protein of human immunodeficiency virus type 1 contains a highly conserved cysteine-rich region, containing seven cysteines from residues 22 through 37. To investigate the importance of noncysteine residues in this region of the Tat protein, we have carried out a mutational analysis, in most cases substituting a single alanine for the wild-type noncysteine residue. Alanine substitution of residue 23, 24, 46, or 47 had no effect on Tat activity in plasmid transfection assays. In contrast, alanine substitutions of all eight noncysteines analyzed, from residues 26 through 41, significantly reduced the activity of the Tat protein, in some cases as drastically as mutations in cysteine residues. The results demonstrate that the precise sequence of the cysteine-rich region is crucial for a fully functional Tat protein.