Internalization and homologous desensitization of the GLP-1 receptor depend on phosphorylation of the receptor carboxyl tail at the same three sites.

Homologous desensitization and internalization of the GLP-1 receptor correlate with phosphorylation of the receptor in a 33-amino acid segment of the cytoplasmic tail. Here, we identify the sites of phosphorylation as being three serine doublets located at positions 441/442, 444/445, and 451/452. The role of phosphorylation on homologous desensitization was assessed after stable expression in fibroblasts of the wild type or of mutant receptors in which phosphorylation sites were changed in various combinations to alanines. We showed that desensitization, as measured by a decrease in the maximal production of cAMP after a first exposure of the cells to GLP-1, was strictly dependent on phosphorylation. Furthermore, the number of phosphorylation sites correlated with the extent of desensitization with no, intermediate, or maximal desensitization observed in the presence of one, two, or three phosphorylation sites, respectively. Internalization of the receptor-ligand complex was assessed by measuring the rate of internalization of bound [125I]GLP-1 or the redistribution of the receptor to an endosomal compartment after agonist binding. Our data demonstrate that internalization was prevented in the absence of receptor phosphorylation and that intermediate rates of endocytosis were obtained with receptors containing one or two phosphorylation sites. Thus, homologous desensitization and internalization require phosphorylation of the receptor at the same three sites. However, the differential quantitative impairment of these two processes in the single and double mutants suggests different molecular mechanisms controlling desensitization and internalization.

Purification and ligand binding of a soluble class I major histocompatibility complex molecule consisting of the first three domains of H-2Kd fused to beta 2-microglobulin expressed in the baculovirus-insect cell system.

A recombinant baculovirus encoding a single-chain murine major histocompatibility complex class I molecule in which the first three domains of H-2Kd are fused to beta 2-microglobulin (beta 2-m) via a 15-amino acid linker has been isolated and used to infect lepidopteran cells. A soluble, 391-amino acid single-chain H-2Kd (SC-Kd) molecule of 48 kDa was synthesized and glycosylated in insect cells and could be purified in the absence of detergents by affinity chromatography using the anti-H-2Kd monoclonal antibody SF1.1.1.1. We tested the ability of SC-Kd to bind antigenic peptides using a direct binding assay based on photoaffinity labeling. The photoreactive derivative was prepared from the H-2Kd-restricted Plasmodium berghei circumsporozoite protein (P.b. CS) peptide 253-260 (YIPSAEKI), a probe that we had previously shown to be unable to bind to the H-2Kd heavy chain in infected cells in the absence of co-expressed beta 2-microglobulin. SC-Kd expressed in insect cells did not require additional mouse beta 2-m to bind the photoprobe, indicating that the covalently attached beta 2-m could substitute for the free molecule. Similarly, binding of the P.b. CS photoaffinity probe to the purified SC-Kd molecule was unaffected by the addition of exogenous beta 2-m. This is in contrast to H-2KdQ10, a soluble H-2Kd molecule in which beta 2-m is noncovalently bound to the soluble heavy chain, whose ability to bind the photoaffinity probe is greatly enhanced in the presence of an excess of exogenous beta 2-m. The binding of the probe to SC-Kd was allele-specific, since labeling was selectively inhibited only by antigenic peptides known to be presented by the H-2Kd molecule.

Three essays in dynamic corporate finance

Summary Throughout my thesis, I elaborate on how real and financing frictions affect corporate decision making under uncertainty, and I explore how firms time their investment and financing decisions given such frictions. While the macroeconomics literature has focused on the impact of real frictions on investment decisions assuming all equity financed firms, the financial economics literature has mainly focused on the study of financing frictions. My thesis therefore assesses the join interaction of real and financing frictions in firms' dynamic investment and financing decisions. My work provides a rationale for the documented poor empirical performance of neoclassical investment models based on the joint effect of real and financing frictions on investment. A major observation relies in how the infrequency of corporate decisions may affect standard empirical tests. My thesis suggests that the book to market sorts commonly used in the empirical asset pricing literature have economic content, as they control for the lumpiness in firms' optimal investment policies. My work also elaborates on the effects of asymmetric information and strategic interaction on firms' investment and financing decisions. I study how firms time their decision to raise public equity when outside investors lack information about their future investment prospects. I derive areal-options model that predicts either cold or hot markets for new stock issues conditional on adverse selection, and I provide a rational approach to study jointly the market timing of corporate decisions and announcement effects in stock returns. My doctoral dissertation therefore contributes to our understanding of how under real and financing frictions may bias standard empirical tests, elaborates on how adverse selection may induce hot and cold markets in new issues' markets, and suggests how the underlying economic behaviour of firms may induce alternative patterns in stock prices.

Identification of three Sorex species with microsatellite markers

Three sibling species of shrews, the common shrew (Sorex araneus), the Valais shrew (S. antinorii) and the Jersey shrew (S. coronatus) are morphologically similar. Different techniques based on karyorypes, morphology, biochemistry and genetic markers have been developed to identify individuals from these taxa. In this paper, we have used multiple microsatellite markers (L13, L14 and L99) to identify 55 dead animals coming from the Tarentaise Valley in France. As some individuals showed an unclear pattern with loci previously thought to be diagnostic (Lugon-Moulin et al. 2000), we have used morphologic measurements (Hausser et al. 1991) to confirm the status of these animals. This analysis clearly showed the limitations of the use of genetic diagnostic markers that have been designed in local populations and then applied to a wider scale. Even if these markers have great advantages over other techniques (i.e. simple to use and do not require samples from living animals), they should always be used with caution. There is always a risk of a locus not being diagnostic in the sampling region or in finding individuals with hybrid genotypes. Additional genetic markers should then be used, simultaneously with other identification techniques, to be sure of the status of the individuals.

Three-dimensional patient-specific dosimetry in radioimmunotherapy with 90Y-ibritumomab-tiuxetan.

Aim of the present article was to perform three-dimensional (3D) single photon emission tomography-based dosimetry in radioimmunotherapy (RIT) with (90)Y-ibritumomab-tiuxetan. A custom MATLAB-based code was used to elaborate 3D images and to compare average 3D doses to lesions and to organs at risk (OARs) with those obtained with planar (2D) dosimetry. Our 3D dosimetry procedure was validated through preliminary phantom studies using a body phantom consisting of a lung insert and six spheres with various sizes. In phantom study, the accuracy of dose determination of our imaging protocol decreased when the object volume decreased below 5 mL, approximately. The poorest results were obtained for the 2.58 mL and 1.30 mL spheres where the dose error evaluated on corrected images with regard to the theoretical dose value was -12.97% and -18.69%, respectively. Our 3D dosimetry protocol was subsequently applied on four patients before RIT with (90)Y-ibritumomab-tiuxetan for a total of 5 lesions and 4 OARs (2 livers, 2 spleens). In patient study, without the implementation of volume recovery technique, tumor absorbed doses calculated with the voxel-based approach were systematically lower than those calculated with the planar protocol, with average underestimation of -39% (range from -13.1% to -62.7%). After volume recovery, dose differences reduce significantly, with average deviation of -14.2% (range from -38.7.4% to +3.4%, 1 overestimation, 4 underestimations). Organ dosimetry in one case overestimated, in the other underestimated the dose delivered to liver and spleen. However, both for 2D and 3D approach, absorbed doses to organs per unit administered activity are comparable with most recent literature findings.

Three essays on organizations and their external environment

Abstract: The literature on the various links between organizations and their external environment is very extensive and fragmented. This thesis is comprised of three separate essays, each examining specific research questions related to these links. The first essay deals with the notion of industry life cycle and how the geographical concentration of an industry is linked to the particular life cycle stage in which the industry finds itself. The aim of this first essay is firstly to verify if the evolution of the Swiss hotel industry fits some of the stylized facts of the industry life cycle. The second aim is to verify if there is evidence of geographical clustering of the hotel industry, and by extension of tourism. The third aim is to verify a hypothesis that industry decline manifests itself mainly by company closures in decentralized locations. The importance for organizational survival and performance of adapting and reacting to environmental changes has long been ascertained. This adaptation requires managers, under conditions of uncertainty, to identify relevant changes in their external environment and to interpret the possible effects of those changes on their organization. Furthermore, it requires finding and adopting organizational responses in reaction to the environmental changes. The second essay explores how managers perceive their environment by reporting the results of two workshops held with managers from the European hotel industry. In the third essay we examine in more detail the role of uncertainty in the interpretation by executives of environmental changes. We integrate existing theories of interpretation and uncertainty into one framework, which we then test using national survey data from the hotel industry. In all three essays we are able to provide some evidence to support our main hypotheses, but.also make suggestions far further research into the topics examined.

Analysis of past and future dam formation and failure in the Santa Cruz River (San Juan province, Argentina)

Around 11.5 * 106 m3 of rock detached from the eastern slope of the Santa Cruz valley (San Juan province, Argentina) in the first fortnight of January 2005. The rockslide?debris avalanche blocked the course, resulting in the development of a lake with maximum length of around 3.5 km. The increase in the inflow rate from 47,000?74,000 m3/d between April and October to 304,000 m3/d between late October and the first fortnight of November, accelerated the growing rate of the lake. On 12 November 2005 the dam failed, releasing 24.6 * 106 m3 of water. The resulting outburst flood caused damages mainly on infrastructure, and affected the facilities of a hydropower dam which was under construction 250 km downstream from the source area. In this work we describe causes and consequences of the natural dam formation and failure, and we dynamically model the 2005 rockslide?debris avalanche with DAN3D. Additionally, as a volume ~ 24 * 106 m3of rocks still remain unstable in the slope, we use the results of the back analysis to forecast the formation of a future natural dam. We analyzed two potential scenarios: a partial slope failure of 6.5 * 106 m3 and a worst case where all the unstable volume remaining in the slope fails. The spreading of those potential events shows that a new blockage of the Santa Cruz River is likely to occur. According to their modeled morphometry and the contributing watershed upstream the blockage area, as the one of 2005, the dams would also be unstable. This study shows the importance of back and forward analysis that can be carried out to obtain critical information for land use planning, hazards mitigation, and emergency management.

Mutations leading to X-linked hypohidrotic ectodermal dysplasia affect three major functional domains in the tumor necrosis factor family member ectodysplasin-A.

Mutations in the epithelial morphogen ectodysplasin-A (EDA), a member of the tumor necrosis factor (TNF) family, are responsible for the human disorder X-linked hypohidrotic ectodermal dysplasia (XLHED) characterized by impaired development of hair, eccrine sweat glands, and teeth. EDA-A1 and EDA-A2 are two splice variants of EDA, which bind distinct EDA-A1 and X-linked EDA-A2 receptors. We identified a series of novel EDA mutations in families with XLHED, allowing the identification of the following three functionally important regions in EDA: a C-terminal TNF homology domain, a collagen domain, and a furin protease recognition sequence. Mutations in the TNF homology domain impair binding of both splice variants to their receptors. Mutations in the collagen domain can inhibit multimerization of the TNF homology region, whereas those in the consensus furin recognition sequence prevent proteolytic cleavage of EDA. Finally, a mutation affecting an intron splice donor site is predicted to eliminate specifically the EDA-A1 but not the EDA-A2 splice variant. Thus a proteolytically processed, oligomeric form of EDA-A1 is required in vivo for proper morphogenesis.

Angiotensin II receptor blockade in normotensive subjects: A direct comparison of three AT1 receptor antagonists.

Use of angiotensin (Ang) II AT1 receptor antagonists for treatment of hypertension is rapidly increasing, yet direct comparisons of the relative efficacy of antagonists to block the renin-angiotensin system in humans are lacking. In this study, the Ang II receptor blockade induced by the recommended starting dose of 3 antagonists was evaluated in normotensive subjects in a double-blind, placebo-controlled, randomized, 4-way crossover study. At 1-week intervals, 12 subjects received a single dose of losartan (50 mg), valsartan (80 mg), irbesartan (150 mg), or placebo. Blockade of the renin-angiotensin system was assessed before and 4, 24, and 30 hours after drug intake by 3 independent methods: inhibition of the blood pressure response to exogenous Ang II, in vitro Ang II receptor assay, and reactive changes in plasma Ang II levels. At 4 hours, losartan blocked 43% of the Ang II-induced systolic blood pressure increase; valsartan, 51%; and irbesartan, 88% (P<0.01 between drugs). The effect of each drug declined with time. At 24 hours, a residual effect was found with all 3 drugs, but at 30 hours, only irbesartan induced a marked, significant blockade versus placebo. Similar results were obtained when Ang II receptor blockade was assessed with an in vitro receptor assay and by the reactive rise in plasma Ang II levels. This study thus demonstrates that the first administration of the recommended starting dose of irbesartan induces a greater and longer lasting Ang II receptor blockade than that of valsartan and losartan in normotensive subjects.

Three small RNAs jointly ensure secondary metabolism and biocontrol in Pseudomonas fluorescens CHA0.

In many Gram-negative bacteria, the GacS/GacA two-component system positively controls the expression of extracellular products or storage compounds. In the plant-beneficial rhizosphere bacterium Pseudomonas fluorescens CHA0, the GacS/GacA system is essential for the production of antibiotic compounds and hence for biological control of root-pathogenic fungi. The small (119-nt) RNA RsmX discovered in this study, together with RsmY and RsmZ, forms a triad of GacA-dependent small RNAs, which sequester the RNA-binding proteins RsmA and RsmE and thereby antagonize translational repression exerted by these proteins in strain CHA0. This small RNA triad was found to be both necessary and sufficient for posttranscriptional derepression of biocontrol factors and for protection of cucumber from Pythium ultimum. The same three small RNAs also positively regulated swarming motility and the synthesis of a quorum-sensing signal, which is unrelated to N-acyl-homoserine lactones, and which autoinduces the Gac/Rsm cascade. Expression of RsmX and RsmY increased in parallel throughout cell growth, whereas RsmZ was produced during the late growth phase. This differential expression is assumed to facilitate fine tuning of GacS/A-controlled cell population density-dependent regulation in P. fluorescens.

Amiloride-sensitive epithelial Na+ channel is made of three homologous subunits.

The amiloride-sensitive epithelial sodium channel constitutes the rate-limiting step for sodium reabsorption in epithelial cells that line the distal part of the renal tubule, the distal colon, the duct of several exocrine glands, and the lung. The activity of this channel is upregulated by vasopressin and aldosterone, hormones involved in the maintenance of sodium balance, blood volume and blood pressure. We have identified the primary structure of the alpha-subunit of the rat epithelial sodium channel by expression cloning in Xenopus laevis oocytes. An identical subunit has recently been reported. Here we identify two other subunits (beta and gamma) by functional complementation of the alpha-subunit of the rat epithelial Na+ channel. The ion-selective permeability, the gating properties and the pharmacological profile of the channel formed by coexpressing the three subunits in oocytes are similar to that of the native channel.

Expression of the proto-oncogene c-fos in three-dimensional fetal brain cell cultures and the lack of correlation with maturation-inducing stimuli.

Previous work has shown that aggregating fetal brain cell cultures are able to attain a highly differentiated state, and that their development is greatly enhanced by growth and/or differentiation factors such as epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and the protein kinase C-activating tumor promoter mezerein. The present study shows that in these 3-dimensional cultures the peptide growth factors EGF and bFGF as well as mezerein are able to induce the expression of the proto-oncogene c-fos. This induction was rapid and transient, in good agreement with observations reported from a wide variety of cell types in vitro. The maximal levels of c-fos mRNA found after stimulation were low in immature cultures and increased greatly as maturation progressed. Of the three factors tested, mezerein was the most potent inducer of c-fos. In contrast to the peptide growth factors EGF and bFGF which were found to induce c-fos only in glial cells, mezerein was stimulatory in glial cells as well as in neurons. A similar cell type specificity has been observed previously for the maturation-enhancing response in immature aggregate cultures. However, in the present study no correlation was found between the degree of c-fos induction and the extent of the maturation-enhancing stimulation. Immature cultures known to be most sensitive and responsive to these maturation-enhancing agents required relatively high doses of peptide growth factors for the induction of c-fos, and the maximal levels of c-fos mRNA elicited were much lower than those in differentiated cultures which did not show any long-term response to these stimuli.(ABSTRACT TRUNCATED AT 250 WORDS)