Up-regulation of the levels of expression and function of a constitutively active mutant of the hamster alpha1B-adrenoceptor by ligands that act as inverse agonists.

The alpha1-adrenergic agonist phenylephrine stimulated phospholipase D (PLD) activity in Rat 1 fibroblasts transfected to express either the wild-type hamster alpha1B-adrenoceptor or a constitutively active mutant (CAM) form of this receptor. The EC50 for agonist stimulation of PLD activity was substantially lower at the CAM receptor than at the wild-type receptor as previously noted for phenylephrine stimulation of phosphoinositidase C activity. Sustained treatment of cells expressing the CAM alpha1B-adrenoceptor with phentolamine resulted in a marked up-regulation in levels of this receptor with half-maximal effects produced within 24 h and with an EC50 of approx. 40 nM. Such an up-regulation could be produced with a range of other ligands generally viewed as alpha1-adrenoceptor antagonists but equivalent treatment of cells expressing the wild-type alpha1B-adrenoceptor was unable to mimic these effects. After sustained treatment of the CAM alpha1B-adrenoceptor expressing cells with phentolamine, basal PLD activity was increased and phenylephrine was now able to stimulate PLD activity to greater levels than in vehicle-treated CAM alpha1B-adrenoceptor-expressing cells. The EC50 for phenylephrine stimulation of PLD activity was not altered, however, by phentolamine pretreatment and the associated up-regulation of the receptor. After phentolamine-induced up-regulation of basal PLD activity, a range of alpha1-antagonists were shown to possess the characteristics of inverse agonists of the CAM alpha1B-adrenoceptor as they were able to substantially decrease the elevated basal PLD activity.

Agenesis of human pancreas due to decreased half-life of insulin promoter factor 1.

Neonatal diabetes mellitus can be transient or permanent. The severe form of permanent neonatal diabetes mellitus can be associated with pancreas agenesis. Normal pancreas development is controlled by a cascade of transcription factors, where insulin promoter factor 1 (IPF1) plays a crucial role. Here, we describe two novel mutations in the IPF1 gene leading to pancreas agenesis. Direct sequence analysis of exons 1 and 2 of the IPF1 gene revealed two point mutations within the homeobox in exon 2. Genetic analysis of the parents showed that each mutation was inherited from one parent. Mutations localized in helices 1 and 2, respectively, of the homeodomain, decreased the protein half-life significantly, leading to intracellular IPF1 levels of 36% and 27% of wild-type levels. Both mutant forms of IPF1 were normally translocated to the nucleus, and their DNA binding activity on different known target promoters was similar to that of the wild-type protein. However, transcriptional activity of both mutant IPF1 proteins, alone or in combination with HNF3 beta/Foxa2, Pbx1, or the heterodimer E47-beta 2 was reduced, findings accounted for by decreased IPF1 steady state levels and not by impaired protein-protein interactions. We conclude that the IPF1 level is critical for human pancreas formation.

Characterisation of heteromers and oligomers in the TNF family of ligands and receptors

TNF family ligands and receptors fulfill a number of functions, mainly in the immune system. For example, the ligands BAFF and APRIL control growth and survival of mature Β cells at various stages of differentiation. TNF family ligands usually form homotrimers, but heteromers have also been described for lymphotoxin α1β2 and for BAFF and APRIL. Interestingly, twenty BAFF homotrimers can assemble into virus-like particles coined BAFF 60-mer, which are superior to BAFF 3-mer regarding their ability to signal in primary Β cells. A screen was performed in 293T cells, by co-transfecting differently tagged ligands, to identify six novel heteromers. The specificity of these novel heteromers, however, did not correspond to that of orphan receptors in the TNFR family. Little is known about heteromers of BAFF and APRIL, in particular their receptor-binding specificity and their ability to signal. A method to produce and purify heteromers of defined stoechiometry was developed, and the resulting reagents were used to demonstrate that BAFF2APRIL, like BAFF, binds to all BAFF receptors - namely BAFFR, TACI and Β CM A -, while APRIL2BAFF and APRIL only binds to TACI and BCMA. Heteromers could signal via their cognate receptors, sometimes as potently and sometimes less potently than homomers, depending on the receptors. A promising system to measure the activity of single-chain homo- and heteromers in vivo was set up: it measures mature Β cell rescue upon administration of single-chain ligands into BAFF-ko mice. To tackle the question of the physiological importance of BAFF 60-mer, a point mutation that prevents assembly of mouse BAFF into 60-mer while retaining its ability to form trimers was identified. This mutation (E247K) was introduced by homologous recombination into mouse embryonic stem cells that are now being used to generate knock-in mice. Results obtained in this work will help to better understand the role of various BAFF and APRIL forms that are elevated in a several autoimmune diseases. - Les ligands et récepteurs de la famille du TNF joue un rôle prédominant dans le système immunitaire. Par exemple, les ligands BAFF et APRIL contrôlent la croissance et la survie des cellules Β matures à différents stades de différenciation. Ces ligands existent souvent sous forme d'homotrimères (3-mer), bien que des héteromères aient été décrits pour la lymphotoxine α1β2 et pour BAFF et APRIL. Dans le cas de BAFF, vingt trimères peuvent, telle une particule virale, s'assembler en 60-mer qui surpasse le 3-mer pour signaler dans des cellules Β primaires. Un crible effectué dans des cellules 293T, par co-transfection de ligands différemment marqués, a permis d'identifier six nouveaux heteromères dont la spécificité n'a, hélas, pas correspondu à celle d'un récepteur orphelin de la famille du TNFR. Les connaissances sur la spécificité de liaison aux récepteurs et la capacité à signaler des heteromères de BAFF et d'APRIL sont fragmentaires. Une méthode pour produire et purifier des heteromères "simple chaîne" de stoechiométrie déterminée a été mise au point, et les réactifs ainsi obtenus utilisés pour démontrer que BAFF2APRIL, comme BAFF, lie tous les récepteurs de BAFF - c'est-à-dire BAFFR, TACI et BCMA -, alors qu'APRIL2BAFF et APRIL ne lient que TACI et BCMA. Les héteromères peuvent transmettre des signaux, parfois aussi bien et parfois plus faiblement que les homomères, selon les récepteurs. Un système prometteur pour mesurer l'activité des ligands simple chaîne in vivo a été mis au point. Il mesure la réapparition de cellules Β matures dans des souris déficientes pour BAFF après administration des ligands. Pour s'attaquer à la question de l'importance physiologique du 60-mer de BAFF, ime mutation empêchant l'assemblage en 60-mer sans affecter la capacité à former des trimères a été identifiée. Cette mutation (E247K) a été introduite par recombinaison homologue dans des cellules souches embryonnaires de souris qui sont utilisées pour obtenir des souris déficientes en BAFF 60-mer. Les résultats de ces travaux contribueront à mieux cerner le rôle des différentes formes de BAFF et d'APRIL produites en excès dans plusieurs maladies auto-immunes.

Altered ligands reveal limited plasticity in the T cell response to a pathogenic epitope.

Experimental leishmaniasis offers a well characterized model of T helper type 1 cell (Th1)-mediated control of infection by an intracellular organism. Susceptible BALB/c mice aberrantly develop Th2 cells in response to infection and are unable to control parasite dissemination. The early CD4(+) T cell response in these mice is oligoclonal and reflects the expansion of Vbeta4/ Valpha8-bearing T cells in response to a single epitope from the parasite Leishmania homologue of mammalian RACK1 (LACK) antigen. Interleukin 4 (IL-4) generated by these cells is believed to direct the subsequent Th2 response. We used T cells from T cell receptor-transgenic mice expressing such a Vbeta4/Valpha8 receptor to characterize altered peptide ligands with similar affinity for I-Ad. Such altered ligands failed to activate IL-4 production from transgenic LACK-specific T cells or following injection into BALB/c mice. Pretreatment of susceptible mice with altered peptide ligands substantially altered the course of subsequent infection. The ability to confer a healer phenotype on otherwise susceptible mice using altered peptides that differed by a single amino acid suggests limited diversity in the endogenous T cell repertoire recognizing this antigen.

Klotho coreceptors inhibit signaling by paracrine fibroblast growth factor 8 subfamily ligands.

It has been recently established that Klotho coreceptors associate with fibroblast growth factor (FGF) receptor tyrosine kinases (FGFRs) to enable signaling by endocrine-acting FGFs. However, the molecular interactions leading to FGF-FGFR-Klotho ternary complex formation remain incompletely understood. Here, we show that in contrast to αKlotho, βKlotho binds its cognate endocrine FGF ligand (FGF19 or FGF21) and FGFR independently through two distinct binding sites. FGF19 and FGF21 use their respective C-terminal tails to bind to a common binding site on βKlotho. Importantly, we also show that Klotho coreceptors engage a conserved hydrophobic groove in the immunoglobulin-like domain III (D3) of the "c" splice isoform of FGFR. Intriguingly, this hydrophobic groove is also used by ligands of the paracrine-acting FGF8 subfamily for receptor binding. Based on this binding site overlap, we conclude that while Klotho coreceptors enhance binding affinity of FGFR for endocrine FGFs, they actively suppress binding of FGF8 subfamily ligands to FGFR.

La "Génération Sandwich" en Suisse romande: mieux comprendre les facteurs associés avec la santé perçue afin de mieux agir en promotion de la santé

La coexistence des charges professionnelles, familiales, et d'aide à des ascendants expose la Génération Sandwich (GS) à des risques potentiels pour sa santé. Toutefois, les connaissances sur la GS sont insuffisantes pour permettre aux infirmières du secteur de la santé au travail de développer des interventions en promotion de la santé basées sur des preuves. La présente étude vise à dresser le portrait des travailleurs de la GS en examinant les liens entre leurs caractéristiques, leurs charges co-existantes et leur santé perçue. Cette recherche repose sur un devis descriptif corrélationnel multivarié. Un questionnaire électronique a permis de récolter les données de 844 employés d'une administration publique suisse. L'examen montre que 23 % de l'échantillon appartient à la GS. Cette appartenance dépend essentiellement de l'âge des ascendants, de la co-résidence avec ces derniers, de la présence d'enfants dans le ménage. Les scores de santé physique des membres de la GS sont meilleurs que ceux de santé mentale. L'hétérogénéité de leurs caractéristiques transparaît dans trois clusters. Enfin, seul le score de santé physique diffère selon le sexe et les groupes. Cette étude fournit des connaissances sur la GS pour fonder des interventions préventives ciblées.

Selectin ligands on leukemic cells

RESUME L'infiltration tissulaire par les cellules leucémiques, responsable de leucostase, est une complication grave de la leucémie aiguë hyperleucocytaire. Elle peut entraîner une détresse respiratoire et des troubles neurologiques de mauvais pronostic. Pendant longtemps, la prolifération intravasculaire des cellules leucémiques et l'augmentation de la viscosité étaient considérées comme en étant responsables, et le traitement reposait sur une cytoréduction rapide par leucaphérèse. Actuellement, l'interaction entre les cellules leucémiques et l'endothélium vasculaire est plutôt considérée comme la cause de ce phénomène. En effet, les cellules leucémiques peuvent induire l'expression des sélectives endothéliales. Les sélectives initient le roulement des leucocytes avant leur adhésion ferme et leur migration dans les tissus. Elles reconnaissent des ligands spécifiques exprimés à la surface des leucocytes, comme PSGL-1 qui est un ligand commun des sélectives. Cependant, plusieurs études suggèrent que d'autres ligands de la E-sélective soient exprimés par les leucocytes. L'interaction des cellules leucémiques avec la E- et la P- sélective est corrélée avec l'expression de la molécule CLA, reconnue par l'anticorps HECA-452. L'immunopurification des ligands de la E-sélective avec cet anticorps a permis d'isoler, des cellules THP1 et U937, une protéine de 170 kDa, ainsi qu'une autre protéine de 250 kDa des cellules U937, en plus de PSGL-1. Ces protéines ont également été purifiées avec la protéine de fusion Esélective/IgM. CD43 et CD44 semblent être des ligands de la E-sélective sur certaines lignées, mais leur interaction avec la E-sélective n'est pas toujours retrouvée. De plus, cette étude a permis de montrer que ces ligands de la E-sélectiné sont exprimés dans les rafts lipidiques, comme PSGL-1 et la L-sélective des neutrophiles. Ces deux nouveaux ligands sont en cours d'identification. Ils pourraient représenter une nouvelle cible dans le traitement de la leucostase, mais aussi lors d'inflammation chronique ou de métastases. ABSTRACT Leukostasis is alife-threatening complication of acute leukemia, that results from tissue infiltration of leukemic blasts that migrate out of blood flow and interfere with normal tissue functions. The process leading to these complications has been attributed to the overcrowding of leukemic cells in the microcirculation. However, leukostasis more likely results from the adhesive interactions between leukemic blasts and the endothelium. Activated endothelium express adhesion molecules like P- and E-selectin, and leukemic cells themselves can induce the expression of E-selectin on endothelial cells. Selectins are essential in initiating the rolling of intravascular cells on endothelium before firm adhesion and transmigration outside of blood vessels. They interact with specific ligands on leukocyte cell surface. P-selectin glycoprotein ligand-1 (PSGL-1) is common ligand for E-, P- and L-selectin. Recently, CD44, ESL-1 and CD44 were shown to cooperate. ìn supporting mouse neutrophil adhesion to E-selectin. Other E-selectin ligands remain to be identified in humans. Leukemic cells were screened in order to characterize human E-selectin ligands. The interactions of E- and P-selectin correlate with the expression of CLA epitope. Therefore, HECA-452 mAb that recognizes CLA was used for immunopurification. Aglycoprotein of 170 kDa was purified from THP1 and U937 cells, and a protein of 250 kDa from U937 cells. These proteins were also purified by affinity binding to E-selectin/IgM chimera. PSGL-1 bound to E-selectin as expected, but CD43 and CD44 were not always adsorbed on E-selectin chimera, depending on cell types. E-selectin ligands were also shown to be in lipid rafts in leukemic cells, like PSGL-1 and L-selectin in human neutrophils. The 170 kDa protein has been sequenced, and three interesting ligands were among the candidates: ESL-1, CD44 and podocalyxin. These ligands are under investigation, and may represent a new therapeutic target in leukostasis, inflammation or cancer metastasis.

Sawhorse-type diruthenium tetracarbonyl complexes containing porphyrin-derived ligands as highly selective photosensitizers for female reproductive cancer cells.

Diruthenium tetracarbonyl complexes of the type [Ru2(CO)4(l2-g2-O2CR)2L2] containing a Ru-Ru backbone with four equatorial carbonyl ligands, two carboxylato bridges, and two axial two-electron ligands in a sawhorse-like geometry have been synthesized with porphyrin-derived substituents in the axial ligands [1: R is CH3, L is 5-(4-pyridyl)-10,15,20-triphenyl-21,23H-porphyrin], in the bridging carboxylato ligands [2: RCO2H is 5-(4-carboxyphenyl)-10,15,20-triphenyl-21,23H-porphyrin, L is PPh3; 3: RCO2H is 5-(4-carboxyphenyl)-10,15,20-triphenyl-21,23H-porphyrin, L is 1,3,5-triaza-7-phosphatricyclo [3.3.1.1]decane], or in both positions [4: RCO2H is 5-(4-carboxyphenyl)-10,15,20-triphenyl-21,23H-porphyrin, L is 5-(4-pyridyl)-10,15,20-triphenyl-21,23H-porphyrin]. Compounds 1-3 were assessed on different types of human cancer cells and normal cells. Their uptake by cells was quantified by fluorescence and checked by fluorescence microscopy. These compounds were taken up by human HeLa cervix and A2780 and Ovcar ovarian carcinoma cells but not by normal cells and other cancer cell lines (A549 pulmonary, Me300 melanoma, PC3 and LnCap prostate, KB head and neck, MDAMB231 and MCF7 breast, or HT29 colon cancer cells). The compounds demonstrated no cytotoxicity in the absence of laser irradiation but exhibited good phototoxicities in HeLa and A2780 cells when exposed to laser light at 652 nm, displaying an LD50 between 1.5 and 6.5 J/cm2 in these two cell lines and more than 15 J/cm2 for the others. Thus, these types of porphyric compound present specificity for cancer cell lines of the female reproductive system and not for normal cells; thus being promising new organometallic photosensitizers.

Selective absence of CD8+ TCRalpha beta+ intestinal epithelial cells in transgenic mice expressing beta2-microglobulin-associated ligands exclusively on thymic cortical epithelium.

Whereas interactions between the TCRalpha beta and self MHC:peptide complexes are clearly required for positive selection of mature CD4(+) and CD8(+) T cells during intrathymic development, the role of self or foreign ligands in maintaining the peripheral T cell repertoire is still controversial. In this report we have utilized keratin 14-beta2-microglobulin (K14-beta2m)-transgenic mice expressing beta2m-associated ligands exclusively on thymic cortical epithelial cells to address the possible influence of TCR:ligand interactions in peripheral CD8(+) T cell homeostasis. Our data indicate that CD8(+) T cells in peripheral lymphoid tissues are present in normal numbers in the absence of self MHC class I:peptide ligands. Surprisingly, however, steady state homeostasis of CD8(+) T cells in the intestinal epithelium is severely affected by the absence of beta2m-associated ligands. Indeed TCRalpha beta(+) IEL subsets expressing CD8alpha beta or CD8alpha alpha are both dramatically reduced in K14-beta2m mice, suggesting that the development, survival or expansion of CD8(+) IEL depends upon interaction of the TCR with MHC class I:peptide or other beta2m-associated ligands elsewhere than on thymic cortical epithelium. Collectively, our data reveal an unexpected difference in the regulation of CD8(+) T cell homeostasis by beta2m-associated ligands in the intestine as compared to peripheral lymphoid organs.

Cutting edge: influence of the TCR Vbeta domain on the selection of semi-invariant NKT cells by endogenous ligands.

Invariant Valpha14 (Valpha14i) NKT cells are a murine CD1d-dependent regulatory T cell subset characterized by a Valpha14-Jalpha18 rearrangement and expression of mostly Vbeta8.2 and Vbeta7. Whereas the TCR Vbeta domain influences the binding avidity of the Valpha14i TCR for CD1d-alpha-galactosylceramide complexes, with Vbeta8.2 conferring higher avidity binding than Vbeta7, a possible impact of the TCR Vbeta domain on Valpha14i NKT cell selection by endogenous ligands has not been studied. In this study, we show that thymic selection of Vbeta7(+), but not Vbeta8.2(+), Valpha14i NKT cells is favored in situations where endogenous ligand concentration or TCRalpha-chain avidity are suboptimal. Furthermore, thymic Vbeta7(+) Valpha14i NKT cells were preferentially selected in vitro in response to CD1d-dependent presentation of endogenous ligands or exogenously added self ligand isoglobotrihexosylceramide. Collectively, our data demonstrate that the TCR Vbeta domain influences the selection of Valpha14i NKT cells by endogenous ligands, presumably because Vbeta7 confers higher avidity binding.

Copeptin, a stable peptide derived from the vasopressin precursor, correlates with the individual stress level.

BACKGROUND: During stress, vasopressin is a potent synergistic factor of CRH as a hypothalamic stimulator of the HPA axis. The measurements of CRH and vasopressin levels are cumbersome because of their instability and short half-life. Copeptin is a more stable peptide stoichiometrically released from the same precursor molecule. The aim of our study was to compare copeptin and cortisol levels in different stress situations. METHODS: Three groups of patients with increasing stress levels were investigated: a) healthy controls without apparent stress (n=20), b) hospitalized medical patients with moderate stress (n=25) and c) surgical patients 30 minutes after extubation, with maximal stress (n=29). In all patients we assessed cortisol and copeptin levels. Copeptin levels were measured with a new sandwich immunoassay. RESULTS: Cortisol levels in controls were (median, IQ range, 486 [397-588] nmol/L), not significantly different as compared to medical patients (438 [371-612] nmol/L, p=0.69). Cortisol levels in surgical patients after extubation were higher (744 [645-1062] nmol/L p<0.01 vs controls and medical patients). Copeptin levels in controls were 4.3 [3.2-5.5] pmol/L, which was lower as compared to medical patients (17.5 [6.4-24.1], p<0.001) and surgical patients after extubation (67.5 [37.8-110.0] pmol/L, p<0.001). The correlation between copeptin levels and cortisol was r=0.46, p<0.001. CONCLUSION: Copeptin is a novel marker of the individual stress level. It more subtly mirrors moderate stress as compared to cortisol values.

Basal and antigen-induced exposure of the proline-rich sequence in CD3ε.

The CD3ε cytoplasmic tail contains a conserved proline-rich sequence (PRS) that influences TCR-CD3 expression and signaling. Although the PRS can bind the SH3.1 domain of the cytosolic adapter Nck, whether the PRS is constitutively available for Nck binding or instead represents a cryptic motif that is exposed via conformational change upon TCR-CD3 engagement (CD3Δc) is currently unresolved. Furthermore, the extent to which a cis-acting CD3ε basic amino acid-rich stretch (BRS), with its unique phosphoinositide-binding capability, might impact PRS accessibility is not clear. In this study, we found that freshly harvested primary thymocytes expressed low to moderate basal levels of Nck-accessible PRS ("open-CD3"), although most TCR-CD3 complexes were inaccessible to Nck ("closed-CD3"). Ag presentation in vivo induced open-CD3, accounting for half of the basal level found in thymocytes from MHC(+) mice. Additional stimulation with either anti-CD3 Abs or peptide-MHC ligands further elevated open-CD3 above basal levels, consistent with a model wherein antigenic engagement induces maximum PRS exposure. We also found that the open-CD3 conformation induced by APCs outlasted the time of ligand occupancy, marking receptors that had been engaged. Finally, CD3ε BRS-phosphoinositide interactions played no role in either adoption of the initial closed-CD3 conformation or induction of open-CD3 by Ab stimulation. Thus, a basal level of open-CD3 is succeeded by a higher, induced level upon TCR-CD3 engagement, involving CD3Δc and prolonged accessibility of the CD3ε PRS to Nck.

Improving binding affinity and stability of peptide ligands by substituting glycines with D-amino acids.

Improving the binding affinity and/or stability of peptide ligands often requires testing of large numbers of variants to identify beneficial mutations. Herein we propose a type of mutation that promises a high success rate. In a bicyclic peptide inhibitor of the cancer-related protease urokinase-type plasminogen activator (uPA), we observed a glycine residue that has a positive ϕ dihedral angle when bound to the target. We hypothesized that replacing it with a D-amino acid, which favors positive ϕ angles, could enhance the binding affinity and/or proteolytic resistance. Mutation of this specific glycine to D-serine in the bicyclic peptide indeed improved inhibitory activity (1.75-fold) and stability (fourfold). X-ray-structure analysis of the inhibitors in complex with uPA showed that the peptide backbone conformation was conserved. Analysis of known cyclic peptide ligands showed that glycine is one of the most frequent amino acids, and that glycines with positive ϕ angles are found in many protein-bound peptides. These results suggest that the glycine-to-D-amino acid mutagenesis strategy could be broadly applied.