Mutations inducing divergent shifts of constitutive activity reveal different modes of binding among catecholamine analogues to the beta(2)-adrenergic receptor.

1. We compared the changes in binding energy generated by two mutations that shift in divergent directions the constitutive activity of the human beta(2) adrenergic receptor (beta(2)AR). 2. A constitutively activating mutant (CAM) and the double alanine replacement (AA mutant) of catechol-binding serines (S204A, S207A) in helix 5 were stably expressed in CHO cell lines, and used to measure the binding affinities of more than 40 adrenergic ligands. Moreover, the efficacy of the same group of compounds was determined as intrinsic activity for maximal adenylyl cyclase stimulation in wild-type beta(2)AR. 3. Although the two mutations had opposite effects on ligand affinity, the extents of change were in both cases largely correlated with the degree of ligand efficacy. This was particularly evident if the extra loss of binding energy due to hydrogen bond deletion in the AA mutant was taken into account. Thus the data demonstrate that there is an overall linkage between the configuration of the binding pocket and the intrinsic equilibrium between active and inactive receptor forms. 4. We also found that AA mutation-induced affinity changes for catecholamine congeners gradually lacking ethanolamine substituents were linearly correlated to the loss of affinity that such modifications of the ligand cause for wild-type receptor. This indicates that the strength of bonds between catechol ring and helix 5 is critically dependent on the rest of interactions of the beta-ethanolamine tail with other residues of the beta(2)-AR binding pocket.

Turnover of plant lineages shapes herbivore phylogenetic beta diversity along ecological gradients.

Understanding drivers of biodiversity patterns is of prime importance in this era of severe environmental crisis. More diverse plant communities have been postulated to represent a larger functional trait-space, more likely to sustain a diverse assembly of herbivore species. Here, we expand this hypothesis to integrate environmental, functional and phylogenetic variation of plant communities as factors explaining the diversity of lepidopteran assemblages along elevation gradients in the Swiss Western Alps. According to expectations, we found that the association between butterflies and their host plants is highly phylogenetically structured. Multiple regression analyses showed the combined effect of climate, functional traits and phylogenetic diversity in structuring butterfly communities. Furthermore, we provide the first evidence that plant phylogenetic beta diversity is the major driver explaining butterfly phylogenetic beta diversity. Along ecological gradients, the bottom up control of herbivore diversity is thus driven by phylogenetically structured turnover of plant traits as well as environmental variables.

Preliminary results on the postmortem measurement of 3-beta-hydroxybutyrate in liver homogenates.

The concentrations of 3-beta-hydroxybutyrate (3HB) in blood and two liver samples were retrospectively examined in a series of medicolegal autopsies. These cases included diabetic ketoacidosis, nondiabetic individuals presenting moderate to severe decompositional changes and nondiabetic medicolegal cases privy of decompositional changes. 3HB concentrations in liver sample homogenates correlate well with blood values in all examined groups. Additionally, decompositional changes were not associated with increases in blood and liver 3HB levels. These results suggest that 3HB can be reliably measured in liver homogenates when blood is not available at autopsy. Furthermore, they suggest that metabolic disturbances potentially leading or contributing to death may be objectified through liver 3HB determination even in decomposed bodies.

Beta(1)-selective agonist (-)-1-(3,4-dimethoxyphenetylamino)-3-(3,4-dihydroxy)-2-propanol [(-)-RO363] differentially interacts with key amino acids responsible for beta(1)-selective binding in resting and active states.

(-)-1-(3,4-Dimethoxyphenetylamino)-3-(3,4-dihydroxy)-2-propanol [(-)-RO363] is a highly selective beta(1)-adrenergic receptor (beta(1)AR) agonist. To study the binding site of beta(1)-selective agonist, chimeric beta(1)/beta(2)ARs and Ala-substituted beta(1)ARs were constructed. Several key residues of beta(1)AR [Leu(110) and Thr(117) in transmembrane domain (TMD) 2], and Phe(359) in TMD 7] were found to be responsible for beta(1)-selective binding of (-)-RO363, as determined by competitive binding. Based on these results, we built a three-dimensional model of the binding domain for (-)-RO363. The model indicated that TMD 2 and TMD 7 of beta(1)AR form a binding pocket; the methoxyphenyl group of N-substituent of (-)-RO363 seems to locate within the cavity surrounded by Leu(110), Thr(117), and Phe(359). The amino acids Leu(110) and Phe(359) interact with the phenyl ring of (-)-RO363, whereas Thr(117) forms hydrogen bond with the methoxy group of (-)-RO363. To examine the interaction of these residues with beta(1)AR in an active state, each of the amino acids was changed to Ala in a constitutively active (CA)-beta(1)AR mutant. The degree of decrease in the affinity of CA-beta(1)AR for (-)-RO363 was essentially the same as that of wild-type beta(1)AR when mutated at Leu(110) and Thr(117). However, the affinity was decreased in Ala-substituted mutant of Phe(359) compared with that of wild-type beta(1)AR. These results indicated that Leu(110) and Thr(117) are necessary for the initial binding of (-)-RO363 with beta(1)-selectivity, and interaction of Phe(359) with the N-substituent of (-)-RO363 in an active state is stronger than in the resting state.

Different internalization properties of the alpha1a- and alpha1b-adrenergic receptor subtypes: the potential role of receptor interaction with beta-arrestins and AP50.

The internalization properties of the alpha1a- and alpha1b-adrenergic receptors (ARs) subtypes transiently expressed in human embryonic kidney (HEK) 293 cells were compared using biotinylation experiments and confocal microscopy. Whereas the alpha1b-AR displayed robust agonist-induced endocytosis, the alpha1a-AR did not. Constitutive internalization of the alpha1a-AR was negligible, whereas the alpha1b-AR displayed significant constitutive internalization and recycling. We investigated the interaction of the alpha1-AR subtypes with beta-arrestins 1 and 2 as well as with the AP50 subunit of the clathrin adaptor complex AP2. The results from both coimmunoprecipitation experiments and beta-arrestin translocation assays indicated that the agonistinduced interaction of the alpha1a-AR with beta-arrestins was much weaker than that of the alpha1b-AR. In addition, the alpha1a-AR did not bind AP50. The alpha1b-AR mutant M8, lacking the main phosphorylation sites in the receptor C tail, was unable to undergo endocytosis and was profoundly impaired in binding beta-arrestins despite its binding to AP50. In contrast, the alpha1b-AR mutant DeltaR8, lacking AP50 binding, bound beta-arrestins efficiently, and displayed delayed endocytosis. RNA interference showed that beta-arrestin 2 plays a prominent role in alpha1b-AR endocytosis. The findings of this study demonstrate differences in internalization between the alpha1a- and alpha1b-AR and provide evidence that the lack of significant endocytosis of the alpha1a-AR is linked to its poor interaction with beta-arrestins as well as with AP50. We also provide evidence that the integrity of the phosphorylation sites in the C tail of the alpha1b-AR is important for receptor/beta-arrestin interaction and that this interaction is the main event triggering receptor internalization.

A comparative study of T-cell receptor V beta usage in non-obese diabetic (NOD) and I-E transgenic NOD mice.

The non-obese diabetic (NOD) mouse is a model for the study of insulin-dependent diabetes mellitus (IDDM). Recently transgenic NOD mice have been derived (NOD-E) that express the major histocompatibility complex (MHC) class II I-E molecule. NOD-E do not become diabetic and show negligible pancreatic insulitis. The possibility pertained that NOD-E mice are protected from disease by a process of T-cell deletion or anergy. This paper describes our attempts to discover whether this was so, by comparing NOD and NOD-E mouse T-cell receptor V beta usage. Splenocytes and lymph node cells were therefore tested for their ability to proliferate in response to monoclonal anti-V beta antibodies. We were unable to show any consistent differences between NOD and NOD-E responses to the panel of antibodies used. Previously proposed V beta were shown to be unlikely candidates for deletion or anergy. T cells present at low frequency (V beta 5+) in both NOD and NOD-E mice were shown to be as capable of expansion in response to antigenic stimulation as were more frequently expressed V beta. Our data therefore do not support deletion or anergy as mechanisms which could account for the observed disease protection in NOD-E mice.

The role of the T-cell receptor (TCR) in alpha beta/gamma delta lineage commitment: clues from intracellular TCR staining.

T cells belong to two mutually exclusive lineages expressing either alpha beta or gamma delta T-cell receptors (TCR). Although alpha beta and gamma delta cells are known to share a common precursor the role of TCR rearrangement and specificity in the lineage commitment process is controversial. Instructive lineage commitment models endow the alpha beta or gamma delta TCR with a deterministic role in lineage choice, whereas separate lineage models invoke TCR-independent lineage commitment followed by TCR-dependent selection and maturation of alpha beta and gamma delta cells. Here we review the published data pertaining to the role of the TCR in alpha beta/gamma delta lineage commitment and provide some additional information obtained from recent intracellular TCR staining studies. We conclude that a variant of the separate lineage model is best able to accommodate all of the available experimental results.

A limited role for beta-selection during gamma delta T cell development.

T cells belong to two distinct lineages expressing either alpha beta or gamma delta TCR. During alpha beta T cell development, it is clearly established that productive rearrangement at the TCR beta locus in immature precursor cells leads to the expression of a pre-TCR complex. Signaling through the pre-TCR results in the selective proliferation and maturation of TCR beta+ cells, a process that is known as beta-selection. However, the potential role of beta-selection during gamma delta T cell development is controversial. Whereas PCR-RFLP and sequencing techniques have provided evidence for a bias toward in-frame VDJ beta rearrangements in gamma delta cells (consistent with beta-selection), gamma delta cells apparently develop normally in mice that are unable to assemble a pre-TCR complex due to a deficiency in TCR beta or pT alpha genes. In this report, we have directly addressed the physiologic significance of beta-selection during gamma delta cell development in normal mice by quantitating intracellular TCR beta protein in gamma delta cells and correlating its presence with cell cycle status. Our results indicate that beta-selection plays a significant (although limited) role in gamma delta cell development by selectively amplifying a minor subset of gamma delta precursor cells with productively rearranged TCR beta genes.

Low density lipoprotein signaling and pancreatic beta cells protection

SummaryLow-density lipoproteins (LDLs) have an important physiological role in organism transporting cholesterol and other fatty substances to target tissues. However, elevated LDL levels in the blood are associated with the formation of arterial plaques and consequently atherosclerosis. It is therefore important to characterize the intracellular pathways induced upon LDL stimulation as they might be involved in the pathological properties of these lipoproteins. It has been previously found that LDL stimulation of mouse embryonic fibroblasts activates p38 mitogen activated protein kinases (MAPKs). This leads to cell spreading and increase in the wound healing capabilities of the cells. These two responses might occur within atherosclerotic plaques.The aim of this project is to reveal the missing links between LDL particle and activation of p38 MAPK kinase. As previously shown in our lab activation of p38 MAPK kinase by the LDL particles occur independently of classical LDL receptor (LDLR). In this study we have shown that scavenger receptor type Β class I (SR-BI) is responsible for the signal transduction from the LDLs to the p38 MAPK. We have also shown that Mitogen activated kinase kinases (MKKs) that can directly activate ρ 38 MAPK in these conditions are MKK3 and MKK6 but not MKK4. We have also tested some of the intermediate components of the pathway like Ras and PI3 kinase but found that they do not play a role.The data obtained in this study showed a part of molecular mechanism responsible for p38 MAPK activation and subsequent wound healing and can contribute to our knowledge on function of the fibroblasts in the development of the atherosclerotic plaques.Diabetes Mellitus is a condition caused by disordered metabolism of blood glucose level. It is one of the most commonly spread disease in the western world, with the incidence reaching 8% of population in United States. Two most common types of diabetes are type 1 and 2 that differs slightly in the mechanism of the development. However in the basis of both types lies the cell death of pancreatic beta cells. The aim of this work is to improve beta cells survival in different pathophysiological settings. This could be extrapolated to the conditions in which Diabetes develops in humans. We decided to use RasGAP- derived fragment Ν with its strong antiapoptotic effect in beta cells. In our lab we have demonstrated that in the mild stress conditions RasGAP can be cleaved by caspases at the position 455 producing two fragments, fragment Ν and fragment C. Fragment Ν exerts

Peroxisome proliferator-activated receptor beta/delta as a therapeutic target for metabolic diseases.

The peroxisome proliferator-activated receptor (PPAR) family comprises three distinct isotypes: PPARalpha, PPARbeta/delta and PPARgamma. PPARs are nuclear hormone receptors that mediate the effects of fatty acids and their derivatives at the transcriptional level. Until recently, the characterisation of the important role of PPARalpha in fatty acid oxidation and of PPARgamma in lipid storage contrasted with the sparse information concerning PPARbeta/delta. However, evidence is now emerging for a role of PPARbeta/delta in tissue repair and energy homeostasis. Experiments with tissue-specific overexpression of PPARbeta/delta or treatment of mice with selective PPARbeta/delta agonists demonstrated that activation of PPARbeta/delta in vivo increases lipid catabolism in skeletal muscle, heart and adipose tissue and improves the serum lipid profile and insulin sensitivity in several animal models. PPARbeta/delta activation also prevents the development of obesity and improves cholesterol homeostasis in obesity-prone mouse models. These new insights into PPARbeta/delta functions suggest that targeting PPARbeta/delta may be helpful for treating disorders associated with the metabolic syndrome. Although these perspectives are promising, several independent and contradictory reports raise concerns about the safety of PPARbeta/delta ligands with respect to tumourigenic activity in the gut. Thus, it appears that further exploration of PPARbeta/delta functions is necessary to better define its potential as a therapeutic target.

Glucagon-like peptide-1 protects beta-cells against apoptosis by increasing the activity of an IGF-2/IGF-1 receptor autocrine loop.

OBJECTIVE: The gluco-incretin hormones glucagon-like peptide (GLP)-1 and gastric inhibitory peptide (GIP) protect beta-cells against cytokine-induced apoptosis. Their action is initiated by binding to specific receptors that activate the cAMP signaling pathway, but the downstream events are not fully elucidated. Here we searched for mechanisms that may underlie this protective effect. RESEARCH DESIGN AND METHODS: We performed comparative transcriptomic analysis of islets from control and GipR(-/-);Glp-1-R(-/-) mice, which have increased sensitivity to cytokine-induced apoptosis. We found that IGF-1 receptor expression was markedly reduced in the mutant islets. Because the IGF-1 receptor signaling pathway is known for its antiapoptotic effect, we explored the relationship between gluco-incretin action, IGF-1 receptor expression and signaling, and apoptosis. RESULTS: We found that GLP-1 robustly stimulated IGF-1 receptor expression and Akt phosphorylation and that increased Akt phosphorylation was dependent on IGF-1 but not insulin receptor expression. We demonstrated that GLP-1-induced Akt phosphorylation required active secretion, indicating the presence of an autocrine activation mechanism; we showed that activation of IGF-1 receptor signaling was dependent on the secretion of IGF-2. We demonstrated, both in MIN6 cell line and primary beta-cells, that reducing IGF-1 receptor or IGF-2 expression or neutralizing secreted IGF-2 suppressed GLP-1-induced protection against apoptosis. CONCLUSIONS: An IGF-2/IGF-1 receptor autocrine loop operates in beta-cells. GLP-1 increases its activity by augmenting IGF-1 receptor expression and by stimulating secretion; this mechanism is required for GLP-1-induced protection against apoptosis. These findings may lead to novel ways of preventing beta-cell loss in the pathogenesis of diabetes.

Long-term, multilineage hematopoiesis occurs in the combined absence of beta-catenin and gamma-catenin

The canonical Wnt signaling pathway plays key roles in stem-cell maintenance, progenitor cell expansion, and lineage decisions. Transcriptional responses induced by Wnt depend on the association of either beta-catenin or gamma-catenin with lymphoid enhancer factor/T cell factor transcription factors. Here we show that hematopoiesis, including thymopoiesis, is normal in the combined absence of beta- and gamma-catenin. Double-deficient hematopoietic stem cells maintain long-term repopulation capacity and multilineage differentiation potential. Unexpectedly, 2 independent ex vivo reporter gene assays show that Wnt signal transmission is maintained in double-deficient hematopoietic stem cells, thymocytes, or peripheral T cells. In contrast, Wnt signaling is strongly reduced in thymocytes lacking TCF-1 or in nonhematopoietic cells devoid of beta-catenin. These data provide the first evidence that hematopoietic cells can transduce canonical Wnt signals in the combined absence of beta- and gamma-catenin

The GTPase RalA regulates different steps of the secretory process in pancreatic beta-cells.

BACKGROUND: RalA and RalB are multifuntional GTPases involved in a variety of cellular processes including proliferation, oncogenic transformation and membrane trafficking. Here we investigated the mechanisms leading to activation of Ral proteins in pancreatic beta-cells and analyzed the impact on different steps of the insulin-secretory process. METHODOLOGY/PRINCIPAL FINDINGS: We found that RalA is the predominant isoform expressed in pancreatic islets and insulin-secreting cell lines. Silencing of this GTPase in INS-1E cells by RNA interference led to a decrease in secretagogue-induced insulin release. Real-time measurements by fluorescence resonance energy transfer revealed that RalA activation in response to secretagogues occurs within 3-5 min and reaches a plateau after 10-15 min. The activation of the GTPase is triggered by increases in intracellular Ca2+ and cAMP and is prevented by the L-type voltage-gated Ca2+ channel blocker Nifedipine and by the protein kinase A inhibitor H89. Defective insulin release in cells lacking RalA is associated with a decrease in the secretory granules docked at the plasma membrane detected by Total Internal Reflection Fluorescence microscopy and with a strong impairment in Phospholipase D1 activation in response to secretagogues. RalA was found to be activated by RalGDS and to be severely hampered upon silencing of this GDP/GTP exchange factor. Accordingly, INS-1E cells lacking RalGDS displayed a reduction in hormone secretion induced by secretagogues and in the number of insulin-containing granules docked at the plasma membrane. CONCLUSIONS/SIGNIFICANCE: Taken together, our data indicate that RalA activation elicited by the exchange factor RalGDS in response to a rise in intracellular Ca2+ and cAMP controls hormone release from pancreatic beta-cell by coordinating the execution of different events in the secretory pathway.

Role of beta-1 integrin in tumor growth and dissemination

SUMMARY Cancer is one of the leading causes of disease-related mortality. In most cases, death is due to the spread of cells from the primary tumor to distant sites causing formation of metastases. To become tumorigenic, cells should acquire ability, including self-sufficiency in growth signals, insensitivity to anti-growth signals, resistance to apoptosis, sustained angiogenesis, limitless replicative potential and tissue invasion and metastasis. Tumor progression depends, in part on the relationship between tumor cells and host tissue stroma, characterized by changes of tumor cell adhesion to their microenvironment and activation of a variety of extracellular proteases that play a role in ECM degradation. integrins are adhesion proteins implicated in tumorigenesis. Their main function is to mediate cell adhesion to the ECM or to other cells and to create a link between the ECM and the cytoskeleton. Tumor cells like normal cells use integrins to attach to ECM, migrate into surrounding tissues and derive survival and growth signals. Integrin-dependent adhesion and migration are thought to play an important role in tumor dissemination. A strategy was designed to address the role of β1 integrin tumor growth and dissemination. Murine mammary carcinoma (TA3) cells were stably transfected with a soluble β1 integrin construct, which is anticipated to play a dominant negative role, being able to associate with different α-subunits expressed on the cell surface but unable to transduce signals to the nucleus. Results from studies based on soluble β1 integrin TA3 transfectants showed that 1) the integrin expression pattern at the cell surface changed with an induction of α2β1 and α5β1 heterodimers; 2) adhesion to collagens, especially collagen I was increased; 3) tumor dissemination after intrape-ritoneal injection in syngeneic mice was abolished and 4) local growth after orthotopic injection was maintained but delayed. Taken together, the data presented here suggest that β1 integrin plays a potentially important role in the regulation of tumor behavior. RESUME Le cancer est une des principales causes de mortalité suite à une maladie. Dans la plupart des cas, la mort est la conséquence de la dissémination de cellules, provenant de la tumeur primaire, dans des endroits distants et causant la formation de métastases. Afin de devenir cancéreuse, une cellule doit acquérir certaines capacités, telles qu'une auto-suffisance en facteurs de croissance, une insensibilité aux facteurs empêchant la croissance cellulaire, une résistance à l'apoptose, une angiogénèse soutenue, un potentiel de réplication illimité et une capacité à pénétrer dans les tissus et à former des colonies métastatiques. La progression d'une tumeur dépend, en partie, de la relation entre les cellules tumorales et les cellules tissulaires de l'hôte. Cette relation est caractérisée par des modifications des cellules tumorales quant à leur adhésion au microenvironnement et à l'activation de protéases qui permettent de dégrader la matrice extracellulaire. Les intégrines sont des protéines impliquées dans le développement tumoral. Leur fonction principale est de réguler l'adhésion des cellules à la matrice extracellulaire, ou à d'autres cellules, et de créer un lien entre cette matrice extracellulaire et le cytosquelette. Les cellules tumorales utilisent également les intégrines pour se lier à la matrice extracellulaire, pour migrer dans les tissus adjacents et pour induire des signaux de croissance et de survie. Ces événements d'adhésion et de migration, qui dépendent des intégrines, jouent un rôle primordial dans la dissémination des cellules cancéreuses. Une stratégie a été élaborée afin de définir le rôle de l'intégrine β1 durant la croissance et la dissémination des cellules tumorales. Des cellules provenant d'un carcinome de la glande mammaire (TA3) ont été transfectées de manière stable avec un vecteur contenant la séquence codante de la partie extracellulaire de l'intégrine β1. L'intégrine tronquée doit être capable de se lier aux sous-unités α exprimées à la surface de la cellule, mais doit être incapable de transmettre un signal à l'intérieur de la cellule. Les résultats obtenus avec les cellules TA3 transfectées contenant l'intégrine β1 soluble montrent que I) le répertoire d'expression des intégrines à la surface de la cellule a changé en faveur des hétérodimères α2β1 et α5β1; 2) l'adhésion aux collagènes, particulièrement au collagène de type I a augmenté; 3) la dissémination des cellules tumorales après une injection intrapéritonéale est empêchée; 4) la croissance tumorale après une injection orthotopique est conservée mais retardée. Ces résultats montrent que l'intégrine β1 joue un rôle primordial dans la régulation du comportement tumoral.