The p53 gene expression and its developmental regulation in schistosomes

We have studied the gene expression, especially of the oncoproteins, and its regulation in schistosomes. Schistosomes have a complex life cycle with defined dimorphic lifestyle. The parasite are so far unique in biology in expressing oncogene products in their adult stage. In order to characterize the expression and developmental regulation, a lambda gt 11 cDNA library and lambda EMBL4 genomic DNA library of each growth stage of Schistosoma mansoni and S. japonicum was constructed, and was screened with various monoclonal antibodies against ongogene products. One positive plaque reacted to anti-p53 antibody (Ab-2, Oncogene Science, Inc.) was further analyzed. This fusion protein was about 120 KDa in molecular weights, and expressed as 1.4 Kb RNA in the adult stage. P53 gene is well-known as the negative regulator of the cell cicle, and the mutations in the gene are turning out to be the most common genetic alterations in human cancers. The comparison of the gene structure among species and stages were being conducted. Chromosome structures, C-band formation, and the results of in situ hybridization using the phage probe would be discussed.

Insights into the regulation of two caspase-activating platforms, the inflammasome and the PIDDosome

Résumé: Les organismes multicellulaires ont adopté diverses stratégies pour répondre aux stress auxquels ils sont exposés. Cette étude a exploré deux de ces stratégies l'inflammation en réponse à une invasion par un pathogène, et l'apoptose ou la survie en réponse aux dommages à l'ADN. L'interleukine-lß (IL-lß) est une importante cytokine inflammatoire. Elle est synthétisée sous forme d'un précurseur inactif et nécessite un clivage par la caspase-1 pour être activée. La caspase-1 elle-même est activée dans un complexe appelé inflammasome. Certains NLRs (Nod-like receptors), IPAF et les NALPs, sont capables de former des inflammasomes fonctionnels. Cette étude s'est intéressée au rôle d'un autre NLR structurellement proche, la protéine NAIP, dans la régulation de la caspase-1 et la maturation de l'IL-1 ß. NAIP est incorporé à l'inflammasome contenant NALP3 et est capable d'inhiber l'activation de la caspase-1 et la maturation de l'IL-lß. Cette fonction inhibitrice dépend des ses domaines BIR et est inhibée par ses LRRs. Le mécanisme exact d'inhibition reste à définir et la régulation de l'activation de NAIP est discutée. La deuxième partie de cette étude concerne la protéine PIDD. Cette protéine est impliquée avec RAIDD dans l'activation de la caspase-2, et est aussi capable, avec l'aide de RIP et de NEMO, d'activer NF-κB en réponse aux dommages à l'ADN. Deux isoformes de PIDD ont déjà été décrites dans la littérature, PIDD (isoforme 1) et LRDD (isoforme 2) et une troisième isoforme est rapportée ici. L'étude de l'expression de ces isoformes a montré qu'elles sont exprimées différemment dans les tissus et dans les lignées cellulaires, et que l'isoforme 3 est induite en réponse à un stress génotoxique. La caractérisation fonctionnelle a établi que les trois isoformes sont capables d'activer NF-κB, donc la survie, mais que seule l'isoforme 1 peut interagir avec RAIDD pour activer la caspase-2 et sensibiliser les cellules à la mort induite par un stress génotoxique. Le domaine intermédiaire de PIDD, situé entre le deuxième ZU5 et le DD est essentiel pour l'interaction entre PIDD et RAIDD et l'activation de la caspase-2 qui en découle. En conclusion, l'épissage différentiel de l'ARNm de PIDD permet la production d'au moins trois protéines possédant des fonctions agonistes ou antagonistes et qui peuvent participer au choix cellulaire entre survie et apoptose en réponse aux dommages à l'ADN. Summary: Multicellular organisms have evolved several strategies to cope with the stresses they encounter. The present study has explored two of these strategies: inflammation in response to a pathogenic invasion, and apoptosis or repair/survival in response to DNA damage. Interleukin-lß (IL-lß) is a key mediator of inflammation. It is synthesized as an inactive precursor and requires cleavage by caspase-1 to be activated. caspase-1 itself is activated in molecular platforms called inflammasomes, which can be formed by members of the NOD-like receptors (NLR) family, like IPAF and NALPs. This study has investigated the role of another NLR, the structurally related protein NAIP, in the regulation of caspase-1 activation and IL-lß maturation. An inhibitory role of NAIP on caspase-1 activation and IL-lß maturation was demonstrated, as well as NAIP incorporation in the NALP3 inflammasome. This inhibitory property relies on NAIP BIR domains and is inhibited by NAIP LRRs. The exact mechanism of NAIP-mediated caspase-1 activation remains to be elucidated and the regulation of NAIP activation is discussed. The second part of this study focused on the caspase-2 activating protein PIDD. This protein is known to mediate caspase-2 activation via RAIDD and to signal NF-κB via RIP and NEMO in response to DNA damage. Two isoforms of PIDD, PIDD (isoform 1) and LRDD (isoform 2), have already been reported and a third isoform is described here. Investigation of the expressional regulation of these isoforms indicated that they are differentially expressed in tissues and cell lines, and that isoform 3 mRNA levels are upregulated in response to genotoxic stress. Functional studies demonstrated that all three isoforms can activate NF-κB in response to DNA damage, but only isoform 1 is able to interact with RAIDD and activate caspase-2, sensitizing cells to genotoxic stress-induced cell death. The intermediate domain located between the second ZUS and the DD is essential for the interaction of PIDD and RAIDD and the subsequent caspase-2 activation. Thus the differential splicing of PIDD mRNA leads to the formation of at least thrée proteins with antagonizing/agonizing functions that could participate in determining cell fate in response to DNA damage.

The imidazoline receptors and the central regulation of the arterial blood pressure: a minireview

Recently, we proposed the hypothesis according to wich the central hypotensive effect of clonidine and related substances could be related to an action upon specific receptors, requiring the imidazoline or imidazoline-like structures, rather than alpha2-adrenoceptors. Since then, direct evidences have been accumulated to confirm the existence of a population of imidazoline specific binding sites in the brainstem of animals and man, more precisely in the Nucleus Reticularis Lateralis (NRL) region of the ventrolateral medulla (VLM), site of the antihypertensive action of clonidine. The purification of the putative endogenous ligand of the imidazoline receptors - named endazoline - is currently being attempted from human brain extracts. This new concept might at last lead to the expected dissociation of the pharmacological mechanisms involved, on the one hand, in the therapeutic antihypertensive effect, and on the other, in their major side-effect, which is sedation. In fact, it has been recently confirmed that hypotension is mediated by the activation of imidazoline preferring receptors (IPR) within the NRL region, while sedation is attributed to the inhibition of alpha2-adrenergic mechanisms in the locus coeruleus, which is involved in the control of the sleep-waking cycle. The IPRmay constitute on interesting target for new drugs in the treatment of arterial hypertension. Finally, dysfunctions of this modulatory system which could be involved in the pathophysiologyof some forms of the hypertensive disease are under investigation.

Regulation of the akt signalling in human skeletal muscle atrophy

Abstract : The term "muscle disuse" is often used to refer collectively to reductions in neuromuscular activity as observed with sedentary lifestyles, reduced weight bearing, cancer, chronic obstructive pulmonary disease, chronic heart failure, spinal cord injury, sarcopenia or exposure to microgravity (spaceflight). Muscle disuse atrophy, caused by accelerated proteolysis, is predominantly due to the activation of the ATP-dependent ubiquitin (Ub) proteasome pathway. The current advances in understanding the molecular factors contributing to the Ub-dependent proteolysis process have been made mostly in rodent models of human disease and denervation with few investigations performed directly in humans. Recently, in mice, the genes Atrogin-1 and MuRF1 have been designated as primary candidates in the control of muscle atrophy. Additionally, the decreased activity of the Akt/GSK-3ß and Akt/mTOR pathways has been associated with a reduction in protein synthesis and contributing to skeletal muscle atrophy. Therefore, it is now commonly accepted that skeletal muscle atrophy is the result of a decreased protein synthesis concomitant with an increase in protein degradation (Glass 2003). Atrogin-1 and MuRF1 are genes expressed exclusively in muscle. In mice, their expression has been shown to be directly correlated with the severity of atrophy. KO-mice experiments showed a major protection against atrophy when either of these genes were deleted. Skeletal muscle hypertrophy is an important function in normal postnatal development and in the adaptive response to exercise. It has been shown, in vitro, that the activation of phosphatidylinositol 3-kinase (PI-3K), by insulin growth factor 1 (IGF-1), stimulates myotubes hypertrophy by activating the downstream pathways, Akt/GSK-3ß and Akt/mTOR. It has also been demonstrated in mice, in vivo, that activation of these signalling pathways causes muscle hypertrophy. Moreover, the latter were recently proposed to also reduce muscle atrophy by inhibiting the FKHR mediated transcription of several muscle atrophy genes; Atrogin-1 and MuRF1. Therefore, these targets present new avenues for developing further the understanding of the molecular mechanisms involved in both skeletal muscle atrophy and hypertrophy. The present study proposed to investigate the regulation of the Akt/GSK-3ß and Akt/mTOR signalling pathways, as well as the expression levels of the "atrogenes", Atrogin-1 and MuRF1, in four human models of skeletal muscle atrophy. In the first study, we measured the regulation of the Akt signalling pathway after 8 weeks of both hypertrophy stimulating resistance training and atrophy stimulation de-training. As expected following resistance training, muscle hypertrophy and an increase in the phosphorylation status of the different members of the Akt pathway was observed. This was paralleled by a concomitant decrease in FOXO1 nuclear protein content. Surprisingly, exercise training also induced an increase in the, expression of the atrophy genes and proteins involved in the ATP-dependant ubiquitin-proteasome system. On the opposite, following the de-training period a muscle atrophy, relative to the post-training muscle size, was measured. At the same time, the phosphorylation levels of Akt and GSK-3ß were reduced while the amount of FOXO1 in the nucleus increased. After the atrophy phase, there was also a reduction in Atrogin-1 and MuRF1 contents. In this study, we demonstrate for the first time in healthy human skeletal muscle, that the regulation of Akt and its downstream targets GSK-3ß, mTOR and FOXO1 are associated with both thé skeletal muscle hypertrophy and atrophy processes. Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by the loss of both upper and lower motor neurons, which leads to severe muscle weakness and atrophy. All measurements were performed in biopsies from 22 ALS patients and 16 healthy controls. ALS patients displayed an increase in Atrogin-1 mRNA and protein content which was associated with a decrease in Akt activity. However there was no difference in the mRNA and phospho-protein content of FOXO1, FOXO3a, p70S6K and GSK-3ß. The transcriptional regulation of human Atrogin-1 may be controlled by an Akt-mediated transcription factor other than FKHR or via an other signalling pathway. Chronic complete spinal cord injury (SCI) is associated with severe muscle atrophy which is linked to co-morbidity factors such as diabetes, obesity, lipid disorders and cardiovascular diseases. Molecular mechanisms associated with chronic complete SCI-related muscle atrophy are not well understood. The aim of the present study was to determine if there was an increase in catabolic signalling targets such as Atrogin-1, MuRF1, FOXO and myostatin, and decreases in anabolic signalling targets such as IGF, Akt, GSK-3ß, mTOR, 4E-BP1 and p-70S6K in chronic complete SCI patients. All measurements were performed in biopsies taken from 8 complete chronic SCI patients and 7 age matched healthy controls. In SCI patients when compared with controls, there was a significant reduction in mRNA levels of Atrogin1, MuRF1 and Myostatin. Protein levels for Atrogin-1, FOX01 and FOX03a were also reduced. IGF-1 and both phosphorylated GSK-3ß and 4E-BP1 were decreased; the latter two in an Akt and mTOR independent manner, respectively. Reductions in Atrogin-1, MuRF1, FOXO and myostatin suggest the existence of an internal mechanism aimed at reducing further loss of muscle proteins during chronic SCI. The downregulation of signalling proteins regulating anabolism such as IGF, GSK3ß and 4E-BP1 would reduce the ability to increase protein synthesis rates in this chronic state of muscle wasting. The molecular mechanisms controlling age-related skeletal muscle loss in humans are poorly understood. The present study aimed to investigate the regulation of several genes and proteins involved in the activation of key signalling pathways promoting muscle hypertrophy such as GH/STAT5/IGF, IGF/Akt/GSK-3ß/4E-BP1 and muscle atrophy such as TNFα/SOCS3 and Akt/FOXO/Atrogin-1 or MuRF1 in muscle biopsies from 13 young and 16 elderly men. In the older, as compared with the young subjects, TNFα and SOCS-3 were increased while growth hormone receptor protein (GHR) and IGF-1 mRNA were both decreased. Akt protein levels were increased however no change in phosphorylated Akt content was observed. GSK-3ß phosphorylation levels were increased while 4E-BP1 was not changed. Nuclear FKHR and FKHRL1 protein levels were decreased, with no changes in their atrophy target genes, Atrogin-1 and MuRF1. Myostatin mRNA and protein levels were significantly elevated. Human sarcopenia may be linked to a reduction in the activity or sensitivity of anabolic signalling proteins such as GHR, IGF and Akt. TNFα, SOCS-3 and myostatin are potential candidates influencing this anabolic perturbation. In conclusion our results support those obtained in rodent or ín vitro models, and demonstrate Akt plays a pivotal role in the control of muscle mass in humans. However, the Akt phosphorylation status was dependant upon the model of muscle atrophy as Akt phosphorylation was reduced in all atrophy models except for SCI. Additionally, the activity pattern of the downstream targets of Akt appears to be different upon the various human models. It seems that under particular conditions such as spinal cord injury or sarcopenia, .the regulation of GSK-3ß, 4eBP1 and p70S6K might be independent of Akt suggesting alternative signalling pathways in the control of these the anabolic response in human skeletal muscle. The regulation of Atrogin-1 and MuRF1 in some of our studies has been shown to be also independent of the well-described Akt/FOXO signalling pathway suggesting that other transcription factors may regulate human Atrogin-1 and MuRF1. These four different models of skeletal muscle atrophy and hypertrophy have brought a better understanding concerning the molecular mechanisms controlling skeletal muscle mass in humans.

RNA-driven cyclin-dependent kinase regulation: when CDK9/cyclin T subunits of P-TEFb meet their ribonucleoprotein partners.

The positive transcription elongation factor (P-TEFb) consists of CDK9, a cyclin-dependent kinase and its cyclin T partner. It is required for transcription of most class II genes. Its activity is regulated by non-coding RNAs. The 7SK cellular RNA turns the HEXIM cellular protein into a P-TEFb inhibitor that binds its cyclin T subunit. Thus, P-TEFb activity responds to variations in global cellular transcriptional activity and to physiological conditions linked to cell differentiation, proliferation or cardiac hypertrophy. In contrast, the Tat activation region RNA plays an activating role. This feature at the 5' end of the human immunodeficiency (HIV) viral transcript associates with the viral protein Tat that in turn binds cyclin T1 and recruits active P-TEFb to the HIV promoter. This results in enhanced P-TEFb activity, which is critical for an efficient production of viral transcripts. Although discovered recently, the regulation of P-TEFb becomes a paradigm for non-coding RNAs that regulate transcription factors. It is also a unique example of RNA-driven regulation of a cyclindependent kinase.

Oil price shocks and labor market fluctuations

We examine the impact of real oil price shocks on labor market flows in the U.S. We first use smooth transition regression (STR) models to investigate to what extent oil prices can be considered as a driving force of labor market fluctuations. Then we develop and calibrate a modified version of Pissarides' (2000) model with energy costs, which we simulate in response to shocks mimicking the behavior of the actual oil price shocks. We find that (i) these shocks are an important driving force of job market flows; (ii) the job finding probability is the main transmission mechanism of such shocks; and (iii) they bring a new amplification mechanism for the volatility and should thus be seen as complementary of labor productivity shocks. Overall we conclude that shocks in oil prices cannot be neglected in explaining cyclical labor adjustments in the U.S.

Immigrants'assimilation process in a segmented labor market

While much of the literature on immigrants' assimilation has focused on countries with a large tradition of receiving immigrants and with flexible labor markets, very little is known on how immigrants adjust to other types of host economies. With its severe dual labor market, and an unprecedented immigration boom, Spain presents a quite unique experience to analyze immigrations' assimilation process. Using data from the 2000 to 2008 Labor Force Survey, we find that immigrants are more occupationally mobile than natives, and that much of this greater flexibility is explained by immigrants' assimilation process soon after arrival. However, we find little evidence of convergence, especially among women and high skilled immigrants. This suggests that instead of integrating, immigrants occupationally segregate, providing evidence consistent with both imperfect substitutability and immigrants' human capital being under-valued. Additional evidence on the assimilation of earnings and the incidence of permanent employment by different skill levels also supports the hypothesis of segmented labor markets.

The Part-time pay penalty in a segmented labor market

This paper is the first to examine the implications of switching to PT work for women's subsequent earnings trajectories, distinguishing by their type of contract: permanent or fixedterm. Using a rich longitudinal Spanish data set from Social Security records of over 76,000 prime-aged women strongly attached to the Spanish labor market, we find that PT work aggravates the segmentation of the labor market insofar there is a PT pay penalty and this penalty is larger and more persistent in the case of women with fixed-term contracts. The paper discusses problems arising in empirical estimation (including a problem not discussed in the literature up to now: the differential measurement error of the LHS variable by PT status), and how to address them. It concludes with policy implications relevant for Continental Europe and its dual structure of employment protection.

Monoamine oxidase A down-regulation contributes to high metanephrine concentration in pheochromocytoma.

CONTEXT: The high diagnostic performance of plasma-free metanephrines (metanephrine and normetanephrine) (MN) for pheochromocytoma (PHEO) results from the tumoral expression of catechol-O-methyltransferase (COMT), the enzyme involved in O-methylation of catecholamines (CAT). Intriguingly, metanephrine, in contrast to epinephrine, is not remarkably secreted during a stress in hypertensive or normotensive subjects, whereas in PHEO patients CAT and MN are both raised to high levels. Because epinephrine and metanephrine are almost exclusively produced by the adrenal medulla, this suggests distinct CAT metabolism in chromaffin cells and pheochromocytes. OBJECTIVE: The objective of the study was to compare CAT metabolism between adrenal medulla and PHEO tissue regarding related enzyme expression including monoamine oxidases (MAO) and COMT. DESIGN: A multicenter comparative study was conducted. STUDY PARTICIPANTS: The study included 21 patients with a histologically confirmed PHEO and eight adrenal glands as control. MAIN OUTCOME MEASURES: CAT, dihydroxyphenol-glycol, 3,4-dihydroxyphenylacetic acid, and MN were measured in adrenal medulla and PHEO tissue. Western blot, quantitative RT-PCR and immunofluorescence studies for MAOA, MAOB, tyrosine hydroxylase, dopamine β-hydroxylase, L-amino acid decarboxylase, and COMT were applied on tissue homogenates and cell preparations. RESULTS: At both the protein and mRNA levels, MAOA and COMT are detected less often in PHEO compared with adrenal medulla, conversely to tyrosine hydroxylase, L-amino acid decarboxylase, and dopamine β-hydroxylase, much more expressed in tumor tissue. MAOB protein is detected less often in tumor but not differently expressed at the mRNA level. Dihydroxyphenol-glycol is virtually absent from tumor, whereas MN, produced by COMT, rises to 4.6-fold compared with adrenal medulla tissue. MAOA down-regulation was observed in 100% of tumors studied, irrespectively of genetic alteration identified; on the other hand, MAOA was strongly expressed in all adrenal medulla collected independently of age, gender, or late sympathetic activation of the deceased donor. CONCLUSION: High concentrations of MN in tumor do not only arise from CAT overproduction but also from low MAOA expression, resulting in higher substrate availability for COMT.

Regulation of adaptive behaviour during fasting by hypothalamic Foxa2.

The lateral hypothalamic area is considered the classic 'feeding centre', regulating food intake, arousal and motivated behaviour through the actions of orexin and melanin-concentrating hormone (MCH). These neuropeptides are inhibited in response to feeding-related signals and are released during fasting. However, the molecular mechanisms that regulate and integrate these signals remain poorly understood. Here we show that the forkhead box transcription factor Foxa2, a downstream target of insulin signalling, regulates the expression of orexin and MCH. During fasting, Foxa2 binds to MCH and orexin promoters and stimulates their expression. In fed and in hyperinsulinemic obese mice, insulin signalling leads to nuclear exclusion of Foxa2 and reduced expression of MCH and orexin. Constitutive activation of Foxa2 in the brain (Nes-Cre/+;Foxa2T156A(flox/flox) genotype) results in increased neuronal MCH and orexin expression and increased food consumption, metabolism and insulin sensitivity. Spontaneous physical activity of these animals in the fed state is significantly increased and is similar to that in fasted mice. Conditional activation of Foxa2 through the T156A mutation expression in the brain of obese mice also resulted in improved glucose homeostasis, decreased fat and increased lean body mass. Our results demonstrate that Foxa2 can act as a metabolic sensor in neurons of the lateral hypothalamic area to integrate metabolic signals, adaptive behaviour and physiological responses.

Regulation of monocyte functional heterogeneity by miR-146a and Relb.

Monocytes serve as a central defense system against infection and injury but can also promote pathological inflammatory responses. Considering the evidence that monocytes exist in at least two subsets committed to divergent functions, we investigated whether distinct factors regulate the balance between monocyte subset responses in vivo. We identified a microRNA (miRNA), miR-146a, which is differentially regulated both in mouse (Ly-6C(hi)/Ly-6C(lo)) and human (CD14(hi)/CD14(lo)CD16(+)) monocyte subsets. The single miRNA controlled the amplitude of the Ly-6C(hi) monocyte response during inflammatory challenge whereas it did not affect Ly-6C(lo) cells. miR-146a-mediated regulation was cell-intrinsic and depended on Relb, a member of the noncanonical NF-κB/Rel family, which we identified as a direct miR-146a target. These observations not only provide mechanistic insights into the molecular events that regulate responses mediated by committed monocyte precursor populations but also identify targets for manipulating Ly-6C(hi) monocyte responses while sparing Ly-6Clo monocyte activity.