Expression of the alpha 1b-adrenergic receptor and G protein subunits in mammalian cell lines using the Semliki Forest virus expression system.

Semliki Forest virus (SFV) vectors have been efficiently used for rapid high level expression of several G protein-coupled receptors. Here we describe the use of SFV vectors to express the alpha 1b-adrenergic receptor (AR) alone or in the presence of the G protein alpha q and/or beta 2 and gamma 2 subunits. Infection of baby hamster kidney (BHK) cells with recombinant SFV-alpha 1b-AR particles resulted in high specific binding activity of the alpha 1b-AR (24 pmol receptor/mg protein). Time-course studies indicated that the highest level of receptor expression was obtained 30 hours post-infection. The stimulation of BHK cells, with epinephrine led to a 5-fold increase in inositol phosphate (IP) accumulation, confirming the functional coupling of the receptor to G protein-mediated activation of phospholipase C. The SFV expression system represents a rapid and reproducible system to study the pharmacological properties and interactions of G protein coupled receptors and of G protein subunits.

Substance use and depression. Comparative course in adolescents.

OBJECTIVE: To examine the relation between depression and substance use in adolescents and the concomitant courses of both disorders. METHODS: Four individual interviews were administered to 85 adolescent substance users aged 14-19 years (mean 17.1 years, SD 1.4) over a 3.5 year period using the Adolescent Drug Abuse Interview (ADAD) and the Beck Depression Inventory (BDI-13). RESULTS: No predictive effect was observed on one dimension over the other, but each dimension was predictive of its own course. A decrease in substance-use severity paralleled a decrease in depressive state. Similarly, stable substance-use rates, either at a low or a high level, tended to be associated with low or high levels of depression, respectively. However, an increase in substance use was not accompanied by an increase in depressive states. Moreover, depression varied greatly between adolescents, and according to gender and age. CONCLUSIONS: Depressive states and substance use in adolescents can vary considerably overtime, and are closely but rather synchronically related. Since most of the adolescents do not seek help for substance-related problems, substance use should be systematically assessed in adolescents presenting with a depressive state.

Rapid evolution of cancer/testis genes on the X chromosome.

BACKGROUND: Cancer/testis (CT) genes are normally expressed only in germ cells, but can be activated in the cancer state. This unusual property, together with the finding that many CT proteins elicit an antigenic response in cancer patients, has established a role for this class of genes as targets in immunotherapy regimes. Many families of CT genes have been identified in the human genome, but their biological function for the most part remains unclear. While it has been shown that some CT genes are under diversifying selection, this question has not been addressed before for the class as a whole. RESULTS: To shed more light on this interesting group of genes, we exploited the generation of a draft chimpanzee (Pan troglodytes) genomic sequence to examine CT genes in an organism that is closely related to human, and generated a high-quality, manually curated set of human:chimpanzee CT gene alignments. We find that the chimpanzee genome contains homologues to most of the human CT families, and that the genes are located on the same chromosome and at a similar copy number to those in human. Comparison of putative human:chimpanzee orthologues indicates that CT genes located on chromosome X are diverging faster and are undergoing stronger diversifying selection than those on the autosomes or than a set of control genes on either chromosome X or autosomes. CONCLUSION: Given their high level of diversifying selection, we suggest that CT genes are primarily responsible for the observed rapid evolution of protein-coding genes on the X chromosome.

Five-year evolution of drug prescribing in a university adult intensive care unit

Introduction: Drug prescription is difficult in ICUs as prescribers are many, drugs expensive and decisions complex. In our ICU, specialist clinicians (SC) are entitled to prescribe a list of specific drugs, negotiated with intensive care physicians (ICP). The objective of this investigation was to assess the 5-year evolution of quantity and costs of drug prescription in our adult ICU and identify the relative costs generated by ICP or SC. Methods: Quantities and costs of drugs delivered on a quarterly basis to the adult ICU of our hospital between 2004 and 2008 were extracted from the pharmacy database by ATC code, an international five-level classification system. Within each ATC first level, drugs with either high level of consumption, high costs or large variations in quantities and costs were singled out and split by type of prescriber, ICP or SC. Cost figures used were drug purchase prices by the hospital pharmacy. Results: Over the 5-year period, both quantities and costs of drugs increased, following a nonsteady, nonparallel pattern. Four ATC codes accounted for 80% of both quantities and costs, with ATC code B (blood and haematopoietic organs) amounting to 63% in quantities and 41% in costs, followed by ATC code J (systemic anti-infective, 20% of the costs), ATC code N (nervous system, 11% of the costs) and ATC code C (cardiovascular system, 8% of the costs). Prescription by SC amounted to 1% in drug quantities, but 19% in drug costs. The rate of increase in quantities and costs was seven times larger for ICP than for SC (Figure 1 overleaf ). Some peak values in costs and quantities were related to a very limited number of patients. Conclusions: A 5-year increase in quantities and costs of drug prescription in an ICU is a matter of concern. Rather unexpectedly, total costs and cost increases were generated mainly by ICP. A careful follow-up is necessary to try influencing this evolution through an institutional policy co-opted by all professional categories involved in the process.

Consuming sports: distinction, univorism and omnivorism

The question of the social usages of culture and its links with social groups continues to be a topic of discussion today. The aim of this article is to contribute to the debate by examining coherence in the choice of physical activities and sports. The study focuses on the upper social groups, questioning, from a macro-sociological standpoint, their possible omnivority and their dissonance of choice with regard to these activities. Based on a quantitative survey of the sports participated in by the French, the study shows that omnivority and massification of activities are major phenomena. However, the upper social groups in France remain distinguishable both in terms of their "high level of omnivority" and their choice of distinctive activities. Dissonance is another of their characteristics but to a lesser extent.

Polysomy 8 defines a clinico-cytogenetic entity representing a subset of myeloid hematologic malignancies associated with a poor prognosis: report on a cohort of 12 patients and review of 105 published cases.

Tetrasomy, pentasomy, and hexasomy 8 (polysomy 8) are relatively rare compared to trisomy 8. Here we report on a series of 12 patients with acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), or myeloproliferative disorder (MPD) associated with polysomy 8 as detected by conventional cytogenetics and fluorescence in situ hybridization (FISH). In an attempt to better characterize the clinical and hematological profile of this cytogenetic entity, our data were combined with those of 105 published patients. Tetrasomy 8 was the most common presentation of polysomy 8. In 60.7% of patients, polysomy 8 occurred as part of complex changes (16.2% with 11q23 rearrangements). No cryptic MLL rearrangements were found in cases in which polysomy 8 was the only karyotypic change. Our study demonstrates the existence of a polysomy 8 syndrome, which represents a subtype of AML, MDS, and MPD characterized by a high incidence of secondary diseases, myelomonocytic or monocytic involvement in AML and poor overall survival (6 months). Age significantly reduced median survival, but associated cytogenetic abnormalities did not modify it. Cytogenetic results further demonstrate an in vitro preferential growth of the cells with a high level of aneuploidy suggesting a selective advantage for polysomy 8 cells.

Chronic exercise preserves lean muscle mass in masters athletes.

Aging is commonly associated with a loss of muscle mass and strength, resulting in falls, functional decline, and the subjective feeling of weakness. Exercise modulates the morbidities of muscle aging. Most studies, however, have examined muscle-loss changes in sedentary aging adults. This leaves the question of whether the changes that are commonly associated with muscle aging reflect the true physiology of muscle aging or whether they reflect disuse atrophy. This study evaluated whether high levels of chronic exercise prevents the loss of lean muscle mass and strength experienced in sedentary aging adults. A cross-section of 40 high-level recreational athletes ("masters athletes") who were aged 40 to 81 years and trained 4 to 5 times per week underwent tests of health/activity, body composition, quadriceps peak torque (PT), and magnetic resonance imaging of bilateral quadriceps. Mid-thigh muscle area, quadriceps area (QA), subcutaneous adipose tissue, and intramuscular adipose tissue were quantified in magnetic resonance imaging using medical image processing, analysis, and visualization software. One-way analysis of variance was used to examine age group differences. Relationships were evaluated using Spearman correlations. Mid-thigh muscle area (P = 0.31) and lean mass (P = 0.15) did not increase with age and were significantly related to retention of mid-thigh muscle area (P < 0.0001). This occurred despite an increase in total body fat percentage (P = 0.003) with age. Mid-thigh muscle area (P = 0.12), QA (P = 0.17), and quadriceps PT did not decline with age. Specific strength (strength per QA) did not decline significantly with age (P = 0.06). As muscle area increased, PT increased significantly (P = 0.008). There was not a significant relationship between intramuscular adipose tissue (P = 0.71) or lean mass (P = 0.4) and PT. This study contradicts the common observation that muscle mass and strength decline as a function of aging alone. Instead, these declines may signal the effect of chronic disuse rather than muscle aging. Evaluation of masters athletes removes disuse as a confounding variable in the study of lower-extremity function and loss of lean muscle mass. This maintenance of muscle mass and strength may decrease or eliminate the falls, functional decline, and loss of independence that are commonly seen in aging adults.

Psychoses a l'adolescence, les neurosciences ameliorent-elles la prediction? [Adolescent psychosis, can neuroscience improve prediction?]

Developments in the field of neuroscience have created a high level of interest in the subject of adolescent psychosis, particularly in relation to prediction and prevention. As the medical practice of adolescent psychosis and its treatment is characterised by a heterogeneity which is both symptomatic and evolutive, the somewhat poor prognosis of chronic development justifies the research performed: apparent indicators of schizophrenic disorders on the one hand and specific endophenotypes on the other are becoming increasingly important. The significant progresses made on the human genome show that the genetic predetermination in current psychiatric pathologies is complex and subject to moderating effects and there is therefore significant potential for nature-nurture interactions (between the environment and the genes). The road to be followed in researching the phenotypic expression of a psychosis gene is long and winding and is susceptible to many external influences at various levels with different effects. Neurobiological, neurophysiological, neuropsychological and neuroanatomical studies help to identify endophenotypes, which allow researchers to create identifying "markers" along this winding road. The endophenotypes could make it possible to redefine the nosological categories and enhance understanding of the physiopathology of schizophrenia. In a predictive approach, large-scale retrospective and prospective studies make it possible to identify risk factors, which are compatible with the neurodevelopmental hypothesis of schizophrenia. However, the predictive value of such markers or risk indicators is not yet sufficiently developed to offer a reliable early-detection method or possible schizophrenia prevention measures. Nonetheless, new developments show promise against the background of a possible future nosographic revolution, based on a paradigm shift. It is perhaps on the basis of homogeneous endophenotypes in particular that we will be able to understand what protects against, or indeed can trigger, psychosis irrespective of the clinical expression or attempts to isolate the common genetic and biological bases according to homogeneous clinical characteristics, which have to date, proved unsuccessful

Novel testis germ cell-specific transcript of the CREB gene contains an alternatively spliced exon with multiple in-frame stop codons.

The gene encoding the cAMP-responsive transcription factor CREB consists of multiple small exons some of which undergo alternative RNA splicing. We describe the finding of a novel transcript of the CREB gene expressed at high levels in the germ cells of the rat testis. The transcript contains an alternatively spliced exon inserted within the sequence encoding the transcriptional transactivation domain of CREB and this exon contains multiple in-frame stop codons. Furthermore, the exon is conserved in both rat and human genes (75% nucleotide identity). Although the function(s) of this RNA or the truncated CREB protein predicted to result from the translation of this unusual transcript is unknown, the high level of expression in the testicular germ cells and remarkable conservation of sequences in rat and human suggests that it may have a unique biological function in these cells.

Frailty and use of health and community services by community-dwelling older men: the Concord Health and Ageing in Men Project.

BACKGROUND: frailty is a concept used to describe older people at high risk of adverse outcomes, including falls, functional decline, hospital or nursing home admission and death. The associations between frailty and use of specific health and community services have not been investigated. METHODS: the cross-sectional relationship between frailty and use of several health and community services in the last 12 months was investigated in 1,674 community-dwelling men aged 70 or older in the Concord Health and Ageing in Men study, a population-based study conducted in Sydney, Australia. Frailty was assessed using a modified version of the Cardiovascular Health Study criteria. RESULTS: overall, 158 (9.4%) subjects were frail, 679 (40.6%) were intermediate (pre-frail) and 837 (50.0%) were robust. Frailty was associated with use of health and community services in the last 12 months, including consulting a doctor, visiting or being visited by a nurse or a physiotherapist, using help with meals or household duties and spending at least one night in a hospital or nursing home. Frail men without disability in activities of daily living were twice more likely to have seen a doctor in the previous 2 weeks than robust men (adjusted odds ratio 2.04, 95% confidence interval 1.21-3.44), independent of age, comorbidity and socio-economic status. CONCLUSION: frailty is strongly associated with use of health and community services in community-dwelling older men. The high level of use of medical services suggests that doctors and nurses could play a key role in implementation of preventive interventions.

Highly inducible synthesis of heterologous proteins in epithelial cells carrying a glucocorticoid-responsive vector.

A glucocorticoid-responsive vector is described which allows for the highly inducible expression of complementary DNAs (cDNAs) in stably transfected mammalian cell lines. This vector, pLK-neo, composed of a variant mouse mammary tumor virus long terminal repeat promoter, containing a hormone regulatory element, a Geneticin resistance-encoding gene in a simian virus 40 transcription unit, and a polylinker insertion site for heterologous cDNAs, was used to express the polymeric immunoglobulin (poly-Ig) receptor and the thymocyte marker, Thy-1, in Madin-Darby canine kidney (MDCK) cells and in murine fibroblast L cells. A high level of poly-Ig receptor or Thy-1 mRNA accumulation was observed in MDCK cells in response to dexamethasone with a parallel ten- to 200-fold increase in protein synthesis depending on the recombinant protein and the transfected cell clone.

Quantitative real-time polymerase chain reaction for detection of adenovirus after T cell-replete hematopoietic cell transplantation: viral load as a marker for invasive disease.

BACKGROUND: The value of adenovirus plasma DNA detection as an indicator for adenovirus disease is unknown in the context of T cell-replete hematopoietic cell transplantation, of which adenovirus disease is an uncommon but serious complication. METHODS: Three groups of 62 T cell-replete hematopoietic cell transplant recipients were selected and tested for adenovirus in plasma by polymerase chain reaction. RESULTS: Adenovirus was detected in 21 (87.5%) of 24 patients with proven adenovirus disease (group 1), in 4 (21%) of 19 patients who shed adenovirus (group 2), and in 1 (10.5%) of 19 uninfected control patients. The maximum viral load was significantly higher in group 1 (median maximum viral load, 6.3x10(6) copies/mL; range, 0 to 1.0x10(9) copies/mL) than in group 2 (median maximum viral load, 0 copies/mL; range, 0 to 1.7x10(8) copies/mL; P<.001) and in group 3 (median maximum viral load, 0 copies/mL; range 0-40 copies/mL; P<.001). All patients in group 2 who developed adenoviremia had symptoms compatible with adenovirus disease (i.e., possible disease). A minimal plasma viral load of 10(3) copies/mL was detected in all patients with proven or possible disease. Adenoviremia was detectable at a median of 19.5 days (range, 8-48 days) and 24 days (range, 9-41 days) before death for patients with proven and possible adenovirus disease, respectively. CONCLUSION: Sustained or high-level adenoviremia appears to be a specific and sensitive indicator of adenovirus disease after T cell-replete hematopoietic cell transplantation. In the context of low prevalence of adenovirus disease, the use of polymerase chain reaction of plasma specimens to detect virus might be a valuable tool to identify and treat patients at risk for viral invasive disease.

Cdc42 explores the cell periphery for mate selection in fission yeast.

How cells polarize in response to external cues is a fundamental biological problem. For mating, yeast cells orient growth toward the source of a pheromone gradient produced by cells of the opposite mating type. Polarized growth depends on the small GTPase Cdc42, a central eukaryotic polarity regulator that controls signaling, cytoskeleton polarization, and vesicle trafficking. However, the mechanisms of polarity establishment and mate selection in complex cellular environments are poorly understood. Here we show that, in fission yeast, low-level pheromone signaling promotes a novel polarization state, where active Cdc42, its GEF Scd1, and scaffold Scd2 form colocalizing dynamic zones that sample the periphery of the cell. Two direct Cdc42 effectors--actin cables marked by myosin V Myo52 and the exocyst complex labeled by Sec6 and Sec8--also dynamically colocalize with active Cdc42. However, these cells do not grow due to a block in the exocytosis of cell wall synthases Bgs1 and Bgs4. High-level pheromone stabilizes active Cdc42 zones and promotes cell wall synthase exocytosis and polarized growth. However, in the absence of prior low-level pheromone signaling, exploration fails, and cells polarize growth at cell poles by default. Consequently, these cells show altered partner choice, mating preferentially with sister rather than nonsister cells. Thus, Cdc42 exploration serves to orient growth for partner selection. This process may also promote genetic diversification.

Conservation genetics and population monitoring of the Alpine Salamander (Salamandra Atra) :

SUMMARYIn the context of the biodiversity crisis, amphibians are experiencing the most severe worldwide decline of all vertebrates and are in urgent need of better management. Efficient conservation strategies rely on sound knowledge of the species biology and of the genetic and demographic processes that might impair their welfare. Nonetheless, these processes are poorly understood in amphibians. Delineating population boundaries remains consequently problematic for these species, while it is of critical importance to define adequate management units for conservation. In this study, our attention focused on the alpine salamander (Salamandra atra), a species that deserves much interest in terms of both conservation biology and evolution. This endemic alpine species shows peculiar life-history traits (viviparity, reduced activity period, slow maturation) and has a slow population turnover, which might be problematic for its persistence in a changing environment. Due to its elusive behaviour (individuals spend most of their time underground and are unavailable for sampling), dynamic processes of gene and individuals were poorly understood for that species. Consequently, its conservation status could hardly be reliably assessed. Similarly the fire salamander (Salamandra salamandra) also poses special challenges for conservation, as no clear demarcation of geographical populations exists and dispersal patterns are poorly known. Through a phylogeographic analysis, we first studied the evolutionary history of the alpine salamander to better document the distribution of the genetic diversity along its geographical range. This study highlighted the presence of multiple divergent lineages in Italy together with a clear genetic divergence between populations from Northern and Dinaric Alps. These signs of cryptic genetic differentiation, which are not accounted for by the current taxonomy of the species, should not be neglected for further definition of conservation units. In addition, our data supported glacial survival of the species in northern peripheral glacial réfugia and nunataks, a pattern rarely documented for long-lived species. Then, we evaluated the level of gene flow between populations at the local scale and tested for asymmetries in male versus female dispersal using both field-based (mark-recapture) and genetic approaches. This study revealed high level of gene flow between populations, which stems mainly from male dispersal. This corroborated the idea that salamanders are much better dispersers than hitherto thought and provided a well- supported example of male-biased dispersal in amphibians. In a third step, based on a mark- recapture survey, we addressed the problem of sampling unavailability in alpine salamanders and evaluated its impact on two monitoring methods. We showed that about three quarters of individuals were unavailable for sampling during sampling sessions, a proportion that can vary with climatic conditions. If not taken into account, these complexities would result in false assumptions on population trends and misdirect conservation efforts. Finally, regarding the daunting task of delineating management units, our attention was drawn on the fire salamander. We conducted a local population genetic study that revealed high levels of gene flow among sampling sites. Management units for this species should consequently be large. Interestingly, despite the presence of several landscape features often reported to act as barriers, genetic breaks occurred at unexpected places. This suggests that landscape features may rather have idiosyncratic effects on population structure. In conclusion, this work brought new insights on both genetic and demographic processes occurring in salamanders. The results suggest that some biological paradigms should be taken with caution when particular species are in focus. Species- specific studies remain thus fundamental for a better understanding of species evolution and conservation, particularly in the context of current global changes.RESUMEDans le contexte de la crise de la biodiversité actuelle, les amphibiens subissent le déclin le plus important de tous les vertébrés et ont urgemment besoin d'une meilleure protection. L'établissement de stratégies de conservation efficaces repose sur des connaissances solides de la biologie des espèces et des processus génétiques et démographiques pouvant menacer leur survie. Ces processus sont néanmoins encore peu étudiés chez les amphibiens.Dans cette étude, notre attention s'est portée sur la salamandre noire (Salamandra atra), une espèce endémique des Alpes dont les traits d'histoire de vie atypiques (viviparité, phase d'activité réduite, lent turnover des populations) pourraient la rendre très vulnérable face aux changements environnementaux. Par ailleurs, en raison de son comportement cryptique (les individus passent la plupart de leur temps sous terre) la dynamique des gènes et des individus est mal comprise chez cette espèce. Il est donc difficile d'évaluer son statut de conservation de manière fiable. La salamandre tachetée {Salamandra salamandra), pour qui il n'existe aucune démarcation géographique apparente des populations, pose également des problèmes en termes de gestion. Dans un premier temps, nous avons étudié l'histoire évolutive de la salamandre noire afin de mieux décrire la distribution de sa diversité génétique au sein de son aire géographique. Cela a permis de mettre en évidence la présence de multiples lignées en Italie, ainsi qu'une nette divergence entre les populations du nord des Alpes et des Alpes dinariques. Ces résultats seront à prendre en compte lorsqu'il s'agira de définir des unités de conservation pour cette espèce. D'autre part, nos données soutiennent l'hypothèse d'une survie glaciaire dans des refuges nordiques périglaciaires ou dans des nunataks, fait rarement documenté pour une espèce longévive. Nous avons ensuite évalué la différentiation génétique des populations à l'échelle locale, ce qui a révélé d'important flux de gènes, ainsi qu'une asymétrie de dispersion en faveur des mâles. Ces résultats corroborent l'idée que les amphibiens dispersent mieux que ce que l'on pensait, et fournissent un exemple robuste de dispersion biaisée en faveur des mâles chez les amphibiens. Nous avons ensuite abordé le problème de Γ inaccessibilité des individus à la capture. Nous avons montré qu'environ trois quarts des individus sont inaccessibles lors des échantillonnages, une proportion qui peut varier en fonction des conditions climatiques. Ignoré, ce processus pourrait entraîner une mauvaise interprétation des fluctuations de populations ainsi qu'une mauvaise allocation des efforts de conservation. Concernant la définition d'unités de gestion pour la salamandre tachetée, nous avons pu mettre en évidence un flux de gènes important entre les sites échantillonnés. Les unités de gestion pour cette espèce devraient donc être étendues. Etonnamment, malgré la présence de nombreuses barrières potentielles au flux de gènes, les démarcations génétiques sont apparues à des endroits inattendus. En conclusion, ce travail a apporté une meilleure compréhension des processus génétiques et démographiques en action chez les salamandres. Les résultats suggèrent que certains paradigmes biologiques devraient être considérés avec précaution quand il s'agit de les appliquer à des espèces particulières. Les études spécifiques demeurent donc fondamentales pour une meilleure compréhension de l'évolution des espèces et leur conservation, tout particulièrement dans le contexte des changements globaux actuels.

Experimental Uveitis can be Maintained in Rabbits for a Period of Six Weeks After a Safe Sensitization Method.

Abstract Purpose: New treatments against long-lasting uveitis need to be tested. Our aim was to develop a six-week model of uveitis in rabbits. Methods: Rabbits were presensitized with an s.c. injection of Mycobacterium tuberculosis H37RA emulsified with TiterMax® Gold adjuvant. Uveitis was induced at day 28 and 50, by intravitreal challenges of antigen suspension. Ocular inflammation was assessed till euthanasia at day 71 after s.c. injection of M. tuberculosis H37RA by: (a) the number of inflammatory cells in aqueous humor (AH); (b) the protein concentration in AH; (c) the clinical score (mean of conjunctival hyperaemia, conjunctival chemosis, oedema and secretion); (d) the microscopical score (mean presence of fibrin and synechiae, aqueous cell density and aqueous flare grade, as scored by slit lamp). Results: At the sites of presensitization injection, rabbits presented flat nodules which progressively vanished. The first challenge induced a significant increase in the four parameters (p < 0.05 the Wilcoxon/Kruskal-Wallis test). The AH contained 764 ± 82 cells/µl and 32 ± 0.77 mg protein/ml. During the following days, inflammatory parameters decreased slightly. The second intravitreal challenge increased inflammation (3564 ± 228 cells/µl AH and 31 ± 1 mg protein/ml), which remained at a high level for a longer period of time. Conclusion: We developed a model of long-term uveitis, which could be maintained in rabbits for at least six weeks. Such a model could be used to test the efficacy of either new drugs or various drug delivery systems intended to deliver active agents during a few months.