Expression cloning of the pancreatic beta cell receptor for the gluco-incretin hormone glucagon-like peptide 1.

Glucagon-like peptide 1 (GLP-1) is a hormone derived from the preproglucagon molecule and is secreted by intestinal L cells. It is the most potent stimulator of glucose-induced insulin secretion and also suppresses in vivo acid secretion by gastric glands. A cDNA for the GLP-1 receptor was isolated by transient expression of a rat pancreatic islet cDNA library into COS cells; this was followed by binding of radiolabeled GLP-1 and screening by photographic emulsion autoradiography. The receptor transfected into COS cells binds GLP-1 with high affinity and is coupled to activation of adenylate cyclase. The receptor binds specifically GLP-1 and does not bind peptides of related structure and similar function, such as glucagon, gastric inhibitory peptide, vasoactive intestinal peptide, or secretin. The receptor is 463 amino acids long and contains seven transmembrane domains. Sequence homology is found only with the receptors for secretin, calcitonin, and parathyroid hormone, which form a newly characterized family of G-coupled receptors.

Glucose competence of the hepatoportal vein sensor requires the presence of an activated glucagon-like peptide-1 receptor.

Activation of the hepatoportal glucose sensors by portal glucose infusion leads to increased glucose clearance and induction of hypoglycemia. Here, we investigated whether glucagon-like peptide-1 (GLP-1) could modulate the activity of these sensors. Mice were therefore infused with saline (S-mice) or glucose (P-mice) through the portal vein at a rate of 25 mg/kg. min. In P-mice, glucose clearance increased to 67.5 +/- 3.7 mg/kg. min as compared with 24.1 +/- 1.5 mg/kg. min in S-mice, and glycemia decreased from 5.0 +/- 0.1 to 3.3 +/- 0.1 mmol/l at the end of the 3-h infusion period. Coinfusion of GLP-1 with glucose into the portal vein at a rate of 5 pmol/kg. min (P-GLP-1 mice) did not increase the glucose clearance rate (57.4 +/- 5.0 ml/kg. min) and hypoglycemia (3.8 +/- 0.1 mmol/l) observed in P-mice. In contrast, coinfusion of glucose and the GLP-1 receptor antagonist exendin-(9-39) into the portal vein at a rate of 0.5 pmol/kg. min (P-Ex mice) reduced glucose clearance to 36.1 +/- 2.6 ml/kg. min and transiently increased glycemia to 9.2 +/- 0.3 mmol/l at 60 min of infusion before it returned to the fasting level (5.6 +/- 0.3 mmol/l) at 3 h. When glucose and exendin-(9-39) were infused through the portal and femoral veins, respectively, glucose clearance increased to 70.0 +/- 4.6 ml/kg. min and glycemia decreased to 3.1 +/- 0.1 mmol/l, indicating that exendin-(9-39) has an effect only when infused into the portal vein. Finally, portal vein infusion of glucose in GLP-1 receptor(-/-) mice failed to increase the glucose clearance rate (26.7 +/- 2.9 ml/kg. min). Glycemia increased to 8.5 +/- 0.5 mmol/l at 60 min and remained elevated until the end of the glucose infusion (8.2 +/- 0.4 mmol/l). Together, our data show that the GLP-1 receptor is part of the hepatoportal glucose sensor and that basal fasting levels of GLP-1 sufficiently activate the receptor to confer maximum glucose competence to the sensor. These data demonstrate an important extrapancreatic effect of GLP-1 in the control of glucose homeostasis.

Girls like pink: explaining sex-typed occupational aspirations amongst young children

There is a high degree of sex-typing in young children's occupational aspirations and this has consequences for subsequent occupational segregation. Sociologists typically attribute early sex-differences in occupational preferences to gender socialization. Yet we still know surprisingly little about the mechanisms involved in the intergenerational transmission of sex-typical preferences and there is considerable theoretical controversy regarding the role of individual agency in the process of preference formation. This study analyzes the determinants of sex-typed occupational aspirations amongst British children aged between 11 and 15. We specify different mechanisms involved in the transmission of sex-typical preferences and propose an innovative definition of individual agency that is anchored in observable psychological traits linked to self-direction. This allows us to perform a simultaneous test of socialization and agency predictors of occupational sex-typing. We find that parental influences on occupational preferences operate mainly through three distinctive channels: 1) the effect that parental socio-economic resources have on the scope of children's occupational aspirations, 2) children's direct imitation of parental occupations, and 3) children's learning of sex-typed roles via the observation of parental behavior. We also find a strong net effect of children's own psychological predispositions -self-esteem in particular- on the incidence of sex-typical occupational preferences. Yet large differences in the occupational aspirations of girls and boys remain unexplained.

Cathepsin B-like and cell death in the unicellular human pathogen Leishmania.

In several studies reporting cell death (CD) in lower eukaryotes and in the human protozoan parasite Leishmania, proteolytic activity was revealed using pan-caspase substrates or inhibitors such as carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-FMK). However, most of the lower eukaryotes do not encode caspase(s) but MCA, which differs from caspase(s) in its substrate specificity and cannot be accountable for the recognition of Z-VAD-FMK. In the present study, we were interested in identifying which enzyme was capturing the Z-VAD substrate. We show that heat shock (HS) induces Leishmania CD and leads to the intracellular binding of Z-VAD-FMK. We excluded binding and inhibition of Z-VAD-FMK to Leishmania major metacaspase (LmjMCA), and identified cysteine proteinase C (LmjCPC), a cathepsin B-like (CPC) enzyme, as the Z-VAD-FMK binding enzyme. We confirmed the specific interaction of Z-VAD-FMK with CPC by showing that Z-VAD binding is absent in a Leishmania mexicana strain in which the cpc gene was deleted. We also show that parasites exposed to various stress conditions release CPC into a soluble fraction. Finally, we confirmed the role of CPC in Leishmania CD by showing that, when exposed to the oxidizing agent hydrogen peroxide (H(2)O(2)), cpc knockout parasites survived better than wild-type parasites (WT). In conclusion, this study identified CPC as the substrate of Z-VAD-FMK in Leishmania and as a potential additional executioner protease in the CD cascade of Leishmania and possibly in other lower eukaryotes.

Recombinant human insulin-like growth factor I (rhIGF-I) for the treatment of amyotrophic lateral sclerosis/motor neuron disease.

BACKGROUND: Recombinant human insulin-like growth factor I (rhIGF-I) is a possible disease modifying therapy for amyotrophic lateral sclerosis (ALS, which is also known as motor neuron disease (MND)). OBJECTIVES: To examine the efficacy of rhIGF-I in affecting disease progression, impact on measures of functional health status, prolonging survival and delaying the use of surrogates (tracheostomy and mechanical ventilation) to sustain survival in ALS. Occurrence of adverse events was also reviewed. SEARCH METHODS: We searched the Cochrane Neuromuscular Disease Group Specialized Register (21 November 2011), CENTRAL (2011, Issue 4), MEDLINE (January 1966 to November 2011) and EMBASE (January 1980 to November 2011) and sought information from the authors of randomised clinical trials and manufacturers of rhIGF-I. SELECTION CRITERIA: We considered all randomised controlled clinical trials involving rhIGF-I treatment of adults with definite or probable ALS according to the El Escorial Criteria. The primary outcome measure was change in Appel Amyotrophic Lateral Sclerosis Rating Scale (AALSRS) total score after nine months of treatment and secondary outcome measures were change in AALSRS at 1, 2, 3, 4, 5, 6, 7, 8, 9 months, change in quality of life (Sickness Impact Profile scale), survival and adverse events. DATA COLLECTION AND ANALYSIS: Each author independently graded the risk of bias in the included studies. The lead author extracted data and the other authors checked them. We generated some missing data by making ruler measurements of data in published graphs. We collected data about adverse events from the included trials. MAIN RESULTS: We identified three randomised controlled trials (RCTs) of rhIGF-I, involving 779 participants, for inclusion in the analysis. In a European trial (183 participants) the mean difference (MD) in change in AALSRS total score after nine months was -3.30 (95% confidence interval (CI) -8.68 to 2.08). In a North American trial (266 participants), the MD after nine months was -6.00 (95% CI -10.99 to -1.01). The combined analysis from both RCTs showed a MD after nine months of -4.75 (95% CI -8.41 to -1.09), a significant difference in favour of the treated group. The secondary outcome measures showed non-significant trends favouring rhIGF-I. There was an increased risk of injection site reactions with rhIGF-I (risk ratio 1.26, 95% CI 1.04 to 1.54). . A second North American trial (330 participants) used a novel primary end point involving manual muscle strength testing. No differences were demonstrated between the treated and placebo groups in this study. All three trials were at high risk of bias. AUTHORS' CONCLUSIONS: Meta-analysis revealed a significant difference in favour of rhIGF-I treatment; however, the quality of the evidence from the two included trials was low. A third study showed no difference between treatment and placebo. There is no evidence for increase in survival with IGF1. All three included trials were at high risk of bias.

Ubiquitin- and ubiquitin-like proteins-conjugating enzymes (E2s) in breast cancer.

Breast cancer is the most common malignancy in women and a significant cause of morbidity and mortality. Sub-types of breast cancer defined by the expression of steroid hormones and Her2/Neu oncogene have distinct prognosis and undergo different therapies. Besides differing in their phenotype, sub-types of breast cancer display various molecular lesions that participate in their pathogenesis. BRCA1 is one of the common hereditary cancer predisposition genes and encodes for an ubiquitin ligase. Ubiquitin ligases or E3 enzymes participate together with ubiquitin activating enzyme and ubiquitin conjugating enzymes in the attachment of ubiquitin (ubiquitination) in target proteins. Ubiquitination is a post-translational modification regulating multiple cell functions. It also plays important roles in carcinogenesis in general and in breast carcinogenesis in particular. Ubiquitin conjugating enzymes are a central component of the ubiquitination machinery and are often perturbed in breast cancer. This paper will discuss ubiquitin and ubiquitin-like proteins conjugating enzymes participating in breast cancer pathogenesis, their relationships with other proteins of the ubiquitination machinery and their role in phenotype of breast cancer sub-types.

Chemical and carbon isotopic evolution of hydrocarbons during prograde metamorphism from 100°C to 550°C: Case study in the Liassic black shale formation of Central Swiss Alps

Hydrocarbon distributions and stable isotope ratios of carbonates (delta(13)C(car), delta(18)O(car)), kerogen (delta(13)C(ker)), extractable organic matter (delta(13)C(EOM)) and individual hydrocarbons of Liassic black shale samples from a prograde metamorphic sequence in the Swiss Alps were used to identify the major organic reactions with increasing metamorphic grade. The studied samples range from the diagenetic zone (< 100 degrees C) to amphibolite facies (similar to 550 degrees C). The samples within the diagenetic zones (< 100 and 150 degrees C) are characterized by the dominance of C-< 20 n-alkanes, suggesting an origin related with marine and/or bacterial inputs. The metamorphic samples (200 to 550 degrees C) have distributions significantly dominated by C-12 and C-13 n-alkanes, C-14, C-16 and C-18 n-alkylcyclopentanes and to a lesser extend C-15, C-17 and C-21 n-alkylcyclohexanes. The progressive C-13-enrichment (up to 3.9 parts per thousand) with metamorphism of the C-> 17 n-alkanes suggests the occurrence of cracking reactions of high molecular weight compounds. The isotopically heavier (up to 5.6 parts per thousand) C-< 17 n-alkanes in metamorphic samples are likely originated by thermal degradation of long-chain homologous with preferential release of isotopically light C-1 and C-2 radicals. The dominance of specific even C-number n-alkylcyclopentanes suggests an origin related to direct cyclization mechanism (without decarboxylation step) of algal or bacterial fatty acids occurring in reducing aqueous metamorphic fluid conditions. The regular increase of the concentrations of n-alkylcycloalkanes vs. C-> 13 n-alkanes with metamorphism suggests progressive thermal release of kerogen-linked fatty acid precursors and degradation of n-alkanes. Changes of the steroid and terpenoid distributions are clearly related to increasing metamorphic temperatures. The absence of 18 alpha(H)-22,29,30-trisnorneohopane (Ts), the occurrence of 17 beta(H)-trisnorhopane, 17 beta(H), 21 alpha(H)-hopanes in the C-29 to C-31 range and 5 alpha(H),14 alpha(H),17 alpha(H)-20R C-27, C-29 steranes in the low diagenetic samples (< 100 degrees C) are characteristic of immature bitumens. The higher thermal stress within the upper diagenetic zone (150 degrees C) is marked by the presence of Ts, the disappearance of 17 beta(H)-trisnorhopane and thermodynamic equilibrium of the 22S/(22S + 22R) homohopane ratios. The increase of the alpha alpha alpha-sterane 20S/(20S + 20R) and 20R beta beta/(beta beta + alpha alpha) ratios (from 0.0 to 0.55 and from 0.0 to 0.40, respectively) in the upper diagenetic zone indicates the occurrence of isomerization reactions already at < 150 degrees C. However, the isomerization at C-20 (R -> S) reaches thermodynamic equilibrium values already at the upper diagenesis (similar to 150 degrees C) whereas the epimerisation at C-14 and C-17 (alpha alpha ->beta beta) arrives to constant values in the lower anchizone (similar to 200 degrees C). The ratios Ts vs. 17 alpha(H)-22,29,30-trisnorneohopane [(Ts/(Ts + Tm)] and 18 alpha(H)-30-norneohopane (C29Ts) vs. 17 alpha(H),21 beta(H)-30-norhopane [C29Ts/(C29Ts + C-29)] increase until the medium anchizone (200 to 250 degrees C) from 0.0 to 0.96 and from 0.0 to 0.44, respectively. An opposite trend owards lower values is observed in the higher metamorphic samples. The occurrence of specific hydrocarbons (e.g., n-alkylcyclopentanes, cadalene, hydrogenated aromatic compounds) in metamorphic samples points to kerogen degradation reactions most probably occurring in the presence of water and under reducing conditions. The changes of hydrocarbon distributions and carbon isotopic compositions of n-alkanes related to metamorphism suggest that the organic geochemistry may help to evaluate the lowest grades of prograde metamorphism. Copyright (c) 2005 Elsevier Ltd.

Human Plasma-derived Polymeric IgA and IgM Antibodies Associate with Secretory Component to Yield Biologically Active Secretory-like Antibodies.

Immunotherapy with monoclonal and polyclonal immunoglobulin is successfully applied to improve many clinical conditions, including infection, autoimmune diseases, or immunodeficiency. Most immunoglobulin products, recombinant or plasma-derived, are based on IgG antibodies, whereas to date, the use of IgA for therapeutic application has remained anecdotal. In particular, purification or production of large quantities of secretory IgA (SIgA) for potential mucosal application has not been achieved. In this work, we sought to investigate whether polymeric IgA (pIgA) recovered from human plasma is able to associate with secretory component (SC) to generate SIgA-like molecules. We found that ∼15% of plasma pIgA carried J chain and displayed selective SC binding capacity either in a mixture with monomeric IgA (mIgA) or after purification. The recombinant SC associated covalently in a 1:1 stoichiometry with pIgA and with similar efficacy as colostrum-derived SC. In comparison with pIgA, the association with SC delayed degradation of SIgA by intestinal proteases. Similar results were obtained with plasma-derived IgM. In vitro, plasma-derived IgA and SIgA neutralized Shigella flexneri used as a model pathogen, resulting in a delay of bacteria-induced damage targeted to polarized Caco-2 cell monolayers. The sum of these novel data demonstrates that association of plasma-derived IgA or IgM with recombinant/colostrum-derived SC is feasible and yields SIgA- and SIgM-like molecules with similar biochemical and functional characteristics as mucosa-derived immunoglobulins.

Characterization of human fibroleukin, a fibrinogen-like protein secreted by T lymphocytes.

We have recently cloned the human homologue of the murine pT49 cDNA (hpT49h), a transcript encoding a protein homologous to the beta- and gamma-chains of fibrinogen. Here, we report the identification of the hpT49h gene product using mAbs generated against a peptide corresponding to the carboxyl-terminal end of the deduced protein and a recombinant protein fragment expressed in Escherichia coli. mAbs 23A6, 7B12, and 3F4 specifically recognized a protein of 70 kDa in reducing SDS-PAGE in the culture supernatant of 293T cells transiently transfected with the full length hpT49h cDNA and freshly isolated PBMC. Under nonreducing conditions, the material migrated with a molecular mass of 250 to 300 kDa, indicating that the 70-kDa protein forms a disulfide bonded complex. Because of its homology with fibrinogen, we have termed this protein fibroleukin. Fibroleukin is spontaneously secreted in vitro by freshly isolated CD4+ and CD8+ T lymphocytes. RT-PCR analysis revealed preferential expression of fibroleukin mRNA in memory T lymphocytes (CD3+/CD45R0+) compared with naive T lymphocytes (CD3+/CD45RA+). Fibroleukin production by PBMC was rapidly lost in culture. Production could be partially maintained in the presence of IFN-gamma, while T lymphocyte activation had no effect. To demonstrate fibroleukin production in vivo, we analyzed colon mucosa by immunohistology. Fibroleukin staining was detected in the extracellular matrix of the T lymphocyte-rich upper portion of the lamina propria mucosa. While the exact function of fibroleukin remains to be defined, these data suggest that fibroleukin may play a role in physiologic lymphocyte functions at mucosal sites.

Direct methods for detecting picorna-like virus from dead and alive Triatomine insects

In this work we report four different destructive and non-destructive methods for detecting picorna-like virus particles in triatomines. The methods are based on direct observation under transmission electron microscope and they consist of four ways to prepare samples of presumable infected material. The samples are prepared processing dead or alive insect parts, or even dry or fresh insect feces. The methods can be used as analytical or preparative techniques, for quantifying virus infection and checking virus integrity as well. In this work the four methods are applied in order to detect Triatoma virus (TrV) particles in T. infestans colonies.

Borrelia-like spirochetes recovered from ticks and small mammals collected in the Atlantic Forest Reserve, Cotia county, State of São Paulo, Brazil

Forty-four marsupials, 77 rodents and 161 ticks were captured in an Atlantic Forest Reserve in Cotia county, State of São Paulo, where human cases of Lyme disease (LD) simile were reported. Twenty-one borrelia-like spirochete isolates were recovered from the mammals' blood and rodent livers or spleens, and triturated ticks inoculated into BSK II medium. Our results suggest that the reservoirs and ticks collected may harbor borrelia-like spirochetes, some of which have an antigenic similarity with the unknown causative agent of LD simile in Brazil, and/or with North American Borrelia burgdorferi s.s.

Like milk or wine: Does firm performance improve with age?

Our empirical literature review shows that little is known about how firm performance changes with age, presumably because of the paucity of data on firm age. For Spanish manufacturing firms, we analyse the firm performance related to firm age between 1998 and 2006. We find evidence that firms improve with age, because ageing firms are observed to have steadily increasing levels of productivity, higher profits, larger size, lower debt ratios, and higher equity ratios. Furthermore, older firms are better able to convert sales growth into subsequent growth of profits and productivity. On the other hand, we also found evidence that firm performance deteriorates with age. Older firms have lower expected growth rates of sales, profits and productivity, they have lower profitability levels (when other variables such as size are controlled for), and also that they appear to be less capable to convert employment growth into growth of sales, profits and productivity. Keywords: firm age, firm growth, LAD, financial structure, vector autoregression JEL CODES: L25, L20

Assessment and analysis of mechanical allodynia-like behavior induced by spared nerve injury (SNI) in the mouse

Rrésumé: La première description dans une publication médicale des douleurs neuropathiques remonte à 1872, le Dr S.W. Mitchell les résumant ainsi [...]" la causalgie est la plus terrible des tortures qu'une lésion nerveuse puisse entraîner "[...]. Par définition, la douleur neuropathique est une douleur chronique faisant suite à une lésion ou dysfonction du système nerveux. Malgré les progrès faits dans la compréhension de ce syndrome, le détail des mécanismes impliqués nous échappe encore et son traitement reste insuffisant car moins de 50% des patients sont soulagés par les thérapies actuelles. Différents modèles expérimentaux ont été élaborés chez l'animal de laboratoire, en particulier des modèles de lésion de nerfs périphériques chez le rat, permettant des investigations tant moléculaires que fonctionnelles des mécanismes impliqués dans le développement de ces douleurs. En revanche, peu de modèles existent chez la souris, alors que cet animal, grâce à la transgénèse, est très fréquemment utilisé pour l'approche fonctionnelle ciblée sur un gène. Dans l'étude présentée ici, nous avons évalué chez la souris C57BL/6 l'adaptation d'un modèle neuropathique, proposé une nouvelle modalité de mesure de la sensibilité douloureuse adaptée à la souris et défini une méthode d'analyse performante des résultats. Ce modèle, dit de lésion avec épargne nerveuse (spared Werve injury, SNI), consiste en la lésion de deux des trois branches du nerf sciatique, soit les nerfs peronier commun et tibial. La troisième branche, le nerf sural est laissé intact et c'est dans le territoire cutané de ce dernier que la sensibilité douloureuse à des stimulations mécaniques est enregistrée. Des filaments calibrés de force croissante sont appliqués sur la surface de la patte impliquée et la fréquence relative de retrait de la patte a été modélisée mathématiquement et analysée par un modèle statistique intégrant tous les paramètres de l'expérience (mixed-effects model). Des variantes chirurgicales lésant séquentiellement les trois branches du nerf sciatique ainsi que la réponse en fonction du sexe de l'animal ont également été évaluées. La lésion SNI entraîne une hypersensibilité mécanique marquée comparativement aux souris avec chirurgie contrôle; cet effet est constant entre les animaux et persiste durant les quatre semaines de l'étude. De subtiles différences entre les variables, y compris une divergence de sensibilité mécanique entre les sexes, ont été démontrées. La nécessité de léser le nerf tibial pour le développement des symptômes a également été documentée par notre méthode d'évaluation et d'analyse. En conclusion, nous avons validé le modèle SNI chez la souris par l'apparition d'un symptôme reproductible et apparenté à l'allodynie mécanique décrite par les patients souffrant de douleurs neuropathiques. Nous avons développé des méthodes d'enregistrement et d'analyse de la sensibilité douloureuse sensibles qui permettent la mise en évidence de facteurs intrinsèques et extrinsèques de variation de la réponse. Le modèle SNI utilisé chez des souris génétiquement modifiées, de par sa précision et reproductibilité, pourra permettre la discrimination de facteurs génétiques et épigénétiques contribuant au développement et à la persistance de douleurs neuropathiques.

Bone anabolic potential of soy isoflavones and plant extracts and genomic changes during differentiation of hPOB-tert osteoblast-like cells

Summary For the nutritional management of bone health and the prevention of osteoporosis it is important to identify nutrients that positively influence the bone remodeling process at the cellular level. Soy isoflavones show promising osteoprotective effects in animals and humans but their mechanism of action in bone cells is yet poorly understood. Firstly, soy tissue cultures were characterized as a new and optimized source of isoflavones. A large variability in the isoflavone content was observed and high-producing strains (46.3 mg/g dry wt isoflavones) were identified. In the Ishikawa cells bioassay, the estrogenicity of isoflavones was confirmed to be 1000 to 10000 less than 17Mestradiol and that of the malonyl forms was shown for the first time (EC50 of 350 nM and 1880 nM for malonylgenistin and malonyldaidzin, respectively). The estrogenic activity of soya tissue culture extracts correlated to their isoflavone content. Secondly, the effects of phytonutrients on BMP-2 gene expression and on the mevalonate synthesis pathway, as key mediators of bone formation, were investigated. Dietary achievable concentrations of genistein and daidzein (10vM), and statins (4xM) but not 17M estradiol (10nM), induced BMP-2 gene expression (by up to 3-fold) and inhibited the cholesterol biosynthetic pathway (by up to 50%) in the human osteoblastic cell line hP0B¬tert. In addition, several plant extracts (Cyperus rotundus, Lindera benzoin and Cnidium monnieri) induced BMP-2 gene expression but this induction was not restricted to the inhibition of the cholesterol synthesis pathway neither to the estrogenicity. Finally, the gene expression profiles during hP0B-tert differentiation induced by vitamin D and dexamethasone were analyzed with the Affymetrix human GeneChip. 1665 different genes and 98 ESTs were significantly regulated. The expression profiles of bone-related genes was largely in agreement with previously documented patterns, supporting the physiological relevance of the genomic results and the hP0B-tert cell line as a valid model of human osteoblast differentiation. The expression of alternative differentiation markers during the osteogenic treatment of hP0B-tert cells indicated that the adipocyte and myoblast differentiation pathways were repressed, confirming that these culture conditions allowed only osteoblast differentiation. The gene ontology analysis identified further sub-groups of genes that may be involved in the bone formation process. Aims of the thesis In order to define new strategies for the nutritional management of bone health and for the prevention of osteoporosis the major goal of the present work was to investigate the potential of phytonutrients to positively modulate the bone formation process at the cellular level and, in particular: 1.To select and optimise alternative plant sources containing high levels of isoflavones with estrogenic activity (Chapter 3). 2.To compare the effects of statins and phytonutrients on BMP-2 gene expression and on the mevalonate synthesis pathway and to select new plant extracts with a bone anabolic potential (Chapter 4). 3.To further characterize the new human periosteal cell line, hP0B-tert, as a bone- formation model, by elucidating its gene expression profile during differentiation induced by vitamin D and dexamethasone (Chapter 5).