A new selective peroxisome proliferator-activated receptor gamma antagonist with antiobesity and antidiabetic activity.

Peroxisome proliferator-activated receptor gamma (PPAR-gamma) plays a key role in adipocyte differentiation and insulin sensitivity. Its synthetic ligands, the thiazolidinediones (TZD), are used as insulin sensitizers in the treatment of type 2 diabetes. These compounds induce both adipocyte differentiation in cell culture models and promote weight gain in rodents and humans. Here, we report on the identification of a new synthetic PPARgamma antagonist, the phosphonophosphate SR-202, which inhibits both TZD-stimulated recruitment of the coactivator steroid receptor coactivator-1 and TZD-induced transcriptional activity of the receptor. In cell culture, SR-202 efficiently antagonizes hormone- and TZD-induced adipocyte differentiation. In vivo, decreasing PPARgamma activity, either by treatment with SR-202 or by invalidation of one allele of the PPARgamma gene, leads to a reduction of both high fat diet-induced adipocyte hypertrophy and insulin resistance. These effects are accompanied by a smaller size of the adipocytes and a reduction of TNFalpha and leptin secretion. Treatment with SR-202 also dramatically improves insulin sensitivity in the diabetic ob/ob mice. Thus, although we cannot exclude that its actions involve additional signaling mechanisms, SR-202 represents a new selective PPARgamma antagonist that is effective both in vitro and in vivo. Because it yields both antiobesity and antidiabetic effects, SR-202 may be a lead for new compounds to be used in the treatment of obesity and type 2 diabetes.

Consequences of modified lipid and glucose metabolism on peripheral nervous system structure and function

284 million people worldwide suffered from type 2 diabetes mellitus (T2DM) in 2010, which will, in approximately half of them, lead to the development of diabetic peripheral neuropathy (DPN). Although DPN is the most common complication of diabetes mellitus and the leading cause of non-traumatic amputations its pathophysiology is still poorly understood. To get more insight into the molecular mechanism underlying DPN in T2DM, I used a rodent model of T2DM, the db/db mice.¦ln vivo electrophysiological recordings of diabetic animals indicated that in addition to reduced nerve conduction velocity db/db mice also present increased nerve excitability. Further ex vivo evaluation of the electrophysiological properties of db/db nerves clearly established a presence of the peripheral nerve hyperexcitability (PNH) phenotype in diabetic animals. Using pharmacological inhibitors we demonstrated that PNH is mostly mediated by the decreased activity of Kv1 channels. ln agreement with these data 1 observed that the diabetic condition led to a reduced presence of the Kv1.2 subunits in juxtaparanodal regions of db/db peripheral nerves whereas its mANA and protein expression levels were not affected. Lmportantly, I confirmed a loss of juxtaparanodal Kv1.2 subunits in nerve biopsies from type 2 diabetic patients. Together these observations indicate that the type 2 diabetic condition leads to potassium-channel mediated changes of nerve excitability thus identifying them as potential drug targets to treat sorne of the DPN related symptoms.¦Schwann cells ensheath and isolate peripheral axons by the production of myelin, which consists of lipids and proteins in a ratio of 2:1. Peripheral myelin protein 2 (= P2, Pmp2 or FABP8) was originally described as one of the most abundant myelin proteins in the peripheral nervous system. P2, which is a member of the fatty acid binding protein (FABP) family, is a 14.8 kDa cytosolic protein expressed on the cytoplasmic side of compact myelin membranes. As indicated by their name, the principal role of FABPs is thought to be the binding and transport of fatty acids.¦To study its role in myelinating glial cells I have recently generated a complete P2 knockout mouse model (P2-/-). I confirmed the loss of P2 in the sciatic nerve of P2-/- mice at the mRNA and protein level. Electrophysiological analysis of the adult (P56) mutant mice revealed a mild but significant reduction in the motor nerve conduction velocity. lnterestingly, this functional change was not accompanied by any detectable alterations in general myelin structure. However, I have observed significant alterations in the mRNA expression level of other FABPs, predominantly FABP9, in the PNS of P2-/- mice as compared to age-matched P2+/+ mice indicating a role of P2 in the glial myelin lipid metabolism.¦Le diabète de type 2 touche 284 million de personnes dans le monde en 2010 et son évolution conduit dans la moitié des cas à une neuropathie périphérique diabétique. Bien que la neuropathie périphérique soit la complication la plus courante du diabète pouvant conduire jusqu'à l'amputation, sa physiopathologie est aujourd'hui encore mal comprise. Dans le but d'améliorer les connaissances moléculaires expliquant les mécanismes de la neuropathie liée au diabète de type 2, j'ai utilisé un modèle murin du diabète de type 2, les souris db/db.¦ln vivo, les enregistrements éléctrophysiologiques des animaux diabétiques montrent qu'en plus d'une diminution de la vitesse de conduction nerveuse, les souris db/db présentent également une augmentation de l'excitabilité nerveuse. Des mesures menées Ex­ vivo ont montré l'existence d'un phénotype d'hyperexcitabilité sur les nerfs périphériques isolés d'animaux diabétiques. Grâce à l'utilisation d'inhibiteurs pharmacologiques, nous avons pu démontrer que l'hyperexcitabilité démontrée était due à une réduction d'activité des canaux Kv1. En accord avec ces données, j'ai observé qu'une situation de diabète conduisait à une diminution des canaux Kv1.2 aux régions juxta-paranodales des nerfs périphériques db/db, alors que l'expression du transcrit et de la protéine restait stable. J'ai également confirmé l'absence de canaux Kv1.2 aux juxta-paranoeuds de biopsies de nerfs de patients diabétiques. L'ensemble de ces observations montrent que les nerfs périphériques chez les patients atteints de diabète de type 2 est due à une diminution des canaux potassiques rapides juxtaparanodaux les identifiant ainsi comme des cibles thérapeutiques potentielles.¦Les cellules de Schwann enveloppent et isolent les axones périphériques d'une membrane spécialisée, la myéline, composée de deux fois plus de lipides que de protéines. La protéine P2 (Pmp2 "peripheral myelin protein 2" ou FABP8 "fatty acid binding protein") est l'une des protéines les plus abondantes au système nerveux périphérique. P2 appartient à la famille de protéines FABP liant et transportant les acides gras et est une protéine cytosolique de 14,8 kDa exprimée du côté cytoplasmique de la myéline compacte.¦Afin d'étudier le rôle de P2 dans les cellules de Schwann myélinisantes, j'ai généré une souris knockout (P2-/-). Après avoir validé l'absence de transcrit et de protéine P2 dans les nerfs sciatiques P2-/-, des mesures électrophysiologiques ont montré une réduction modérée mais significative de la vitesse de conduction du nerf moteur périphérique. Il est important de noter que ces changements fonctionnels n'ont pas pu être associés à quelconque changement dans la structure de la myéline. Cependant, j'ai observé dans les nerfs périphériques P2-/-, une altération significative du niveau d'expression d'ARNm d'autres FABPs et en particulier FABP9. Ce dernier résultat démontre l'importance du rôle de la protéine P2 dans le métabolisme lipidique de la myéline.

Nitrate reductase activity and spad readings in leaf tissues of guinea grass submitted to nitrogen and potassium rates

Nitrogen and K deficiency are among the most yield limiting factors in Brazilian pastures. The lack of these nutrients can hamper the chlorophyll biosynthesis and N content in plant tissues. A greenhouse experiment was carried out to evaluate the relationship among N and K concentrations, the indirect determination of chlorophyll content (SPAD readings), nitrate reductase activity (RNO3-) in newly expanded leaf lamina (NL) and the dry matter yield for plant tops of Mombaça grass (Panicum maximum Jacq.). A fractionated 5² factorial design was used, with 13 combinations of N and K rates in the nutrient solution. The experimental units were arranged in a randomized block design, with four replications. Plants were harvested twice. The first harvest occurred 36 days after seedling transplanting and the second 29 days after the first. Significance occurred for the interaction between the N and K rates to SPAD readings and to RNO3- assessment taken on the NL during the first growth. Besides, RNO3- and SPAD readings increased only with the NL N concentration, reaching the highest values of both variables up to about 25 g kg-1, but were ratively constant at higher leaf N. Significant relationships either between SPAD readings or RNO3- activity and shoot dry mass weight were also observed. The critical levels of N concentration in the NL were, respectively, 22 and 17g kg-1 in the first and second harvest. Thus, SPAD instrument and RNO3- assessment can be used as complementary tools to evaluate the N status in forage grass.

Ownership structure and innovation: Is there a real link?

This work focuses on the study of the relationship between ownership and control structure of the company and its innovative activity. Its aim consists of analysing the role that may be played by determinants within the company related to ownership structure when the decision to incur research and development activities is taken as well as on the output of this innovate process. Among these determinants we may think of issues such as who owns the firm and how the control of decision-making is distributed, the nature of this control and the level of concentration of ownership, among others. The study is carried out for the year 2001 using a representative sample of Spanish manufacturing industries.

Soil structure transformations from ferralic to nitic horizons on a toposequence in southeastern Brazil

The soil structure transformation from ferralic to nitic horizons was studied in a toposequence on quaternary red clayey sediments and diabase in Piracicaba (SP), Brazil. Morphological and micromorphological studies, image analysis, soil water characteristic curves and monitoring of (total) soil water potential head were used. The presence of polyconcave vughs, clayskins and planar voids shows that the vertical and lateral transition and structural transformation from ferralic to nitic horizons is given by the coalescence of the microaggregates, probably due to tensions created in a drier period in the past. Changes to a more humid climate with a defined dry season and alternate drying and wetting cycles resulted in the fissuration of the previously coalesced material, forming polyhedral aggregates and microaggregates. Simultaneously, clay illuviation filled the voids and together with the compacting action of the biological activity of these soils contributed to the coalescence of microaggregates.

Structural changes in latosols of the cerrado region: I - relationships between soil physical properties and least limiting water range

The agricultural potential of Latosols of the Brazilian Cerrado region is high, but when intensively cultivated under inappropriate management systems, the porosity can be seriously reduced, leading to rapid soil degradation. Consequently, accelerated erosion and sedimentation of springs and creeks have been observed. Therefore, the objective of this study was to evaluate structural changes of Latosols in Rio Verde, Goiás, based on the Least Limiting Water Range (LLWR), and relationships between LLWR and other physical properties. Soil samples were collected from the B horizons of five oxidic Latosols representing the textural variability of the Latosols of the Cerrado biome. LLWR and other soil physical properties were determined at various soil compaction degrees induced by uniaxial compression. Soil compaction caused effects varying from enhanced plant growth due to higher water retention, to severe restriction of edaphic functions. Also, inverse relationships were observed between clay content and bulk density values (Bd) under different structural conditions. Bd values corresponding to critical soil macroporosity (BdcMAC) were more restrictive to a sustainable use of the studied Latosols than the critical Bd corresponding to LLWR (BdcLLWR). The high tolerable compression potential of these oxidic Latosols was related to the high aeration porosity associated to the granular structure.

Unique spectrum of activity of prosimian TRIM5alpha against exogenous and endogenous retroviruses.

Lentiviruses, the genus of retrovirus that includes HIV-1, rarely endogenize. Some lemurs uniquely possess an endogenous lentivirus called PSIV ("prosimian immunodeficiency virus"). Thus, lemurs provide the opportunity to study the activity of host defense factors, such as TRIM5α, in the setting of germ line invasion. We characterized the activities of TRIM5α proteins from two distant lemurs against exogenous retroviruses and a chimeric PSIV. TRIM5α from gray mouse lemur, which carries PSIV in its genome, exhibited the narrowest restriction activity. One allelic variant of gray mouse lemur TRIM5α restricted only N-tropic murine leukemia virus (N-MLV), while a second variant restricted N-MLV and, uniquely, B-tropic MLV (B-MLV); both variants poorly blocked PSIV. In contrast, TRIM5α from ring-tailed lemur, which does not contain PSIV in its genome, revealed one of the broadest antiviral activities reported to date against lentiviruses, including PSIV. Investigation into the antiviral specificity of ring-tailed lemur TRIM5α demonstrated a major contribution of a 32-amino-acid expansion in variable region 2 (v2) of the B30.2/SPRY domain to the breadth of restriction. Data on lemur TRIM5α and the prediction of ancestral simian sequences hint at an evolutionary scenario where antiretroviral specificity is prominently defined by the lineage-specific expansion of the variable loops of B30.2/SPRY.

Sibling relationships in Dutch and immigrant families

This study examines differences in sibling relationships among native Dutch and immigrant groups in the Netherlands. It uses a large national dataset to compare adult sibling relationships among Moroccan, Turkish, Caribbean and native Dutch groups, focusing on the varying importance of gender composition and age structure for the sibling relationship in the ethnic minority groups and the native Dutch families. Results show that, on average, ethnic minorities in the Netherlands have more involved sibling relationships in adulthood, with more contact, more emotional support, practical support (except Turks and Antilleans), a higher relationship quality, but also more conflict (among the Turks and Antilleans) compared to the Dutch. Gender constellation and hierarchical position were not of equal influence in all groups, although no clear patterns emerged.

Impact of Pseudomonas fluorescens strain CHA0 and a derivative with improved biocontrol activity on the culturable resident bacterial community on cucumber roots

Information on the effects of released wild-type or genetically engineered bacteria on resident bacterial communities is important to assess the potential risks associated with the introduction of these organisms into agroecosystems. The rifampicin-resistant biocontrol strain Pseudomonas fluorescens CHA0-Rif and its derivative CHA0-Rif/pME3424, which has improved biocontrol activity and enhanced production of the antibiotics 2,4-diacetylphloroglucinol (Phl) and pyoluteorin (Plt), were introduced into soil microcosms and the culturable bacterial community developing on cucumber roots was investigated 10 and 52 days later. The introduction of either of the two strains led to a transiently enhanced metabolic activity of the bacterial community on glucose dimers and polymers as measured with BIOLOG GN plates, but natural succession between the two sampling dates changed the metabolic activity of the bacterial community more than did the inoculants. The introduced strains did not significantly affect the abundance of dominant genotypic groups of culturable bacteria discriminated by restriction analysis of amplified 16S rDNA of 2500 individual isolates. About 30-50% of the resident bacteria were very sensitive to Phl and Plt, but neither the wild-type nor CHA0-Rif/pME3424 changed the proportion of sensitive and resistant bacteria in situ. In microcosms with a synthetic bacterial community, both biocontrol strains reduced the population of a strain of Pseudomonas but did not affect the abundance of four other bacterial strains including two highly antibiotic-sensitive isolates. We conclude that detectable perturbations in the metabolic activity of the resident bacterial community caused by the biocontrol strain CHA0-Rif are (i) transient, (ii) similar for the genetically improved derivative CHA0-Rif/pME3424 and (iii) less pronounced than changes in the community structure during plant growth.

Development and validation of a symptom-based activity index for adults with eosinophilic esophagitis.

BACKGROUND & AIMS: Standardized instruments are needed to assess the activity of eosinophilic esophagitis (EoE) and to provide end points for clinical trials and observational studies. We aimed to develop and validate a patient-reported outcome (PRO) instrument and score, based on items that could account for variations in patient assessments of disease severity. We also evaluated relationships between patient assessment of disease severity and EoE-associated endoscopic, histologic, and laboratory findings. METHODS: We collected information from 186 patients with EoE in Switzerland and the United States (69.4% male; median age, 43 y) via surveys (n = 135), focus groups (n = 27), and semistructured interviews (n = 24). Items were generated for the instruments to assess biologic activity based on physician input. Linear regression was used to quantify the extent to which variations in patient-reported disease characteristics could account for variations in patient assessment of EoE severity. The PRO instrument was used prospectively in 153 adult patients with EoE (72.5% male; median age, 38 y), and validated in an independent group of 120 patients with EoE (60.8% male; median age, 40.5 y). RESULTS: Seven PRO factors that are used to assess characteristics of dysphagia, behavioral adaptations to living with dysphagia, and pain while swallowing accounted for 67% of the variation in patient assessment of disease severity. Based on statistical consideration and patient input, a 7-day recall period was selected. Highly active EoE, based on endoscopic and histologic findings, was associated with an increase in patient-assessed disease severity. In the validation study, the mean difference between patient assessment of EoE severity (range, 0-10) and PRO score (range, 0-8.52) was 0.15. CONCLUSIONS: We developed and validated an EoE scoring system based on 7 PRO items that assess symptoms over a 7-day recall period. Clinicaltrials.gov number: NCT00939263.

Constitutive activity and inverse agonism at the alpha1adrenoceptors.

Mutations of G protein-coupled receptors (GPCR) can increase their constitutive (agonist-independent) activity. Some of these mutations have been artificially introduced by site-directed mutagenesis, others occur spontaneously in human diseases. The alpha(1B)adrenoceptor was the first GPCR in which point mutations were shown to trigger receptor activation. This article briefly summarizes some of the findings reported in the last several years on constitutive activity of the alpha(1)adrenoceptor subtypes, the location where mutations have been found in the receptors, the spontaneous activity of native receptors in recombinant as well as physiological systems. In addition, it will highlight how the analysis of the pharmacological and molecular properties of the constitutively active adrenoceptor mutants provided an important contribution to our understanding of the molecular mechanisms underlying the mechanism of receptor activation and inverse agonism.

MORPHOLOGICAL AND MICROMORPHOLOGICAL CHANGES IN THE STRUCTURE OF A RHODIC HAPLUDOX AS A RESULT OF AGRICULTURAL MANAGEMENT

In evaluation of soil quality for agricultural use, soil structure is one of the most important properties, which is influenced not only by climate, biological activity, and management practices but also by mechanical and physico-chemical forces acting in the soil. The purpose of this study was to evaluate the influence of conventional agricultural management on the structure and microstructure of a Latossolo Vermelho distroférrico típico (Rhodic Hapludox) in an experimental area planted to maize. Soil morphology was described using the crop profile method by identifying the distinct structural volumes called Morphologically Homogeneous Units (MHUs). For comparison, we also described a profile in an adjacent area without agricultural use and under natural regrowth referred to as Memory. We took undisturbed samples from the main MHUs so as to form thin sections and blocks of soil for micromorphological and micromorphometrical analyses. Results from the application of the crop profile method showed the occurrence of the following structural types: loose (L), fragmented (F) and continuous (C) in both profiles analyzed. In the Memory soil profile, the fragmented structures were classified as Fptμ∆+tf and Fmt∆μ, whose micromorphology shows an enaulic-porphyric (porous) relative distribution with a great deal of biological activity as indicated by the presence of vughs and channels. Lower down, from 0.20 to 0.35 m, there was a continuous soil volume (sub-type C∆μ), with a subangular block microstructure and an enaulic-porphyric relative distribution, though in this case more compact and with aggregate coalescence and less biological activity. The micromorphometrical study of the soil of the Memory Plot showed the predominance of complex pores in NAM (15.03 %), Fmt∆μ (11.72 %), and Fptμ∆+tf (7.73 %), and rounded pores in C∆μ (8.21 %). In the soil under conventional agricultural management, we observed fragmented structures similar to the Memory Plot from 0.02 to 0.20 m, followed by a volume with a compact continuous structure (C∆μ), without visible porosity and with few roots. In the MHUs under conventional management, reduction in the packing pores (40 %) was observed, mainly in the continuous units (C). The microstructure had well-defined blocks, with the occurrence of planar pores and less evidence of biological activity. In conclusion, the morphological and micromorphological analyses of the soil profiles studied offered complementary information regarding soil structural quality, especially concerning the changes in pore types as result of mechanical stress undergone by the soil.

Early Changes in Soil Metabolic Diversity and Bacterial Community Structure in Sugarcane under Two Harvest Management Systems

Preharvest burning is widely used in Brazil for sugarcane cropping. However, due to environmental restrictions, harvest without burning is becoming the predominant option. Consequently, changes in the microbial community are expected from crop residue accumulation on the soil surface, as well as alterations in soil metabolic diversity as of the first harvest. Because biological properties respond quickly and can be used to monitor environmental changes, we evaluated soil metabolic diversity and bacterial community structure after the first harvest under sugarcane management without burning compared to management with preharvest burning. Soil samples were collected under three sugarcane varieties (SP813250, SP801842 and RB72454) and two harvest management systems (without and with preharvest burning). Microbial biomass C (MBC), carbon (C) substrate utilization profiles, bacterial community structure (based on profiles of 16S rRNA gene amplicons), and soil chemical properties were determined. MBC was not different among the treatments. C-substrate utilization and metabolic diversity were lower in soil without burning, except for the evenness index of C-substrate utilization. Soil samples under the variety SP801842 showed the greatest changes in substrate utilization and metabolic diversity, but showed no differences in bacterial community structure, regardless of the harvest management system. In conclusion, combined analysis of soil chemical and microbiological data can detect early changes in microbial metabolic capacity and diversity, with lower values in management without burning. However, after the first harvest, there were no changes in the soil bacterial community structure detected by PCR-DGGE under the sugarcane variety SP801842. Therefore, the metabolic profile is a more sensitive indicator of early changes in the soil microbial community caused by the harvest management system.

Scaffold attachment factor B1 directly interacts with nuclear receptors in living cells and represses transcriptional activity.

Transcriptional activity relies on coregulators that modify the chromatin structure and serve as bridging factors between transcription factors and the basal transcription machinery. Using the DE domain of human peroxisome proliferator-activated receptor gamma (PPARgamma) as bait in a yeast two-hybrid screen of a human adipose tissue library, we isolated the scaffold attachment factor B1 (SAFB1/HET/HAP), which was previously shown to be a corepressor of estrogen receptor alpha. We show here that SAFB1 has a very broad tissue expression profile in human and is also expressed all along mouse embryogenesis. SAFB1 interacts in pull-down assays not only with PPARgamma but also with all nuclear receptors tested so far, albeit with different affinities. The association of SAFB1 and PPARgamma in vivo is further demonstrated by fluorescence resonance energy transfer (FRET) experiments in living cells. We finally show that SAFB1 is a rather general corepressor for nuclear receptors. Its change in expression during the early phases of adipocyte and enterocyte differentiation suggests that SAFB1 potentially influences cell proliferation and differentiation decisions.

Stable alpha-synuclein oligomers strongly inhibit chaperone activity of the Hsp70 system by weak interactions with J-domain co-chaperones.

α-Synuclein aggregation and accumulation in Lewy bodies are implicated in progressive loss of dopaminergic neurons in Parkinson disease and related disorders. In neurons, the Hsp70s and their Hsp40-like J-domain co-chaperones are the only known components of chaperone network that can use ATP to convert cytotoxic protein aggregates into harmless natively refolded polypeptides. Here we developed a protocol for preparing a homogeneous population of highly stable β-sheet enriched toroid-shaped α-Syn oligomers with a diameter typical of toxic pore-forming oligomers. These oligomers were partially resistant to in vitro unfolding by the bacterial Hsp70 chaperone system (DnaK, DnaJ, GrpE). Moreover, both bacterial and human Hsp70/Hsp40 unfolding/refolding activities of model chaperone substrates were strongly inhibited by the oligomers but, remarkably, not by unstructured α-Syn monomers even in large excess. The oligomers acted as a specific competitive inhibitor of the J-domain co-chaperones, indicating that J-domain co-chaperones may preferably bind to exposed bulky misfolded structures in misfolded proteins and, thus, complement Hsp70s that bind to extended segments. Together, our findings suggest that inhibition of the Hsp70/Hsp40 chaperone system by α-Syn oligomers may contribute to the disruption of protein homeostasis in dopaminergic neurons, leading to apoptosis and tissue loss in Parkinson disease and related neurodegenerative diseases.