USP13 enzyme regulates Siah2 ligase stability and activity via noncatalytic ubiquitin-binding domains

The RING finger E3 ubiquitin ligase Siah2 is implicated in control of diverse cellular biological events, including MAPK signaling and hypoxia. Here we demonstrate that Siah2 is subject to regulation by the deubiquitinating enzyme USP13. Overexpression of USP13 increases Siah2 stability by attenuating its autodegradation. Consequently, the ability of Siah2 to target its substrates prolyl hydroxylase 3 and Spry2 (Sprouty2) for ubiquitin-mediated proteasomal degradation is attenuated. Conversely, inhibition of USP13 expression with corresponding shRNA decreases the stability of both Siah2 and its substrate Spry2. Thus, USP13 limits Siah2 autodegradation and its ubiquitin ligase activity against its target substrates. Strikingly, the effect of USP13 on Siah2 is not mediated by its isopeptidase activity: mutations in its ubiquitin-binding sequences positioned within the ubiquitin-specific processing protease and ubiquitin-binding domains, but not within putative catalytic sites, abolish USP13 binding to and effect on Siah2 autodegradation and targeted ubiquitination. Notably, USP13 expression is attenuated in melanoma cells maintained under hypoxia, thereby relieving Siah2 inhibition and increasing its activity under low oxygen levels. Significantly, on melanoma tissue microarray, high nuclear expression of USP13 coincided with high nuclear expression of Siah2. Overall, this study identifies a new layer of Siah2 regulation mediated by USP13 binding to ubiquitinated Siah2 protein with a concomitant inhibitory effect on its activity under normoxia.     

Linking Environmental Forcing and Trophic Supply to Benthic Communities in the Vercelli Seamount Area (Tyrrhenian Sea)

Seamounts and their influence on the surrounding environment are currently being extensively debated but, surprisingly, scant information is available for the Mediterranean area. Furthermore, although the deep Tyrrhenian Sea is characterised by a complex bottom morphology and peculiar hydrodynamic features, which would suggest a variable influence on the benthic domain, few studies have been carried out there, especially for soft-bottom macrofaunal assemblages. In order to fill this gap, the structure of the meio-and macrofaunal assemblages of the Vercelli Seamount and the surrounding deep area (northern Tyrrhenian Sea – western Mediterranean) were studied in relation to environmental features. Sediment was collected with a box-corer from the seamount summit and flanks and at two far-field sites in spring 2009, in order to analyse the metazoan communities, the sediment texture and the sedimentary organic matter. At the summit station, the heterogeneity of the habitat, the shallowness of the site and the higher trophic supply (water column phytopigments and macroalgal detritus, for instance) supported a very rich macrofaunal community, with high abundance, biomass and diversity. In fact, its trophic features resembled those observed in coastal environments next to seagrass meadows. At the flank and far-field stations, sediment heterogeneity and depth especially influenced the meiofaunal distribution. From a trophic point of view, the low content of the valuable sedimentary proteins that was found confirmed the general oligotrophy of the Tyrrhenian Sea, and exerted a limiting influence on the abundance and biomass of the assemblages. In this scenario, the rather refractory sedimentary carbohydrates became a food source for metazoans, which increased their abundance and biomass at the stations where the hydrolytic-enzyme-mediated turnover of carbohydrates was faster, highlighting high lability.     

Kinetics of tert-[35S]Butylbicyclophosphorothionate Binding in the Cerebral Cortex of Newborn and Adult Rats: Effects of GABA and Receptor Desensitization

Abstract: The effects of GABA on the kinetics of tert -[³⁵S]butylbicyclophosphorothionate ([³⁵S]TBPS) binding to the convulsant site of GABA_A receptors were studied in membrane suspensions from the cerebral cortex of newborn (1-day-old) and adult (90-day-old) rats. TBPS dissociation was biphasic in neonates and adults, indicating that more than one interconvertible state of [³⁵S]TBPS binding sites may be present in the cerebral cortex. In the absence of GABA, the fast ( t _1/2, 11 min) and slow ( t _1/2, 77 min) components of TBPS dissociation in newborn rats were approximately fourfold slower than in adults. The acceleration of the dissociation rates caused by 2 µ M GABA, however, was more robust in neonates than in adults (six- to ninefold vs. twofold increase, respectively). Moreover, the dissociation rates of TBPS in membranes preincubated with 2 µ M GABA (dissociation started by adding 40 µ M picrotoxin) were two- to fourfold slower than in membranes preincubated without GABA (dissociation started by adding 40 µ M picrotoxin plus 2 µ M GABA). Taken together, these results suggest that (1) the closed state of GABA_A receptors is associated with a more effective steric barrier for the binding of TBPS in neonates compared with adults, (2) GABA produces a larger acceleration of the binding kinetics of TBPS in neonates than in adults, and (3) long incubations with GABA may cause receptor desensitization, which in turn slows down the dissociation rates of TBPS.     

Discovery and optimization of RO-85, a novel drug-like,potent, and selective P2X3 receptor antagonist

Despite the extensive literature describing the role of the ATP-gated P2X₃ receptors in a variety of physiological processes the potential of antagonists as therapeutic agents has been limited by the lack of drug-like selective molecules. In this paper we report the discovery and optimization of RO-85, a novel drug-like, potent and selective P2X₃ antagonist. High-throughput screening of the Roche compound collection identified a small hit series of heterocyclic amides from a large parallel synthesis library. Rapid optimization, facilitated by high-throughput synthesis, focusing on increasing potency and improving drug-likeness resulted in the discovery of RO-85.     

Nitrogen storage and remobilization in Brassica napus L. during the growth cycle: identification, characterization and immunolocalization of a putative taproot storage glycoprotein.

In taproot of oilseed rape (Brassica napus L.), a 23 kDa polypeptide has been recently identified as a putative vegetative storage protein (VSP) because of its accumulation during flowering and its specific mobilization to sustain grain filling when N uptake is strongly reduced. The objectives were to characterize this protein more precisely and to study the effect of environmental factors (N availability, daylength, temperature, water deficit, wounding) or endogenous signals (methyl jasmonate, abscisic acid) that might change the N source/sink relationships within the plant, and may therefore trigger its accumulation. The 23 kDa putative VSP has two isoforms, is glycosylated and both isoforms share the same N-terminal sequence which had been used to produce specific polyclonal antibodies. Low levels of an immunoreactive protein of 24 kDa were found in leaves and flowers. In taproot, the 23 kDa putative VSP seems to accumulate only in the vacuoles of peripheral cortical parenchyma cells, around the phloem vessels. Among all treatments tested, the accumulation of this protein could only be induced by abscisic acid and methyl jasmonate. When compared to control plants, application of methyl jasmonate reduced N uptake by 89% after 15 d, induced a strong remobilization of N from senescing leaves and a concomitant accumulation of the 23 kDa putative VSP. These results suggested that, in rape, the 23 kDa protein is used as a storage buffer between N losses from senescing leaves promoted by methyl jasmonate and grain filling.     

Role of phage ϕ1 in two strains of <Emphasis Type="Italic">Salmonella</Emphasis> Rissen,sensitive and resistant to phage ϕ1

Background

The study describes the Salmonella Rissen phage ?1 isolated from the ?1-sensitive Salmonella Rissen strain R^W. The same phage was then used to select the resistant strain R^R?1+, which can harbour or not ?1.

Results

Following this approach, we found that ?1, upon excision from R^W cells with mitomycin, behaves as a temperate phage: lyses host cells and generates phage particles; instead, upon spontaneous excision from R^R?1+ cells, it does not generate phage particles; causes loss of phage resistance; switches the O-antigen from the smooth to the rough phenotype, and favors the transition of Salmonella Rissen from the planktonic to the biofilm growth.The R^W and R^R?1+ strains differ by 10 genes; of these, only two (phosphomannomutase_1 and phosphomannomutase_2; both involved in the mannose synthesis pathway) display significant differences at the expression levels. This result suggests that phage resistance is associated with these two genes.

Conclusions

Phage ?1 displays the unusual property of behaving as template as well as lytic phage. This feature was used by the phage to modulate several phases of Salmonella Rissen lifestyle.

     

Subtle variation in size and shape of the whole forewing and the red band among co‐mimics revealed by geometric morphometric analysis in Heliconius butterflies

Heliconius are unpalatable butterflies that exhibit remarkable intra‐ and interspecific variation in wing color pattern, specifically warning coloration. Species that have converged on the same pattern are often clustered in Müllerian mimicry rings. Overall, wing color patterns are nearly identical among co‐mimics. However, fine‐scale differences exist, indicating that factors in addition to natural selection may underlie wing phenotype. Here, we investigate differences in shape and size of the forewing and the red band in the Heliconius postman mimicry ring (H. erato phyllis and the co‐mimics H. besckei, H. melpomene burchelli, and H. melpomene nanna) using a landmark‐based approach. If phenotypic evolution is driven entirely by predation pressure, we expect nonsignificant differences among co‐mimics in terms of wing shape. Also, a reinforcement of wing pattern (i.e., greater similarity) could occur when co‐mimics are in sympatry. We also examined variation in the red forewing band because this trait is critical for both mimicry and sexual communication. Morphometric results revealed significant but small differences among species, particularly in the shape of the forewing of co‐mimics. Although we did not observe greater similarity when co‐mimics were in sympatry, nearly identical patterns provided evidence of convergence for mimicry. In contrast, mimetic pairs could be distinguished based on the shape (but not the size) of the red band, suggesting an “advergence” process. In addition, sexual dimorphism in the red band shape (but not size) was found for all lineages. Thus, we infer that natural selection due to predation by birds might not be the only mechanism responsible for variation in color patterns, and sexual selection could be an important driver of wing phenotypic evolution in this mimicry ring.     

Characteristics of Patients with Smear-Negative Pulmonary Tuberculosis (TB) in a Region with High TB and HIV Prevalence

Introduction

Smear-negative pulmonary TB (SNPT) represents 30–60% of all pulmonary TB cases. The mortality of these patients can reach 25% in populations with high prevalence of HIV infection, and 10–20% of TB transmission at the population level are attributable to SNPT cases.

Methods

We conducted a retrospective study to evaluate epidemiological, clinical, and radiological characteristics of patients with SNPT and to compare these with patients who were diagnosed as having smear-positive pulmonary TB (SPPT). All adult patients (≥ 18 years old) with a positive culture for Mycobacterium tuberculosis, and a diagnosis of pulmonary TB were included in the study.

Results

198 patients met the inclusion criteria (positive culture for Mycobacterium tuberculosis) and were included in the analysis. Of these patients, 69 (34.8%) were smear positive (SPPT) and 129 (65.2%) were smear negative (SNPT). In univariate analysis, cough, dyspnea, and hemoptysis were less frequent in SNPT patients in comparison with SPPT patients. In a multivariate model, having no cough and no radiographic pattern typical of TB were the characteristics independently associated with a diagnosis of SNPT.

Conclusions

We found a very high prevalence of SNPT among patients with TB in a setting with high TB and HIV prevalence. The absence of cough in the presence of other symptoms suggestive of TB, and having no radiographic pattern typical of TB where independent predictors of SNPT.     

Effects of some indoor environmental factors on respiratory symptoms and lung function in a sample of young non smokers in North Italy

Summary We evaluated the effects of some indoor environmental factors in a non smoking subsample (n=381, age 8–19 years) of the general population living in the Po River Delta. Each subject completed an interviewer-administered standardized questionnaire on respiratory symptoms and risk factors. Acceptable maneuvers of forced vital capacity and slope of alveolar plateau of nitrogen were obtained in 96% and 59% of the subjects, respectively. In the houses there were more frequently natural gas for cooking (86%) than bottled gas (14%) and central heating (82%) than stove (18%). As regards passive smoking exposure, 18% of subjects had both parents smoking, 50% had one parent smoking. Significantly higher prevalence rates of wheeze, dyspnea, diagnosis of asthma were found in subjects of both sexes using bottled gas for cooking in comparison to those using natural gas, when also exposed to passive smoking. An insignificant trend towards higher symptom rates was shown by those using stove, instead of central heating. Lung function was affected only in females: those with both parents smoking had reduced forced expirograms, those with bottled gas for cooking or stove for heating had a decreased peak expiratory flow. Interactions of stove and passive smoking on peak expiratory flow and on slope of alveolar plateau were statistically significant. These findings confirm the mild adverse respiratory effects of certain home environment factors shown by other epidemiologic surveys in North Europe and in the USA. They have been a basis for the implementation, under the auspices of National Research Council and Electric Energy Authority, of future specific studies in which continuous monitoring of indoor pollutants and repeated recording of symptoms and lung function in North and Central Italy will be performed.