Progressive Purkinje Cell Degeneration in tambaleante Mutant Mice Is a Consequence of a Missense Mutation in HERC1 E3 Ubiquitin Ligase

The HERC gene family encodes proteins with two characteristic domains: HECT and RCC1-like. Proteins with HECT domain shave been described to function as ubiquitin ligases, and those that contain RCC1-like domains have been reported to function as GTPases regulators. These two activities are essential in a number of important cellular processes such as cell cycle, cell signaling, and membrane trafficking. Mutations affecting these domains have been found associated with retinitis pigmentosa, amyotrophic lateral sclerosis, and cancer. In humans, six HERC genes have been reported which encode two subgroups of HERC proteins: large (HERC1-2) and small (HERC3-6). The giant HERC1 protein was the first to be identified. It has been involved in membrane trafficking and cell proliferation/growth through its interactions with clathrin, M2-pyruvate kinase, and TSC2 proteins. Mutations affecting other members of the HERC family have been found to be associated with sterility and growth retardation. Here, we report the characterization of a recessive mutation named tambaleante, which causes progressive Purkinje cell degeneration leading to severe ataxia with reduced growth and lifespan in homozygous mice aged over two months. We mapped this mutation in mouse chromosome 9 and then performed positional cloning. We found a GuA transition at position 1448, causing a Gly to Glu substitution (Gly483Glu) in the highly conserved N- terminal RCC1-like domain of the HERC1 protein. Successful transgenic rescue, with either a mouse BAC containing the normal copy of Herc1 or with the human HERC1 cDNA, validated our findings. Histological and biochemical studies revealed extensive autophagy associated with an increase of the mutant protein level and a decrease of mTOR activity. Our observations concerning this first mutation in the Herc1 gene contribute to the functional annotation of the encoded E3 ubiquitin ligase and underline the crucial and unexpected role of this protein in Purkinje cell physiology.

Insulin-Like growth-factor 1 myocardial expression decreases in chronic alcohol consumption

Background Chronic alcohol ingestion may cause severe biochemical and pathophysiological derangements to skeletal muscle. Unfortunately, these alcohol-induced events may also prime skeletal muscle for worsened, delayed, or possibly incomplete repair following acute injury. As alcoholics may be at increased risk for skeletal muscle injury, our goals were to identify the effects of chronic alcohol ingestion on components of skeletal muscle regeneration. To accomplish this, age- and gender-matched C57Bl/6 mice were provided normal drinking water or water that contained 20% alcohol (v/v) for 1820 wk. Subgroups of mice were injected with a 1.2% barium chloride (BaCl2) solution into the tibialis anterior (TA) muscle to initiate degeneration and regeneration processes. Body weights and voluntary wheel running distances were recorded during the course of recovery. Muscles were harvested at 2, 7 or 14 days post-injection and assessed for markers of inflammation and oxidant stress, fiber cross-sectional areas, levels of growth and fibrotic factors, and fibrosis. Results Body weights of injured, alcohol-fed mice were reduced during the first week of recovery. These mice also ran significantly shorter distances over the two weeks following injury compared to uninjured, alcoholics. Injured TA muscles from alcohol-fed mice had increased TNFα and IL6 gene levels compared to controls 2 days after injury. Total protein oxidant stress and alterations to glutathione homeostasis were also evident at 7 and 14 days after injury. Ciliary neurotrophic factor (CNTF) induction was delayed in injured muscles from alcohol-fed mice which may explain, in part, why fiber cross-sectional area failed to normalize 14 days following injury. Gene levels of TGFβ1 were induced early following injury before normalizing in muscle from alcohol-fed mice compared to controls. However, TGFβ1 protein content was consistently elevated in injured muscle regardless of diet. Fibrosis was increased in injured, muscle from alcohol-fed mice at 7 and 14 days of recovery compared to injured controls. Conclusions Chronic alcohol ingestion appears to delay the normal regenerative response following significant skeletal muscle injury. This is evidenced by reduced cross-sectional areas of regenerated fibers, increased fibrosis, and altered temporal expression of well-described growth and fibrotic factors.

Insulin-Like growth-factor 1 myocardial expression decreases in chronic alcohol consumption

Background Chronic alcohol ingestion may cause severe biochemical and pathophysiological derangements to skeletal muscle. Unfortunately, these alcohol-induced events may also prime skeletal muscle for worsened, delayed, or possibly incomplete repair following acute injury. As alcoholics may be at increased risk for skeletal muscle injury, our goals were to identify the effects of chronic alcohol ingestion on components of skeletal muscle regeneration. To accomplish this, age- and gender-matched C57Bl/6 mice were provided normal drinking water or water that contained 20% alcohol (v/v) for 18-20 wk. Subgroups of mice were injected with a 1.2% barium chloride (BaCl2) solution into the tibialis anterior (TA) muscle to initiate degeneration and regeneration processes. Body weights and voluntary wheel running distances were recorded during the course of recovery. Muscles were harvested at 2, 7 or 14 days post-injection and assessed for markers of inflammation and oxidant stress, fiber cross-sectional areas, levels of growth and fibrotic factors, and fibrosis. Results Body weights of injured, alcohol-fed mice were reduced during the first week of recovery. These mice also ran significantly shorter distances over the two weeks following injury compared to uninjured, alcoholics. Injured TA muscles from alcohol-fed mice had increased TNFα and IL6 gene levels compared to controls 2 days after injury. Total protein oxidant stress and alterations to glutathione homeostasis were also evident at 7 and 14 days after injury. Ciliary neurotrophic factor (CNTF) induction was delayed in injured muscles from alcohol-fed mice which may explain, in part, why fiber cross-sectional area failed to normalize 14 days following injury. Gene levels of TGFβ1 were induced early following injury before normalizing in muscle from alcohol-fed mice compared to controls. However, TGFβ1 protein content was consistently elevated in injured muscle regardless of diet. Fibrosis was increased in injured, muscle from alcohol-fed mice at 7 and 14 days of recovery compared to injured controls. Conclusions Chronic alcohol ingestion appears to delay the normal regenerative response following significant skeletal muscle injury. This is evidenced by reduced cross-sectional areas of regenerated fibers, increased fibrosis, and altered temporal expression of well-described growth and fibrotic factors.

Regulació del factor de transcripció NFAT5 per les quinases WNK1, SPAK i OSR1 i cerca de quinases activadores de la pròpia WNK1 en resposta a estrés osmòtic

Estudi elaborat a partir d’una estada a la School of Life Sciences de la University of Dundee, Gran Bretanya, entre gener i març del 2007.L'estrès osmòtic causa rà pidament l'activació de la quinasa WNK1, que fosforila i activa a continuació les quinases SPAK i OSR1, que alhora regulen canals i transportadors d’ions preexistents a la membrana cel•lular. El factor de transcripció NFAT5 és el principal regulador de la resposta cel•lular transcripcional secundà ria a hipertonicitat i s’ha descrit que les quinases p38, Fyn, PKA, ERK/MEK i ATM estan involucrades en la seva regulació post-traduccional. No obstant, com que la funció d’aquestes quinases no explica totalment els mecanismes d'activació de NFAT5, s’ha estudiat si l’activitat transcripcional de NFAT5 pot estar regulada per WNK1, SPAK o OSR1. Així doncs, es va observar que l’activitat d’un reporter dependent de NFAT5 no es veu afectada per la presència de cap de les quinases anteriors, en la seva forma wild-type o dominant negatiu. D’altra banda, es va estudiar quin domini de WNK1 és necessari per a que pugui respondre a hipertonicitat i quines quinases poden estar involucrades en la fosforilació de la serina 382 de WNK1. En conclusió, les dades obtingudes apunten que l’activació de WNK1 en resposta a estrès osmòtic requereix la seva fosforilació en la serina 382 per quinases upstream com PAK2 o RSK i que també és necessari un dels seus dominis coiled-coil, almenys els aminoà cids 558 i 561. Aquests processos, però, semblen ser independents de l’activació de NFAT5 en resposta a hipertonicitat.   

Robust Factor Analysis for Compositional Data

Factor analysis as frequent technique for multivariate data inspection is widely used also for compositional data analysis. The usual way is to use a centered logratio (clr)transformation to obtain the random vector y of dimension D. The factor model istheny = Λf + e (1)with the factors f of dimension k & D, the error term e, and the loadings matrix Λ.Using the usual model assumptions (see, e.g., Basilevsky, 1994), the factor analysismodel (1) can be written asCov(y) = ΛΛT + ψ (2)where ψ = Cov(e) has a diagonal form. The diagonal elements of ψ as well as theloadings matrix Λ are estimated from an estimation of Cov(y).Given observed clr transformed data Y as realizations of the random vectory. Outliers or deviations from the idealized model assumptions of factor analysiscan severely effect the parameter estimation. As a way out, robust estimation ofthe covariance matrix of Y will lead to robust estimates of Λ and ψ in (2), seePison et al. (2003). Well known robust covariance estimators with good statisticalproperties, like the MCD or the S-estimators (see, e.g. Maronna et al., 2006), relyon a full-rank data matrix Y which is not the case for clr transformed data (see,e.g., Aitchison, 1986).The isometric logratio (ilr) transformation (Egozcue et al., 2003) solves thissingularity problem. The data matrix Y is transformed to a matrix Z by usingan orthonormal basis of lower dimension. Using the ilr transformed data, a robustcovariance matrix C(Z) can be estimated. The result can be back-transformed tothe clr space byC(Y ) = V C(Z)V Twhere the matrix V with orthonormal columns comes from the relation betweenthe clr and the ilr transformation. Now the parameters in the model (2) can beestimated (Basilevsky, 1994) and the results have a direct interpretation since thelinks to the original variables are still preserved.The above procedure will be applied to data from geochemistry. Our specialinterest is on comparing the results with those of Reimann et al. (2002) for the Kolaproject data

Avoiding transcription factor competition at promoter level increases the chances of obtaining oscillations

Background: The ultimate goal of synthetic biology is the conception and construction of genetic circuits that are reliable with respect to their designed function (e.g. oscillators, switches). This task remains still to be attained due to the inherent synergy of the biological building blocks and to an insufficient feedback between experiments and mathematical models. Nevertheless, the progress in these directions has been substantial. Results: It has been emphasized in the literature that the architecture of a genetic oscillator must include positive (activating) and negative (inhibiting) genetic interactions in order to yield robust oscillations. Our results point out that the oscillatory capacity is not only affected by the interaction polarity but by how it is implemented at promoter level. For a chosen oscillator architecture, we show by means of numerical simulations that the existence or lack of competition between activator and inhibitor at promoter level affects the probability of producing oscillations and also leaves characteristic fingerprints on the associated period/amplitude features. Conclusions: In comparison with non-competitive binding at promoters, competition drastically reduces the region of the parameters space characterized by oscillatory solutions. Moreover, while competition leads to pulse-like oscillations with long-tail distribution in period and amplitude for various parameters or noisy conditions, the non-competitive scenario shows a characteristic frequency and confined amplitude values. Our study also situates the competition mechanism in the context of existing genetic oscillators, with emphasis on the Atkinson oscillator.

Monte Carlo Study of the Growth of L12 ordered domains in fcc A3B binary alloys

A Monte Carlo study of the late time growth of L12-ordered domains in a fcc A3B binary alloy is presented. The energy of the alloy has been modeled by a nearest-neighbor interaction Ising Hamiltonian. The system exhibits a fourfold degenerated ground state and two kinds of interfaces separating ordered domains: flat and curved antiphase boundaries. Two different dynamics are used in the simulations: the standard atom-atom exchange mechanism and the more realistic vacancy-atom exchange mechanism. The results obtained by both methods are compared. In particular we study the time evolution of the excess energy, the structure factor and the mean distance between walls. In the case of atom-atom exchange mechanism anisotropic growth has been found: two characteristic lengths are needed in order to describe the evolution. Contrarily, with the vacancyatom exchange mechanism scaling with a single length holds. Results are contrasted with existing experiments in Cu3Au and theories for anisotropic growth.

Hypocretins regulate the anxiogenic-like effects of nicotine and induce reinstatement of nicotine-seeking behavior

Emerging evidence suggests that the hypocretinergic system is involved in addictive behavior. In this study, we investigated the role of these hypothalamic neuropeptides in anxiety-like responses of nicotine and stress-induced reinstatement of nicotine-seeking behavior. Acute nicotine (0.8 mg/kg, s.c.) induced anxiogenic-like effects in the elevated plus-maze and activated the paraventricular nucleus of thehypothalamus (PVN) as revealed by c-Fos expression. Pretreatment with the hypocretin receptor 1 (Hcrtr-1) antagonist SB334867 orpreprohypocretin gene deletion blocked both nicotine effects. In the PVN, SB334867 also prevented the activation of corticotrophinreleasing factor (CRF) and arginine-vasopressin (AVP) neurons, which expressed Hcrtr-1. In addition, an increase of the percentage of c-Fos-positive hypocretin cells in the perifornical and dorsomedial hypothalamic (PFA/DMH) areas was found after nicotine (0.8 mg/kg,s.c.) administration. Intracerebroventricular infusion of hypocretin-1 (Hcrt-1) (0.75 nmol/1 l) or footshock stress reinstated a previouslyextinguished nicotine-seeking behavior. The effects of Hcrt-1 were blocked by SB334867, but not by the CRF1 receptor antagonistantalarmin. Moreover, SB334867 did not block CRF-dependent footshock-induced reinstatement of nicotine-seeking while antalarmin was effective in preventing this nicotine motivational response. Therefore, the Hcrt system interacts with CRF and AVP neurons in the PVN and modulates the anxiogenic-like effects of nicotine whereas Hcrt and CRF play a different role in the reinstatement of nicotineseeking.Indeed, Hcrt-1 reinstates nicotine-seeking through a mechanism independent of CRF activation whereas CRF mediates the reinstatement induced by stress.

Predictions of the MCMI-III personality disorders from NEO-PI-R domains and facets: Comparison between American and Spanish samples

Este estudio ex post facto analiza las relaciones entre las dimensiones y facetas del NEO-PI-R y los 14 trastornos de personalidad del MCMI-III en una muestra no clínica española (N = 674). Se exploran las diferencias y similitudes con los resul- tados de Dyce y O’Connor en una muestra americana con los mismos instrumentos. Como se esperaba, los análisis factoriales de facetas reteniendo cinco factores mostraron un modelo de relaciones muy similar entre ambas muestras, con un coeficiente de la congruencia total de 0,92, y coeficientes de congruencia de factor aceptables, salvo para el factor Apertura (0,68). En consonancia con las predicciones de Widiger y Widiger et al. los porcentajes de correlaciones significativas estaban alrededor de 60% en ambas muestras, y la mayoría coincidían. El análisis de regresión múltiple con dimensiones también reveló un gran parecido entre los resultados americanos y españoles, Neuroticismo fue el predictor más relacionado con los trastornos de personalidad. Se encontraron diferencias en las regresiones por facetas, aunque la varianza explicada fue prácticamente la misma que en las dimensiones. Se discute la validez transcultural y el valor predictivo del NEO-PI-R sobre los trastornos de personalidad del MCMI-III, junto con las ventajas relativas de las facetas sobre las dimensiones.

Lipid rafts act as specialised domains for tetanus toxin binding and internalisation in neurons

Tetanus (TeNT) is a zinc protease that blocks neurotransmission by cleaving the synaptic protein vesicle-associated membrane protein/synaptobrevin. Although its intracellular catalytic activity is well established, the mechanism by which this neurotoxin interacts with the neuronal surface is not known. In this study, we characterize p15s, the first plasma membrane TeNT binding proteins and we show that they are glycosylphosphatidylinositol-anchored glycoproteins in nerve growth factor (NGF)-differentiated PC12 cells, spinal cord cells, and purified motor neurons. We identify p15 as neuronal Thy-1 in NGF-differentiated PC12 cells. Fluorescence lifetime imaging microscopy measurements confirm the close association of the binding domain of TeNT and Thy-1 at the plasma membrane. We find that TeNT is recruited to detergent-insoluble lipid microdomains on the surface of neuronal cells. Finally, we show that cholesterol depletion affects a raft subpool and blocks the internalization and intracellular activity of the toxin. Our results indicate that TeNT interacts with target cells by binding to lipid rafts and that cholesterol is required for TeNT internalization and/or trafficking in neurons.

Sustained CTL activation by murine pulmonary epithelial cells promotes the development of COPD-like disease

Chronic obstructive pulmonary disease (COPD) is a lethal progressive lung disease culminating in permanent airway obstruction and alveolar enlargement. Previous studies suggest CTL involvement in COPD progression; however, their precise role remains unknown. Here, we investigated whether the CTL activation receptor NK cell group 2D (NKG2D) contributes to the development of COPD. Using primary murine lung epithelium isolated from mice chronically exposed to cigarette smoke and cultured epithelial cells exposed to cigarette smoke extract in vitro, we demonstrated induced expression of the NKG2D ligand retinoic acid early tran - script 1 (RAET1)as well as NKG2D-mediated cytotoxicity. Furthermore, a genetic model of inducible RAET1 expression on mouse pulmonary epithelial cells yielded a severe emphysematous phenotype characterized by epithelial apoptosis and increased CTL activation, which was reversed by blocking NKG2D activation. We also assessed whether NKG2D ligand expression corresponded with pulmonary disease in human patients by staining airway and peripheral lung tissues from never smokers, smokers with normal lung function, and current and former smokers with COPD. NKG2D ligand expression was independent of NKG2D receptor expression in COPD patients, demonstrating that ligand expression is the limiting factor in CTL activation. These results demonstrate that aberrant, persistent NKG2D ligand expression in the pulmonary epithelium contributes to the development of COPD pathologies.

Recombination dynamics in ZnO nanowires: Surfaces states versus mode quality factor

In this work, we investigate the influence of finite size on the recombinations dynamics of ZnO nanowires. We demonstrate that diameter as well as lenght of nanowires determine the lifetime of the neutral donor bound excitons. Our findings suggest that while the length is mainly responsible for different mode quality factors of the cavity-like nanowires, the diameter determines the influence of surface states as alternative recombinations channels for the optical modes trapped in the nanocavity. In addition, comparing nanowires grown using different catalyst we show that the surfaces states strongly depend on each precursor characteristics.

Epidermal growth factor secreted from submandibular salivary glands interferes with the lipolytic effect of adrenaline in mice

We had described that epidermal growth factor (EGF) interfered with the lipolytic effect of catecholamines in isolated adipocytes. Since catecholamines stimulate the release of EGF from submandibular salivary glands to blood plasma in male mice, we studied whether EGF affected also the lipolytic response to adrenaline in whole animals. We studied the effect of adrenaline in sialoadenectomized and sham-operated mice receiving or not a high dose of EGF following adrenaline injection. There was no difference in plasma EGF concentration between sham-operated and sialoadenectomized animals receiving saline. After adrenaline administration plasma EGF increased by 20-fold in sham-operated but did not increase in sialoadenectomized mice. Indeed, the increase was much higher (more than 100-fold) in mice receiving exogenous EGF. The effect of adrenaline on plasma concentration of both glycerol and nonesterified fatty acids was higher as lower was plasma EGF concentration. Isolated adipocytes obtained from sham-operated or sialoadenectomized mice had identical lipolytic response to adrenaline. The lipolytic response of adipocytes to isoproterenol was decreased by addition of EGF. To study whether the interference with the in vivo lipolytic effect of adrenaline had further metabolic consequences, we measured plasma b-hydroxybutyrate concentration in plasma. There was no difference in the response to adrenaline between sham-operated and sialoadenectomized mice in spite of the difference in plasma nonsterified fatty acid concentration. Studies in isolated hepatocytes indicated that ketogenesis run at near maximal rate in this range of substrate concentration. These results suggest that EGF in the physiological range decreases the lipolytic effect of adrenaline but does not compromise further metabolic events like the enhancement of ketogenesis.

Finding and characterizing Partially Methylated Domains in human haematopoietic cells

DNA cytosine methylation has been demonstrated to be a central epigenetic modification that has essential roles in a myriad of cellular processes. Some examples of these include gene regulation, DNA-protein interactions, cellular differentiation, X-inactivation, maintenance of genome integrity by suppressing transposable elements and viruses, embryogenesis, genomic imprinting and tumourigenesis. This list is increasingly growing thanks to recent advances in genome-wide technologies, like Whole Genome Bisulfite Sequencing (WGBS-Seq). The development of this technology in research has allowed the identification of new features of the DNA methylation landscape that was not possible using previous technologies, like Partially Methylated Domains (PMDs). PMDs have been found in several cell lines, as well as in both healthy and cancer primary samples. They have been described as regions with high variability in methylation levels across individual CpG sites and intermediate methylation levels on average with respect to the genome. Here, we performed an extensive search of PMDs in a big dataset of different haematopoietic primary cells from both myeloid and lymphoid lineages. We found and characterized significant PMDs in plasma B cells, confirming that PMDs are a phenomenon that is restricted to certain differentiated cells. Additionally, we found loci aberrantly hypomethylated in a myeloma sample which overlapped with plasma B cell PMDs. Genome-wide comparison of the myeloma and plasma B cell sample revealed that this is probably also the case for other loci.

Empowering local democracy in Catalonia: tools and policy domains to implement a top-down solution

This article is the result of an ongoing research into a variety of features of Spanish local government. It aims, in particular, at providing a profile of the tools implemented by local authorities to improve local democracy in Catalonia. The main hypothesis of the work is that, even though the Spanish local model is constrained by a shared and unique set of legal regulations, local institutions in Catalonia have developed their own model of local participation. And the range of instruments like these is still now increasing. More specifically, the scope of this research is twofold. On the one hand, different types of instruments for public deliberation in the Catalan local administration system are identified and presented, based on the place they take in the policy cycle. On the other hand, we focus on policy domains and the quality of the decision-making processes. Researching the stability of the participation tools or whether local democracy prefers more 'ad hoc' processes allows us to analyze the boundaries/limits of local democracy in Catalonia. The main idea underlying this paper is that, despite the existence of a single legal model regulating municipalities in Catalonia, local authorities tend to use their legally granted selfmanagement capacities to design their own instruments which end up presenting perceivable distinct features, stressing democracy in different policy domains, and in diverse policy cycles. Therefore, this paper is intended to identify such models and to provide factors (variables) so that an explanatory model can be built.