A calcium-dependent nuclease from apoptotic rat thymocytes is homologous with cyclophilin : recombinant cyclophilins A, B, and C have nuclease activity

Apoptosis is an important physiological process that involves the deletion of specific cells in a controlled and timely manner. A biochemical hallmark typifying apoptosis in normal lymphocytes is DNA cleavage caused by a calcium-dependent nuclease. We have previously identified and purified an 18-kDa nuclease (NUC18) from glucocorticoid-treated rat thymocytes whose activity is associated with this apoptotic DNA fragmentation. Partial protein sequencing of pure NUC18 has generated two peptide sequences that have a remarkable similarity to rat cyclophilin A and other members of the cyclophilin family

The INFN-grid testbed

The Italian INFN-Grid Project is committed to set-up, run and manage an unprecedented nation-wide Grid infrastructure. The implementation and use of this INFN-Grid Testbed is presented and discussed. Particular care and attention are devoted to those activities, relevant for the management of the Testbed, carried out by the INFN within international Grid Projects

INDIGO-DataCloud: a Platform to Facilitate Seamless Access to E-Infrastructures

[EN] This paper describes the achievements of the H2020 project INDIGO-DataCloud. The project has provided e-infrastructures with tools, applications and cloud framework enhancements to manage the demanding requirements of scientific communities, either locally or through enhanced interfaces. The middleware developed allows to federate hybrid resources, to easily write, port and run scientific applications to the cloud. In particular, we have extended existing PaaS (Platform as a Service) solutions, allowing public and private e-infrastructures, including those provided by EGI, EUDAT, and Helix Nebula, to integrate their existing services and make them available through AAI services compliant with GEANT interfederation policies, thus guaranteeing transparency and trust in the provisioning of such services. Our middleware facilitates the execution of applications using containers on Cloud and Grid based infrastructures, as well as on HPC clusters. 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Role of apoptosis-inducing factor (AIF) in programmed nuclear death during conjugation in Tetrahymena thermophila

<p>Abstract</p> <p>Background</p> <p>Programmed nuclear death (PND), which is also referred to as nuclear apoptosis, is a remarkable process that occurs in ciliates during sexual reproduction (conjugation). In <it>Tetrahymena thermophila</it>, when the new macronucleus differentiates, the parental macronucleus is selectively eliminated from the cytoplasm of the progeny, concomitant with apoptotic nuclear events. However, the molecular mechanisms underlying these events are not well understood. The parental macronucleus is engulfed by a large autophagosome, which contains numerous mitochondria that have lost their membrane potential. In animals, mitochondrial depolarization precedes apoptotic cell death, which involves DNA fragmentation and subsequent nuclear degradation.</p> <p>Results</p> <p>We focused on the role of mitochondrial apoptosis-inducing factor (AIF) during PND in <it>Tetrahymena</it>. The disruption of <it>AIF </it>delays the normal progression of PND, specifically, nuclear condensation and kilobase-size DNA fragmentation. AIF is localized in <it>Tetrahymena </it>mitochondria and is released into the macronucleus prior to nuclear condensation. In addition, AIF associates and co-operates with the mitochondrial DNase to facilitate the degradation of kilobase-size DNA, which is followed by oligonucleosome-size DNA laddering.</p> <p>Conclusions</p> <p>Our results suggest that <it>Tetrahymena </it>AIF plays an important role in the degradation of DNA at an early stage of PND, which supports the notion that the mitochondrion-initiated apoptotic DNA degradation pathway is widely conserved among eukaryotes.</p

Proposed Role for COUP-TFII in Regulating Fetal Leydig Cell Steroidogenesis, Perturbation of Which Leads to Masculinization Disorders in Rodents

Reproductive disorders that are common/increasing in prevalence in human males may arise because of deficient androgen production/action during a fetal ‘masculinization programming window’. We identify a potentially important role for Chicken Ovalbumin Upstream Promoter-Transcription Factor II (COUP-TFII) in Leydig cell (LC) steroidogenesis that may partly explain this. In rats, fetal LC size and intratesticular testosterone (ITT) increased ∼3-fold between e15.5-e21.5 which associated with a progressive decrease in the percentage of LC expressing COUP-TFII. Exposure of fetuses to dibutyl phthalate (DBP), which induces masculinization disorders, dose-dependently prevented the age-related decrease in LC COUP-TFII expression and the normal increases in LC size and ITT. We show that nuclear COUP-TFII expression in fetal rat LC relates inversely to LC expression of steroidogenic factor-1 (SF-1)-dependent genes (StAR, Cyp11a1, Cyp17a1) with overlapping binding sites for SF-1 and COUP-TFII in their promoter regions, but does not affect an SF-1 dependent LC gene (3β-HSD) without overlapping sites. We also show that once COUP-TFII expression in LC has switched off, it is re-induced by DBP exposure, coincident with suppression of ITT. Furthermore, other treatments that reduce fetal ITT in rats (dexamethasone, diethylstilbestrol (DES)) also maintain/induce LC nuclear expression of COUP-TFII. In contrast to rats, in mice DBP neither causes persistence of fetal LC COUP-TFII nor reduces ITT, whereas DES-exposure of mice maintains COUP-TFII expression in fetal LC and decreases ITT, as in rats. These findings suggest that lifting of repression by COUP-TFII may be an important mechanism that promotes increased testosterone production by fetal LC to drive masculinization. As we also show an age-related decline in expression of COUP-TFII in human fetal LC, this mechanism may also be functional in humans, and its susceptibility to disruption by environmental chemicals, stress and pregnancy hormones could explain the origin of some human male reproductive disorders

The Herbicide Atrazine Activates Endocrine Gene Networks via Non-Steroidal NR5A Nuclear Receptors in Fish and Mammalian Cells

Atrazine (ATR) remains a widely used broadleaf herbicide in the United States despite the fact that this s-chlorotriazine has been linked to reproductive abnormalities in fish and amphibians. Here, using zebrafish we report that environmentally relevant ATR concentrations elevated zcyp19a1 expression encoding aromatase (2.2 µg/L), and increased the ratio of female to male fish (22 µg/L). ATR selectively increased zcyp19a1, a known gene target of the nuclear receptor SF-1 (NR5A1), whereas zcyp19a2, which is estrogen responsive, remained unchanged. Remarkably, in mammalian cells ATR functions in a cell-specific manner to upregulate SF-1 targets and other genes critical for steroid synthesis and reproduction, including Cyp19A1, StAR, Cyp11A1, hCG, FSTL3, LHß, INHα, αGSU, and 11ß-HSD2. Our data appear to eliminate the possibility that ATR directly affects SF-1 DNA- or ligand-binding. Instead, we suggest that the stimulatory effects of ATR on the NR5A receptor subfamily (SF-1, LRH-1, and zff1d) are likely mediated by receptor phosphorylation, amplification of cAMP and PI3K signaling, and possibly an increase in the cAMP-responsive cellular kinase SGK-1, which is known to be upregulated in infertile women. Taken together, we propose that this pervasive and persistent environmental chemical alters hormone networks via convergence of NR5A activity and cAMP signaling, to potentially disrupt normal endocrine development and function in lower and higher vertebrates

Combinations of physiologic estrogens with xenoestrogens alter calcium and kinase responses, prolactin release, and membrane estrogen receptor trafficking in rat pituitary cells

<p>Abstract</p> <p>Background</p> <p>Xenoestrogens such as alkylphenols and the structurally related plastic byproduct bisphenol A have recently been shown to act potently via nongenomic signaling pathways and the membrane version of estrogen receptor-α. Though the responses to these compounds are typically measured individually, they usually contaminate organisms that already have endogenous estrogens present. Therefore, we used quantitative medium-throughput screening assays to measure the effects of physiologic estrogens in combination with these xenoestrogens.</p> <p>Methods</p> <p>We studied the effects of low concentrations of endogenous estrogens (estradiol, estriol, and estrone) at 10 pM (representing pre-development levels), and 1 nM (representing higher cycle-dependent and pregnancy levels) in combinations with the same levels of xenoestrogens in GH₃/B6/F10 pituitary cells. These levels of xenoestrogens represent extremely low contamination levels. We monitored calcium entry into cells using Fura-2 fluorescence imaging of single cells. Prolactin release was measured by radio-immunoassay. Extracellular-regulated kinase (1 and 2) phospho-activations and the levels of three estrogen receptors in the cell membrane (ERα, ERβ, and GPER) were measured using a quantitative plate immunoassay of fixed cells either permeabilized or nonpermeabilized (respectively).</p> <p>Results</p> <p>All xenoestrogens caused responses at these concentrations, and had disruptive effects on the actions of physiologic estrogens. Xenoestrogens reduced the % of cells that responded to estradiol via calcium channel opening. They also inhibited the activation (phosphorylation) of extracellular-regulated kinases at some concentrations. They either inhibited or enhanced rapid prolactin release, depending upon concentration. These latter two dose-responses were nonmonotonic, a characteristic of nongenomic estrogenic responses.</p> <p>Conclusions</p> <p>Responses mediated by endogenous estrogens representing different life stages are vulnerable to very low concentrations of these structurally related xenoestrogens. Because of their non-classical dose-responses, they must be studied in detail to pinpoint effective concentrations and the directions of response changes.</p