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Summary Mammalian embryogenesis is a complex process controlled by transcription factors that regulate the balance between pluripotency and differentiation. Transcription factor aryl hydrocarbon receptor (AhR) regulates OCT4/POU5F1 and NANOG, both essential controllers of pluripotency, stemness and early embryo development. Molecular mechanisms controlling OCT4/POU5F1 and NANOG during embryogenesis remain unidentified. We show that AhR regulates pluripotency factors and maintains the metabolic activity required for proper embryo differentiation. AhR-lacking embryos (AhR−/−) showed a pluripotent phenotype characterized by a delayed expression of trophectoderm differentiation markers. Accordingly, central pluripotency factors OCT4/POU5F1 and NANOG were overexpressed in AhR−/− embryos at initial developmental stages. An altered intracellular localization of these factors was observed in the absence of AhR and, importantly, Oct4 had an opposite expression pattern with respect to AhR from the two-cell stage to blastocyst, suggesting a negative regulation of OCT4/POU5F by AhR. We propose that AhR is a regulator of pluripotency and differentiation in early mouse embryogenesis., Highlights • AhR regulates pluripotency factors OCT4 and NANOG during early embryo differentiation • AhR lacking embryos (AhR−/−) show a pluripotent phenotype • Pluripotent phenotype of AhR−/− embryos show enhanced glycolytic metabolism, In this article, Fernández-Salguero and colleagues show that transcription factor AhR regulates pluripotency factors and maintains the metabolic activity required for proper embryo differentiation. AhR-lacking embryos (AhR−/−) showed a pluripotent phenotype characterized by a delayed expression of trophectoderm differentiation markers. Accordingly, central pluripotency factors were overexpressed in AhR−/− embryos at initial developmental stages.

© 2021 The Authors., Skin integrity requires constant maintenance of a quiescent, yet responsive, population of stem cells. While interfollicular epidermal progenitors control normal homeostasis, hair follicle stem cells residing within the bulge provide regenerative potential during hair cycle and in response to wounding. The aryl hydrocarbon receptor (AhR) modulates cell plasticity and differentiation and its overactivation results in severe skin lesions in humans. However, its physiological role in skin homeostasis and hair growth is unknown. Reconstitution assays grafting primary keratinocytes and dermal fibroblasts into nude mice and 3-D epidermal equivalents revealed a positive role for AhR in skin regeneration, epidermal differentiation, and stem cell maintenance. Furthermore, lack of receptor expression in AhR−/− mice delayed morphogenesis and impaired hair regrowth with a phenotype closely correlating with a reduction in suprabasal bulge stem cells (α6lowCD34+). Moreover, RNA-microarray and RT-qPCR analyses of fluorescence-activated cell sorting (FACS)-isolated bulge stem cells revealed that AhR depletion impaired transcriptional signatures typical of both epidermal progenitors and bulge stem cells but upregulated differentiation markers likely compromising their undifferentiated phenotype. Altogether, our findings support that AhR controls skin regeneration and homeostasis by ensuring epidermal stem cell identity and highlights this receptor as potential target for the treatment of cutaneous pathologies., This work was supported by grants to P.M.F.S. from the Spanish Ministry of Economy and Competitiveness (SAF2017-82597-R and PID2020-114846RB-I00) and from the Junta de Extremadura (GR18006, IB160210, and IB20014). E.M.R.L. was supported by the RTICC and the Junta de Extremadura. All Spanish funding is cosponsored by the European Union FEDER program.

Aging impairs organismal homeostasis leading to multiple pathologies. Yet, the mechanisms and molecular intermediates involved are largely unknown. Here, we report that aged aryl hydrocarbon receptor-null mice (

Mammalian embryogenesis is a complex process controlled by transcription factors that dynamically regulate the balance between pluripotency and differentiation. Transcription factor AhR is known to regulate Oct4/Pou5f1 and Nanog, both essential genes in pluripotency, stemness and early embryo development. Yet, the molecular mechanisms controlling Oct4/Pou5f1 and Nanog during embryo development remain largely unidentified. Here, we show that AhR is required for proper embryo differentiation by regulating pluripotency factors and by maintaining adequate metabolic activity. AhR lacking embryos (AhR-/-) showed a more pluripotent phenotype characterized by a delayed expression of differentiation markers of the first and second cell divisions. Accordingly, central pluripotency factors OCT4/POU5F1, NANOG, and SOX2 were overexpressed in AhR-/- embryos at initial developmental stages. An altered intracellular localization of these factors was observed in absence of AhR and, importantly, OCT4 had an opposite expression pattern with respect to AhR from the 2-cell stage to blastocyst, suggesting a negative regulatory mechanism of OCT4/POU5F by AhR. Hippo signalling, rather than being repressed, was upregulated in very early AhR-/- embryos, possibly contributing to their undifferentiation at later stages. Consistently, AhR-null blastocysts overexpressed the early marker of inner cell mass (ICM) differentiation Sox17 whereas downregulated extraembryonic differentiation-driving genes Cdx2 and Gata3. Moreover, the persistent pluripotent phenotype of AhR-/- embryos was supported by an enhanced glycolytic metabolism and a reduction in mitochondrial activity. We propose that AhR is a regulator of pluripotency and differentiation in early mouse embryogenesis and that its deficiency may underline the reduced viability and increased resorptions of AhR-null mice.

© The Author(s) 2020., Transcriptional repression of Nanog is an important hallmark of stem cell differentiation. Chromatin modifications have been linked to the epigenetic profile of the Nanog gene, but whether chromatin organization actually plays a causal role in Nanog regulation is still unclear. Here, we report that the formation of a chromatin loop in the Nanog locus is concomitant to its transcriptional downregulation during human NTERA-2 cell differentiation. We found that two Alu elements flanking the Nanog gene were bound by the aryl hydrocarbon receptor (AhR) and the insulator protein CTCF during cell differentiation. Such binding altered the profile of repressive histone modifications near Nanog likely leading to gene insulation through the formation of a chromatin loop between the two Alu elements. Using a dCAS9-guided proteomic screening, we found that interaction of the histone methyltransferase PRMT1 and the chromatin assembly factor CHAF1B with the Alu elements flanking Nanog was required for chromatin loop formation and Nanog repression. Therefore, our results uncover a chromatin-driven, retrotransposon-regulated mechanism for the control of Nanog expression during cell differentiation., This work was supported by grants to P.M.F-S. from the Spanish Ministry of Economy and Competitiveness (SAF2014-51813-R and SAF2017-82597-R) and from the Junta de Extremadura (GR15008 and IB160210). Research at P.M.F-S. laboratory was also funded by the Red Temática de Investigación Cooperativa en Cáncer (RTICC), Carlos III Institute, Spanish Ministry of Economy and Competitiveness (RD12/0036/0032). A.C.R. was funded by a long-term FEBS Fellowship. L.M. work was funded by MINECO Grants BIO2012-39980 and BIO2015-70978-R. All Spanish funding is co-sponsored by the European Union FEDER program.

The aryl hydrocarbon receptor (AhR) is well-known for its major contributions to the cellular responses against environmental toxins and carcinogens. Notably, AhR has also emerged as a key transcription factor controlling many physiological processes including cell proliferation and apoptosis, differentiation, adhesion and migration, pluripotency and stemness. These novel functions have broadened our understanding of the signalling pathways and molecular intermediates interacting with AhR under both homeostatic and pathological conditions. Recent discoveries link AhR with the function of essential organs such as liver, skin and gonads, and with complex organismal structures including the immune and cardiovascular systems. The identification of potential endogenous ligands able to regulate AhR activity, opens the possibility of designing ad hoc molecules with pharmacological and/or therapeutic value to treat human diseases in which AhR may have a causal role. Integration of experimental data from in vitro and in vivo studies with "omic" analyses of human patients affected with cancer, immune diseases, inflammation or neurological disorders will likely contribute to validate the clinical relevance of AhR and the possible benefits of modulating its activity by pharmacologically-driven strategies. In this review, we will highlight signalling pathways involved in human diseases that could be targetable by AhR modulators and discuss the feasibility of using such molecules in therapy. The pros and cons of AhR-aimed approaches will be also mentioned.

Non-small cell lung adenocarcinoma (NSCLC) bearing K-RasG12D mutations is one of the most prevalent types of lung cancer worldwide. Aryl hydrocarbon receptor (AHR) expression varies in human lung tumors and has been associated with either increased or reduced lung metastasis. In the mouse, Ahr also adjusts lung regeneration upon injury by limiting the expansion of resident stem cells. Here, we show that the loss of Ahr enhances K-RasG12D-driven NSCLC in mice through the amplification of stem cell subpopulations. Consistent with this, we show that K-RasG12D, Ahr−/− lungs contain larger numbers of cells expressing markers for both progenitor Clara (SCGB1A1 and CC10) and alveolar type-II (SFTPC) cells when compared to K-RasG12D, Ahr+/+-driven tumors. They also have elevated numbers of cells positive for pluripotent stem cells markers such as SOX2, ALDH1, EPCAM, LGR5 and PORCN. Typical pluripotency genes Nanog, Sox2 and c-Myc were also upregulated in K-RasG12D, Ahr−/− lung tumors as found by RNAseq analysis. In line with this, purified K-RasG12D/+, Ahr−/− lung cells generate larger numbers of organoids in culture that can subsequently differentiate into bronchioalveolar structures enriched in both pluripotency and stemness genes. Collectively, these data indicate that Ahr antagonizes K-RasG12D-driven NSCLC by restricting the number of cancer-initiating stem cells. They also suggest that Ahr expression might represent a good prognostic marker to determine the progression of K-RasG12D-positive NSCLC patients.

The aryl hydrocarbon receptor (AhR) has roles in cell proliferation, differentiation and organ homeostasis, including the liver. AhR depletion induces undifferentiation and pluripotency in normal and transformed cells. Here, AhR-null mice (AhR−/−) were used to explore whether AhR controls liver regeneration and carcinogenesis by restricting the expansion of stem-like cells and the expression of pluripotency genes. Short-term CCl4 liver damage was earlier and more efficiently repaired in AhR−/− than in AhR+/+ mice. Stem-like CK14 + and TBX3 + and pluripotency-expressing OCT4 + and NANOG + cells expanded sooner in AhR−/− than in AhR+/+ regenerating livers. Stem-like side population cells (SP) isolated from AhR−/− livers had increased β-catenin (β-Cat) signaling with overexpression of Axin2, Dkk1 and Cyclin D1. Interestingly, β-Cat, Axin2 and Dkk1 also increased during regeneration but more notably in AhR-null livers. Liver carcinogenesis induced by diethylnitrosamine (DEN) produced large carcinomas in all AhR−/− mice but mostly premalignant adenomas in less than half of AhR+/+ mice. AhR-null tumoral tissue, but not their surrounding non-tumoral parenchyma, had nuclear β-Cat and Axin2 overexpression. OCT4 and NANOG were nevertheless similarly expressed in AhR+/+ and AhR−/− lesions. We suggest that AhR may serve to adjust liver repair and to block tumorigenesis by modulating stem-like cells and β-Cat signaling.

Summary Aryl hydrocarbon receptor (AhR) deficiency alters tissue homeostasis. However, how AhR regulates organ maturation and differentiation remains mostly unknown. Liver differentiation entails a polyploidization process fundamental for cell growth, metabolism, and stress responses. Here, we report that AhR regulates polyploidization during the preweaning-to-adult mouse liver maturation. Preweaning AhR-null (AhR−/−) livers had smaller hepatocytes, hypercellularity, altered cell cycle regulation, and enhanced proliferation. Those phenotypes persisted in adult AhR−/− mice and correlated with compromised polyploidy, predominance of diploid hepatocytes, and enlarged centrosomes. Phosphatidylinositol-3-phosphate kinase (PI3K), extracellular signal-regulated kinase (ERK), and Wnt/β-catenin signaling remained upregulated from preweaning to adult AhR-null liver, likely increasing mammalian target of rapamycin (mTOR) activation. Metabolomics revealed the deregulation of mitochondrial oxidative phosphorylation intermediates succinate and fumarate in AhR−/− liver. Consistently, PI3K, ERK, and Wnt/β-catenin inhibition partially rescued polyploidy in AhR−/− mice. Thus, AhR may integrate survival, proliferation, and metabolism for liver polyploidization. Since tumor cells tend to be polyploid, AhR modulation could have therapeutic value in the liver., Graphical Abstract, Highlights • AhR is required for liver polyploidization during preweaning-to-adult transition • INS-R/PI3K/AKT, ERK, Wnt/β-Cat and mTOR are downregulated during liver polyploidization • Reduced polyploidy relates with enhanced mitochondrial metabolism in AhR-null liver • Understanding how AhR modulates polyploidy may provide strategies against cancer, Developmental Biology; Cancer Systems Biology; Metabolomics

Recent experimental evidences from cellular systems and from mammalian and non-mammalian animal models highlight novel functions for the aryl hydrocarbon/dioxin receptor (AhR) in maintaining cell differentiation and tissue homeostasis. Notably, AhR depletion stimulates an undifferentiated and pluripotent phenotype likely associated to a mesenchymal transition in epithelial cells and to increased primary tumorigenesis and metastasis in melanoma. In this work, we have used a lung model of epithelial regeneration to investigate whether AhR regulates proper tissue repair by adjusting the expansion of undifferentiated stem-like cells. AhR-null mice developed a faster and more efficient repair of the lung bronchiolar epithelium upon naphthalene injury that required increased cell proliferation and the earlier activation of stem-like Clara, Basal and neuroepithelial cells precursors. Increased basal content in multipotent Sca1/CD31/CD4 cells and in cells expressing pluripotency factors NANOG and OCT4 could also improve re-epithelialization in AhR-null lungs. The reduced response of AhR-deficient lungs to Sonic Hedgehog (Shh) repression shortly after injury may also help their improved bronchiolar epithelium repair. These results support a role for AhR in the regenerative response against toxins, and open the possibility of modulating its activation level to favor recovery from lesions caused by environmental contaminants., This work was supported by grants to P.M.F-S. from the Spanish Ministry of Economy and Competitiveness (SAF2014-51813-R) and from the Junta de Extremadura (GR15008 and IB16210). Research at P.M.F-S. laboratory was also funded by the Red Temática de Investigación Cooperativa en Cáncer (RTICC), Carlos III Institute, Spanish Ministry of Economy and Competitiveness (RD12/0036/0032). A.M.-H. was supported by the Junta de Extremadura and N.M.-M. and A.N.-P. by the Ministerio de Economía y Competitividad. B.P. was supported by Junta de Extremadura. All Spanish funding is co-sponsored by the European Union FEDER program.

Previous studies suggested that the aryl hydrocarbon receptor (AhR) contributes to mice reproduction and fertility. However, the mechanisms involved remain mostly unknown. Retrotransposon silencing by Piwi-interacting RNAs (piRNAs) is essential for germ cell maturation and, remarkably, AhR has been identified as a regulator of murine B1-SINE retrotransposons. Here, using littermate AhR and AhR mice, we report that AhR regulates the general course of spermatogenesis and oogenesis by a mechanism likely to be associated with piRNA-associated proteins, piRNAs and retrotransposons. piRNA-associated proteins MVH and Miwi are upregulated in leptotene to pachytene spermatocytes with a more precocious timing in AhR than in AhR testes. piRNAs and transcripts from B1-SINE, LINE-1 and IAP retrotransposons increased at these meiotic stages in AhR-null testes. Moreover, B1-SINE transcripts colocalize with MVH and Miwi in leptonema and pachynema spermatocytes. Une pectedly, AhR males have increased sperm counts, higher sperm functionality and enhanced fertility than AhR mice. In contrast, piRNA-associated proteins and B1-SINE and IAP-derived transcripts are reduced in adult AhR2/2 ovaries. Accordingly, AhR-null female mice have lower numbers of follicles when compared with AhR\+/\+ mice. Thus, AhR deficiency differentially affects testis and ovary development possibly by a process involving piRNA-associated proteins, piRNAs and transposable elements., This work was supported by grants to P.M.F-S. from the Spanish Ministry of Science and Innovation (BFU2011-22678) and Economy and Competitiveness (SAF2014-51813-R) and from the Junta de Extremadura (GR15008). Research at the F.J.P. laboratory was supported by grant no. AGL2013-43211-R from the Spanish Ministry of Economy and Competitiveness. Research at the P.M.F-S laboratory was also supported by the Red Tematica de Investigacion Cooperativa en Cancer (RTICC), Carlos III Institute, Spanish Ministry of Economy and Competitiveness (RD12/0036/0032). E.R.L., F.J.G-R and E.B. were supported by the RTICC, MICINN and Junta de Extremadura, respectively. N.M.M. was an FPI fellow of the Spanish Ministry of Education. P.M.M. was supported by a pre-doctoral grant from the Spanish Ministry of Education, Culture and Sport (FPU13/03991) C.O.F. was supported by a postdoctoral Juan de la Cierva (IJCI-2014-21671) grant from the Spanish Ministry of Economy and Competitiveness. A.M.P. and E.L. were supported by grants from the Spanish Ministry of Economy and Competitiveness (BFU2014-59307-R), MEIONet and the Junta de Castilla y Leon. All Spanish funding is co-sponsored by the European Union FEDER programme.

Cell differentiation is a central process in development and in cancer growth and dissemination. OCT4 (POU5F1) and NANOG are essential for cell stemness and pluripotency; yet, the mechanisms that regulate their expression remain largely unknown. Repetitive elements account for almost half of the Human Genome; still, their role in gene regulation is poorly understood. Here, we show that the dioxin receptor (AHR) leads to differentiation of human carcinoma cells through the transcriptional upregulation of Alu retrotransposons, whose RNA transcripts can repress pluripotency genes. Despite the genome-wide presence of Alu elements, we provide evidences that those located at the NANOG and OCT4 promoters bind AHR, are transcribed by RNA polymerase-III and repress NANOG and OCT4 in differentiated cells. OCT4 and NANOG repression likely involves processing of Alu-derived transcripts through the miRNA machinery involving the Microprocessor and RISC. Consistently, stable AHR knockdown led to basal undifferentiation, impaired Alus transcription and blockade of OCT4 and NANOG repression. We suggest that transcripts produced from AHR-regulated Alu retrotransposons may control the expression of stemness genes OCT4 and NANOG during differentiation of carcinoma cells. The control of discrete Alu elements by specific transcription factors may have a dynamic role in genome regulation under physiological and diseased conditions., Ministerio de Economía y Competitividad [BFU2011-22678, SAF2014-51813-R to P.M.F-S.]; Junta de Extremadura [GR10008, GR15008]; Red Temática de Investigación Cooperativa en Cáncer (RTICC); Carlos III Institute; Spanish Ministry of Economy and Competitiveness [RD12/0036/0032]; FPI Fellowship from the Junta de Extremadura (to A.M.H.); Marie Curie IRG project (FP7-PEOPLE-2007-4-3-IRG: SOMATIC LINE-1, in part to A.M.); CICE-FEDER-P09-CTS-4980, CICE-FEDER-P12-CTS-2256, Plan Nacional de I+D+I 2008–2011 and 2013–2016 (FIS-FEDER-PI11/01489, FIS-FEDER-PI14/02152), PCIN-2014-115-ERA-NET NEURON II (to J.L.G.P.); European Research Council [ERC-Consolidator ERC-STG-2012-233764]; International Early Career Scientist grant from the Howard Hughes Medical Institute [IECS-55007420]; European Union FEDER program. Funding for open access charge: Ministerio de Economía y Competitividad [BFU2011-22678, SAF2014-51813-R to P.M.F-S.].

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a xenobiotic agent with high persistency that induces neurotoxic effects altering neurodevelopment and behavior. The molecular mechanisms and the signaling pathways involved in TCDD-mediated neurotoxicity, together with the search of its molecular targets in neurons are under intense study. We have previously shown that high nanomolar concentrations of TCDD for incubation times of minutes induce apoptosis in SHSY5Y human neuroblastoma cells by the disruption of calcium homeostasis, affecting membrane structural integrity. In this work, we have analyzed the effect of low nanomolar concentrations of TCDD for incubation times of hours to define the role of aryl hydrocarbon receptor which can be activated at those concentrations. TCDD induces toxicity in SHSY5Y human neuroblastoma cells under these experimental conditions with an EC50 value of approximately 3nM at 24h of incubation time. Transient transfection of a hairpin RNA for AhR protects against TCDD neurotoxicity, suggesting that AhR is mediating the dioxin effect. Altogether, these results support the hypothesis that TCDD toxicity in SHSY5Y neuroblastoma cells depends on dioxin concentration and time of incubation, with a main role of aryl hydrocarbon receptor at low nanomolar TCDD concentrations.

The persistent xenobiotic agent 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces neurotoxic effects that alters neurodevelopment and behavior both during development and adulthood. There are many ongoing efforts to determine the molecular mechanisms of TCDD-mediated neurotoxicity, the signaling pathways involved and its molecular targets in neurons. In this work, we have used SHSY5Y human neuroblastoma cells to characterize the TCDD-induced toxicity. TCDD produces a loss of viability linked to an increased caspase-3 activity, PARP-1 fragmentation, DNA laddering, nuclear fragmentation and hypodiploid (apoptotic) DNA content, in a similar way than staurosporine, a prototypical molecule of apoptosis induction. In addition, TCDD produces a decrease of mitochondrial membrane potential and an increase of intracellular calcium concentration (P < 0.05). Finally, based on the high lipophilic properties of the dioxin, we test the TCDD effect on the membrane integrity using sarcoplasmic reticulum vesicles as a model. TCDD produces calcium efflux through the membrane and an anisotropy decrease (P < 0.05) that reflects an increase in membrane fluidity. Altogether these results support the hypothesis that TCDD toxicity in SHSY5Y neuroblastoma cells provokes the disruption of calcium homeostasis, probably affecting membrane structural integrity, leading to an apoptotic process.

J. Neurochem. (2011) 10.1111/j.1471-4159.2011.07291.x Abstract 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a prototypical environmental contaminant with neurotoxic properties that alters neurodevelopment and behavior. TCDD is a ligand of the aryl hydrocarbon receptor (AhR), which is a key signaling molecule to fully understand the toxic and carcinogenic properties of dioxin. Much effort is underway to unravel the molecular mechanisms and the signaling pathways involved in TCDD-induced neurotoxicity, and to define its molecular targets in neurons. We have used cerebellar granule cells (CGC) from wild-type (AhR+/+) and AhR-null (AhR−/−) mice to characterize the cell death that takes place in neurons after TCDD toxicity. TCDD induced cell death in CGC cultures from wild-type mice with an EC50 of 127 ± 21 nM. On the contrary, when CGC neurons from AhR-null mice were treated with TCDD no significant cell death was observed. The role of AhR in TCDD-induced death was further assessed by using the antagonists resveratrol and α-naphtoflavone, which readily protected against TCDD toxicity in AhR+/+ CGC cultures. AhR+/+ CGC cultures treated with TCDD showed nuclear fragmentation, DNA laddering, and increased caspase 3 activity, similarly to what was found by the use of staurosporine, a well-established inducer of apoptosis. Finally, the AhR pathway was active in CGC because TCDD could induce the expression of the target gene cytochrome P450 1A2 in AhR+/+ CGC cultures. All together these results support the hypothesis that TCDD toxicity in CGC neurons involves the AhR and that it takes place mainly through an apoptotic process. AhR could be then considered a novel target in neurotoxicity and neurodegeneration whose down-modulation could block certain xenobiotic-related adverse effects in CNS.

13 páginas, 12 figuras.-- et al., The dioxin receptor (AhR) modulates cell plasticity and migration, although the signaling involved remains unknown. Here, we report a mechanism that integrates AhR into these cytoskeleton-related functions. Immortalized and mouse embryonic fibroblasts lacking AhR (AhR-/-) had increased cell area due to spread cytoplasms that reverted to wild-type morphology upon AhR re-expression. The AhR-null phenotype included increased F-actin stress fibers, depolarized focal adhesions, and enhanced spreading and adhesion. The cytoskeleton alterations of AhR- /- cells were due to down-regulation of constitutive Vav3 expression, a guanosine diphosphate/guanosine triphosphate exchange factor for Rho/Rac GTPases and a novel transcriptional target of AhR. AhR was recruited to the vav3 promoter and maintained constitutive mRNA expression in a ligand-independent manner. Consistently, AhR-/- fibroblasts had reduced Racl activity and increased activation of the RhoA/Rho kinase (Rock) pathway. Pharmacological inhibition of Racl shifted AhR+/+ fibroblasts to the null phenotype, whereas Rock inhibition changed AhR-null cells to the AhR+/+ morphology. Knock-down of vav3 transcripts by small interfering RNA induced cytoskeleton defects and changes in adhesion and spreading mimicking those of AhR-null cells. Moreover, vav3-/- MEFs, as AhR-/- mouse embryonic fibroblasts, had increased cell area and enhanced stress fibers. By modulating Vav3-dependent signaling, AhR could regulate cell shape, adhesion, and migration under physiological conditions and, perhaps, in certain pathological states. © 2009 by The American Society for Cell Biology., This work was supported by grants to P.M.F.-S. from the Spanish Ministry of Education and Sciences (SMES) (SAF2005-00130, SAF2008-0462), the Junta de Extremadura (2PR04A060) and the Red Temática de Investigación Cooperativa en Cáncer (RTICC) (RD06/0020/1016, Fondo de Investigaciones Sanitarias (FIS), Carlos III Institute, Spanish Ministry of Health) and by grants to X.R.B. from the US National Cancer Institute/NIH (5R01-CA73735-11), the Spanish Ministry of Education and Science (MES) (SAF2006-01789), the Castilla-León Autonomous Government (SA053A05), and the Red Temática de Investigación Cooperativa en Cáncer (RTICC) (RD06/0020/0001, Fondo de Investigaciones Sanitarias (FIS), Carlos III Institute, Spanish Ministry of Health). J.M.C.-G. and A.C.R. were supported by fellowships from the Junta de Extremadura and the SMES, respectively. V. S. has been partially supported by the SMES Juan de la Cierva program and the National Institutes of Health. All Spanish funding is cosponsored by the European Union FEDER program.

N-Methyl-d-aspartate (NMDA) receptors play a critical role in the brain stimulating synaptic plasticity and mediating neurodegeneration; a neuroprotective role has also been described, but its molecular mechanisms in hippocampus are under study. Here, we report that in primary cultures of rat hippocampal neurons exposure to low micromolar NMDA concentrations are neuroprotective against excitotoxic insults, while high micromolar NMDA concentrations provoke neuronal death. Molecular analysis reveals that a toxic concentration of NMDA induced a transient phosphorylation of cAMP-response element-binding protein (pCREB) in 2 min that rapidly decreased below basal levels. In contrast, a nontoxic NMDA concentration gave up to longer (20 min) rise of pCREB, suggesting that neuroprotection could be associated to a relatively prolonged presence of pCREB in the neurons. In support of this tenet, rolipram, an inhibitor of phosphodiesterase IV that increases the levels of cAMP and pCREB, protected against NMDA-induced neuronal death. Similar results were obtained with dibutyrate-cAMP (a cAMP analogue with membrane permeability) that also abrogated NMDA excitotoxicity. Conversely, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline sulfonamide (H89), an inhibitor of protein kinase A (PKA), that prevents the formation of pCREB induced by nontoxic NMDA concentrations, reverted the neuroprotection achieved by preincubation of low micromolar NMDA concentrations. These results substantiate the notion that induction of pCREB via PKA plays an important role in NMDA-mediated neuroprotection.

[Background] The dioxin (AhR) receptor can have oncogenic or tumor suppressor activities depending on the phenotype of the target cell. We have shown that AhR knockdown promotes melanoma primary tumorigenesis and lung metastasis in the mouse and that human metastatic melanomas had reduced AhR levels with respect to benign nevi., [Methods] Mouse melanoma B16F10 cells were engineered by retroviral transduction to stably downregulate AhR expression, Aldh1a1 expression or both. They were characterized for Aldh1a1 activity, stem cell markers and migration and invasion in vitro. Their tumorigenicity in vivo was analyzed using xenografts and lung metastasis assays as well as in vivo imaging., [Results] Depletion of aldehyde dehydrogenase 1a1 (Aldh1a1) impairs the pro-tumorigenic and pro-metastatic advantage of melanoma cells lacking AhR expression (sh-AhR). Thus, Aldh1a1 knockdown in sh-AhR cells (sh-AhR + sh-Aldh1a1) diminished their migration and invasion potentials and blocked tumor growth and metastasis to the lungs in immunocompetent AhR+/+ recipient mice. However, Aldh1a1 downmodulation in AhR-expressing B16F10 cells did not significantly affect tumor growth in vivo. Aldh1a1 knockdown reduced the high levels of CD133 + /CD29 + /CD44 + cells, melanosphere size and the expression of the pluripotency marker Sox2 in sh-AhR cells. Interestingly, Sox2 increased Aldh1a1 expression in sh-AhR but not in sh-AhR + sh-Aldh1a1 cells, suggesting that Aldh1a1 and Sox2 may be co-regulated in melanoma cells. In vivo imaging revealed that mice inoculated with AhR + Aldh1a1 knockdown cells had reduced tumor burden and enhanced survival than those receiving Aldh1a1-expressing sh-AhR cells., [Conclusions] Aldh1a1 overactivation in an AhR-deficient background enhances melanoma progression. Since AhR may antagonize the protumoral effects of Aldh1a1, the AhR low -Aldh1a1 high phenotype could be indicative of bad outcome in melanoma., This work was supported by grants to P.M.F-S. from the Spanish Ministry of Economy and Competitiveness (BFU2011-22678 and SAF2014-51813-R) and from the Gobierno de Extremadura (GR10008) and to J.M.M. from the Agencia Extremeña de Cooperación Internacional para el Desarrollo (AEXCID-13IA002, Gobierno de Extremadura). Research at P.M.F-S and F.J.O. laboratories has been also funded by the Red Temática de Investigación Cooperativa en Cáncer (RTICC), Fondo de Investigaciones Sanitarias (FIS), Carlos III Institute, Spanish Ministry of Health (RD12/0036/0032 and RD12/0036/0026, respectively). M.C.T. was a F.P.I. fellow from the Spanish Ministry of Education and Sciences. All Spanish funding is co-sponsored by the European Union FEDER program. The support and help of the Servicio de Técnicas Aplicadas a las Biociencia (STAB) of the Universidad de Extremadura is greatly acknowledged.

Synthetic peptides patterned after the C-terminus of synaptosomal associated protein of 25 kDa (SNAP25) efficiently abrogate regulated exocytosis. In contrast, the use of SNAP25 N-terminal-derived peptides to modulate SNAP receptors (SNARE) complex assembly and neurosecretion has not been explored. Here, we show that the N-terminus of SNAP25, specially the segment that encompasses 22Ala-44Ile, is essential for the formation of the SNARE complex. Peptides patterned after this protein domain are potent inhibitors of SNARE complex formation. The inhibitory activity correlated with their propensity to adopt an alpha-helical secondary structure. These peptides abrogated SNARE complex formation only when added previous to the onset of aggregate assembly. Analysis of the mechanism of action revealed that these peptides disrupted the binary complex formed by SNAP25 and syntaxin. The identified peptides inhibited Ca2+-dependent exocytosis from detergent-permeabilized excitable cells. Noteworthy, these amino acid sequences markedly protected intact hippocampal neurones against hypoglycaemia-induced, glutamate-mediated excitotoxicity with a potency that rivalled that displayed by botulinum neurotoxins. Our findings indicate that peptides patterned after the N-terminus of SNAP25 are potent inhibitors of SNARE complex formation and neuronal exocytosis. Because of their activity in intact neurones, these cell permeable peptides may be hits for antispasmodic and analgesic drug development.

Nitric oxide (NO) is responsible for cytochrome P450 (CYP450) loss during isolation and cytokine treatment of primary rat hepatocytes. As P450s mediate the metabolism of toxic chemicals, their inhibition could compromise the cells competence to eliminate toxins, a condition potentially relevant in neurological diseases involving constitutive activation of nitric oxide synthase (NOS) and NO over-production. Here, we have investigated the correlation between NO accumulation and CYP1A2 down-regulation during maturation of mouse cerebellar granule cells (CGC). As neurons matured in culture, the inducible levels of CYP1A2 protein and catalytic activity decreased to almost undetectable values. In parallel, a significant increase in NO concentration was observed. Neuronal NOS remained constitutively active during maturation, thus contributing to NO accumulation. The NOS inhibitor l-NAME, restored CYP1A2 catalytic activity up to 9 days in vitro, supporting a role for NO in the inhibition process. Maturation was also followed by increased NMDA receptor activity and intracellular Ca2+ concentration. We suggest that maintained NOS activity during CGC maturation could lead to NO accumulation and to decreased CYP1A2 inducibility. Increased NMDA receptor activity and Ca2+ entry could contribute to this process. Thus, neurodegeneration could diminish the induction of specific P450s and impair the metabolism of foreign and/or endogenous chemicals in the CNS.

11 pages, 9 figures, 1 table.-- PMID: 12065713 [PubMed]., Excitotoxicity has been implicated in the etiology of ischemic stroke, chronic neurodegenerative disorders, and very recently, in glioma growth. Thus, the development of novel neuroprotectant molecules that reduce excitotoxic brain damage is vigorously pursued. We have used an ionic current block-based cellular assay to screen a synthetic combinatorial library of trimers of N-alkylglycines on the N-methyl-D-aspartate (NMDA) receptor, a well known molecular target involved in excitotoxicity. We report the identification of a family of N-alkylglycines that selectively blocked the NMDA receptor. Notably, compound 3,3-diphenylpropyl-N-glycinamide (referred to as N20C) inhibited NMDA receptor channel activity with micromolar affinity, fast on-off blockade kinetics, and strong voltage dependence. Molecule N20C did not act as a competitive glutamate or glycine antagonist. In contrast, saturation of the blocker binding site with N20C prevented dizolcipine (MK-801) blockade of the NMDA receptor, implying that both drugs bind to the same receptor site. The N-alkylglycine efficiently prevented in vitro excitotoxic neurodegeneration of cerebellar and hippocampal neurons in culture. Attenuation of neuronal glutamate/NMDA-induced Ca(2+) overload and subsequent modulation of the glutamate-nitric oxide-cGMP pathway seems to underlie N20C neuroprotection. Noteworthy, this molecule exhibited significant in vivo neuroprotectant activity against an acute, severe, excitotoxic insult. Taken together, these findings indicate that N-alkylglycine N20C is a novel, low molecular weight, moderate-affinity NMDA receptor open channel blocker with in vitro and in vivo neuroprotective activity, which, in due turn, may become a tolerated drug for the treatment of neurodegenerative diseases and cancer., This work was supported by grants from La Fundación La Caixa (to A.F.-M.), Fundació La Marató de TV3 (to A.M. and V.F.), the Spanish Interministerial Commission of Science (Centro de Investigación Cientı́fica y Tecnológica) and Technology and the European Commission (to A.F.-M. and E.P.-P.), and the Centro de Investigación Cientı́fica y Tecnológica (to A.F.-M. and A.M.).

We have examined the effect of diabetes and pharmacological insulin treatment on the content of glycogen phosphorylase and glycogen associated with the sarcoplasmic reticulum–glycogenolytic complex from rat skeletal muscle. Diabetes was induced in rats by streptozotocin injection. Enzymatic activities were measured using spectrophotometric methods. Glycogen phosphorylase was determined measuring the pyridoxal-5' -phosphate content and using polyacrylamide gel electrophoresis. Glycogen content was measured by enzymatic and the phenol sulfuric methods. The content of glycogen phosphorylase associated with the sarcoplasmic reticulum glycogenolytic complex gradually arises after diabetes induction. The content of glycogen phosphorylase was restored to a control value by pharmacological insulin treatment. In addition, the content of glycogen in preparations of sarcoplasmic reticulum–glycogenolytic complex of diabetic animals was also increased, whereas the content of glycogen in total muscle of diabetic rats was similar to that of the control rats. The absolute and relative amount of glycogen associated with sarcoplasmic reticulum seemed to increase in diabetic animals. These effects on the compartmentalisation of glycogen were suppressed by insulin treatment. Additionally, the rate of conversion of glycogen phosphorylase b to a, an index of the phosphorylase kinase activity, was 50 % lower in diabetic rats, increasing the dephosphorylated form of glycogen phosphorylase and, as a consequence, its association with sarcoplasmic reticulum membranes. These results suggest that under diabetic conditions, both glycogen phosphorylase and a small percentage of muscle glycogen are relocalized in the sarcoplasmic reticulum–glycogenolytic complex. [Diabetologia (2001) 44: 1238–1246]

The Ca(2+)-ATPase from sarcoplasmic reticulum (SR) membranes couples the Ca(2+) transport to ATP hydrolysis through phosphorylation in its cytoplasmic catalytic domain. Interactions between protein domains and the role of monomer-monomer interactions remain unclear. Here, we report a differential scanning calorimetric study of the thermal unfolding of this protein. In the pH range 6-8, thermal unfolding of the Ca(2+)-ATPase in glycogen phosphorylase-free SR membranes shows a major endothermic peak with a critical temperature midpoint ranging between 51 and 55 degrees C, depending on pH, Ca(2+), Mg(2+)-ADP and KCl concentrations. The enthalpy change of the overall unfolding process ranged between 250 and 300 kcal/mol of Ca(2+)-ATPase monomer. Thermal denaturation of the Ca(2+)-ATPase in SR membranes is well fitted to an irreversible process that can be rationalized in terms of a non-two state process, N (native)right harpoon over left harpoon I (intermediate)-->D (denatured). Thermodynamic analysis show that this protein has a compact structure, implying a tight structural interconnection between catalytic and Ca(2+) transport domains. The apparent cooperative unit, defined by the van 't Hoff enthalpy to the overall unfolding enthalpy ratio, increased from 1.1 at pH 6 to 1.8 at pH 8, showing that monomer-monomer interactions are stronger at weakly basic pH than at weakly acidic pH. While micromolar Ca(2+) concentrations had only a weak effect on the cooperativity of the unfolding process, this is clearly increased by millimolar Mg(2+)-ADP. In addition, high ionic strength lowered the apparent cooperative unit to approximately 1.0 in the pH range 6-8. Taken together, these results suggest that protein-protein interactions are altered by variables that modulate the catalytic activity of this enzyme.

The Ca 2+ ,Mg 2+ -ATPase from sarcoplasmic reticulum couples ATP hydrolysis to Ca 2+ transport toward the lumen of the muscular vesicular system. Combined structural and functional studies suggest that the Ca 2+ binding sites are formed by six amino acids of the same polypeptide and that cation translocation may take place through a channel inside a monomer of the ATPase. However, calorimetric, fluorescent, and kinetic studies suggest that the ATPase may assemble into functional oligomers of as yet unknown stoichiometry. We have addressed this question and attempted to determine the ATPase stoichiometry using a biophysical approach based on the analysis of the ATPase inhibition by fluorescein 5′-isothiocyanate in the presence of increasing ATP concentrations. For native SR membranes, our inhibition data are well described by a model consisting of two interacting nucleotide-binding sites per oligomer. This stoichiometry was disrupted in detergent C 12 E 8 -solubilized ATPase. Thus, these findings suggest that interacting nucleotide binding sites of the ATPase may appear as dimers, and imply that interactions of the globular cytoplasmic domains would play a modulatory role of the protein enzymatic activity.

Excitotoxic neuronal death, associated with neurodegeneration and stroke, is triggered primarily by massive Ca2+ influx arising from overactivation of glutamate receptor channels of the N-methyl-D-aspartate (NMDA) subtype. To search for channel blockers, synthetic combinatorial libraries were assayed for block of agonist-evoked currents by the human NR1-NR2A NMDA receptor subunits expressed in amphibian oocytes. A set of arginine-rich hexapeptides selectively blocked the NMDA receptor channel with IC50 approximately 100 nM, a potency similar to clinically tolerated blockers such as memantine, and only marginally blocked on non-NMDA glutamate receptors. These peptides prevent neuronal cell death elicited by an excitotoxic insult on hippocampal cultures.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent toxicant that alters normal brain development and produces cognitive disability and motor dysfunction. However, after decades of intense study, the molecular mechanisms of TCDD-induced neurotoxicity, the signaling pathways involved and its molecular targets in neurons still remain unknown. TCDD acts as an exogenous ligand of the aryl hydrocarbon receptor (AhR) that becomes a key signaling molecule in the regulation of the toxic and carcinogenic properties of TCDD. We have used NGF-differentiated pheochromocytoma (dPC12) cells to determine the type of cell death that takes place by TCDD toxicity. TCDD induced cell death in dPC12 cultures with an EC(50) of 218+/-24 nM, similar to that obtained in undifferentiated PC12 cells, 171+/-31 nM. Nuclear fragmentation was observed after TCDD incubation in parallel to an increase in caspase-3 activity. Staurosporine, which readily induced apoptosis in dPC12 cells, showed a similar increase in caspase-3 activity and the characteristic pattern of nuclear fragmentation. Flow cytometry measurements showed that dPC12 cells in the presence of TCDD were positive for annexin V labeling but negative for propidium iodide staining. In addition, TCDD increased the area of the peak corresponding to hypodiploid (apoptotic) DNA content. All together these results support the hypothesis that TCDD toxicity in dPC12 cells takes place mainly through an apoptotic process.

Zinc is a basic trace element that plays important roles in brain and, consequently, its homeostasis needs to be critically controlled. High zinc concentrations in the interneuron synaptic space may induce neuronal death through mechanisms still partially solved. Undifferentiated pheochromocytoma (PC12) cells have been used to study zinc toxicity. As these cells can be differentiated into neuronal-like cells, the results obtained from differentiated cultures are more useful to understand zinc toxicity in neurons. In this paper, we show by flow cytometry that nerve growth factor (NGF) induces PC12 cells differentiation characterized by cell cycle arrest in the G1/G0 phase, similarly to that observed in serum-deprived cultures. Zinc induces cell death in NGF-differentiated PC12 cultures with an EC(50) value of 143+/-14 microM, which reveals a higher sensitivity with respect to undifferentiated PC12 cultures (EC(50), 308+/-32 microM) and a similar response to that obtained in hippocampal neurons (134+/-12 microM). Thus, the differentiation process appeared responsible for such increase in sensitivity. To further support this tenet, when the NGF differentiation was impaired in presence of 10 microM MK-801, a selective blocker of the N-methyl-d-aspartate (NMDA) receptor that plays a role in the differentiation process, the higher sensitivity to zinc was reverted to an EC(50) value of 241+/-26 microM. Flow cytometry experiments showed that NGF-differentiated PC12 cells in presence of zinc were positive for propidium iodide but not for annexin-V labeling. These results, together with data from fluorescent labeling of nuclear fragmentation, caspase-3 activation, and reactive oxygen species generation, support the view that zinc toxicity in NGF-differentiated PC12 cells takes place mainly through a necrotic process.

Resveratrol (RES), a chemopreventive molecule, inhibits the proliferation of tumor cells of different etiologies. We previously showed that RES alters the cell cycle and induces apoptosis in MCF-7 breast tumor cells by interfering with the estrogen receptor (ERaalpha)-dependent phosphoinositide 3-kinase (PI3K) pathway. Here, we analyzed signaling downstream of PI3K, to understand the mechanisms of RES-induced apoptosis. Apoptotic death by RES in MCF-7 was mediated by Bcl-2 downregulation since overexpression of this protein abolished apoptosis. Decreased Bcl-2 levels were not related to cytochrome c release, activation of caspases 3/8 or poly(ADP-ribose) polymerase proteolysis. However, RES decreased mitochondrial membrane potential and increased reactive oxygen species and nitric oxide production. NF-kappaB, a regulator of Bcl-2 expression, and calpain protease activity, a regulator of NF-kappaB, were both inhibited by RES. The patterns for NF-kappaB and calpain activities followed that of PI3K and were inhibited by LY294002. NF-kappaB inhibition coincided with diminished MMP-9 activity and cell migration. These data suggest that RES-induced apoptosis in MCF-7 could involve an oxidative, caspase-independent mechanism, whereby inhibition of PI3K signaling converges to Bcl-2 through NF-kappaB and calpain protease activity. Therefore, Bcl-2 and NF-kappaB could be considered potential targets for the chemopreventive activity of RES in estrogen-responsive tumor cells.

Synthetic peptides patterned after the C-terminus of synaptosomal associated protein of 25 kDa (SNAP25) efficiently abrogate regulated exocytosis. In contrast, the use of SNAP25 N-terminal-derived peptides to modulate SNAP receptors (SNARE) complex assembly and neurosecretion has not been explored. Here, we show that the N-terminus of SNAP25, specially the segment that encompasses 22Ala-44Ile, is essential for the formation of the SNARE complex. Peptides patterned after this protein domain are potent inhibitors of SNARE complex formation. The inhibitory activity correlated with their propensity to adopt an alpha-helical secondary structure. These peptides abrogated SNARE complex formation only when added previous to the onset of aggregate assembly. Analysis of the mechanism of action revealed that these peptides disrupted the binary complex formed by SNAP25 and syntaxin. The identified peptides inhibited Ca2+-dependent exocytosis from detergent-permeabilized excitable cells. Noteworthy, these amino acid sequences markedly protected intact hippocampal neurones against hypoglycaemia-induced, glutamate-mediated excitotoxicity with a potency that rivalled that displayed by botulinum neurotoxins. Our findings indicate that peptides patterned after the N-terminus of SNAP25 are potent inhibitors of SNARE complex formation and neuronal exocytosis. Because of their activity in intact neurones, these cell permeable peptides may be hits for antispasmodic and analgesic drug development.

Synthetic peptides patterned after the proteins involved in vesicle fusion [the so-called SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) proteins] are potent inhibitors of SNARE complex assembly and neuronal exocytosis. It is noteworthy that the identification of peptide sequences not related to the SNARE proteins has not been accomplished yet; this is due, in part, to the structural constraints and the specificity of the protein interactions that govern the formation of the SNARE complex. Here we have addressed this question and used a combinatorial approach to identify peptides that modulate the assembly of the SNARE core complex and inhibit neuronal exocytosis. An α-helix-constrained, mixture-based, 17-mer combinatorial peptide library composed of 137180 sequences was synthesized in a positional scanning format. Peptide mixtures were assayed for their ability to prevent the formation of the in vitro-reconstituted SDS-resistant SNARE core complex. Library deconvolution identified eight peptides that inhibited the assembly of the SNARE core complex. Notably, the most potent 17-mer peptide (acetyl-SAAEAFAKLYAEAFAKG-NH2) abolished both Ca2+-evoked catecholamine secretion from detergent-permeabilized chromaffin cells and l-glutamate release from intact hippocampal primary cultures. Collectively, these findings indicate that amino acid sequences that prevent SNARE complex formation are not restricted to those that mimic domains of SNARE proteins, thus expanding the diversity of molecules that target neuronal exocytosis. Because of the implication of neurosecretion in the aetiology of several human neurological disorders, these newly identified peptides may be considered hits for the development of novel anti-spasmodic drugs.

Excessive activation of glutamate receptors of the N-methyl-D-aspartate (NMDA) subtype is considered a relevant initial step underlying different neurodegenerative diseases. Recently, with the approval of memantine to treat Alzheimer dementia, NMDA receptors have regained clinical interest. Accordingly, the development and validation of NMDA receptor antagonists is being reconsidered. We recently identified a family of trialkylglycines that act as channel blockers of the NMDAreceptor. Their neuroprotective activity against excitotoxic insults remains elusive. To address this issue, we first characterized the contribution of glutamate receptor subtypes to hippocampal death in culture as a function of days in culture in vitro (DIV). Whereas at 7 DIV neither NMDA nor glutamate produced a significant neuronal death, at 14 and 21 DIV, NMDA produced the death of 40% of the neurons exposed to this receptor agonist that was fully protected by MK-801. Similar results were obtained for L-glutamate at 14 DIV. In contrast, when neurons at 21 DIV were used, glutamate killed 51.1 +/- 4.9% of the neuronal population. This neuronal death was only partially prevented by MK-801, and fully abrogated by a combination of MK-801 and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Glucose deprivation injured 37.1 +/- 9.2% of the neurons through a mechanism sensitive to MK-801. The family of recently identified N-alkylglycines tested protected neurons against NMDA and glucosedeprivation toxicity, but not against glutamate toxicity. Noteworthy, N-alkylglicines with a moderate protection against NMDA-induced toxicity strongly protected from beta-amyloid toxicity. Collectively, these findings imply both NMDA and non-NMDA receptors in excitotoxicity of hippocampal neurons, and suggest that blockade of NMDA receptors alone may not suffice to efficiently abrogate neurodegeneration.

The Ca 2+ -ATPase from sarcoplasmic reticulum couples the hydrolysis of one molecule of ATP to the transport of two Ca 2+ ions in skeletal muscle fibers. Here, we study the accessibility of the fluorescein covalently attached to the Lys 515 at the nucleotide binding domain of the ATPase to the small collisional quencher iodide at pH 6 and 8, as well as the effect of ligand binding (La 3+ , La 3+ -nucleotide, and Ca 2+ ). Our results indicate that bound fluorescein is significantly more accessible at pH 6 than at pH 8, suggesting that pH modulates the structure of the nucleotide binding domain of the ATPase. This notion was further substantiated by the finding that La 3+ -nucleotide only interacted with the catalytic center at acidic pH. Notably, the differential accessibility of the nucleotide binding domain at acidic and basic pH cannot be rationalized in terms of the ATPase E1/E2 conformational equilibrium since a shift of the ATPase toward the E1 (plus Ca 2+ ) or E2 (plus EGTA) did not affect the accessibility of fluorescein-labeled ATPase to the quencher. Taken together, these findings show the presence of structural flexibility in the FITC binding site and suggest a structural modulation of the Ca 2+ -ATPase nucleotide binding domain by pH and La 3+ binding through long-range linkage mechanisms.

The incidence of cerebrovascular accidents (CVA) in the context of cardiac catheterization (CC) varies from 0.07% to 0.4%, according to the literature [1,2]. And it increases to 0.8% if revascularization techniques are performed. Most cases of stroke manifest within the first 24 h, though the condition may also be diagnosed in later phases. The clinical manifestations range greatly from vision disorders (diplopia, vision loss) to motor-sensory alterations. The risk profile, according to different observational studies includes both patient factors (age, sex, weight, etc.) and factors dependent upon the technique (duration of catheterization, access route, the performance of percutaneous transluminal coronary angioplasty (PTCA), etc.), and varies with the type of CVA involved [1]. We present the case of a patient subjected to CC who developed sudden onset binocular diplopia secondary to selective involvement of the fourth cranial nerve nucleus.

The thermal unfolding of monomeric and delipidated Ca(2+)-ATPase, solubilized in C12E8, can be appropriately described as a non-two-state irreversible denaturation, with only one endothermic peak. In the Ca2+ concentration range (0-0.5 mM) which stimulates the ATPase activity of solubilized monomeric ATPase, Ca2+ shifts the critical temperature midpoint of the denaturation process (Tm) from 42 to 50 degrees C without segregation of the endothermic peak into two separate components. Because 20 mM Mg2+ only shifts the Tm from 42 to 44 degrees C, we conclude that the effect of Ca2+ upon the Tm is likely to be due to binding to the high affinity Ca2+ sites in the ATPase. The effect of Ca2+ upon the enthalpy of denaturation is biphasic, suggesting the presence of low affinity Ca2+ sites (K0.5 in the millimolar range) in monomeric and solubilized ATPase.

Vanilloid receptor subunit 1 (VR1) is a nonselective cation channel that integrates multiple pain-producing stimuli. VR1 channels are blocked with high efficacy by the well established noncompetitive antagonist ruthenium red and exhibit high permeability to divalent cations. The molecular determinants that define these functional properties remain elusive. We have addressed this question and evaluated by site-specific neutralization the contribution on pore properties of acidic residues located in the putative VR1 pore region. Mutant receptors expressed in Xenopus oocytes exhibited capsaicin-operated ionic currents akin to those of wild type channels. Incorporation of glutamine residues at Glu(648) and Glu(651) rendered minor effects on VR1 pore attributes, while Glu(636) slightly modulated pore blockade. In contrast, replacement of Asp(646) by asparagine decreased 10-fold ruthenium red blockade efficacy and reduced 4-fold the relative permeability of the divalent cation Mg(2+) with respect to Na(+) without changing the selectivity of monovalent cations. At variance with wild type channels and E636Q, E648Q, and E651Q mutant receptors, ruthenium red blockade of D646N mutants was weakly sensitive to extracellular pH acidification. Collectively, our results suggest that Asp(646) is a molecular determinant of VR1 pore properties and imply that this residue may form a ring of negative charges that structures a high affinity binding site for cationic molecules at the extracellular entryway.