Analysis of Schizosaccharomyces pombe mediator reveals a set of essential subunits conserved between yeast and metazoan cells

DEFF Research Database (Denmark)

Spåhr, H; Samuelsen, C O; Baraznenok, V

2001-01-01

. cerevisiae share an essential protein module, which associates with nonessential speciesspecific subunits. In support of this view, sequence analysis of the conserved yeast Mediator components Med4 and Med8 reveals sequence homology to the metazoan Mediator components Trap36 and Arc32. Therefore, 8 of 10...... essential genes conserved between S. pombe and S. cerevisiae also have a metazoan homolog, indicating that an evolutionary conserved Mediator core is present in all eukaryotic cells. Our data suggest a closer functional relationship between yeast and metazoan Mediator than previously anticipated....

The essential role of AMPA receptor GluR2 subunit RNA editing in the normal and diseased brain

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Amanda Lorraine Wright

2012-04-01

Full Text Available AMPA receptors are comprised of different combinations of GluR1-GluR4 (also known as GluA1-GluA4 and GluR-A to GluR-D subunits. The GluR2 subunit is subject to Q/R site RNA editing by the ADAR2 enzyme, which converts a codon for glutamine (Q, present in the GluR2 gene, to a codon for arginine (R found in the mRNA. AMPA receptors are calcium (Ca2+-permeable if they contain the unedited GluR2(Q subunit or if they lack the GluR2 subunit. While most AMPA receptors in the brain contain the edited GluR2(R subunit and are therefore Ca2+-impermeable, recent evidence suggests that Ca2+-permeable GluR2-lacking AMPA receptors are important in synaptic plasticity and learning. However, the presence of Ca2+-permeable AMPA receptors containing unedited GluR2 leads to excitotoxic cell loss. Recent studies have indicated that RNA editing of GluR2 is deregulated in diseases, such as amyotrophic lateral sclerosis (ALS, as well in acute neurodegenerative conditions, such as ischemia. More recently, studies have investigated the regulation of RNA editing and possible causes for its deregulation during disease. In this review, we will explore the role of GluR2 RNA editing in the healthy and diseased brain and outline new insights into the mechanisms that control this process.

Effects of visual deprivation during brain development on expression of AMPA receptor subunits in rat’s hippocampus

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Sayyed Alireza Talaei

2015-06-01

Conclusion: Dark rearing of rats during critical period of brain development changes the relative expression and also arrangement of both AMPA receptor subunits, GluR1 and GluR2 in the hippocampus, age dependently.

Characterization of the regulatory subunit from brain cyclic AMP-dependent protein kinase II

International Nuclear Information System (INIS)

Stein, J.C.

1985-01-01

Tryptic peptides derived from the regulatory subunits of brain and heart cAMP-dependent protein kinase II were mapped by reverse phase HPLC. At 280 nm, 15 unique peptides were found only in the heart RII digest, while 5 other peptides were obtained only from brain RII. At 210 nm, 13 brain-RII specific and 15 heart-RII specific tryptic peptides were identified and resolved. Two-dimensional mapping analyses revealed that several 37 P-labeled tryptic fragments derived from the autophosphorylation and the photoaffinity labeled cAMP-binding sites of brain RII were separate and distinct from the 32 P-peptides isolated from similarly treated heart RII. The tryptic phosphopeptide containing the autophosphorylation site in brain RII was purified. The sequence and phosphorylation site is: Arg-Ala-Ser(P)-Val-Cys-Ala-Glu-Ala-Tyr-Asn-Pro-Asp-Glu-Glu-Glu-Asp-Asp-Ala-Glu. Astrocytes and neurons exhibit high levels of the brain RII enzyme, while oligodendrocytes contain the heart RII enzyme. Monoclonal antibodies to bovine cerebral cortex RII were made and characterized. The antibodies elucidated a subtle difference between membrane-associated and cytosolic RII from cerebral cortex

ASIC2 Subunits Target Acid-Sensing Ion Channels to the Synapse via an Association with PSD-95

OpenAIRE

Zha, Xiang-ming; Costa, Vivian; Harding, Anne Marie S.; Reznikov, Leah; Benson, Christopher J.; Welsh, Michael J.

2009-01-01

Acid-sensing ion channel-1a (ASIC1a) mediates H+-gated current to influence normal brain physiology and impact several models of disease. Although ASIC2 subunits are widely expressed in brain and modulate ASIC1a current, their function remains poorly understood. We identified ASIC2a in dendrites, dendritic spines, and brain synaptosomes. This localization largely relied on ASIC2a binding to PSD-95 and matched that of ASIC1a, which does not co-immunoprecipitate with PSD-95. We found that ASIC2...

Amplification of TLO Mediator Subunit Genes Facilitate Filamentous Growth in Candida Spp.

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Liu, Zhongle; Moran, Gary P.; Myers, Lawrence C.

2016-01-01

Filamentous growth is a hallmark of C. albicans pathogenicity compared to less-virulent ascomycetes. A multitude of transcription factors regulate filamentous growth in response to specific environmental cues. Our work, however, suggests the evolutionary history of C. albicans that resulted in its filamentous growth plasticity may be tied to a change in the general transcription machinery rather than transcription factors and their specific targets. A key genomic difference between C. albicans and its less-virulent relatives, including its closest relative C. dubliniensis, is the unique expansion of the TLO (TeLOmere-associated) gene family in C. albicans. Individual Tlo proteins are fungal-specific subunits of Mediator, a large multi-subunit eukaryotic transcriptional co-activator complex. This amplification results in a large pool of ‘free,’ non-Mediator associated, Tlo protein present in C. albicans, but not in C. dubliniensis or other ascomycetes with attenuated virulence. We show that engineering a large ‘free’ pool of the C. dubliniensis Tlo2 (CdTlo2) protein in C. dubliniensis, through overexpression, results in a number of filamentation phenotypes typically associated only with C. albicans. The amplitude of these phenotypes is proportional to the amount of overexpressed CdTlo2 protein. Overexpression of other C. dubliniensis and C. albicans Tlo proteins do result in these phenotypes. Tlo proteins and their orthologs contain a Mediator interaction domain, and a potent transcriptional activation domain. Nuclear localization of the CdTlo2 activation domain, facilitated naturally by the Tlo Mediator binding domain or artificially through an appended nuclear localization signal, is sufficient for the CdTlo2 overexpression phenotypes. A C. albicans med3 null mutant causes multiple defects including the inability to localize Tlo proteins to the nucleus and reduced virulence in a murine systemic infection model. Our data supports a model in which the

Amplification of TLO Mediator Subunit Genes Facilitate Filamentous Growth in Candida Spp.

Directory of Open Access Journals (Sweden)

Zhongle Liu

2016-10-01

Full Text Available Filamentous growth is a hallmark of C. albicans pathogenicity compared to less-virulent ascomycetes. A multitude of transcription factors regulate filamentous growth in response to specific environmental cues. Our work, however, suggests the evolutionary history of C. albicans that resulted in its filamentous growth plasticity may be tied to a change in the general transcription machinery rather than transcription factors and their specific targets. A key genomic difference between C. albicans and its less-virulent relatives, including its closest relative C. dubliniensis, is the unique expansion of the TLO (TeLOmere-associated gene family in C. albicans. Individual Tlo proteins are fungal-specific subunits of Mediator, a large multi-subunit eukaryotic transcriptional co-activator complex. This amplification results in a large pool of 'free,' non-Mediator associated, Tlo protein present in C. albicans, but not in C. dubliniensis or other ascomycetes with attenuated virulence. We show that engineering a large 'free' pool of the C. dubliniensis Tlo2 (CdTlo2 protein in C. dubliniensis, through overexpression, results in a number of filamentation phenotypes typically associated only with C. albicans. The amplitude of these phenotypes is proportional to the amount of overexpressed CdTlo2 protein. Overexpression of other C. dubliniensis and C. albicans Tlo proteins do result in these phenotypes. Tlo proteins and their orthologs contain a Mediator interaction domain, and a potent transcriptional activation domain. Nuclear localization of the CdTlo2 activation domain, facilitated naturally by the Tlo Mediator binding domain or artificially through an appended nuclear localization signal, is sufficient for the CdTlo2 overexpression phenotypes. A C. albicans med3 null mutant causes multiple defects including the inability to localize Tlo proteins to the nucleus and reduced virulence in a murine systemic infection model. Our data supports a model in which

Mediator Complex Subunits MED2, MED5, MED16, and MED23 Genetically Interact in the Regulation of Phenylpropanoid Biosynthesis.

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Dolan, Whitney L; Dilkes, Brian P; Stout, Jake M; Bonawitz, Nicholas D; Chapple, Clint

2017-12-01

The phenylpropanoid pathway is a major global carbon sink and is important for plant fitness and the engineering of bioenergy feedstocks. In Arabidopsis thaliana , disruption of two subunits of the transcriptional regulatory Mediator complex, MED5a and MED5b, results in an increase in phenylpropanoid accumulation. By contrast, the semidominant MED5b mutation reduced epidermal fluorescence4-3 ( ref4-3 ) results in dwarfism and constitutively repressed phenylpropanoid accumulation. Here, we report the results of a forward genetic screen for suppressors of ref4-3. We identified 13 independent lines that restore growth and/or phenylpropanoid accumulation in the ref4-3 background. Two of the suppressors restore growth without restoring soluble phenylpropanoid accumulation, indicating that the growth and metabolic phenotypes of the ref4-3 mutant can be genetically disentangled. Whole-genome sequencing revealed that all but one of the suppressors carry mutations in MED5b or other Mediator subunits. RNA-seq analysis showed that the ref4-3 mutation causes widespread changes in gene expression, including the upregulation of negative regulators of the phenylpropanoid pathway, and that the suppressors reverse many of these changes. Together, our data highlight the interdependence of individual Mediator subunits and provide greater insight into the transcriptional regulation of phenylpropanoid biosynthesis by the Mediator complex. © 2017 American Society of Plant Biologists. All rights reserved.

Brain and Peripheral Atypical Inflammatory Mediators Potentiate Neuroinflammation and Neurodegeneration.

Science.gov (United States)

Kempuraj, Duraisamy; Thangavel, Ramasamy; Selvakumar, Govindhasamy P; Zaheer, Smita; Ahmed, Mohammad E; Raikwar, Sudhanshu P; Zahoor, Haris; Saeed, Daniyal; Natteru, Prashant A; Iyer, Shankar; Zaheer, Asgar

2017-01-01

Neuroinflammatory response is primarily a protective mechanism in the brain. However, excessive and chronic inflammatory responses can lead to deleterious effects involving immune cells, brain cells and signaling molecules. Neuroinflammation induces and accelerates pathogenesis of Parkinson's disease (PD), Alzheimer's disease (AD) and Multiple sclerosis (MS). Neuroinflammatory pathways are indicated as novel therapeutic targets for these diseases. Mast cells are immune cells of hematopoietic origin that regulate inflammation and upon activation release many proinflammatory mediators in systemic and central nervous system (CNS) inflammatory conditions. In addition, inflammatory mediators released from activated glial cells induce neurodegeneration in the brain. Systemic inflammation-derived proinflammatory cytokines/chemokines and other factors cause a breach in the blood brain-barrier (BBB) thereby allowing for the entry of immune/inflammatory cells including mast cell progenitors, mast cells and proinflammatory cytokines and chemokines into the brain. These peripheral-derived factors and intrinsically generated cytokines/chemokines, α-synuclein, corticotropin-releasing hormone (CRH), substance P (SP), beta amyloid 1-42 (Aβ1-42) peptide and amyloid precursor proteins can activate glial cells, T-cells and mast cells in the brain can induce additional release of inflammatory and neurotoxic molecules contributing to chronic neuroinflammation and neuronal death. The glia maturation factor (GMF), a proinflammatory protein discovered in our laboratory released from glia, activates mast cells to release inflammatory cytokines and chemokines. Chronic increase in the proinflammatory mediators induces neurotoxic Aβ and plaque formation in AD brains and neurodegeneration in PD brains. Glial cells, mast cells and T-cells can reactivate each other in neuroinflammatory conditions in the brain and augment neuroinflammation. Further, inflammatory mediators from the brain can

Differential expression of Mediator complex subunit MED15 in testicular germ cell tumors.

Science.gov (United States)

Klümper, Niklas; Syring, Isabella; Offermann, Anne; Shaikhibrahim, Zaki; Vogel, Wenzel; Müller, Stefan C; Ellinger, Jörg; Strauß, Arne; Radzun, Heinz Joachim; Ströbel, Philipp; Brägelmann, Johannes; Perner, Sven; Bremmer, Felix

2015-09-17

Testicular germ cell tumors (TGCT) are the most common cancer entities in young men with increasing incidence observed in the last decades. For therapeutic management it is important, that TGCT are divided into several histological subtypes. MED15 is part of the multiprotein Mediator complex which presents an integrative hub for transcriptional regulation and is known to be deregulated in several malignancies, such as prostate cancer and bladder cancer role, whereas the role of the Mediator complex in TGCT has not been investigated so far. Aim of the study was to investigate the implication of MED15 in TGCT development and its stratification into histological subtypes. Immunohistochemical staining (IHC) against Mediator complex subunit MED15 was conducted on a TGCT cohort containing tumor-free testis (n = 35), intratubular germ cell neoplasia unclassified (IGCNU, n = 14), seminomas (SEM, n = 107) and non-seminomatous germ cell tumors (NSGCT, n = 42), further subdivided into embryonic carcinomas (EC, n = 30), yolk sac tumors (YST, n = 5), chorionic carcinomas (CC, n = 5) and teratomas (TER, n = 2). Quantification of MED15 protein expression was performed through IHC followed by semi-quantitative image analysis using the Definiens software. In tumor-free seminiferous tubules, MED15 protein expression was absent or only low expressed in spermatogonia. Interestingly, the precursor lesions IGCNU exhibited heterogeneous but partly very strong MED15 expression. SEM weakly express the Mediator complex subunit MED15, whereas NSGCT and especially EC show significantly enhanced expression compared to tumor-free testis. In conclusion, MED15 is differentially expressed in tumor-free testis and TGCT. While MED15 is absent or low in tumor-free testis and SEM, NSGCT highly express MED15, hinting at the diagnostic potential of this marker to distinguish between SEM and NSGCT. Further, the precursor lesion IGCNU showed increased nuclear MED15

Reconstitution of active human core Mediator complex reveals a critical role of the MED14 subunit.

Science.gov (United States)

Cevher, Murat A; Shi, Yi; Li, Dan; Chait, Brian T; Malik, Sohail; Roeder, Robert G

2014-12-01

The evolutionarily conserved Mediator complex is a critical coactivator for RNA polymerase II (Pol II)-mediated transcription. Here we report the reconstitution of a functional 15-subunit human core Mediator complex and its characterization by functional assays and chemical cross-linking coupled to MS (CX-MS). Whereas the reconstituted head and middle modules can stably associate, basal and coactivator functions are acquired only after incorporation of MED14 into the bimodular complex. This results from a dramatically enhanced ability of MED14-containing complexes to associate with Pol II. Altogether, our analyses identify MED14 as both an architectural and a functional backbone of the Mediator complex. We further establish a conditional requirement for metazoan-specific MED26 that becomes evident in the presence of heterologous nuclear factors. This general approach paves the way for systematic dissection of the multiple layers of functionality associated with the Mediator complex.

Redox regulation of the MED28 and MED32 mediator subunits is important for development and senescence.

Science.gov (United States)

Shaikhali, Jehad; Davoine, Céline; Björklund, Stefan; Wingsle, Gunnar

2016-05-01

Mediator is a conserved multi-protein complex that acts as a bridge between promoter-bound transcriptional regulators and RNA polymerase II. While redox signaling is important in adjusting plant metabolism and development, the involvement of Mediator in redox homeostasis and regulation only recently started to emerge. Our previous results show that the MED10a, MED28, and MED32 Mediator subunits form various types of covalent oligomers linked by intermolecular disulfide bonds in vitro. To link that with biological significance we have characterized Arabidopsis med32 and med28 mutants and found that they are affected in root development and senescence, phenotypes possibly associated to redox changes.

The Contrasting Role of the Mediator Subunit MED30 in the Progression of Bladder Cancer.

Science.gov (United States)

Syring, Isabella; Weiten, Richard; Müller, Tim; Schmidt, Doris; Steiner, Susanne; Kristiansen, Glen; Müller, Stefan C; Ellinger, Jörg

2017-12-01

The Mediator complex is a key regulator of gene transcription, and several studies have demonstrated altered expression of particular subunits in diverse human diseases, especially cancer. To date, nothing is known about the role of MED30 in bladder cancer. We, therefore, performed an RNA expression and survival analysis of the subunit MED30 in 537 samples of bladder cancer by using the database cBioPortal. To validate these data on the protein level, we practiced immunohistochemical staining against MED30 on a tissue microarray containing 210 samples of all tumour stages and performed survival analyses. For functional analysis, the siRNA-mediated knockdown of MED30 was performed in the cell lines T24 and TCCSUP followed by proliferation, migration, and invasion assays. On the mRNA and protein levels, higher expression of MED30 is associated with better patient survival. In accordance with this, advanced T- and N-stages showed lower expression of MED30. In contrast, knockdown of MED30 led to reduction of the tumour parameters proliferation, migration, and invasion in the BCa cell lines. MED30 appears to be integrated in the progression of the urothelial tumour in the bladder. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Reconstitution of active human core Mediator complex reveals a pivotal role of the MED14 subunit

Science.gov (United States)

Cevher, Murat A.; Shi, Yi; Li, Dan; Chait, Brian T.; Malik, Sohail; Roeder, Robert G.

2014-01-01

The evolutionarily conserved Mediator complex is a critical coactivator for RNA polymerase II (Pol II)-mediated transcription. Here, we report the reconstitution of a functional 15-subunit human core Mediator complex and its characterization by functional assays and chemical cross-linking coupled to mass spectrometry (CX-MS). Whereas the reconstituted head and middle modules can stably associate, only with incorporation of MED14 into the bi-modular complex does it acquire basal and coactivator functions. This results from a dramatically enhanced ability of MED14-containing complexes to associate with Pol II. Altogether, our analyses identify MED14 as both an architectural and a functional backbone of the Mediator complex. We further establish a conditional requirement for metazoan-specific MED26 that becomes evident in the presence of heterologous nuclear factors. This general approach paves the way for systematically dissecting the multiple layers of functionalities associated with the Mediator complex. PMID:25383669

TRAP230/ARC240 and TRAP240/ARC250 Mediator subunits are functionally conserved through evolution

DEFF Research Database (Denmark)

Samuelsen, Camilla O; Baraznenok, Vera; Khorosjutina, Olga

2003-01-01

In Saccharomyces cerevisiae Mediator, a subgroup of proteins (Srb8, Srb9, Srb10, and Srb11) form a module, which is involved in negative regulation of transcription. Homologues of Srb10 and Srb11 are found in some mammalian Mediator preparations, whereas no clear homologues have been reported...... for Srb8 and Srb9. Here, we identify a TRAP240/ARC250 homologue in Schizosaccharomyces pombe and demonstrate that this protein, spTrap240, is stably associated with a larger form of Mediator, which also contains conserved homologues of Srb8, Srb10, and Srb11. We find that spTrap240 and Sch. pombe Srb8 (sp......Srb8) regulate the same distinct subset of genes and have indistinguishable phenotypic characteristics. Importantly, Mediator containing the spSrb8/spTrap240/spSrb10/spSrb11 subunits is isolated only in free form, devoid of RNA polymerase II. In contrast, Mediator lacking this module associates...

The nematode homologue of Mediator complex subunit 28, F28F8.5, is a critical regulator of C. elegans development.

Science.gov (United States)

Kostrouchová, Markéta; Kostrouch, David; Chughtai, Ahmed A; Kaššák, Filip; Novotný, Jan P; Kostrouchová, Veronika; Benda, Aleš; Krause, Michael W; Saudek, Vladimír; Kostrouchová, Marta; Kostrouch, Zdeněk

2017-01-01

The evolutionarily conserved Mediator complex is a critical player in regulating transcription. Comprised of approximately two dozen proteins, the Mediator integrates diverse regulatory signals through direct protein-protein interactions that, in turn, modulate the influence of Mediator on RNA Polymerase II activity. One Mediator subunit, MED28, is known to interact with cytoplasmic structural proteins, providing a potential direct link between cytoplasmic dynamics and the control of gene transcription. Although identified in many animals and plants, MED28 is not present in yeast; no bona fide MED28 has been described previously in Caenorhabditis elegans. Here, we identify bioinformatically F28F8.5, an uncharacterized predicted protein, as the nematode homologue of MED28. As in other Metazoa, F28F8.5 has dual nuclear and cytoplasmic localization and plays critical roles in the regulation of development. F28F8.5 is a vital gene and its null mutants have severely malformed gonads and do not reproduce. F28F8.5 interacts on the protein level with the Mediator subunits MDT-6 and MDT-30. Our results indicate that F28F8.5 is an orthologue of MED28 and suggest that the potential to link cytoplasmic and nuclear events is conserved between MED28 vertebrate and nematode orthologues.

Cardiomyocyte-Specific Ablation of Med1 Subunit of the Mediator Complex Causes Lethal Dilated Cardiomyopathy in Mice.

Science.gov (United States)

Jia, Yuzhi; Chang, Hsiang-Chun; Schipma, Matthew J; Liu, Jing; Shete, Varsha; Liu, Ning; Sato, Tatsuya; Thorp, Edward B; Barger, Philip M; Zhu, Yi-Jun; Viswakarma, Navin; Kanwar, Yashpal S; Ardehali, Hossein; Thimmapaya, Bayar; Reddy, Janardan K

2016-01-01

Mediator, an evolutionarily conserved multi-protein complex consisting of about 30 subunits, is a key component of the polymerase II mediated gene transcription. Germline deletion of the Mediator subunit 1 (Med1) of the Mediator in mice results in mid-gestational embryonic lethality with developmental impairment of multiple organs including heart. Here we show that cardiomyocyte-specific deletion of Med1 in mice (csMed1-/-) during late gestational and early postnatal development by intercrossing Med1fl/fl mice to α-MyHC-Cre transgenic mice results in lethality within 10 days after weaning due to dilated cardiomyopathy-related ventricular dilation and heart failure. The csMed1-/- mouse heart manifests mitochondrial damage, increased apoptosis and interstitial fibrosis. Global gene expression analysis revealed that loss of Med1 in heart down-regulates more than 200 genes including Acadm, Cacna1s, Atp2a2, Ryr2, Pde1c, Pln, PGC1α, and PGC1β that are critical for calcium signaling, cardiac muscle contraction, arrhythmogenic right ventricular cardiomyopathy, dilated cardiomyopathy and peroxisome proliferator-activated receptor regulated energy metabolism. Many genes essential for oxidative phosphorylation and proper mitochondrial function such as genes coding for the succinate dehydrogenase subunits of the mitochondrial complex II are also down-regulated in csMed1-/- heart contributing to myocardial injury. Data also showed up-regulation of about 180 genes including Tgfb2, Ace, Atf3, Ctgf, Angpt14, Col9a2, Wisp2, Nppa, Nppb, and Actn1 that are linked to cardiac muscle contraction, cardiac hypertrophy, cardiac fibrosis and myocardial injury. Furthermore, we demonstrate that cardiac specific deletion of Med1 in adult mice using tamoxifen-inducible Cre approach (TmcsMed1-/-), results in rapid development of cardiomyopathy and death within 4 weeks. We found that the key findings of the csMed1-/- studies described above are highly reproducible in TmcsMed1-/- mouse heart

Properties and expression of Na+/K+-ATPase α-subunit isoforms in the brain of the swamp eel, Monopterus albus, which has unusually high brain ammonia tolerance.

Directory of Open Access Journals (Sweden)

Xiu L Chen

Full Text Available The swamp eel, Monopterus albus, can survive in high concentrations of ammonia (>75 mmol l(-1 and accumulate ammonia to high concentrations in its brain (4.5 µmol g(-1. Na(+/K(+-ATPase (Nka is an essential transporter in brain cells, and since NH4(+ can substitute for K(+ to activate Nka, we hypothesized that the brain of M. albus expressed multiple forms of Nka α-subunits, some of which might have high K(+ specificity. Thus, this study aimed to clone and sequence the nka α-subunits from the brain of M. albus, and to determine the effects of ammonia exposure on their mRNA expression and overall protein abundance. The effectiveness of NH4(+ to activate brain Nka from M. albus and Mus musculus was also examined by comparing their Na(+/K(+-ATPase and Na(+/NH4(+-ATPase activities over a range of K(+/NH4(+ concentrations. The full length cDNA coding sequences of three nkaα (nkaα1, nkaα3a and nkaα3b were identified in the brain of M. albus, but nkaα2 expression was undetectable. Exposure to 50 mmol l(-1 NH4Cl for 1 day or 6 days resulted in significant decreases in the mRNA expression of nkaα1, nkaα3a and nkaα3b. The overall Nka protein abundance also decreased significantly after 6 days of ammonia exposure. For M. albus, brain Na(+/NH4(+-ATPase activities were significantly lower than the Na(+/K(+-ATPase activities assayed at various NH4(+/K(+ concentrations. Furthermore, the effectiveness of NH4(+ to activate Nka from the brain of M. albus was significantly lower than that from the brain of M. musculus, which is ammonia-sensitive. Hence, the (1 lack of nkaα2 expression, (2 high K(+ specificity of K(+ binding sites of Nkaα1, Nkaα3a and Nkaα3b, and (3 down-regulation of mRNA expression of all three nkaα isoforms and the overall Nka protein abundance in response to ammonia exposure might be some of the contributing factors to the high brain ammonia tolerance in M. albus.

The nematode homologue of Mediator complex subunit 28, F28F8.5, is a critical regulator of C. elegans development

Directory of Open Access Journals (Sweden)

Markéta Kostrouchová

2017-06-01

Full Text Available The evolutionarily conserved Mediator complex is a critical player in regulating transcription. Comprised of approximately two dozen proteins, the Mediator integrates diverse regulatory signals through direct protein-protein interactions that, in turn, modulate the influence of Mediator on RNA Polymerase II activity. One Mediator subunit, MED28, is known to interact with cytoplasmic structural proteins, providing a potential direct link between cytoplasmic dynamics and the control of gene transcription. Although identified in many animals and plants, MED28 is not present in yeast; no bona fide MED28 has been described previously in Caenorhabditis elegans. Here, we identify bioinformatically F28F8.5, an uncharacterized predicted protein, as the nematode homologue of MED28. As in other Metazoa, F28F8.5 has dual nuclear and cytoplasmic localization and plays critical roles in the regulation of development. F28F8.5 is a vital gene and its null mutants have severely malformed gonads and do not reproduce. F28F8.5 interacts on the protein level with the Mediator subunits MDT-6 and MDT-30. Our results indicate that F28F8.5 is an orthologue of MED28 and suggest that the potential to link cytoplasmic and nuclear events is conserved between MED28 vertebrate and nematode orthologues.

The Mediator subunit MDT-15 confers metabolic adaptation to ingested material.

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Stefan Taubert

2008-02-01

Full Text Available In eukaryotes, RNA polymerase II (Pol(II dependent gene expression requires accessory factors termed transcriptional coregulators. One coregulator that universally contributes to Pol(II-dependent transcription is the Mediator, a multisubunit complex that is targeted by many transcriptional regulatory factors. For example, the Caenorhabditis elegans Mediator subunit MDT-15 confers the regulatory actions of the sterol response element binding protein SBP-1 and the nuclear hormone receptor NHR-49 on fatty acid metabolism. Here, we demonstrate that MDT-15 displays a broader spectrum of activities, and that it integrates metabolic responses to materials ingested by C. elegans. Depletion of MDT-15 protein or mutation of the mdt-15 gene abrogated induction of specific detoxification genes in response to certain xenobiotics or heavy metals, rendering these animals hypersensitive to toxin exposure. Intriguingly, MDT-15 appeared to selectively affect stress responses related to ingestion, as MDT-15 functional defects did not abrogate other stress responses, e.g., thermotolerance. Together with our previous finding that MDT-15:NHR-49 regulatory complexes coordinate a sector of the fasting response, we propose a model whereby MDT-15 integrates several transcriptional regulatory pathways to monitor both the availability and quality of ingested materials, including nutrients and xenobiotic compounds.

Contribution of the major and minor subunits to fimbria-mediated adherence of Haemophilus influenzae to human epithelial cells and erythrocytes

NARCIS (Netherlands)

van Ham, S. M.; van Alphen, L.; Mooi, F. R.; van Putten, J. P.

1995-01-01

Fimbriae are colonization factors of the human pathogen Haemophilus influenzae in that they mediate bacterial adherence to human eukaryotic cells. The contribution of the major (HifA) and putative minor (HifD and HifE) subunits of H. influenzae fimbriae to fimbria-specific adherence was studied by

Kv2 Ion Channels Determine the Expression and Localization of the Associated AMIGO-1 Cell Adhesion Molecule in Adult Brain Neurons

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Hannah I. Bishop

2018-01-01

Full Text Available Voltage-gated K+ (Kv channels play important roles in regulating neuronal excitability. Kv channels comprise four principal α subunits, and transmembrane and/or cytoplasmic auxiliary subunits that modify diverse aspects of channel function. AMIGO-1, which mediates homophilic cell adhesion underlying neurite outgrowth and fasciculation during development, has recently been shown to be an auxiliary subunit of adult brain Kv2.1-containing Kv channels. We show that AMIGO-1 is extensively colocalized with both Kv2.1 and its paralog Kv2.2 in brain neurons across diverse mammals, and that in adult brain, there is no apparent population of AMIGO-1 outside of that colocalized with these Kv2 α subunits. AMIGO-1 is coclustered with Kv2 α subunits at specific plasma membrane (PM sites associated with hypolemmal subsurface cisternae at neuronal ER:PM junctions. This distinct PM clustering of AMIGO-1 is not observed in brain neurons of mice lacking Kv2 α subunit expression. Moreover, in heterologous cells, coexpression of either Kv2.1 or Kv2.2 is sufficient to drive clustering of the otherwise uniformly expressed AMIGO-1. Kv2 α subunit coexpression also increases biosynthetic intracellular trafficking and PM expression of AMIGO-1 in heterologous cells, and analyses of Kv2.1 and Kv2.2 knockout mice show selective loss of AMIGO-1 expression and localization in neurons lacking the respective Kv2 α subunit. Together, these data suggest that in mammalian brain neurons, AMIGO-1 is exclusively associated with Kv2 α subunits, and that Kv2 α subunits are obligatory in determining the correct pattern of AMIGO-1 expression, PM trafficking and clustering.

The residence time of GABA(A)Rs at inhibitory synapses is determined by direct binding of the receptor α1 subunit to gephyrin

DEFF Research Database (Denmark)

Mukherjee, Jayanta; Kretschmannova, Karla; Gouzer, Geraldine

2011-01-01

The majority of fast synaptic inhibition in the brain is mediated by benzodiazepine-sensitive α1-subunit-containing GABA type A receptors (GABA(A)Rs); however, our knowledge of the mechanisms neurons use to regulate their synaptic accumulation is rudimentary. Using immunoprecipitation, we....... Mutating residues 360-375 decreases both the accumulation of α1-containing GABA(A)Rs at gephyrin-positive inhibitory synapses in hippocampal neurons and the amplitude of mIPSCs. We also demonstrate that the affinity of gephyrin for the α1 subunit is modulated by Thr375, a putative phosphorylation site....... Mutation of Thr375 to a phosphomimetic, negatively charged amino acid decreases both the affinity of the α1 subunit for gephyrin, and therefore receptor accumulation at synapses, and the amplitude of mIPSCs. Finally, single-particle tracking reveals that gephyrin reduces the diffusion of α1-subunit...

Managing brain extracellular K+ during neuronal activity: The physiological role of the Na+/K+-ATPase subunit isoforms

Directory of Open Access Journals (Sweden)

Brian Roland eLarsen

2016-04-01

Full Text Available AbstractDuring neuronal activity in the brain, extracellular K+ rises and is subsequently removed to prevent a widespread depolarization. One of the key players in regulating extracellular K+ is the Na+/K+-ATPase, although the relative involvement and physiological impact of the different subunit isoform compositions of the Na+/K+-ATPase remain unresolved. The various cell types in the brain serve a certain temporal contribution in the face of network activity; astrocytes respond directly to the immediate release of K+ from neurons, whereas the neurons themselves become the primary K+ absorbers as activity ends. The kinetic characteristics of the catalytic α subunit isoforms of the Na+/K+-ATPase are, partly, determined by the accessory β subunit with which they combine. The isoform combinations expressed by astrocytes and neurons, respectively, appear to be in line with the kinetic characteristics required to fulfill their distinct physiological roles in clearance of K+ from the extracellular space in the face of neuronal activity.Understanding the nature, impact and effects of the various Na+/K+-ATPase isoform combinations in K+ management in the central nervous system might reveal insights into pathological conditions such as epilepsy, migraine, and spreading depolarization following cerebral ischemia. In addition, particular neurological diseases occur as a result of mutations in the α2- (familial hemiplegic migraine type 2 and α3 isoforms (rapid-onset dystonia parkinsonism/alternating hemiplegia of childhood. This review addresses aspects of the Na+/K+-ATPase in the regulation of extracellular K+ in the central nervous system as well as the related pathophysiology. Understanding the physiological setting in non-pathological tissue would provide a better understanding of the pathological events occurring during disease.

Protein kinase A regulatory subunit distribution in medulloblastoma

International Nuclear Information System (INIS)

Mucignat-Caretta, Carla; Denaro, Luca; Redaelli, Marco; D'Avella, Domenico; Caretta, Antonio

2010-01-01

Previous studies showed a differential distribution of the four regulatory subunits of cAMP-dependent protein kinases inside the brain, that changed in rodent gliomas: therefore, the distribution of these proteins inside the brain can give information on the functional state of the cells. Our goal was to examine human brain tumors to provide evidence for a differential distribution of protein kinase A in different tumors. The distribution of detergent insoluble regulatory (R1 and R2) and catalytic subunits of cAMP dependent kinases was examined in pediatric brain tumors by immunohistochemistry and fluorescent cAMP analogues binding. R2 is organized in large single dots in medulloblastomas, while it has a different appearance in other tumors. Fluorescent cAMP labelling was observed only in medulloblastoma. A different distribution of cAMP dependent protein kinases has been observed in medulloblastoma

Effects of Traumatic Stress Induced in the Juvenile Period on the Expression of Gamma-Aminobutyric Acid Receptor Type A Subunits in Adult Rat Brain

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Cui Yan Lu

2017-01-01

Full Text Available Studies have found that early traumatic experience significantly increases the risk of posttraumatic stress disorder (PTSD. Gamma-aminobutyric acid (GABA deficits were proposed to be implicated in development of PTSD, but the alterations of GABA receptor A (GABAAR subunits induced by early traumatic stress have not been fully elucidated. Furthermore, previous studies suggested that exercise could be more effective than medications in reducing severity of anxiety and depression but the mechanism is unclear. This study used inescapable foot-shock to induce PTSD in juvenile rats and examined their emotional changes using open-field test and elevated plus maze, memory changes using Morris water maze, and the expression of GABAAR subunits (γ2, α2, and α5 in subregions of the brain in the adulthood using western blotting and immunohistochemistry. We aimed to observe the role of GABAAR subunits changes induced by juvenile trauma in the pathogenesis of subsequent PTSD in adulthood. In addition, we investigated the protective effects of exercise for 6 weeks and benzodiazepine (clonazepam for 2 weeks. This study found that juvenile traumatic stress induced chronic anxiety and spatial memory loss and reduced expression of GABAAR subunits in the adult rat brains. Furthermore, exercise led to significant improvement as compared to short-term BZ treatment.

Myristoylated α subunits of guanine nucleotide-binding regulatory proteins

International Nuclear Information System (INIS)

Buss, J.E.; Mumby, S.M.; Casey, P.J.; Gilman, A.G.; Sefton, B.M.

1987-01-01

Antisera directed against specific subunits of guanine nucleotide-binding regulatory proteins (G proteins) were used to immunoprecipitate these polypeptides from metabolically labeled cells. This technique detects, in extracts of a human astrocytoma cell line, the α subunits of G/sub s/ (stimulatory) (α 45 and α 52 ), a 41-kDa subunit of G/sub i/ (inhibitory) (α 41 ), a 40-kDa protein (α 40 ), and the 36-kDa β subunit. No protein that comigrated with the α subunit of G 0 (unknown function) (α 39 ) was detected. In cells grown in the presence of [ 3 H]myristic acid, α 41 and α 40 contained 3 H label, while the β subunit did not. Chemical analysis of lipids attached covalently to purified α 41 and α 39 from bovine brain also revealed myristic acid. Similar analysis of brain G protein β and γ subunits and of G/sub t/ (Transducin) subunits (α, β, and γ) failed to reveal fatty acids. The fatty acid associated with α 41 , α 40 , and α 39 was stable to treatment with base, suggesting that the lipid is linked to the polypeptide via an amide bond. These GTP binding proteins are thus identified as members of a select group of proteins that contains myristic acid covalently attached to the peptide backbone. Myristate may play an important role in stabilizing interactions of G proteins with phospholipid or with membrane-bound proteins

Novel isoforms of the TFIID subunit TAF4 modulate nuclear receptor-mediated transcriptional activity

International Nuclear Information System (INIS)

Brunkhorst, Adrian; Neuman, Toomas; Hall, Anita; Arenas, Ernest; Bartfai, Tamas; Hermanson, Ola; Metsis, Madis

2004-01-01

The transcription factor TFIID consists of TATA-binding protein (TBP) and TBP-associated factors (TAFs). TAFs are essential for modulation of transcriptional activity but the regulation of TAFs is complex and many important aspects remain unclear. In this study, we have identified and characterized five novel truncated forms of the TFIID subunit TAF4 (TAF II 135). Analysis of the mouse gene structure revealed that all truncations were the results of alternative splicing and resulted in the loss of domains or parts of domains implicated in TAF4 functional interactions. Results from transcriptional assays showed that several of the TAF4 isoforms exerted dominant negative effects on TAF4 activity in nuclear receptor-mediated transcriptional activation. In addition, alternative TAF4 isoforms could be detected in specific cell types. Our results indicate an additional level of complexity in TAF4-mediated regulation of transcription and suggest context-specific roles for these new TAF4 isoforms in transcriptional regulation in vivo

A dominant mutation in mediator of paramutation2, one of three second-largest subunits of a plant-specific RNA polymerase, disrupts multiple siRNA silencing processes.

Science.gov (United States)

Sidorenko, Lyudmila; Dorweiler, Jane E; Cigan, A Mark; Arteaga-Vazquez, Mario; Vyas, Meenal; Kermicle, Jerry; Jurcin, Diane; Brzeski, Jan; Cai, Yu; Chandler, Vicki L

2009-11-01

Paramutation involves homologous sequence communication that leads to meiotically heritable transcriptional silencing. We demonstrate that mop2 (mediator of paramutation2), which alters paramutation at multiple loci, encodes a gene similar to Arabidopsis NRPD2/E2, the second-largest subunit of plant-specific RNA polymerases IV and V. In Arabidopsis, Pol-IV and Pol-V play major roles in RNA-mediated silencing and a single second-largest subunit is shared between Pol-IV and Pol-V. Maize encodes three second-largest subunit genes: all three genes potentially encode full length proteins with highly conserved polymerase domains, and each are expressed in multiple overlapping tissues. The isolation of a recessive paramutation mutation in mop2 from a forward genetic screen suggests limited or no functional redundancy of these three genes. Potential alternative Pol-IV/Pol-V-like complexes could provide maize with a greater diversification of RNA-mediated transcriptional silencing machinery relative to Arabidopsis. Mop2-1 disrupts paramutation at multiple loci when heterozygous, whereas previously silenced alleles are only up-regulated when Mop2-1 is homozygous. The dramatic reduction in b1 tandem repeat siRNAs, but no disruption of silencing in Mop2-1 heterozygotes, suggests the major role for tandem repeat siRNAs is not to maintain silencing. Instead, we hypothesize the tandem repeat siRNAs mediate the establishment of the heritable silent state-a process fully disrupted in Mop2-1 heterozygotes. The dominant Mop2-1 mutation, which has a single nucleotide change in a domain highly conserved among all polymerases (E. coli to eukaryotes), disrupts both siRNA biogenesis (Pol-IV-like) and potentially processes downstream (Pol-V-like). These results suggest either the wild-type protein is a subunit in both complexes or the dominant mutant protein disrupts both complexes. Dominant mutations in the same domain in E. coli RNA polymerase suggest a model for Mop2-1 dominance

Laccase/Mediator Systems

NARCIS (Netherlands)

Hilgers, Roelant; Vincken, Jean Paul; Gruppen, Harry; Kabel, Mirjam A.

2018-01-01

Laccase-mediator systems (LMS) have been widely studied for their capacity to oxidize the nonphenolic subunits of lignin (70-90% of the polymer). The phenolic subunits (10-30% of the polymer), which can also be oxidized without mediators, have received considerably less attention. Consequently, it

p75 Neurotrophin Receptor Cleavage by α- and γ-Secretases Is Required for Neurotrophin-mediated Proliferation of Brain Tumor-initiating Cells*

Science.gov (United States)

Forsyth, Peter A.; Krishna, Niveditha; Lawn, Samuel; Valadez, J. Gerardo; Qu, Xiaotao; Fenstermacher, David A.; Fournier, Michelle; Potthast, Lisa; Chinnaiyan, Prakash; Gibney, Geoffrey T.; Zeinieh, Michele; Barker, Philip A.; Carter, Bruce D.; Cooper, Michael K.; Kenchappa, Rajappa S.

2014-01-01

Malignant gliomas are highly invasive, proliferative, and resistant to treatment. Previously, we have shown that p75 neurotrophin receptor (p75NTR) is a novel mediator of invasion of human glioma cells. However, the role of p75NTR in glioma proliferation is unknown. Here we used brain tumor-initiating cells (BTICs) and show that BTICs express neurotrophin receptors (p75NTR, TrkA, TrkB, and TrkC) and their ligands (NGF, brain-derived neurotrophic factor, and neurotrophin 3) and secrete NGF. Down-regulation of p75NTR significantly decreased proliferation of BTICs. Conversely, exogenouous NGF stimulated BTIC proliferation through α- and γ-secretase-mediated p75NTR cleavage and release of its intracellular domain (ICD). In contrast, overexpression of the p75NTR ICD induced proliferation. Interestingly, inhibition of Trk signaling blocked NGF-stimulated BTIC proliferation and p75NTR cleavage, indicating a role of Trk in p75NTR signaling. Further, blocking p75NTR cleavage attenuated Akt activation in BTICs, suggesting role of Akt in p75NTR-mediated proliferation. We also found that p75NTR, α-secretases, and the four subunits of the γ-secretase enzyme were elevated in glioblastoma multiformes patients. Importantly, the ICD of p75NTR was commonly found in malignant glioma patient specimens, suggesting that the receptor is activated and cleaved in patient tumors. These results suggest that p75NTR proteolysis is required for BTIC proliferation and is a novel potential clinical target. PMID:24519935

p75 neurotrophin receptor cleavage by α- and γ-secretases is required for neurotrophin-mediated proliferation of brain tumor-initiating cells.

Science.gov (United States)

Forsyth, Peter A; Krishna, Niveditha; Lawn, Samuel; Valadez, J Gerardo; Qu, Xiaotao; Fenstermacher, David A; Fournier, Michelle; Potthast, Lisa; Chinnaiyan, Prakash; Gibney, Geoffrey T; Zeinieh, Michele; Barker, Philip A; Carter, Bruce D; Cooper, Michael K; Kenchappa, Rajappa S

2014-03-21

Malignant gliomas are highly invasive, proliferative, and resistant to treatment. Previously, we have shown that p75 neurotrophin receptor (p75NTR) is a novel mediator of invasion of human glioma cells. However, the role of p75NTR in glioma proliferation is unknown. Here we used brain tumor-initiating cells (BTICs) and show that BTICs express neurotrophin receptors (p75NTR, TrkA, TrkB, and TrkC) and their ligands (NGF, brain-derived neurotrophic factor, and neurotrophin 3) and secrete NGF. Down-regulation of p75NTR significantly decreased proliferation of BTICs. Conversely, exogenouous NGF stimulated BTIC proliferation through α- and γ-secretase-mediated p75NTR cleavage and release of its intracellular domain (ICD). In contrast, overexpression of the p75NTR ICD induced proliferation. Interestingly, inhibition of Trk signaling blocked NGF-stimulated BTIC proliferation and p75NTR cleavage, indicating a role of Trk in p75NTR signaling. Further, blocking p75NTR cleavage attenuated Akt activation in BTICs, suggesting role of Akt in p75NTR-mediated proliferation. We also found that p75NTR, α-secretases, and the four subunits of the γ-secretase enzyme were elevated in glioblastoma multiformes patients. Importantly, the ICD of p75NTR was commonly found in malignant glioma patient specimens, suggesting that the receptor is activated and cleaved in patient tumors. These results suggest that p75NTR proteolysis is required for BTIC proliferation and is a novel potential clinical target.

Age-related changes in functional postsynaptic nicotinic acetylcholine receptor subunits in neurons of the laterodorsal tegmental nucleus, a nucleus important in drug addiction.

Science.gov (United States)

Christensen, Mark H; Kohlmeier, Kristi A

2016-03-01

The earlier an individual initiates cigarette smoking, the higher the likelihood of development of dependency to nicotine, the addictive ingredient in cigarettes. One possible mechanism underlying this higher addiction liability is an ontogenetically differential cellular response induced by nicotine in neurons mediating the reinforcing or euphoric effects of this drug, which could arise from age-related differences in the composition of nicotinic acetylcholine receptor (nAChR) subunits. In the current study, we examined whether the subunit composition of nAChRs differed between neurons within the laterodorsal tegmentum (LDT), a nucleus importantly involved in drug addiction associated behaviours, across two periods of ontogeny in which nicotine-mediated excitatory responses were shown to depend on age. To this end, whole-cell patch-clamp recordings in mouse brain slices from identified LDT neurons, in combination with nAChR subunit-specific receptor antagonists, were conducted. Comparison of the contribution of different nAChR subunits to acetylcholine (ACh)-induced inward currents indicated that the contributions of the β2 and/or β4 and α7 nAChR subunits alter across age. Taken together, we conclude that across a limited ontogenetic period, there is plasticity in the subunit composition of nAChRs in LDT neurons. In addition, our data indicate, for the first time, functional presence of α6 nAChR subunits in LDT neurons within the age ranges studied. Changes in subunit composition of nAChRs across ontogeny could contribute to the age-related differential excitability induced by nicotine. Differences in the subunit composition of nAChRs within the LDT would be expected to contribute to ontogenetic-dependent outflow from the LDT to target regions, which include reward-related circuitry. © 2014 Society for the Study of Addiction.

Generation of functional inhibitory synapses incorporating defined combinations of GABA(A or glycine receptor subunits

Directory of Open Access Journals (Sweden)

Christine Laura Dixon

2015-12-01

Full Text Available Fast inhibitory neurotransmission in the brain is mediated by wide range of GABAA receptor (GABAAR and glycine receptor (GlyR isoforms, each with different physiological and pharmacological properties. Because multiple isoforms are expressed simultaneously in most neurons, it is difficult to define the properties of inhibitory postsynaptic currents mediated by individual isoforms in vivo. Although recombinant expression systems permit the expression of individual isoforms in isolation, they require exogenous agonist application which cannot mimic the dynamic neurotransmitter profile characteristic of native synapses. We describe a neuron-HEK293 cell co-culture technique for generating inhibitory synapses incorporating defined combinations of GABAAR or GlyR subunits. Primary neuronal cultures, prepared from embryonic rat cerebral cortex or spinal cord, are used to provide presynaptic GABAergic and glycinergic terminals, respectively. When the cultures are mature, HEK293 cells expressing the subunits of interest plus neuroligin 2A are plated onto the neurons, which rapidly form synapses onto HEK293 cells. Patch clamp electrophysiology is then used to analyze the physiological and pharmacological properties of the inhibitory postsynaptic currents mediated by the recombinant receptors. The method is suitable for investigating the kinetic properties or the effects of drugs on inhibitory postsynaptic currents mediated by defined GABAAR or GlyR isoforms of interest, the effects of hereditary disease mutations on the formation and function of both types of synapses, and synaptogenesis and synaptic clustering mechanisms. The entire cell preparation procedure takes 2 – 5 weeks.

Brain mediators of the effects of noxious heat on pain.

Science.gov (United States)

Atlas, Lauren Y; Lindquist, Martin A; Bolger, Niall; Wager, Tor D

2014-08-01

Recent human neuroimaging studies have investigated the neural correlates of either noxious stimulus intensity or reported pain. Although useful, analyzing brain relationships with stimulus intensity and behavior separately does not address how sensation and pain are linked in the central nervous system. In this study, we used multi-level mediation analysis to identify brain mediators of pain--regions in which trial-by-trial responses to heat explained variability in the relationship between noxious stimulus intensity (across 4 levels) and pain. This approach has the potential to identify multiple circuits with complementary roles in pain genesis. Brain mediators of noxious heat effects on pain included targets of ascending nociceptive pathways (anterior cingulate, insula, SII, and medial thalamus) and also prefrontal and subcortical regions not associated with nociceptive pathways per se. Cluster analysis revealed that mediators were grouped into several distinct functional networks, including the following: somatosensory, paralimbic, and striatal-cerebellar networks that increased with stimulus intensity; and 2 networks co-localized with "default mode" regions in which stimulus intensity-related decreases mediated increased pain. We also identified "thermosensory" regions that responded to increasing noxious heat but did not predict pain reports. Finally, several regions did not respond to noxious input, but their activity predicted pain; these included ventromedial prefrontal cortex, dorsolateral prefrontal cortex, cerebellar regions, and supplementary motor cortices. These regions likely underlie both nociceptive and non-nociceptive processes that contribute to pain, such as attention and decision-making processes. Overall, these results elucidate how multiple distinct brain systems jointly contribute to the central generation of pain. Copyright © 2014 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.

The Tlo Proteins Are Stoichiometric Components of Candida albicans Mediator Anchored via the Med3 Subunit

Science.gov (United States)

Zhang, Anda; Petrov, Kostadin O.; Hyun, Emily R.; Liu, Zhongle; Gerber, Scott A.

2012-01-01

The amplification of the TLO (for telomere-associated) genes in Candida albicans, compared to its less pathogenic, close relative Candida dubliniensis, suggests a role in virulence. Little, however, is known about the function of the Tlo proteins. We have purified the Mediator coactivator complex from C. albicans (caMediator) and found that Tlo proteins are a stoichiometric component of caMediator. Many members of the Tlo family are expressed, and each is a unique member of caMediator. Protein expression analysis of individual Tlo proteins, as well as the purification of tagged Tlo proteins, demonstrate that there is a large free population of Tlo proteins in addition to the Mediator-associated population. Coexpression and copurification of Tloα12 and caMed3 in Escherichia coli established a direct physical interaction between the two proteins. We have also made a C. albicans med3Δ/Δ strain and purified an intact Mediator from this strain. The analysis of the composition of the med3Δ Mediator shows that it lacks a Tlo subunit. Regarding Mediator function, the med3Δ/Δ strain serves as a substitute for the difficult-to-make tloΔ/Δ C. albicans strain. A potential role of the TLO and MED3 genes in virulence is supported by the inability of the med3Δ/Δ strain to form normal germ tubes. This study of caMediator structure provides initial clues to the mechanism of action of the Tlo genes and a platform for further mechanistic studies of caMediator's involvement in gene regulatory patterns that underlie pathogenesis. PMID:22562472

Med1 subunit of the mediator complex in nuclear receptor-regulated energy metabolism, liver regeneration, and hepatocarcinogenesis.

Science.gov (United States)

Jia, Yuzhi; Viswakarma, Navin; Reddy, Janardan K

2014-01-01

Several nuclear receptors regulate diverse metabolic functions that impact on critical biological processes, such as development, differentiation, cellular regeneration, and neoplastic conversion. In the liver, some members of the nuclear receptor family, such as peroxisome proliferator-activated receptors (PPARs), constitutive androstane receptor (CAR), farnesoid X receptor (FXR), liver X receptor (LXR), pregnane X receptor (PXR), glucocorticoid receptor (GR), and others, regulate energy homeostasis, the formation and excretion of bile acids, and detoxification of xenobiotics. Excess energy burning resulting from increases in fatty acid oxidation systems in liver generates reactive oxygen species, and the resulting oxidative damage influences liver regeneration and liver tumor development. These nuclear receptors are important sensors of exogenous activators as well as receptor-specific endogenous ligands. In this regard, gene knockout mouse models revealed that some lipid-metabolizing enzymes generate PPARα-activating ligands, while others such as ACOX1 (fatty acyl-CoA oxidase1) inactivate these endogenous PPARα activators. In the absence of ACOX1, the unmetabolized ACOX1 substrates cause sustained activation of PPARα, and the resulting increase in energy burning leads to hepatocarcinogenesis. Ligand-activated nuclear receptors recruit the multisubunit Mediator complex for RNA polymerase II-dependent gene transcription. Evidence indicates that the Med1 subunit of the Mediator is essential for PPARα, PPARγ, CAR, and GR signaling in liver. Med1 null hepatocytes fail to respond to PPARα activators in that these cells do not show induction of peroxisome proliferation and increases in fatty acid oxidation enzymes. Med1-deficient hepatocytes show no increase in cell proliferation and do not give rise to liver tumors. Identification of nuclear receptor-specific coactivators and Mediator subunits should further our understanding of the complexities of metabolic

MEDIATOR18 and MEDIATOR20 confer susceptibility to Fusarium oxysporum in Arabidopsis thaliana

OpenAIRE

Fallath, Thorya; Kidd, Brendan N.; Stiller, Jiri; Davoine, Celine; Bj?rklund, Stefan; Manners, John M.; Kazan, Kemal; Schenk, Peer M.

2017-01-01

The conserved protein complex known as Mediator conveys transcriptional signals by acting as an intermediary between transcription factors and RNA polymerase II. As a result, Mediator subunits play multiple roles in regulating developmental as well as abiotic and biotic stress pathways. In this report we identify the head domain subunits MEDIATOR18 and MEDIATOR20 as important susceptibility factors for Fusarium oxysporum infection in Arabidopsis thaliana. Mutants of MED18 and MED20 display do...

An in vivo requirement for the mediator subunit med14 in the maintenance of stem cell populations.

Science.gov (United States)

Burrows, Jeffrey T A; Pearson, Bret J; Scott, Ian C

2015-04-14

The Mediator complex has recently been shown to be a key player in the maintenance of embryonic and induced pluripotent stem cells. However, the in vivo consequences of loss of many Mediator subunits are unknown. We identified med14 as the gene affected in the zebrafish logelei (log) mutant, which displayed a morphological arrest by 2 days of development. Surprisingly, microarray analysis showed that transcription was not broadly affected in log mutants. Indeed, log cells transplanted into a wild-type environment were able to survive into adulthood. In planarians, RNAi knockdown demonstrated a requirement for med14 and many other Mediator components in adult stem cell maintenance and regeneration. Multiple stem/progenitor cell populations were observed to be reduced or absent in zebrafish med14 mutant embryos. Taken together, our results show a critical, evolutionarily conserved, in vivo function for Med14 (and Mediator) in stem cell maintenance, distinct from a general role in transcription. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Involvement of N-methyl-D-aspartate receptor subunits in zinc-mediated modification of CA1 long-term potentiation in the developing hippocampus.

Science.gov (United States)

Takeda, Atsushi; Itagaki, Kosuke; Ando, Masaki; Oku, Naoto

2012-03-01

Zinc is an endogenous N-methyl-D-aspartate (NMDA) receptor blocker. It is possible that zinc-mediated modification of hippocampal CA1 long-term potentiation (LTP) is linked to the expression of NMDA receptor subunits, which varies with postnatal development. In the present study, the effect of ZnCl(2) and CaEDTA, a membrane-impermeable zinc chelator, on CA1 LTP induction was examined in hippocampal slices from immature (3-week-old) and young (6-week-old) rats. Tetanus (10-100 Hz, 1 sec)-induced CA1 LTP was more greatly enhanced in 3-week-old rats. CA1 LTP was inhibited in the presence of 2-amino-5-phosphonovalerate (APV), an NMDA receptor antagonist, and CaEDTA in 3-week-old rats, as in the case of 6-week-old rats reported previously. In 3-week-old rats, on the other hand, 5 μM ZnCl(2) attenuated NMDA receptor-mediated EPSPs more than in 6-week-old rats and significantly attenuated CA1 LTP. Moreover, 5 μM ZnCl(2) significantly attenuated CA1 LTP in the presence of (2R,4S)-4-(3-phosphonopropyl)-2-piperidinecarboxylic acid (PPPA), an NR2A antagonist, in 3-week-old rats, but not that in the presence of ifenprodil, an NR2B antagonist, suggesting that zinc-mediated attenuation of CA1 LTP is associated with the preferential expression of NR2B subunit in 3-week-old rats. In 6-week-old rats, however, 5 μM ZnCl(2) significantly potentiated CA1 LTP and also CA1 LTP in the presence of PPPA. The present study demonstrates that endogenous zinc may participate in the induction of CA1 LTP. It is likely that the changes in expression of NMDA receptor subunits are involved in the zinc-mediated modification of CA1 LTP in the developing hippocampus. Copyright © 2011 Wiley Periodicals, Inc.

Brain-derived neurotrophic factor ameliorates brain stem cardiovascular dysregulation during experimental temporal lobe status epilepticus.

Directory of Open Access Journals (Sweden)

Ching-Yi Tsai

Full Text Available BACKGROUND: Status epilepticus (SE is an acute, prolonged epileptic crisis with a mortality rate of 20-30%; the underlying mechanism is not completely understood. We assessed the hypothesis that brain stem cardiovascular dysregulation occurs during SE because of oxidative stress in rostral ventrolateral medulla (RVLM, a key nucleus of the baroreflex loop; to be ameliorated by brain-derived neurotrophic factor (BDNF via an antioxidant action. METHODOLOGY/PRINCIPAL FINDINGS: In a clinically relevant experimental model of temporal lobe SE (TLSE using Sprague-Dawley rats, sustained hippocampal seizure activity was accompanied by progressive hypotension that was preceded by a reduction in baroreflex-mediated sympathetic vasomotor tone; heart rate and baroreflex-mediated cardiac responses remained unaltered. Biochemical experiments further showed concurrent augmentation of superoxide anion, phosphorylated p47(phox subunit of NADPH oxidase and mRNA or protein levels of BDNF, tropomyosin receptor kinase B (TrkB, angiotensin AT1 receptor subtype (AT1R, nitric oxide synthase II (NOS II or peroxynitrite in RVLM. Whereas pretreatment by microinjection bilaterally into RVLM of a superoxide dismutase mimetic (tempol, a specific antagonist of NADPH oxidase (apocynin or an AT1R antagonist (losartan blunted significantly the augmented superoxide anion or phosphorylated p47(phox subunit in RVLM, hypotension and the reduced baroreflex-mediated sympathetic vasomotor tone during experimental TLSE, pretreatment with a recombinant human TrkB-Fc fusion protein or an antisense bdnf oligonucleotide significantly potentiated all those events, alongside peroxynitrite. However, none of the pretreatments affected the insignificant changes in heart rate and baroreflex-mediated cardiac responses. CONCLUSIONS/SIGNIFICANCE: We conclude that formation of peroxynitrite by a reaction between superoxide anion generated by NADPH oxidase in RVLM on activation by AT1R and NOS II

Positive modulation of delta-subunit containing GABAA receptors in mouse neurons

DEFF Research Database (Denmark)

Vardya, Irina; Hoestgaard-Jensen, Kirsten; Nieto-Gonzalez, Jose Luis

2012-01-01

δ-subunit containing extrasynaptic GABA(A) receptors are potential targets for modifying neuronal activity in a range of brain disorders. With the aim of gaining more insight in synaptic and extrasynaptic inhibition, we used a new positive modulator, AA29504, of δ-subunit containing GABA(A) recep......δ-subunit containing extrasynaptic GABA(A) receptors are potential targets for modifying neuronal activity in a range of brain disorders. With the aim of gaining more insight in synaptic and extrasynaptic inhibition, we used a new positive modulator, AA29504, of δ-subunit containing GABA......(A) receptors in mouse neurons in vitro and in vivo. Whole-cell patch-clamp recordings were carried out in the dentate gyrus in mouse brain slices. In granule cells, AA29504 (1 μM) caused a 4.2-fold potentiation of a tonic current induced by THIP (1 μM), while interneurons showed a potentiation of 2.6-fold......-free environment using Ca²⁺ imaging in cultured neurons, AA29504 showed GABA(A) receptor agonism in the absence of agonist. Finally, AA29504 exerted dose-dependent stress-reducing and anxiolytic effects in mice in vivo. We propose that AA29504 potentiates δ-containing GABA(A) receptors to enhance tonic inhibition...

Behavioural endophenotypes in mice lacking the auxiliary GABAB receptor subunit KCTD16.

Science.gov (United States)

Cathomas, Flurin; Sigrist, Hannes; Schmid, Luca; Seifritz, Erich; Gassmann, Martin; Bettler, Bernhard; Pryce, Christopher R

2017-01-15

Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the brain and is implicated in the pathophysiology of a number of neuropsychiatric disorders. The GABA B receptors are G-protein coupled receptors consisting of principle subunits and auxiliary potassium channel tetramerization domain (KCTD) subunits. The KCTD subunits 8, 12, 12b and 16 are cytosolic proteins that determine the kinetics of the GABA B receptor response. Previously, we demonstrated that Kctd12 null mutant mice (Kctd12 -/- ) exhibit increased auditory fear learning and that Kctd12 +/- mice show altered circadian activity, as well as increased intrinsic excitability in hippocampal pyramidal neurons. KCTD16 has been demonstrated to influence neuronal excitability by regulating GABA B receptor-mediated gating of postsynaptic ion channels. In the present study we investigated for behavioural endophenotypes in Kctd16 -/- and Kctd16 +/- mice. Compared with wild-type (WT) littermates, auditory and contextual fear conditioning were normal in both Kctd16 -/- and Kctd16 +/- mice. When fear memory was tested on the following day, Kctd16 -/- mice exhibited less extinction of auditory fear memory relative to WT and Kctd16 +/- mice, as well as more contextual fear memory relative to WT and, in particular, Kctd16 +/- mice. Relative to WT, both Kctd16 +/- and Kctd16 -/- mice exhibited normal circadian activity. This study adds to the evidence that auxillary KCTD subunits of GABA B receptors contribute to the regulation of behaviours that could constitute endophenotypes for hyper-reactivity to aversive stimuli in neuropsychiatric disorders. Copyright © 2016 Elsevier B.V. All rights reserved.

Human cDNA clones for an α subunit of G/sub i/ signal-transduction protein

International Nuclear Information System (INIS)

Bray, P.; Carter, A.; Guo, V.; Puckett, C.; Kamholz, J.; Spiegel, A.; Nirenberg, M.

1987-01-01

Two cDNA clones were obtained from a λgt11 cDNA human brain library that correspond to α/sub i/ subunits of G signal-transduction proteins (where α/sub i/ subunits refer to the α subunits of G proteins that inhibit adenylate cyclase). The nucleotide sequence of human brain α/sub i/ is highly homologous to that of bovine brain α/sub i/ and the predicted amino acid sequences are identical. However, human and bovine brain α/sub i/ cDNAs differ significantly from α/sub i/ cDNAs from human monocytes, rat glioma, and mouse macrophages in amino acid (88% homology) and nucleotide (71-75% homology) sequences. In addition, the nucleotide sequences of the 3' untranslated regions of human and bovine brain α/sub i/ cDNAs differ markedly from the sequences of human monocyte, rat glioma, and mouse macrophage α/sub i/ cDNAs. These results suggest there are at least two classes of α/sub i/ mRNA

Role of the Na(+)/K(+)-ATPase beta-subunit in peptide-mediated transdermal drug delivery.

Science.gov (United States)

Wang, Changli; Ruan, Renquan; Zhang, Li; Zhang, Yunjiao; Zhou, Wei; Lin, Jun; Ding, Weiping; Wen, Longping

2015-04-06

In this work, we discovered that the Na(+)/K(+)-ATPase beta-subunit (ATP1B1) on epidermal cells plays a key role in the peptide-mediated transdermal delivery of macromolecular drugs. First, using a yeast two-hybrid assay, we screened candidate proteins that have specific affinity for the short peptide TD1 (ACSSSPSKHCG) identified in our previous work. Then, we verified the specific binding of TD1 to ATP1B1 in yeast and mammalian cells by a pull-down ELISA and an immunoprecipitation assay. Finally, we confirmed that TD1 mainly interacted with the C-terminus of ATP1B1. Our results showed that the interaction between TD1 and ATP1B1 affected not only the expression and localization of ATP1B1, but also the epidermal structure. In addition, this interaction could be antagonized by the exogenous competitor ATP1B1 or be inhibited by ouabain, which results in the decreased delivery of macromolecular drugs across the skin. The discovery of a critical role of ATP1B1 in the peptide-mediated transdermal drug delivery is of great significance for the future development of new transdermal peptide enhancers.

Distribution of AMPA-type glutamate receptor subunits in the chick visual system

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Pires R.S.

1997-01-01

Full Text Available Several glutamate receptor (GluR subunits have been characterized during the past few years. In the present study, subunit-specific antisera were used to determine the distribution of the AMPA-type glutamate receptor subunits GluR1-4 in retinorecipient areas of the chick brain. Six white leghorn chicks (Gallus gallus, 7-15 days old, unknown sex were deeply anesthetized and perfused with 4% buffered paraformaldehyde and brain sections were stained using immunoperoxidase techniques. The AMPA-type glutamate receptor subunits GluR1, GluR2/3 and GluR4 were present in several retinorecipient areas, with varying degrees of colocalization. For example, perikarya in layers 2, 3, and 5 of the optic tectum contained GluR1, whereas GluR2/3 subunits appeared mainly in neurons of layer 13. The GluR4 subunit was only detected in a few cells of the tectal layer 13. GluR1 and GluR2/3 were observed in neurons of the nucleus geniculatus lateralis ventralis, whereas GluR4 was only present in its neuropil. Somata in the accessory optic nucleus appeared to contain GluR2/3 and GluR4, whereas GluR1 was the dominant subunit in the neuropil of this nucleus. These results suggest that different subpopulations of visual neurons might express different combinations of AMPA-type GluR subunits, which in turn might generate different synaptic responses to glutamate derived from retinal ganglion cell axons

Constitutively active signaling by the G protein βγ-subunit mediates intrinsically increased phosphodiesterase-4 activity in human asthmatic airway smooth muscle cells.

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Aihua Hu

Full Text Available Signaling by the Gβγ subunit of Gi protein, leading to downstream c-Src-induced activation of the Ras/c-Raf1/MEK-ERK1/2 signaling pathway and its upregulation of phosphodiesterase-4 (PDE4 activity, was recently shown to mediate the heightened contractility in proasthmatic sensitized isolated airway smooth muscle (ASM, as well as allergen-induced airway hyperresponsiveness and inflammation in an in vivo animal model of allergic asthma. This study investigated whether cultured human ASM (HASM cells derived from asthmatic donor lungs exhibit constitutively increased PDE activity that is attributed to intrinsically upregulated Gβγ signaling coupled to c-Src activation of the Ras/MEK/ERK1/2 cascade. We show that, relative to normal cells, asthmatic HASM cells constitutively exhibit markedly increased intrinsic PDE4 activity coupled to heightened Gβγ-regulated phosphorylation of c-Src and ERK1/2, and direct co-localization of the latter with the PDE4D isoform. These signaling events and their induction of heightened PDE activity are acutely suppressed by treating asthmatic HASM cells with a Gβγ inhibitor. Importantly, along with increased Gβγ activation, asthmatic HASM cells also exhibit constitutively increased direct binding of the small Rap1 GTPase-activating protein, Rap1GAP, to the α-subunit of Gi protein, which serves to cooperatively facilitate Ras activation and, thereby, enable enhanced Gβγ-regulated ERK1/2-stimulated PDE activity. Collectively, these data are the first to identify that intrinsically increased signaling via the Gβγ subunit, facilitated by Rap1GAP recruitment to the α-subunit, mediates the constitutively increased PDE4 activity detected in asthmatic HASM cells. These new findings support the notion that interventions targeted at suppressing Gβγ signaling may lead to novel approaches to treat asthma.

Brain sites mediating corticosteroid feedback inhibition of stimulated ACTH secretion

International Nuclear Information System (INIS)

Jacobson, L.

1989-01-01

There is substantial evidence that the brain mediates stress-induced and circadian increases in ACTH secretion and that corticosteroid concentrations which normalize basal plasma ACTH are insufficient to normalize ACTH responses to circadian or stressful stimuli in adrenalectomized rats. To identify brain sites mediating corticosteroid inhibition of stimulated ACTH secretion, two approaches were used. The first compared brain [ 14 C]-2-deoxyglucose uptake in rats with differential ACTH responses to stress. Relative to sham-adrenalectomized (SHAM) rats, adrenalectomized rats replaced with low, constant corticosterone levels via a subcutaneous corticosterone pellet (B-PELLET) exhibited elevated and prolonged ACTH responses to a variety of stimuli. Adrenalectomized rate given a circadian corticosterone rhythm via corticosterone in their drinking water exhibited elevated ACTH levels immediately after stress, but unlike B-PELLET rats, terminated stress induced ACTH secretion normally relative to SHAMS. Therefore, the abnormal ACTH responses to stress in B-PELLET rats were due to the lack of both circadian variations and stress-induced increases in corticosterone. Hypoxia was selected as a standardized stimulus for correlating brain [ 14 C]-2-deoxyglucose uptake with ACTH secretion. In intact rats, increases in plasma ACTH and decreases in arterial PO 2 correlated with the severity of hypoxia at arterial PCO 2 below 60 mm Hg. Hypoxia PELLET vs. SHAM rats. However, in preliminary experiments, although hypoxia increased brain 2-deoxyglucose uptake in most brain regions, plasma ACTH correlated poorly with 2-deoxyglucose uptake at 12% and 10% O 2

The function of the Mediator complex in plant immunity.

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An, Chuanfu; Mou, Zhonglin

2013-03-01

Upon pathogen infection, plants undergo dramatic transcriptome reprogramming to shift from normal growth and development to immune response. During this rapid process, the multiprotein Mediator complex has been recognized as an important player to fine-tune gene-specific and pathway-specific transcriptional reprogramming by acting as an adaptor/coregulator between sequence-specific transcription factor and RNA polymerase II (RNAPII). Here, we review current understanding of the role of five functionally characterized Mediator subunits (MED8, MED15, MED16, MED21 and MED25) in plant immunity. All these Mediator subunits positively regulate resistance against leaf-infecting biotrophic bacteria or necrotrophic fungi. While MED21 appears to regulate defense against fungal pathogens via relaying signals from upstream regulators and chromatin modification to RNAPII, the other four Mediator subunits locate at different positions of the defense network to convey phytohormone signal(s). Fully understanding the role of Mediator in plant immunity needs to characterize more Mediator subunits in both Arabidopsis and other plant species. Identification of interacting proteins of Mediator subunits will further help to reveal their specific regulatory mechanisms in plant immunity.

Cellular mechanisms of estradiol-mediated sexual differentiation of the brain.

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Wright, Christopher L; Schwarz, Jaclyn S; Dean, Shannon L; McCarthy, Margaret M

2010-09-01

Gonadal steroids organize the developing brain during a perinatal sensitive period and have enduring consequences for adult behavior. In male rodents testicular androgens are aromatized in neurons to estrogens and initiate multiple distinct cellular processes that ultimately determine the masculine phenotype. Within specific brain regions, overall cell number and dendritic morphology are the principal targets for hormonal organization. Recent advances have been made in elucidating the cellular mechanisms by which the neurological underpinnings of sexually dimorphic physiology and behavior are determined. These include estradiol-mediated prostaglandin synthesis, presynaptic release of glutamate, postsynaptic changes in glutamate receptors and changes in cell adhesion molecules. Sex differences in cell death are mediated by hormonal modulation of survival and death factors such as TNFalpha and Bcl-2/BAX. Copyright 2010 Elsevier Ltd. All rights reserved.

Mediator kinase module and human tumorigenesis.

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Clark, Alison D; Oldenbroek, Marieke; Boyer, Thomas G

2015-01-01

Mediator is a conserved multi-subunit signal processor through which regulatory informatiosn conveyed by gene-specific transcription factors is transduced to RNA Polymerase II (Pol II). In humans, MED13, MED12, CDK8 and Cyclin C (CycC) comprise a four-subunit "kinase" module that exists in variable association with a 26-subunit Mediator core. Genetic and biochemical studies have established the Mediator kinase module as a major ingress of developmental and oncogenic signaling through Mediator, and much of its function in signal-dependent gene regulation derives from its resident CDK8 kinase activity. For example, CDK8-targeted substrate phosphorylation impacts transcription factor half-life, Pol II activity and chromatin chemistry and functional status. Recent structural and biochemical studies have revealed a precise network of physical and functional subunit interactions required for proper kinase module activity. Accordingly, pathologic change in this activity through altered expression or mutation of constituent kinase module subunits can have profound consequences for altered signaling and tumor formation. Herein, we review the structural organization, biological function and oncogenic potential of the Mediator kinase module. We focus principally on tumor-associated alterations in kinase module subunits for which mechanistic relationships as opposed to strictly correlative associations are established. These considerations point to an emerging picture of the Mediator kinase module as an oncogenic unit, one in which pathogenic activation/deactivation through component change drives tumor formation through perturbation of signal-dependent gene regulation. It follows that therapeutic strategies to combat CDK8-driven tumors will involve targeted modulation of CDK8 activity or pharmacologic manipulation of dysregulated CDK8-dependent signaling pathways.

Papillae formation on trichome cell walls requires the function of the mediator complex subunit Med25.

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Fornero, Christy; Suo, Bangxia; Zahde, Mais; Juveland, Katelyn; Kirik, Viktor

2017-11-01

Glassy Hair 1 (GLH1) gene that promotes papillae formation on trichome cell walls was identified as a subunit of the transcriptional mediator complex MED25. The MED25 gene is shown to be expressed in trichomes. The expression of the trichome development marker genes GLABRA2 (GL2) and Ethylene Receptor2 (ETR2) is not affected in the glh1 mutant. Presented data suggest that Arabidopsis MED25 mediator component is likely involved in the transcription of genes promoting papillae deposition in trichomes. The plant cell wall plays an important role in communication, defense, organization and support. The importance of each of these functions varies by cell type. Specialized cells, such as Arabidopsis trichomes, exhibit distinct cell wall characteristics including papillae. To better understand the molecular processes important for papillae deposition on the cell wall surface, we identified the GLASSY HAIR 1 (GLH1) gene, which is necessary for papillae formation. We found that a splice-site mutation in the component of the transcriptional mediator complex MED25 gene is responsible for the near papillae-less phenotype of the glh1 mutant. The MED25 gene is expressed in trichomes. Reporters for trichome developmental marker genes GLABRA2 (GL2) and Ethylene Receptor2 (ETR2) were not affected in the glh1 mutant. Collectively, the presented results show that MED25 is necessary for papillae formation on the cell wall surface of leaf trichomes and suggest that the Arabidopsis MED25 mediator component is likely involved in the transcription of a subset of genes that promote papillae deposition in trichomes.

Nose-to-brain delivery of macromolecules mediated by cell-penetrating peptides

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Tingting Lin

2016-07-01

Full Text Available Brain delivery of macromolecular therapeutics (e.g., proteins remains an unsolved problem because of the formidable blood–brain barrier (BBB. Although a direct pathway of nose-to-brain transfer provides an answer to circumventing the BBB and has already been intensively investigated for brain delivery of small drugs, new challenges arise for intranasal delivery of proteins because of their larger size and hydrophilicity. In order to overcome the barriers and take advantage of available pathways (e.g., epithelial tight junctions, uptake by olfactory neurons, transport into brain tissues, and intra-brain diffusion, a low molecular weight protamine (LMWP cell-penetrating peptide was utilized to facilitate nose-to-brain transport. Cell-penetrating peptides (CPP have been widely used to mediate macromolecular delivery through many kinds of biobarriers. Our results show that conjugates of LMWP–proteins are able to effectively penetrate into the brain after intranasal administration. The CPP-based intranasal method highlights a promising solution for protein therapy of brain diseases.

Nose-to-brain delivery of macromolecules mediated by cell-penetrating peptides

Institute of Scientific and Technical Information of China (English)

Tingting Lin; Ergang Liu; Huining He; Meong Cheol Shin; Cheol Moon; Victor C.Yang; Yongzhuo Huang

2016-01-01

Brain delivery of macromolecular therapeutics(e.g., proteins) remains an unsolved problem because of the formidable blood–brain barrier(BBB). Although a direct pathway of nose-to-brain transfer provides an answer to circumventing the BBB and has already been intensively investigated for brain delivery of small drugs,new challenges arise for intranasal delivery of proteins because of their larger size and hydrophilicity. In order to overcome the barriers and take advantage of available pathways(e.g., epithelial tight junctions, uptake by olfactory neurons, transport into brain tissues, and intra-brain diffusion), a low molecular weight protamine(LMWP) cell-penetrating peptide was utilized to facilitate nose-to-brain transport. Cell-penetrating peptides(CPP)have been widely used to mediate macromolecular delivery through many kinds of biobarriers. Our results show that conjugates of LMWP–proteins are able to effectively penetrate into the brain after intranasal administration.The CPP-based intranasal method highlights a promising solution for protein therapy of brain diseases.

Partial agonists and subunit selectivity at NMDA receptors

DEFF Research Database (Denmark)

Risgaard, Rune; Hansen, Kasper Bø; Clausen, Rasmus Prætorius

2010-01-01

Subunit-selective ligands for glutamate receptors remains an area of interest as glutamate is the major excitatory neurotransmitter in the brain and involved in a number of diseased states in the central nervous system (CNS). Few subtype-selective ligands are known, especially among the N...

Prevalence and clinical significance of mediator complex subunit 12 mutations in 362 Han Chinese samples with uterine leiomyoma.

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Wu, Juan; Zou, Yang; Luo, Yong; Guo, Jiu-Bai; Liu, Fa-Ying; Zhou, Jiang-Yan; Zhang, Zi-Yu; Wan, Lei; Huang, Ou-Ping

2017-07-01

Uterine leiomyomas (ULs) are the most common gynecological benign tumors originating from the myometrium. Prevalent mutations in the mediator complex subunit 12 (MED12) gene have been identified in ULs, and functional evidence has revealed that these mutations may promote the development of ULs. However, whether MED12 mutations are associated with certain clinical characteristics in ULs remains largely unknown. In the present study, the potential mutations of MED12 and its paralogous gene, mediator complex subunit 12-like (MED12L), were screened in 362 UL tumors from Han Chinese patients. A total of 158 out of 362 UL tumors (43.6%) were identified as harboring MED12 somatic mutations, and the majority of these mutations were restricted to the 44th residue. MED12 mutations were also observed in 2 out of 145 (1.4%) adjacent control myometrium. Furthermore, the mutation spectrum of MED12 in the concurrent leiomyomas was noticeably different. Correlation analysis of MED12 mutations with the available clinical features indicated that patients with mutated MED12 tended to have smaller cervical diameters. By contrast, no MED12L mutation was identified in the present samples. In summary, the present study demonstrated the presence of prevalent MED12 somatic mutations in UL samples, and the MED12 mutation was associated with smaller cervical diameters. The low mutation frequency of MED12 in adjacent control myometrium indicated that MED12 mutation may be an early event in the pathogenesis of ULs. Furthermore, MED12 mutation status in concurrent tumors from multiple leiomyomas supported several prior observations that the majority of these tumors arose independently.

Receptor-Mediated Endocytosis and Brain Delivery of Therapeutic Biologics

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Guangqing Xiao

2013-01-01

Full Text Available Transport of macromolecules across the blood-brain-barrier (BBB requires both specific and nonspecific interactions between macromolecules and proteins/receptors expressed on the luminal and/or the abluminal surfaces of the brain capillary endothelial cells. Endocytosis and transcytosis play important roles in the distribution of macromolecules. Due to the tight junction of BBB, brain delivery of traditional therapeutic proteins with large molecular weight is generally not possible. There are multiple pathways through which macromolecules can be taken up into cells through both specific and nonspecific interactions with proteins/receptors on the cell surface. This review is focused on the current knowledge of receptor-mediated endocytosis/transcytosis and brain delivery using the Angiopep-2-conjugated system and the molecular Trojan horses. In addition, the role of neonatal Fc receptor (FcRn in regulating the efflux of Immunoglobulin G (IgG from brain to blood, and approaches to improve the pharmacokinetics of therapeutic biologics by generating Fc fusion proteins, and increasing the pH dependent binding affinity between Fc and FcRn, are discussed.

MEDIATOR18 and MEDIATOR20 confer susceptibility to Fusarium oxysporum in Arabidopsis thaliana

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Stiller, Jiri; Davoine, Celine; Björklund, Stefan; Manners, John M.; Kazan, Kemal; Schenk, Peer M.

2017-01-01

The conserved protein complex known as Mediator conveys transcriptional signals by acting as an intermediary between transcription factors and RNA polymerase II. As a result, Mediator subunits play multiple roles in regulating developmental as well as abiotic and biotic stress pathways. In this report we identify the head domain subunits MEDIATOR18 and MEDIATOR20 as important susceptibility factors for Fusarium oxysporum infection in Arabidopsis thaliana. Mutants of MED18 and MED20 display down-regulation of genes associated with jasmonate signaling and biosynthesis while up-regulation of salicylic acid associated pathogenesis related genes and reactive oxygen producing and scavenging genes. We propose that MED18 and MED20 form a sub-domain within Mediator that controls the balance of salicylic acid and jasmonate associated defense pathways. PMID:28441405

F4+ enterotoxigenic Escherichia coli (ETEC) adhesion mediated by the major fimbrial subunit FaeG.

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Xia, Pengpeng; Song, Yujie; Zou, Yajie; Yang, Ying; Zhu, Guoqiang

2015-09-01

The FaeG subunit is the major constituent of F4(+) fimbriae, associated with glycoprotein and/or glycolipid receptor recognition and majorly contributes to the pathogen attachment to the host cells. To investigate the key factor involved in the fimbrial binding of F4(+) Escherichia coli, both the recombinant E. coli SE5000 strains carrying the fae operon gene clusters that express the different types of fimbriae in vitro, named as rF4ab, rF4ac, and rF4ad, respectively, corresponding to the fimbrial types F4ab, F4ac, and F4ad, and the three isogenic in-frame faeG gene deletion mutants were constructed. The adhesion assays and adhesion inhibition assays showed that ΔfaeG mutants had a significant reduction in the binding to porcine brush border as well as the intestinal epithelial cell lines, while the complemented strain ΔfaeG/pfaeG restored the adhesion function. The recombinant bacterial strains rF4ab, rF4ac, and rF4ad have the same binding property as wild-type F4(+) E. coli strains do and improvement in terms of binding to porcine brush border and the intestinal epithelial cells, and the adherence was blocked by the monoclonal antibody anti-F4 fimbriae. These data demonstrate that the fimbrial binding of F4(+) E. coli is directly mediated by the major FaeG subunit. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Molecular architecture of the yeast Mediator complex

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Robinson, Philip J; Trnka, Michael J; Pellarin, Riccardo; Greenberg, Charles H; Bushnell, David A; Davis, Ralph; Burlingame, Alma L; Sali, Andrej; Kornberg, Roger D

2015-01-01

The 21-subunit Mediator complex transduces regulatory information from enhancers to promoters, and performs an essential role in the initiation of transcription in all eukaryotes. Structural information on two-thirds of the complex has been limited to coarse subunit mapping onto 2-D images from electron micrographs. We have performed chemical cross-linking and mass spectrometry, and combined the results with information from X-ray crystallography, homology modeling, and cryo-electron microscopy by an integrative modeling approach to determine a 3-D model of the entire Mediator complex. The approach is validated by the use of X-ray crystal structures as internal controls and by consistency with previous results from electron microscopy and yeast two-hybrid screens. The model shows the locations and orientations of all Mediator subunits, as well as subunit interfaces and some secondary structural elements. Segments of 20–40 amino acid residues are placed with an average precision of 20 Å. The model reveals roles of individual subunits in the organization of the complex. DOI: http://dx.doi.org/10.7554/eLife.08719.001 PMID:26402457

Near-infrared deep brain stimulation via upconversion nanoparticle–mediated optogenetics

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Chen, Shuo; Weitemier, Adam Z.; Zeng, Xiao; He, Linmeng; Wang, Xiyu; Tao, Yanqiu; Huang, Arthur J. Y.; Hashimotodani, Yuki; Kano, Masanobu; Iwasaki, Hirohide; Parajuli, Laxmi Kumar; Okabe, Shigeo; Teh, Daniel B. Loong; All, Angelo H.; Tsutsui-Kimura, Iku; Tanaka, Kenji F.; Liu, Xiaogang; McHugh, Thomas J.

2018-02-01

Optogenetics has revolutionized the experimental interrogation of neural circuits and holds promise for the treatment of neurological disorders. It is limited, however, because visible light cannot penetrate deep inside brain tissue. Upconversion nanoparticles (UCNPs) absorb tissue-penetrating near-infrared (NIR) light and emit wavelength-specific visible light. Here, we demonstrate that molecularly tailored UCNPs can serve as optogenetic actuators of transcranial NIR light to stimulate deep brain neurons. Transcranial NIR UCNP-mediated optogenetics evoked dopamine release from genetically tagged neurons in the ventral tegmental area, induced brain oscillations through activation of inhibitory neurons in the medial septum, silenced seizure by inhibition of hippocampal excitatory cells, and triggered memory recall. UCNP technology will enable less-invasive optical neuronal activity manipulation with the potential for remote therapy.

Modulation of NMDA Receptor Properties and Synaptic Transmission by the NR3A Subunit in Mouse Hippocampal and Cerebrocortical Neurons

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Tong, Gary; Takahashi, Hiroto; Tu, Shichun; Shin, Yeonsook; Talantova, Maria; Zago, Wagner; Xia, Peng; Nie, Zhiguo; Goetz, Thomas; Zhang, Dongxian; Lipton, Stuart A.; Nakanishi, Nobuki

2015-01-01

Expression of the NR3A subunit with NR1/NR2 in Xenopus oocytes or mammalian cell lines leads to a reduction in N-methyl-D-aspartate (NMDA)-induced currents and decreased Mg2+ sensitivity and Ca2+ permeability compared with NR1/NR2 receptors. Consistent with these findings, neurons from NR3A knockout (KO) mice exhibit enhanced NMDA-induced currents. Recombinant NR3A can also form excitatory glycine receptors with NR1 in the absence of NR2. However, the effects of NR3A on channel properties in neurons and synaptic transmission have not been fully elucidated. To study physiological roles of NR3A subunits, we generated NR3A transgenic (Tg) mice. Cultured NR3A Tg neurons exhibited two populations of NMDA receptor (NMDAR) channels, reduced Mg2+ sensitivity, and decreased Ca2+ permeability in response to NMDA/glycine, but glycine alone did not elicit excitatory currents. In addition, NMDAR-mediated excitatory postsynaptic currents (EPSCs) in NR3A Tg hippocampal slices showed reduced Mg2+ sensitivity, consistent with the notion that NR3A subunits incorporated into synaptic NMDARs. To study the function of endogenous NR3A subunits, we compared NMDAR-mediated EPSCs in NR3A KO and WT control mice. In NR3A KO mice, the ratio of the amplitudes of the NMDAR-mediated component to α-amino-3-hydroxy-5-methyl-4-isox-azolepropionic acid receptor-mediated component of the EPSC was significantly larger than that seen in WT littermates. This result suggests that NR3A subunits contributed to the NMDAR-mediated component of the EPSC in WT mice. Taken together, these results show that NR3A subunits contribute to NMDAR responses from both synaptic and extra-synaptic receptors, likely composed of NR1, NR2, and NR3 subunits. PMID:18003876

Evolution of disorder in Mediator complex and its functional relevance.

Science.gov (United States)

Nagulapalli, Malini; Maji, Sourobh; Dwivedi, Nidhi; Dahiya, Pradeep; Thakur, Jitendra K

2016-02-29

Mediator, an important component of eukaryotic transcriptional machinery, is a huge multisubunit complex. Though the complex is known to be conserved across all the eukaryotic kingdoms, the evolutionary topology of its subunits has never been studied. In this study, we profiled disorder in the Mediator subunits of 146 eukaryotes belonging to three kingdoms viz., metazoans, plants and fungi, and attempted to find correlation between the evolution of Mediator complex and its disorder. Our analysis suggests that disorder in Mediator complex have played a crucial role in the evolutionary diversification of complexity of eukaryotic organisms. Conserved intrinsic disordered regions (IDRs) were identified in only six subunits in the three kingdoms whereas unique patterns of IDRs were identified in other Mediator subunits. Acquisition of novel molecular recognition features (MoRFs) through evolution of new subunits or through elongation of the existing subunits was evident in metazoans and plants. A new concept of 'junction-MoRF' has been introduced. Evolutionary link between CBP and Med15 has been provided which explain the evolution of extended-IDR in CBP from Med15 KIX-IDR junction-MoRF suggesting role of junction-MoRF in evolution and modulation of protein-protein interaction repertoire. This study can be informative and helpful in understanding the conserved and flexible nature of Mediator complex across eukaryotic kingdoms. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Molecular Mechanisms Responsible for Neuron-Derived Conditioned Medium (NCM-Mediated Protection of Ischemic Brain.

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Chi-Hsin Lin

Full Text Available The protective value of neuron-derived conditioned medium (NCM in cerebral ischemia and the underlying mechanism(s responsible for NCM-mediated brain protection against cerebral ischemia were investigated in the study. NCM was first collected from the neuronal culture growing under the in vitro ischemic condition (glucose-, oxygen- and serum-deprivation or GOSD for 2, 4 or 6 h. Through the focal cerebral ischemia (bilateral CCAO/unilateral MCAO animal model, we discovered that ischemia/reperfusion (I/R-induced brain infarction was significantly reduced by NCM, given directly into the cistern magna at the end of 90 min of CCAO/MCAO. Immunoblocking and chemical blocking strategies were applied in the in vitro ischemic studies to show that NCM supplement could protect microglia, astrocytes and neurons from GOSD-induced cell death, in a growth factor (TGFβ1, NT-3 and GDNF and p-ERK dependent manner. Brain injection with TGFβ1, NT3, GDNF and ERK agonist (DADS alone or in combination, therefore also significantly decreased the infarct volume of ischemic brain. Moreover, NCM could inhibit ROS but stimulate IL-1β release from GOSD-treated microglia and limit the infiltration of IL-β-positive microglia into the core area of ischemic brain, revealing the anti-oxidant and anti-inflammatory activities of NCM. In overall, NCM-mediated brain protection against cerebral ischemia has been demonstrated for the first time in S.D. rats, due to its anti-apoptotic, anti-oxidant and potentially anti-glutamate activities (NCM-induced IL-1β can inhibit the glutamate-mediated neurotoxicity and restriction upon the infiltration of inflammatory microglia into the core area of ischemic brain. The therapeutic potentials of NCM, TGFβ1, GDNF, NT-3 and DADS in the control of cerebral ischemia in human therefore have been suggested and require further investigation.

Electrophysiology and Beyond: Multiple roles of Na+ channel β subunits in development and disease

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Patino, Gustavo A.; Isom, Lori L.

2010-01-01

Voltage-gated Na+ channel (VGSC) β subunits are not “auxiliary.” These multifunctional molecules not only modulate Na+ current (INa), but also function as cell adhesion molecules (CAMs) – playing roles in aggregation, migration, invasion, neurite outgrowth, and axonal fasciculation. β subunits are integral members of VGSC signaling complexes at nodes of Ranvier, axon initial segments, and cardiac intercalated disks, regulating action potential propagation through critical intermolecular and cell-cell communication events. At least in vitro, many β subunit cell adhesive functions occur both in the presence and absence of pore-forming VGSC α subunits, and in vivo β subunits are expressed in excitable as well as non-excitable cells, thus β subunits may play important functional roles on their own, in the absence of α subunits. VGSC β1 subunits are essential for life and appear to be especially important during brain development. Mutations in β subunit genes result in a variety of human neurological and cardiovascular diseases. Moreover, some cancer cells exhibit alterations in β subunit expression during metastasis. In short, these proteins, originally thought of as merely accessory to α subunits, are critical players in their own right in human health and disease. Here we discuss the role of VGSC β subunits in the nervous system. PMID:20600605

A Special Extract of Bacopa monnieri (CDRI-08 Restores Learning and Memory by Upregulating Expression of the NMDA Receptor Subunit GluN2B in the Brain of Scopolamine-Induced Amnesic Mice

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Rakesh Rai

2015-01-01

Full Text Available In the present communication, we have investigated effects of the CDRI-08, a well characterized extract of Bacopa monnieri, on expression of the GluN2B subunit of NMDAR in various brain regions of the scopolamine-induced amnesic mice. Our behavioral data reveal that scopolamine-treated amnesic mice exhibit significant decline in the spatial memory compared to the normal control mice. Our RT-PCR and immunoblotting data revealed that the scopolamine treatment resulted in a significant downregulation of the NMDAR GluN2B subunit expression in prefrontal cortex and hippocampus. Our enzyme assay data revealed that scopolamine caused a significant increase in the acetylcholinesterase activity in both the brain regions. Further, oral administration of the CDRI-08 to scopolamine-treated amnesic mice restored the spatial memory which was found to be associated with significant upregulation of the GluN2B subunit expression and decline in the acetylcholinesterase activity in prefrontal cortex as well as hippocampus towards their levels in the normal control mice. Our study provides the evidence for the mechanism underlying role of the Bacopa monnieri extract (CDRI-08 in restoring spatial memory in amnesic mice, which may have therapeutic implications.

GABA receptor subunit distribution and FMRP-mGluR5 signaling abnormalities in the cerebellum of subjects with schizophrenia, mood disorders, and autism.

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Fatemi, S Hossein; Folsom, Timothy D

2015-09-01

Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the brain. GABAergic receptor abnormalities have been documented in several major psychiatric disorders including schizophrenia, mood disorders, and autism. Abnormal expression of mRNA and protein for multiple GABA receptors has also been observed in multiple brain regions leading to alterations in the balance between excitatory/inhibitory signaling in the brain with potential profound consequences for normal cognition and maintenance of mood and perception. Altered expression of GABAA receptor subunits has been documented in fragile X mental retardation 1 (FMR1) knockout mice, suggesting that loss of its protein product, fragile X mental retardation protein (FMRP), impacts GABAA subunit expression. Recent postmortem studies from our laboratory have shown reduced expression of FMRP in the brains of subjects with schizophrenia, bipolar disorder, major depression, and autism. FMRP acts as a translational repressor and, under normal conditions, inhibits metabotropic glutamate receptor 5 (mGluR5)-mediated signaling. In fragile X syndrome (FXS), the absence of FMRP is hypothesized to lead to unregulated mGluR5 signaling, ultimately resulting in the behavioral and intellectual impairments associated with this disorder. Our laboratory has identified changes in mGluR5 expression in autism, schizophrenia, and mood disorders. In the current review article, we discuss our postmortem data on GABA receptors, FMRP, and mGluR5 levels and compare our results with other laboratories. Finally, we discuss the interactions between these molecules and the potential for new therapeutic interventions that target these interconnected signaling systems. Copyright © 2014 Elsevier B.V. All rights reserved.

Δ9-THC-Caused Synaptic and Memory Impairments Are Mediated through COX-2 Signaling

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Chen, Rongqing; Zhang, Jian; Fan, Ni; Teng, Zhao-qian; Wu, Yan; Yang, Hongwei; Tang, Ya-ping; Sun, Hao; Song, Yunping; Chen, Chu

2013-01-01

Marijuana has been used for thousands of years as a treatment for medical conditions. However, untoward side effects limit its medical value. Here we show that synaptic and cognitive impairments following repeated exposure to Δ9-tetrahydrocannabinol (Δ9-THC) are associated with the induction of cyclooxygenase-2 (COX-2), an inducible enzyme that converts arachidonic acid to prostanoids, in the brain. COX-2 induction by Δ9-THC is mediated via CB1 receptor-coupled G-protein βγ subunits. Pharmaco...

Brain structure mediates the association between height and cognitive ability.

Science.gov (United States)

Vuoksimaa, Eero; Panizzon, Matthew S; Franz, Carol E; Fennema-Notestine, Christine; Hagler, Donald J; Lyons, Michael J; Dale, Anders M; Kremen, William S

2018-05-11

Height and general cognitive ability are positively associated, but the underlying mechanisms of this relationship are not well understood. Both height and general cognitive ability are positively associated with brain size. Still, the neural substrate of the height-cognitive ability association is unclear. We used a sample of 515 middle-aged male twins with structural magnetic resonance imaging data to investigate whether the association between height and cognitive ability is mediated by cortical size. In addition to cortical volume, we used genetically, ontogenetically and phylogenetically distinct cortical metrics of total cortical surface area and mean cortical thickness. Height was positively associated with general cognitive ability and total cortical volume and cortical surface area, but not with mean cortical thickness. Mediation models indicated that the well-replicated height-general cognitive ability association is accounted for by individual differences in total cortical volume and cortical surface area (highly heritable metrics related to global brain size), and that the genetic association between cortical surface area and general cognitive ability underlies the phenotypic height-general cognitive ability relationship.

Dual functions of a small regulatory subunit in the mitochondrial calcium uniporter complex.

Science.gov (United States)

Tsai, Ming-Feng; Phillips, Charles B; Ranaghan, Matthew; Tsai, Chen-Wei; Wu, Yujiao; Willliams, Carole; Miller, Christopher

2016-04-21

Mitochondrial Ca(2+) uptake, a process crucial for bioenergetics and Ca(2+) signaling, is catalyzed by the mitochondrial calcium uniporter. The uniporter is a multi-subunit Ca(2+)-activated Ca(2+) channel, with the Ca(2+) pore formed by the MCU protein and Ca(2+)-dependent activation mediated by MICU subunits. Recently, a mitochondrial inner membrane protein EMRE was identified as a uniporter subunit absolutely required for Ca(2+) permeation. However, the molecular mechanism and regulatory purpose of EMRE remain largely unexplored. Here, we determine the transmembrane orientation of EMRE, and show that its known MCU-activating function is mediated by the interaction of transmembrane helices from both proteins. We also reveal a second function of EMRE: to maintain tight MICU regulation of the MCU pore, a role that requires EMRE to bind MICU1 using its conserved C-terminal polyaspartate tail. This dual functionality of EMRE ensures that all transport-competent uniporters are tightly regulated, responding appropriately to a dynamic intracellular Ca(2+) landscape.

Brain Regions Related to Impulsivity Mediate the Effects of Early Adversity on Antisocial Behavior.

Science.gov (United States)

Mackey, Scott; Chaarani, Bader; Kan, Kees-Jan; Spechler, Philip A; Orr, Catherine; Banaschewski, Tobias; Barker, Gareth; Bokde, Arun L W; Bromberg, Uli; Büchel, Christian; Cattrell, Anna; Conrod, Patricia J; Desrivières, Sylvane; Flor, Herta; Frouin, Vincent; Gallinat, Jürgen; Gowland, Penny; Heinz, Andreas; Ittermann, Bernd; Paillère Martinot, Marie-Laure; Artiges, Eric; Nees, Frauke; Papadopoulos-Orfanos, Dimitri; Poustka, Luise; Smolka, Michael N; Jurk, Sarah; Walter, Henrik; Whelan, Robert; Schumann, Gunter; Althoff, Robert R; Garavan, Hugh

2017-08-15

Individual differences in impulsivity and early adversity are known to be strong predictors of adolescent antisocial behavior. However, the neurobiological bases of impulsivity and their relation to antisocial behavior and adversity are poorly understood. Impulsivity was estimated with a temporal discounting task. Voxel-based morphometry was used to determine the brain structural correlates of temporal discounting in a large cohort (n = 1830) of 14- to 15-year-old children. Mediation analysis was then used to determine whether the volumes of brain regions associated with temporal discounting mediate the relation between adverse life events (e.g., family conflict, serious accidents) and antisocial behaviors (e.g., precocious sexual activity, bullying, illicit substance use). Greater temporal discounting (more impulsivity) was associated with 1) lower volume in frontomedial cortex and bilateral insula and 2) greater volume in a subcortical region encompassing the ventral striatum, hypothalamus and anterior thalamus. The volume ratio between these cortical and subcortical regions was found to partially mediate the relation between adverse life events and antisocial behavior. Temporal discounting is related to regions of the brain involved in reward processing and interoception. The results support a developmental imbalance model of impulsivity and are consistent with the idea that negative environmental factors can alter the developing brain in ways that promote antisocial behavior. Copyright © 2016 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

Pharmacologic Effects in vivo in Brain by Vector-Mediated Peptide Drug Delivery

Science.gov (United States)

Bickel, Ulrich; Yoshikawa, Takayoshi; Landaw, Elliot M.; Faull, Kym F.; Pardridge, William M.

1993-04-01

Pharmacologic effects in brain caused by systemic administration of neuropeptides are prevented by poor transport of the peptide through the brain vascular endothelium, which comprises the blood-brain barrier in vivo. In the present study, successful application of a chimeric peptide approach to enhance drug delivery through the blood-brain barrier for the purpose of achieving a central nervous system pharmacologic effect is described. The chimeric peptide was formed by linkage of a potent vasoactive intestinal peptide (VIP) analogue, which had been monobiotinylated, to a drug transport vector. The vector consisted of a covalent conjugate of avidin and the OX26 monoclonal antibody to the transferrin receptor. Owing to the high concentration of transferrin receptors on brain capillary endothelia, OX26 targets brain and undergoes receptor-mediated transcytosis through the blood-brain barrier. Systemic infusion of low doses (12 μg/kg) of the VIP chimeric peptide in rats resulted in an in vivo central nervous system pharmacologic effect: a 65% increase in cerebral blood flow. Biotinylated VIP analogue without the brain transport vector was ineffective.

Bisphenol-A rapidly enhanced passive avoidance memory and phosphorylation of NMDA receptor subunits in hippocampus of young rats

International Nuclear Information System (INIS)

Xu Xiaohong; Li Tao; Luo Qingqing; Hong Xing; Xie Lingdan; Tian Dong

2011-01-01

Bisphenol-A (BPA), an endocrine disruptor, is found to influence development of brain and behaviors in rodents. The previous study indicated that perinatal exposure to BPA impaired learning-memory and inhibited N-methyl-D-aspartate receptor (NMDAR) subunits expressions in hippocampus during the postnatal development in rats; and in cultured hippocampal neurons, BPA rapidly promotes dynamic changes in dendritic morphology through estrogen receptor-mediated pathway by concomitant phosphorylation of NMDAR subunit NR2B. In the present study, we examined the rapid effect of BPA on passive avoidance memory and NMDAR in the developing hippocampus of Sprague-Dawley rats at the age of postnatal day 18. The results showed that BPA or estradiol benzoate (EB) rapidly extended the latency to step down from the platform 1 h after footshock and increased the phosphorylation levels of NR1, NR2B, and mitogen-activated extracellular signal-regulated kinase (ERK) in hippocampus within 1 h. While 24 h after BPA or EB treatment, the improved memory and the increased phosphorylation levels of NR1, NR2B, ERK disappeared. Furthermore, pre-treatment with an estrogen receptors (ERs) antagonist, ICI182,780, or an ERK-activating kinase inhibitor, U0126, significantly attenuated EB- or BPA-induced phosphorylations of NR1, NR2B, and ERK within 1 h. These data suggest that BPA rapidly enhanced short-term passive avoidance memory in the developing rats. A non-genomic effect via ERs may mediate the modulation of the phosphorylation of NMDAR subunits NR1 and NR2B through ERK signaling pathway. - Highlights: → BPA rapidly extended the latency to step down from platform 1 h after footshock. → BPA rapidly increased pNR1, pNR2B, and pERK in hippocampus within 1 h. → ERs antagonist or MEK inhibitor attenuated BPA-induced pNR1, pNR2B, and pERK.

Activation of Brain Somatostatin Signaling Suppresses CRF Receptor-Mediated Stress Response.

Science.gov (United States)

Stengel, Andreas; Taché, Yvette F

2017-01-01

Corticotropin-releasing factor (CRF) is the hallmark brain peptide triggering the response to stress and mediates-in addition to the stimulation of the hypothalamus-pituitary-adrenal (HPA) axis-other hormonal, behavioral, autonomic and visceral components. Earlier reports indicate that somatostatin-28 injected intracerebroventricularly counteracts the acute stress-induced ACTH and catecholamine release. Mounting evidence now supports that activation of brain somatostatin signaling exerts a broader anti-stress effect by blunting the endocrine, autonomic, behavioral (with a focus on food intake) and visceral gastrointestinal motor responses through the involvement of distinct somatostatin receptor subtypes.

Architecture of the RNA polymerase II-Mediator core initiation complex.

Science.gov (United States)

Plaschka, C; Larivière, L; Wenzeck, L; Seizl, M; Hemann, M; Tegunov, D; Petrotchenko, E V; Borchers, C H; Baumeister, W; Herzog, F; Villa, E; Cramer, P

2015-02-19

The conserved co-activator complex Mediator enables regulated transcription initiation by RNA polymerase (Pol) II. Here we reconstitute an active 15-subunit core Mediator (cMed) comprising all essential Mediator subunits from Saccharomyces cerevisiae. The cryo-electron microscopic structure of cMed bound to a core initiation complex was determined at 9.7 Å resolution. cMed binds Pol II around the Rpb4-Rpb7 stalk near the carboxy-terminal domain (CTD). The Mediator head module binds the Pol II dock and the TFIIB ribbon and stabilizes the initiation complex. The Mediator middle module extends to the Pol II foot with a 'plank' that may influence polymerase conformation. The Mediator subunit Med14 forms a 'beam' between the head and middle modules and connects to the tail module that is predicted to bind transcription activators located on upstream DNA. The Mediator 'arm' and 'hook' domains contribute to a 'cradle' that may position the CTD and TFIIH kinase to stimulate Pol II phosphorylation.

mTHPC-mediated photodynamic diagnosis of malignant brain tumors

International Nuclear Information System (INIS)

Zimmermann, A.

2001-03-01

Radical tumor resection is the basis for prolonged survival of patients suffering from malignant brain tumors such as glioblastoma multiform. We have carried out a phase II study involving 22 patients with malignant brain tumors to assess the feasibility and the effectiveness of the combination of intraoperative photodynamic diagnosis (PDD) and fluorescence-guided resection (FGR) mediated by the second generation photosensitizer meta-tetrahydroxyphenylchlorin (mTHPC). In addition, intraoperative photodynamic therapy (PDT) was performed. Several commercially available fluorescence diagnostic systems were investigated for their applicability for clinical practice. We have adapted and optimized a diagnostic system which includes a surgical microscope, an excitation light source (filtered to 370-440 nm), a video camera detection system, and a spectrometer for clear identification of the mTHPC fluorescence emission at 652 nm. Especially in regions of faint fluorescence it turned out to be essential to maximize the spectral information by optimizing and matching the spectral properties of all components, such as excitation source, camera and color filters. In summary, based on 138 tissue samples derived from 22 tumor specimens we have been able to achieve a sensitivity of 87.9 % and a specificity of 95.7 %. This study demonstrates that mTHPC-mediated intraoperative fluorescence-guided resection followed by photodynamic therapy is a feasible concept. (author)

Leptin reverses corticosterone-induced inhibition of neural stem cell proliferation through activating the NR2B subunits of NMDA receptors

Energy Technology Data Exchange (ETDEWEB)

Shi, Wen-Zhu [Anesthesia and Operation Center, Hainan Branch of Chinese PLA General Hospital, Hainan 572013 (China); Anesthesia and Operation Center, Chinese PLA General Hospital, Beijing 100853 (China); Miao, Yu-Liang [Department of Anesthesiology, PLA No. 306 Hospital, Beijing 100101 (China); Guo, Wen-Zhi [Department of Anesthesiology, Beijing Military General Hospital of Chinese People’s Liberation Army, Beijing 100700 (China); Wu, Wei, E-mail: wwzwgk@163.com [Department of Head and Neck Surgery of Otolaryngology, PLA No. 306 Hospital, Beijing 100101 (China); Li, Bao-Wei [Department of Head and Neck Surgery of Otolaryngology, PLA No. 306 Hospital, Beijing 100101 (China); An, Li-Na [Department of Anesthesiology, Armed Police General Hospital, Beijing 100039 (China); Fang, Wei-Wu [Department of Anesthesiology, PLA No. 306 Hospital, Beijing 100101 (China); Mi, Wei-Dong, E-mail: elite2005gg@163.com [Anesthesia and Operation Center, Chinese PLA General Hospital, Beijing 100853 (China)

2014-04-25

Highlights: • Leptin promotes the proliferation of neural stem cells isolated from embryonic mouse hippocampus. • Leptin reverses corticosterone-induced inhibition of neural stem cell proliferation. • The effects of leptin are partially mediated by upregulating NR2B subunits. - Abstract: Corticosterone inhibits the proliferation of hippocampal neural stem cells (NSCs). The removal of corticosterone-induced inhibition of NSCs proliferation has been reported to contribute to neural regeneration. Leptin has been shown to regulate brain development, improve angiogenesis, and promote neural regeneration; however, its effects on corticosterone-induced inhibition of NSCs proliferation remain unclear. Here we reported that leptin significantly promoted the proliferation of hippocampal NSCs in a concentration-dependent pattern. Also, leptin efficiently reversed the inhibition of NSCs proliferation induced by corticosterone. Interestingly, pre-treatment with non-specific NMDA antagonist MK-801, specific NR2B antagonist Ro 25-6981, or small interfering RNA (siRNA) targeting NR2B, significantly blocked the effect of leptin on corticosterone-induced inhibition of NSCs proliferation. Furthermore, corticosterone significantly reduced the protein expression of NR2B, whereas pre-treatment with leptin greatly reversed the attenuation of NR2B expression caused by corticosterone in cultured hippocampal NSCs. Our findings demonstrate that leptin reverses the corticosterone-induced inhibition of NSCs proliferation. This process is, at least partially mediated by increased expression of NR2B subunits of NMDA receptors.

Heterodimerization with the β1 subunit directs the α2 subunit of nitric oxide-sensitive guanylyl cyclase to calcium-insensitive cell-cell contacts in HEK293 cells: Interaction with Lin7a.

Science.gov (United States)

Hochheiser, Julia; Haase, Tobias; Busker, Mareike; Sömmer, Anne; Kreienkamp, Hans-Jürgen; Behrends, Sönke

2016-12-15

Nitric oxide-sensitive guanylyl cyclase is a heterodimeric enzyme consisting of an α and a β subunit. Two different α subunits (α 1 and α 2 ) give rise to two heterodimeric enzymes α 1 /β 1 and α 2 /β 1 . Both coexist in a wide range of tissues including blood vessels and the lung, but expression of the α 2 /β 1 form is generally much lower and approaches levels similar to the α 1 /β 1 form in the brain only. In the present paper, we show that the α 2 /β 1 form interacts with Lin7a in mouse brain synaptosomes based on co-precipitation analysis. In HEK293 cells, we found that the overexpressed α 2 /β 1 form, but not the α 1 /β 1 form is directed to calcium-insensitive cell-cell contacts. The isolated PDZ binding motif of an amino-terminally truncated α 2 subunit was sufficient for cell-cell contact localization. For the full length α 2 subunit with the PDZ binding motif this was only the case in the heterodimer configuration with the β 1 subunit, but not as isolated α 2 subunit. We conclude that the PDZ binding motif of the α 2 subunit is only accessible in the heterodimer conformation of the mature nitric oxide-sensitive enzyme. Interaction with Lin7a, a small scaffold protein important for synaptic function and cell polarity, can direct this complex to nectin based cell-cell contacts via MPP3 in HEK293 cells. We conclude that heterodimerization is a prerequisite for further protein-protein interactions that direct the α 2 /β 1 form to strategic sites of the cell membrane with adjacent neighbouring cells. Drugs increasing the nitric oxide-sensitivity of this specific form may be particularly effective. Copyright © 2016 Elsevier Inc. All rights reserved.

Social Competence in Childhood Brain Tumor Survivors: Feasibility and Preliminary Outcomes of a Peer-Mediated Intervention

Science.gov (United States)

Devine, Katie A.; Bukowski, William M.; Sahler, Olle Jane Z.; Ohman-Strickland, Pamela; Smith, Tristram H.; Lown, E. Anne; Patenaude, Andrea Farkas; Korones, David N.; Noll, Robert B.

2016-01-01

Objective Evaluate the acceptability, feasibility, and preliminary outcomes of a peer-mediated intervention to improve social competence of brain tumor survivors and classmates. Methods Twelve childhood brain tumor survivors and 217 classroom peers in intervention (n = 8) or comparison (n = 4) classrooms completed measures of social acceptance and reputation at two time points in the year. The intervention (5–8 sessions over 4–6 weeks) taught peer leaders skills for engaging classmates. Individual and classroom outcomes were analyzed with ANCOVA. Results Recruitment rates of families of brain tumor survivors (81%) and schools (100%) were adequate. Peer leaders reported satisfaction with the intervention. Preliminary outcome data trended toward some benefit in increasing the number of friend nominations for survivors of brain tumors but no changes in other peer-reported metrics. Preliminary results also suggested some positive effects on classroom levels of victimization and rejection. Conclusions A peer-mediated intervention was acceptable to families of brain tumor survivors and feasible to implement in schools. Findings warrant a larger trial to evaluate improvements for children with brain tumors and their peers. PMID:27355881

Basic residues in the 74-83 and 191-198 segments of protein kinase CK2 catalytic subunit are implicated in negative but not in positive regulation by the beta-subunit

DEFF Research Database (Denmark)

Sarno, S; Vaglio, P; Marin, O

1997-01-01

by the beta-subunit many fold more than that of alpha wild type, while extrastimulation by beta mutant D55L56E57A, observable with alpha wild type, is abolished with these mutants. These data support the conclusion that down regulation by the acidic residues clustered in the N-terminal moiety of beta...... is mediated by basic residues in the 74-83 and in the 191-198 sequences of the alpha-subunit. These are also implicated in substrate recognition consistent with the concept that the N-terminal acidic region of the beta subunit operates as a pseudosubstrate. In contrast, another CK2alpha mutant, V66A, is more...

The Mediator complex: a central integrator of transcription

Science.gov (United States)

Allen, Benjamin L.; Taatjes, Dylan J.

2016-01-01

The RNA polymerase II (pol II) enzyme transcribes all protein-coding and most non-coding RNA genes and is globally regulated by Mediator, a large, conformationally flexible protein complex with variable subunit composition (for example, a four-subunit CDK8 module can reversibly associate). These biochemical characteristics are fundamentally important for Mediator's ability to control various processes important for transcription, including organization of chromatin architecture and regulation of pol II pre-initiation, initiation, re-initiation, pausing, and elongation. Although Mediator exists in all eukaryotes, a variety of Mediator functions appear to be specific to metazoans, indicative of more diverse regulatory requirements. PMID:25693131

Oncogenic exon 2 mutations in Mediator subunit MED12 disrupt allosteric activation of cyclin C-CDK8/19.

Science.gov (United States)

Park, Min Ju; Shen, Hailian; Spaeth, Jason M; Tolvanen, Jaana H; Failor, Courtney; Knudtson, Jennifer F; McLaughlin, Jessica; Halder, Sunil K; Yang, Qiwei; Bulun, Serdar E; Al-Hendy, Ayman; Schenken, Robert S; Aaltonen, Lauri A; Boyer, Thomas G

2018-03-30

Somatic mutations in exon 2 of the RNA polymerase II transcriptional Mediator subunit MED12 occur at high frequency in uterine fibroids (UFs) and breast fibroepithelial tumors as well as recurrently, albeit less frequently, in malignant uterine leimyosarcomas, chronic lymphocytic leukemias, and colorectal cancers. Previously, we reported that UF-linked mutations in MED12 disrupt its ability to activate cyclin C (CycC)-dependent kinase 8 (CDK8) in Mediator, implicating impaired Mediator-associated CDK8 activity in the molecular pathogenesis of these clinically significant lesions. Notably, the CDK8 paralog CDK19 is also expressed in myometrium, and both CDK8 and CDK19 assemble into Mediator in a mutually exclusive manner, suggesting that CDK19 activity may also be germane to the pathogenesis of MED12 mutation-induced UFs. However, whether and how UF-linked mutations in MED12 affect CDK19 activation is unknown. Herein, we show that MED12 allosterically activates CDK19 and that UF-linked exon 2 mutations in MED12 disrupt its CDK19 stimulatory activity. Furthermore, we find that within the Mediator kinase module, MED13 directly binds to the MED12 C terminus, thereby suppressing an apparent UF mutation-induced conformational change in MED12 that otherwise disrupts its association with CycC-CDK8/19. Thus, in the presence of MED13, mutant MED12 can bind, but cannot activate, CycC-CDK8/19. These findings indicate that MED12 binding is necessary but not sufficient for CycC-CDK8/19 activation and reveal an additional step in the MED12-dependent activation process, one critically dependent on MED12 residues altered by UF-linked exon 2 mutations. These findings confirm that UF-linked mutations in MED12 disrupt composite Mediator-associated kinase activity and identify CDK8/19 as prospective therapeutic targets in UFs. © 2018 Park et al.

Symptom severity and life satisfaction in brain injury: The mediating role of disability acceptance and social self-efficacy.

Science.gov (United States)

Ditchman, Nicole; Sung, Connie; Easton, Amanda B; Johnson, Kristina S; Batchos, Elisabeth

2017-01-01

Although the negative impact of symptom severity on subjective well-being outcomes has been established among individuals with brain injury, the mediating and protective role that positive human traits might have on this relationship has not been adequately explored. The purpose of this study was to examine the impact of social self-efficacy and disability acceptance on the relationship between symptom severity and life satisfaction among individuals with brain injury. Hierarchical regression analysis and correlation techniques were used to test a hypothesized dual-mediation model of life satisfaction in a sample of 105 adults with acquired brain injury. Results indicated that social self-efficacy and disability acceptance fully mediated the relationship between symptom severity and life satisfaction, lending support for a dual-mediation model with disability acceptance being the strongest contributor. These findings suggest there may be considerable value for rehabilitation providers to develop strengths-based service strategies and/or specialized intervention programs that focus on capitalizing these positive human traits to promote life satisfaction and well-being for clients with brain injury. Implications for clinical practice and future research direction are also discussed.

Structural basis of subunit selectivity for competitive NMDA receptor antagonists with preference for GluN2A over GluN2B subunits

Energy Technology Data Exchange (ETDEWEB)

Lind, Genevieve E.; Mou, Tung-Chung; Tamborini, Lucia; Pomper, Martin G.; De Micheli, Carlo; Conti, Paola; Pinto, Andrea; Hansen, Kasper B. (JHU); (Milan); (Montana)

2017-07-31

NMDA-type glutamate receptors are ligand-gated ion channels that contribute to excitatory neurotransmission in the central nervous system (CNS). Most NMDA receptors comprise two glycine-binding GluN1 and two glutamate-binding GluN2 subunits (GluN2A–D). We describe highly potent (S)-5-[(R)-2-amino-2-carboxyethyl]-4,5-dihydro-1H-pyrazole-3-carboxylic acid (ACEPC) competitive GluN2 antagonists, of which ST3 has a binding affinity of 52 nM at GluN1/2A and 782 nM at GluN1/2B receptors. This 15-fold preference of ST3 for GluN1/2A over GluN1/2B is improved compared with NVP-AAM077, a widely used GluN2A-selective antagonist, which we show has 11-fold preference for GluN1/2A over GluN1/2B. Crystal structures of the GluN1/2A agonist binding domain (ABD) heterodimer with bound ACEPC antagonists reveal a binding mode in which the ligands occupy a cavity that extends toward the subunit interface between GluN1 and GluN2A ABDs. Mutational analyses show that the GluN2A preference of ST3 is primarily mediated by four nonconserved residues that are not directly contacting the ligand, but positioned within 12 Å of the glutamate binding site. Two of these residues influence the cavity occupied by ST3 in a manner that results in favorable binding to GluN2A, but occludes binding to GluN2B. Thus, we reveal opportunities for the design of subunit-selective competitive NMDA receptor antagonists by identifying a cavity for ligand binding in which variations exist between GluN2A and GluN2B subunits. This structural insight suggests that subunit selectivity of glutamate-site antagonists can be mediated by mechanisms in addition to direct contributions of contact residues to binding affinity.

Dis3- and exosome subunit-responsive 3′ mRNA instability elements

International Nuclear Information System (INIS)

Kiss, Daniel L.; Hou, Dezhi; Gross, Robert H.; Andrulis, Erik D.

2012-01-01

Highlights: ► Successful use of a novel RNA-specific bioinformatic tool, RNA SCOPE. ► Identified novel 3′ UTR cis-acting element that destabilizes a reporter mRNA. ► Show exosome subunits are required for cis-acting element-mediated mRNA instability. ► Define precise sequence requirements of novel cis-acting element. ► Show that microarray-defined exosome subunit-regulated mRNAs have novel element. -- Abstract: Eukaryotic RNA turnover is regulated in part by the exosome, a nuclear and cytoplasmic complex of ribonucleases (RNases) and RNA-binding proteins. The major RNase of the complex is thought to be Dis3, a multi-functional 3′–5′ exoribonuclease and endoribonuclease. Although it is known that Dis3 and core exosome subunits are recruited to transcriptionally active genes and to messenger RNA (mRNA) substrates, this recruitment is thought to occur indirectly. We sought to discover cis-acting elements that recruit Dis3 or other exosome subunits. Using a bioinformatic tool called RNA SCOPE to screen the 3′ untranslated regions of up-regulated transcripts from our published Dis3 depletion-derived transcriptomic data set, we identified several motifs as candidate instability elements. Secondary screening using a luciferase reporter system revealed that one cassette—harboring four elements—destabilized the reporter transcript. RNAi-based depletion of Dis3, Rrp6, Rrp4, Rrp40, or Rrp46 diminished the efficacy of cassette-mediated destabilization. Truncation analysis of the cassette showed that two exosome subunit-sensitive elements (ESSEs) destabilized the reporter. Point-directed mutagenesis of ESSE abrogated the destabilization effect. An examination of the transcriptomic data from exosome subunit depletion-based microarrays revealed that mRNAs with ESSEs are found in every up-regulated mRNA data set but are underrepresented or missing from the down-regulated data sets. Taken together, our findings imply a potentially novel mechanism of m

Multiple thyrotropin β-subunit and thyrotropin receptor-related genes arose during vertebrate evolution.

Directory of Open Access Journals (Sweden)

Gersende Maugars

Full Text Available Thyroid-stimulating hormone (TSH is composed of a specific β subunit and an α subunit that is shared with the two pituitary gonadotropins. The three β subunits derive from a common ancestral gene through two genome duplications (1R and 2R that took place before the radiation of vertebrates. Analysis of genomic data from phylogenetically relevant species allowed us to identify an additional Tshβ subunit-related gene that was generated through 2R. This gene, named Tshβ2, present in cartilaginous fish, little skate and elephant shark, and in early lobe-finned fish, coelacanth and lungfish, was lost in ray-finned fish and tetrapods. The absence of a second type of TSH receptor (Tshr gene in these species suggests that both TSHs act through the same receptor. A novel Tshβ sister gene, named Tshβ3, was generated through the third genomic duplication (3R that occurred early in the teleost lineage. Tshβ3 is present in most teleost groups but was lostin tedraodontiforms. The 3R also generated a second Tshr, named Tshrb. Interestingly, the new Tshrb was translocated from its original chromosomic position after the emergence of eels and was then maintained in its new position. Tshrb was lost in tetraodontiforms and in ostariophysians including zebrafish although the latter species have two TSHs, suggesting that TSHRb may be dispensable. The tissue distribution of duplicated Tshβs and Tshrs was studied in the European eel. The endocrine thyrotropic function in the eel would be essentially mediated by the classical Tshβ and Tshra, which are mainly expressed in the pituitary and thyroid, respectively. Tshβ3 and Tshrb showed a similar distribution pattern in the brain, pituitary, ovary and adipose tissue, suggesting a possible paracrine/autocrine mode of action in these non-thyroidal tissues. Further studies will be needed to determine the binding specificity of the two receptors and how these two TSH systems are interrelated.

Body distributioin of RGD-mediated liposome in brain-targeting drug delivery.

Science.gov (United States)

Qin, Jing; Chen, DaWei; Hu, Haiyang; Qiao, MingXi; Zhao, XiuLi; Chen, Baoyu

2007-09-01

RGD conjugation liposomes (RGD-liposomes) were evaluated for brain-targeting drug delivery. The flow cytometric in vitro study demonstrated that RGD-liposomes could bind to monocytes and neutrophils effectively. Ferulic acid (4-hydroxy-3-methoxycinnamic, FA) was loaded into liposomes. Rats were subjected to intrastriatal microinjections of 100 units of human recombinant IL-1beta to produce brain inflammation and caudal vein injection of three formulations (FA solution, FA liposome and RGD-coated FA liposome). Animals were sacrificed 15, 30, 60 and 120 min after administration to study the body distribution of the FA in the three formulations. HPLC was used to determine the concentration of FA in vivo with salicylic acid as internal standard. The results of body distribution indicated that RGD-coated liposomes could be mediated into the brain with a 6-fold FA concentration compared to FA solution and 3-fold in comparison to uncoated liposome. Brain targeted delivery was achieved and a reduction in dosage might be allowed.

Evidence for Multiple Mediator Complexes in Yeast Independently Recruited by Activated Heat Shock Factor.

Science.gov (United States)

Anandhakumar, Jayamani; Moustafa, Yara W; Chowdhary, Surabhi; Kainth, Amoldeep S; Gross, David S

2016-07-15

Mediator is an evolutionarily conserved coactivator complex essential for RNA polymerase II transcription. Although it has been generally assumed that in Saccharomyces cerevisiae, Mediator is a stable trimodular complex, its structural state in vivo remains unclear. Using the "anchor away" (AA) technique to conditionally deplete select subunits within Mediator and its reversibly associated Cdk8 kinase module (CKM), we provide evidence that Mediator's tail module is highly dynamic and that a subcomplex consisting of Med2, Med3, and Med15 can be independently recruited to the regulatory regions of heat shock factor 1 (Hsf1)-activated genes. Fluorescence microscopy of a scaffold subunit (Med14)-anchored strain confirmed parallel cytoplasmic sequestration of core subunits located outside the tail triad. In addition, and contrary to current models, we provide evidence that Hsf1 can recruit the CKM independently of core Mediator and that core Mediator has a role in regulating postinitiation events. Collectively, our results suggest that yeast Mediator is not monolithic but potentially has a dynamic complexity heretofore unappreciated. Multiple species, including CKM-Mediator, the 21-subunit core complex, the Med2-Med3-Med15 tail triad, and the four-subunit CKM, can be independently recruited by activated Hsf1 to its target genes in AA strains. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

A multimodal RAGE-specific inhibitor reduces amyloid β–mediated brain disorder in a mouse model of Alzheimer disease

Science.gov (United States)

Deane, Rashid; Singh, Itender; Sagare, Abhay P.; Bell, Robert D.; Ross, Nathan T.; LaRue, Barbra; Love, Rachal; Perry, Sheldon; Paquette, Nicole; Deane, Richard J.; Thiyagarajan, Meenakshisundaram; Zarcone, Troy; Fritz, Gunter; Friedman, Alan E.; Miller, Benjamin L.; Zlokovic, Berislav V.

2012-01-01

In Alzheimer disease (AD), amyloid β peptide (Aβ) accumulates in plaques in the brain. Receptor for advanced glycation end products (RAGE) mediates Aβ-induced perturbations in cerebral vessels, neurons, and microglia in AD. Here, we identified a high-affinity RAGE-specific inhibitor (FPS-ZM1) that blocked Aβ binding to the V domain of RAGE and inhibited Aβ40- and Aβ42-induced cellular stress in RAGE-expressing cells in vitro and in the mouse brain in vivo. FPS-ZM1 was nontoxic to mice and readily crossed the blood-brain barrier (BBB). In aged APPsw/0 mice overexpressing human Aβ-precursor protein, a transgenic mouse model of AD with established Aβ pathology, FPS-ZM1 inhibited RAGE-mediated influx of circulating Aβ40 and Aβ42 into the brain. In brain, FPS-ZM1 bound exclusively to RAGE, which inhibited β-secretase activity and Aβ production and suppressed microglia activation and the neuroinflammatory response. Blockade of RAGE actions at the BBB and in the brain reduced Aβ40 and Aβ42 levels in brain markedly and normalized cognitive performance and cerebral blood flow responses in aged APPsw/0 mice. Our data suggest that FPS-ZM1 is a potent multimodal RAGE blocker that effectively controls progression of Aβ-mediated brain disorder and that it may have the potential to be a disease-modifying agent for AD. PMID:22406537

Distinct brain systems mediate the effects of nociceptive input and self-regulation on pain.

Directory of Open Access Journals (Sweden)

Choong-Wan Woo

2015-01-01

Full Text Available Cognitive self-regulation can strongly modulate pain and emotion. However, it is unclear whether self-regulation primarily influences primary nociceptive and affective processes or evaluative ones. In this study, participants engaged in self-regulation to increase or decrease pain while experiencing multiple levels of painful heat during functional magnetic resonance imaging (fMRI imaging. Both heat intensity and self-regulation strongly influenced reported pain, but they did so via two distinct brain pathways. The effects of stimulus intensity were mediated by the neurologic pain signature (NPS, an a priori distributed brain network shown to predict physical pain with over 90% sensitivity and specificity across four studies. Self-regulation did not influence NPS responses; instead, its effects were mediated through functional connections between the nucleus accumbens and ventromedial prefrontal cortex. This pathway was unresponsive to noxious input, and has been broadly implicated in valuation, emotional appraisal, and functional outcomes in pain and other types of affective processes. These findings provide evidence that pain reports are associated with two dissociable functional systems: nociceptive/affective aspects mediated by the NPS, and evaluative/functional aspects mediated by a fronto-striatal system.

Liposome-Based Adjuvants for Subunit Vaccines: Formulation Strategies for Subunit Antigens and Immunostimulators

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Signe Tandrup Schmidt

2016-03-01

Full Text Available The development of subunit vaccines has become very attractive in recent years due to their superior safety profiles as compared to traditional vaccines based on live attenuated or whole inactivated pathogens, and there is an unmet medical need for improved vaccines and vaccines against pathogens for which no effective vaccines exist. The subunit vaccine technology exploits pathogen subunits as antigens, e.g., recombinant proteins or synthetic peptides, allowing for highly specific immune responses against the pathogens. However, such antigens are usually not sufficiently immunogenic to induce protective immunity, and they are often combined with adjuvants to ensure robust immune responses. Adjuvants are capable of enhancing and/or modulating immune responses by exposing antigens to antigen-presenting cells (APCs concomitantly with conferring immune activation signals. Few adjuvant systems have been licensed for use in human vaccines, and they mainly stimulate humoral immunity. Thus, there is an unmet demand for the development of safe and efficient adjuvant systems that can also stimulate cell-mediated immunity (CMI. Adjuvants constitute a heterogeneous group of compounds, which can broadly be classified into delivery systems or immunostimulators. Liposomes are versatile delivery systems for antigens, and they can carefully be customized towards desired immune profiles by combining them with immunostimulators and optimizing their composition, physicochemical properties and antigen-loading mode. Immunostimulators represent highly diverse classes of molecules, e.g., lipids, nucleic acids, proteins and peptides, and they are ligands for pattern-recognition receptors (PRRs, which are differentially expressed on APC subsets. Different formulation strategies might thus be required for incorporation of immunostimulators and antigens, respectively, into liposomes, and the choice of immunostimulator should ideally be based on knowledge regarding the

Determinants of RNA polymerase alpha subunit for interaction with beta, beta', and sigma subunits: hydroxyl-radical protein footprinting.

OpenAIRE

Heyduk, T; Heyduk, E; Severinov, K; Tang, H; Ebright, R H

1996-01-01

Escherichia coli RNA polymerase (RNAP) alpha subunit serves as the initiator for RNAP assembly, which proceeds according to the pathway 2 alpha-->alpha 2-->alpha 2 beta-->alpha 2 beta beta'-->alpha 2 beta beta' sigma. In this work, we have used hydroxyl-radical protein footprinting to define determinants of alpha for interaction with beta, beta', and sigma. Our results indicate that amino acids 30-75 of alpha are protected from hydroxyl-radical-mediated proteolysis upon interaction with beta ...

The Mediator complex of Caenorhabditis elegans: insights into the developmental and physiological roles of a conserved transcriptional coregulator.

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Grants, Jennifer M; Goh, Grace Y S; Taubert, Stefan

2015-02-27

The Mediator multiprotein complex ('Mediator') is an important transcriptional coregulator that is evolutionarily conserved throughout eukaryotes. Although some Mediator subunits are essential for the transcription of all protein-coding genes, others influence the expression of only subsets of genes and participate selectively in cellular signaling pathways. Here, we review the current knowledge of Mediator subunit function in the nematode Caenorhabditis elegans, a metazoan in which established and emerging genetic technologies facilitate the study of developmental and physiological regulation in vivo. In this nematode, unbiased genetic screens have revealed critical roles for Mediator components in core developmental pathways such as epidermal growth factor (EGF) and Wnt/β-catenin signaling. More recently, important roles for C. elegans Mediator subunits have emerged in the regulation of lipid metabolism and of systemic stress responses, engaging conserved transcription factors such as nuclear hormone receptors (NHRs). We emphasize instances where similar functions for individual Mediator subunits exist in mammals, highlighting parallels between Mediator subunit action in nematode development and in human cancer biology. We also discuss a parallel between the association of the Mediator subunit MED12 with several human disorders and the role of its C. elegans ortholog mdt-12 as a regulatory hub that interacts with numerous signaling pathways. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

LRP1 in Brain Vascular Smooth Muscle Cells Mediates Local Clearance of Alzheimer's Amyloid-β

OpenAIRE

Kanekiyo, Takahisa; Liu, Chia-Chen; Shinohara, Mitsuru; Li, Jie; Bu, Guojun

2012-01-01

Impaired clearance of amyloid-β (Aβ) is a major pathogenic event for Alzheimer’s disease (AD). Aβ depositions in brain parenchyma as senile plaques and along cerebrovasculature as cerebral amyloid angiopathy (CAA) are hallmarks of AD. A major pathway that mediates brain Aβ clearance is the cerebrovascular system where Aβ is eliminated through the blood-brain barrier (BBB) and/or degraded by cerebrovascular cells along the interstitial fluid drainage pathway. An Aβ clearance receptor, the low-...

Helicobacter pylori VacA toxin/subunit p34: targeting of an anion channel to the inner mitochondrial membrane.

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Grazyna Domańska

2010-04-01

Full Text Available The vacuolating toxin VacA, released by Helicobacter pylori, is an important virulence factor in the pathogenesis of gastritis and gastroduodenal ulcers. VacA contains two subunits: The p58 subunit mediates entry into target cells, and the p34 subunit mediates targeting to mitochondria and is essential for toxicity. In this study we found that targeting to mitochondria is dependent on a unique signal sequence of 32 uncharged amino acid residues at the p34 N-terminus. Mitochondrial import of p34 is mediated by the import receptor Tom20 and the import channel of the outer membrane TOM complex, leading to insertion of p34 into the mitochondrial inner membrane. p34 assembles in homo-hexamers of extraordinary high stability. CD spectra of the purified protein indicate a content of >40% beta-strands, similar to pore-forming beta-barrel proteins. p34 forms an anion channel with a conductivity of about 12 pS in 1.5 M KCl buffer. Oligomerization and channel formation are independent both of the 32 uncharged N-terminal residues and of the p58 subunit of the toxin. The conductivity is efficiently blocked by 5-nitro-2-(3-phenylpropylaminobenzoic acid (NPPB, a reagent known to inhibit VacA-mediated apoptosis. We conclude that p34 essentially acts as a small pore-forming toxin, targeted to the mitochondrial inner membrane by a special hydrophobic N-terminal signal.

NMDARs Mediate the Role of Monoamine Oxidase A in Pathological Aggression

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Bortolato, Marco; Godar, Sean C.; Melis, Miriam; Soggiu, Alessio; Roncada, Paola; Casu, Angelo; Flore, Giovanna; Chen, Kevin; Frau, Roberto; Urbani, Andrea; Castelli, M. Paola; Devoto, Paola; Shih, Jean C.

2012-01-01

Converging evidence shows that monoamine oxidase A (MAO A), the key enzyme catalyzing serotonin (5-hydroxytryptamine; 5-HT) and norepinephrine (NE) degradation, is a primary factor in the pathophysiology of antisocial and aggressive behavior. Accordingly, male MAO A-deficient humans and mice exhibit an extreme predisposition to aggressive outbursts in response to stress. As NMDARs regulate the emotional reactivity to social and environmental stimuli, we hypothesized their involvement in the modulation of aggression mediated by MAO A. In comparison with WT male mice, MAO A KO counterparts exhibited increases in 5-HT and NE levels across all brain regions, but no difference in glutamate concentrations and NMDAR binding. Notably, the prefrontal cortex (PFC) of MAO A KO mice exhibited higher expression of NR2A and NR2B, as well as lower levels of glycosylated NR1 subunits. In line with these changes, the current amplitude and decay time of NMDARs in PFC was significantly reduced. Furthermore, the currents of these receptors were hypersensitive to the action of the antagonists of the NMDAR complex (dizocilpine), as well as NR2A (PEAQX) and NR2B (Ro 25–6981) subunits. Notably, systemic administration of these agents selectively countered the enhanced aggression in MAO A KO mice, at doses that did not inherently affect motor activity. Our findings suggest that the role of MAO A in pathological aggression may be mediated by changes in NMDAR subunit composition in the PFC, and point to a critical function of this receptor in the molecular bases of antisocial personality. PMID:22723698

Lysosomal Storage of Subunit c of Mitochondrial ATP Synthase in Brain-Specific Atp13a2-Deficient Mice.

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Sato, Shigeto; Koike, Masato; Funayama, Manabu; Ezaki, Junji; Fukuda, Takahiro; Ueno, Takashi; Uchiyama, Yasuo; Hattori, Nobutaka

2016-12-01

Kufor-Rakeb syndrome (KRS) is an autosomal recessive form of early-onset parkinsonism linked to the PARK9 locus. The causative gene for KRS is Atp13a2, which encodes a lysosomal type 5 P-type ATPase. We recently showed that KRS/PARK9-linked mutations lead to several lysosomal alterations, including reduced proteolytic processing of cathepsin D in vitro. However, it remains unknown how deficiency of Atp13a2 is connected to lysosomal impairments. To address this issue, we analyzed brain tissues of Atp13a2 conditional-knockout mice, which exhibited characteristic features of neuronal ceroid lipofuscinosis, including accumulation of lipofuscin positive for subunit c of mitochondrial ATP synthase, suggesting that a common pathogenic mechanism underlies both neuronal ceroid lipofuscinosis and Parkinson disease. Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Distinct forms of the β subunit of GTP-binding regulatory proteins identified by molecular cloning

International Nuclear Information System (INIS)

Fong, H.K.W.; Amatruda, T.T. III; Birren, B.W.; Simon, M.I.

1987-01-01

Two distinct β subunits of guanine nucleotide-binding regulatory proteins have been identified by cDNA cloning and are referred to as β 1 and β 1 subunits. The bovine transducin β subunit (β 1 ) has been cloned previously. The author now isolated and analyzed cDNA clones that encode the β 2 subunit from bovine adrenal, bovine brain, and a human myeloid leukemia cell line, HL-60. The 340-residue M/sub r/ 37,329 Β 2 protein is 90% identical with β 1 in predicted amino acid sequence, and it is also organized as a series of repetitive homologous segments. The major mRNA that encodes the bovine β 2 subunit is 1.7 kilobases in length. It is expressed at lower levels than β 1 subunit mRNA in all tissues examined. The β 1 and β 2 messages are expressed in cloned human cell lines. Hybridization of cDNA probes to bovine DNA showed that β 1 and β 2 are encoded by separate genes. The amino acid sequences for the bovine and human β 2 subunit are identical, as are the amino acid sequences for the bovine and human β 1 subunit. This evolutionary conservation suggests that the two β subunits have different roles in the signal transduction process

Inhibition of Ku70 acetylation by INHAT subunit SET/TAF-Iβ regulates Ku70-mediated DNA damage response.

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Kim, Kee-Beom; Kim, Dong-Wook; Park, Jin Woo; Jeon, Young-Joo; Kim, Daehwan; Rhee, Sangmyung; Chae, Jung-Il; Seo, Sang-Beom

2014-07-01

DNA double-strand breaks (DSBs) can cause either cell death or genomic instability. The Ku heterodimer Ku70/80 is required for the NHEJ (non-homologous end-joining) DNA DSB repair pathway. The INHAT (inhibitor of histone acetyltransferases) complex subunit, SET/TAF-Iβ, can inhibit p300- and PCAF-mediated acetylation of both histone and p53, thereby repressing general transcription and that of p53 target genes. Here, we show that SET/TAF-Iβ interacts with Ku70/80, and that this interaction inhibits CBP- and PCAF-mediated Ku70 acetylation in an INHAT domain-dependent manner. Notably, DNA damage by UV disrupted the interaction between SET/TAF-Iβ and Ku70. Furthermore, we demonstrate that overexpressed SET/TAF-Iβ inhibits recruitment of Ku70/80 to DNA damage sites. We propose that dysregulation of SET/TAF-Iβ expression prevents repair of damaged DNA and also contributes to cellular proliferation. All together, our findings indicate that SET/TAF-Iβ interacts with Ku70/80 in the nucleus and inhibits Ku70 acetylation. Upon DNA damage, SET/TAF-Iβ dissociates from the Ku complex and releases Ku70/Ku80, which are then recruited to DNA DSB sites via the NHEJ DNA repair pathway.

PRDM16 enhances nuclear receptor-dependent transcription of the brown fat-specific Ucp1 gene through interactions with Mediator subunit MED1.

Science.gov (United States)

Iida, Satoshi; Chen, Wei; Nakadai, Tomoyoshi; Ohkuma, Yoshiaki; Roeder, Robert G

2015-02-01

PR domain-containing 16 (PRDM16) induces expression of brown fat-specific genes in brown and beige adipocytes, although the underlying transcription-related mechanisms remain largely unknown. Here, in vitro studies show that PRDM16, through its zinc finger domains, directly interacts with the MED1 subunit of the Mediator complex, is recruited to the enhancer of the brown fat-specific uncoupling protein 1 (Ucp1) gene through this interaction, and enhances thyroid hormone receptor (TR)-driven transcription in a biochemically defined system in a Mediator-dependent manner, thus providing a direct link to the general transcription machinery. Complementary cell-based studies show that upon forskolin treatment, PRDM16 induces Ucp1 expression in undifferentiated murine embryonic fibroblasts, that this induction depends on MED1 and TR, and, consistent with a direct effect, that PRDM16 is recruited to the Ucp1 enhancer. Related studies have defined MED1 and PRDM16 interaction domains important for Ucp1 versus Ppargc1a induction by PRDM16. These results reveal novel mechanisms for PRDM16 function through the Mediator complex. © 2015 Iida et al.; Published by Cold Spring Harbor Laboratory Press.

Interaction of the regulatory subunit of the cAMP-dependent protein kinase with PATZ1 (ZNF278)

International Nuclear Information System (INIS)

Yang, Weng-Lang; Ravatn, Roald; Kudoh, Kazuya; Alabanza, Leah; Chin, Khew-Voon

2010-01-01

The effects of cAMP in cell are predominantly mediated by the cAMP-dependent protein kinase (PKA), which is composed of two genetically distinct subunits, catalytic (C) and regulatory (R), forming a tetrameric holoenzyme R 2 C 2 . The only known function for the R subunit is that of inhibiting the activity of the C subunit kinase. It has been shown that overexpression of RIα, but not the C subunit kinase, is associated with neoplastic transformation. In addition, it has also been demonstrated that mutation in the RIα, but not the C subunit is associated with increased resistance to the DNA-damaging anticancer drug cisplatin, thus suggesting that the RIα subunit of PKA may have functions independent of the kinase. We show here that the RIα subunit interacts with a BTB/POZ domain zinc-finger transcription factor, PATZ1 (ZNF278), and co-expression with RIα results in its sequestration in the cytoplasm. The cytoplasmic/nuclear translocation is inducible by cAMP. C-terminus deletion abolishes PATZ1 interaction with RIα and results in its localization in the nucleus. PATZ1 transactivates the cMyc promoter and the presence of cAMP and co-expression with RIα modulates its transactivation. Moreover, PATZ1 is aberrantly expressed in cancer. Taken together, our results showed a potentially novel mechanism of cAMP signaling mediated through the interaction of RIα with PATZ1 that is independent of the kinase activity of PKA, and the aberrant expression of PATZ1 in cancer point to its role in cell growth regulation.

Highly conserved small subunit residues influence rubisco large subunit catalysis.

Science.gov (United States)

Genkov, Todor; Spreitzer, Robert J

2009-10-30

The chloroplast enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the rate-limiting step of photosynthetic CO(2) fixation. With a deeper understanding of its structure-function relationships and competitive inhibition by O(2), it may be possible to engineer an increase in agricultural productivity and renewable energy. The chloroplast-encoded large subunits form the active site, but the nuclear-encoded small subunits can also influence catalytic efficiency and CO(2)/O(2) specificity. To further define the role of the small subunit in Rubisco function, the 10 most conserved residues in all small subunits were substituted with alanine by transformation of a Chlamydomonas reinhardtii mutant that lacks the small subunit gene family. All the mutant strains were able to grow photosynthetically, indicating that none of the residues is essential for function. Three of the substitutions have little or no effect (S16A, P19A, and E92A), one primarily affects holoenzyme stability (L18A), and the remainder affect catalysis with or without some level of associated structural instability (Y32A, E43A, W73A, L78A, P79A, and F81A). Y32A and E43A cause decreases in CO(2)/O(2) specificity. Based on the x-ray crystal structure of Chlamydomonas Rubisco, all but one (Glu-92) of the conserved residues are in contact with large subunits and cluster near the amino- or carboxyl-terminal ends of large subunit alpha-helix 8, which is a structural element of the alpha/beta-barrel active site. Small subunit residues Glu-43 and Trp-73 identify a possible structural connection between active site alpha-helix 8 and the highly variable small subunit loop between beta-strands A and B, which can also influence Rubisco CO(2)/O(2) specificity.

CSNAP Is a Stoichiometric Subunit of the COP9 Signalosome

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Shelly Rozen

2015-10-01

Full Text Available The highly conserved COP9 signalosome (CSN complex is a key regulator of all cullin-RING-ubiquitin ligases (CRLs, the largest family of E3 ubiquitin ligases. Until now, it was accepted that the CSN is composed of eight canonical components. Here, we report the discovery of an additional integral and stoichiometric subunit that had thus far evaded detection, and we named it CSNAP (CSN acidic protein. We show that CSNAP binds CSN3, CSN5, and CSN6, and its incorporation into the CSN complex is mediated through the C-terminal region involving conserved aromatic residues. Moreover, depletion of this small protein leads to reduced proliferation and a flattened and enlarged morphology. Finally, on the basis of sequence and structural properties shared by both CSNAP and DSS1, a component of the related 19S lid proteasome complex, we propose that CSNAP, the ninth CSN subunit, is the missing paralogous subunit of DSS1.

Tuning of the Na,K-ATPase by the beta subunit

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Hilbers, Florian; Kopec, Wojciech; Isaksen, Toke Jost; Holm, Thomas Hellesøe; Lykke-Hartmann, Karin; Nissen, Poul; Khandelia, Himanshu; Poulsen, Hanne

2016-02-01

The vital gradients of Na+ and K+ across the plasma membrane of animal cells are maintained by the Na,K-ATPase, an αβ enzyme complex, whose α subunit carries out the ion transport and ATP hydrolysis. The specific roles of the β subunit isoforms are less clear, though β2 is essential for motor physiology in mammals. Here, we show that compared to β1 and β3, β2 stabilizes the Na+-occluded E1P state relative to the outward-open E2P state, and that the effect is mediated by its transmembrane domain. Molecular dynamics simulations further demonstrate that the tilt angle of the β transmembrane helix correlates with its functional effect, suggesting that the relative orientation of β modulates ion binding at the α subunit. β2 is primarily expressed in granule neurons and glomeruli in the cerebellum, and we propose that its unique functional characteristics are important to respond appropriately to the cerebellar Na+ and K+ gradients.

Emerging functions of multi-protein complex Mediator with special emphasis on plants.

Science.gov (United States)

Malik, Naveen; Agarwal, Pinky; Tyagi, Akhilesh

2017-10-01

Mediator is a multi-subunit protein complex which is involved in transcriptional regulation in yeast and other eukaryotes. As a co-activator, it connects information from transcriptional activators/repressors to transcriptional machinery including RNA polymerase II and general transcription factors. It is not only involved in transcription initiation but also has important roles to play in transcription elongation and termination. Functional attributes of different Mediator subunits have been largely defined in yeast and mammalian systems earlier, while such studies in plants have gained momentum recently. Mediator regulates various processes related to plant development and is also involved in biotic and abiotic stress response. Thus, plant Mediator, like yeast and mammalian Mediator complex, is indispensable for plant growth and survival. Interaction of its multiple subunits with other regulatory proteins and their ectopic expression or knockdown in model plant like Arabidopsis and certain crop plants are paving the way to biochemical analysis and unravel molecular mechanisms of action of Mediator in plants.

Mediator structure and rearrangements required for holoenzyme formation.

Science.gov (United States)

Tsai, Kuang-Lei; Yu, Xiaodi; Gopalan, Sneha; Chao, Ti-Chun; Zhang, Ying; Florens, Laurence; Washburn, Michael P; Murakami, Kenji; Conaway, Ronald C; Conaway, Joan W; Asturias, Francisco J

2017-04-13

The conserved Mediator co-activator complex has an essential role in the regulation of RNA polymerase II transcription in all eukaryotes. Understanding the structure and interactions of Mediator is crucial for determining how the complex influences transcription initiation and conveys regulatory information to the basal transcription machinery. Here we present a 4.4 Å resolution cryo-electron microscopy map of Schizosaccharomyces pombe Mediator in which conserved Mediator subunits are individually resolved. The essential Med14 subunit works as a central backbone that connects the Mediator head, middle and tail modules. Comparison with a 7.8 Å resolution cryo-electron microscopy map of a Mediator-RNA polymerase II holoenzyme reveals that changes in the structure of Med14 facilitate a large-scale Mediator rearrangement that is essential for holoenzyme formation. Our study suggests that access to different conformations and crosstalk between structural elements are essential for the Mediator regulation mechanism, and could explain the capacity of the complex to integrate multiple regulatory signals.

Sphingosine 1-phosphate receptor 5 mediates the immune quiescence of the human brain endothelial barrier

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van Doorn Ruben

2012-06-01

Full Text Available Abstract Background The sphingosine 1-phosphate (S1P receptor modulator FTY720P (Gilenya® potently reduces relapse rate and lesion activity in the neuroinflammatory disorder multiple sclerosis. Although most of its efficacy has been shown to be related to immunosuppression through the induction of lymphopenia, it has been suggested that a number of its beneficial effects are related to altered endothelial and blood–brain barrier (BBB functionality. However, to date it remains unknown whether brain endothelial S1P receptors are involved in the maintenance of the function of the BBB thereby mediating immune quiescence of the brain. Here we demonstrate that the brain endothelial receptor S1P5 largely contributes to the maintenance of brain endothelial barrier function. Methods We analyzed the expression of S1P5 in human post-mortem tissues using immunohistochemistry. The function of S1P5 at the BBB was assessed in cultured human brain endothelial cells (ECs using agonists and lentivirus-mediated knockdown of S1P5. Subsequent analyses of different aspects of the brain EC barrier included the formation of a tight barrier, the expression of BBB proteins and markers of inflammation and monocyte transmigration. Results We show that activation of S1P5 on cultured human brain ECs by a selective agonist elicits enhanced barrier integrity and reduced transendothelial migration of monocytes in vitro. These results were corroborated by genetically silencing S1P5 in brain ECs. Interestingly, functional studies with these cells revealed that S1P5 strongly contributes to brain EC barrier function and underlies the expression of specific BBB endothelial characteristics such as tight junctions and permeability. In addition, S1P5 maintains the immunoquiescent state of brain ECs with low expression levels of leukocyte adhesion molecules and inflammatory chemokines and cytokines through lowering the activation of the transcription factor NFκB. Conclusion Our

Tuning of the Na,K-ATPase by the beta subunit

DEFF Research Database (Denmark)

Hilbers, Florian; Kopec, Wojciech; Isaksen, Toke Jost

2016-01-01

The vital gradients of Na(+) and K(+) across the plasma membrane of animal cells are maintained by the Na,K-ATPase, an αβ enzyme complex, whose α subunit carries out the ion transport and ATP hydrolysis. The specific roles of the β subunit isoforms are less clear, though β2 is essential for motor...... to the cerebellar Na(+) and K(+) gradients....... physiology in mammals. Here, we show that compared to β1 and β3, β2 stabilizes the Na(+)-occluded E1P state relative to the outward-open E2P state, and that the effect is mediated by its transmembrane domain. Molecular dynamics simulations further demonstrate that the tilt angle of the β transmembrane helix...

High Affinity IgE-Fc Receptor alpha and gamma Subunit Interactions

International Nuclear Information System (INIS)

Rashid, A.; Housden, J. E. M.; Sabban, S.; Helm, B.

2014-01-01

Objective: To explore the relationships between the subunits (alpha, beta and gamma) of the high affinity IgE receptor (Fc and RI) and its ability to mediate transmembrane signaling. Study Design: Experimental study. Place and Duration of Study: Department of Molecular Biology and Biotechnology, University of Sheffield, UK, from 2008 to 2009. Methodology: The approach employed was to create a chimera (human alpha-gamma-gamma) using the extracellular (EC) domain of the human high affinity IgE receptor. The alpha subunit (huFc and RIalpha) of IgE receptor was spliced onto the rodent gamma TM and cytoplasmic domain (CD). This was transfected into the Rat Basophilic Leukemia cell line in order to assess the possibility of selectively activating cells transfected with this single pass construct for antigen induced mediator release. Results: The RBLs cell lines transfected with the huFc and RIalpha/gamma/gamma cDNA constructs were assessed for the cell surface expression of the huFc and RIalpha subunit and the response to the antigenic stimulus by looking for degranulation and intracellular Ca2+ mobilisation. The results obtained showed the absence of huFc and RIalpha subunit expression on the surface of transfected cells as seen by flowcytometric studies, beta-hexosaminidase assays and intracellular calcium mobilisation studies. Conclusion: In the present study the grounds for non-expression of huFc and RIalpha/gamma/gamma cDNA remains elusive but may be due to the fact that the human-rodent chimeric receptors are assembled differently than the endogenous rodent receptors as seen in study in which COS 7 cells were transfected with human/rat chimeric complexes. (author)

MC EMiNEM maps the interaction landscape of the Mediator.

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Theresa Niederberger

Full Text Available The Mediator is a highly conserved, large multiprotein complex that is involved essentially in the regulation of eukaryotic mRNA transcription. It acts as a general transcription factor by integrating regulatory signals from gene-specific activators or repressors to the RNA Polymerase II. The internal network of interactions between Mediator subunits that conveys these signals is largely unknown. Here, we introduce MC EMiNEM, a novel method for the retrieval of functional dependencies between proteins that have pleiotropic effects on mRNA transcription. MC EMiNEM is based on Nested Effects Models (NEMs, a class of probabilistic graphical models that extends the idea of hierarchical clustering. It combines mode-hopping Monte Carlo (MC sampling with an Expectation-Maximization (EM algorithm for NEMs to increase sensitivity compared to existing methods. A meta-analysis of four Mediator perturbation studies in Saccharomyces cerevisiae, three of which are unpublished, provides new insight into the Mediator signaling network. In addition to the known modular organization of the Mediator subunits, MC EMiNEM reveals a hierarchical ordering of its internal information flow, which is putatively transmitted through structural changes within the complex. We identify the N-terminus of Med7 as a peripheral entity, entailing only local structural changes upon perturbation, while the C-terminus of Med7 and Med19 appear to play a central role. MC EMiNEM associates Mediator subunits to most directly affected genes, which, in conjunction with gene set enrichment analysis, allows us to construct an interaction map of Mediator subunits and transcription factors.

MC EMiNEM maps the interaction landscape of the Mediator.

Science.gov (United States)

Niederberger, Theresa; Etzold, Stefanie; Lidschreiber, Michael; Maier, Kerstin C; Martin, Dietmar E; Fröhlich, Holger; Cramer, Patrick; Tresch, Achim

2012-01-01

The Mediator is a highly conserved, large multiprotein complex that is involved essentially in the regulation of eukaryotic mRNA transcription. It acts as a general transcription factor by integrating regulatory signals from gene-specific activators or repressors to the RNA Polymerase II. The internal network of interactions between Mediator subunits that conveys these signals is largely unknown. Here, we introduce MC EMiNEM, a novel method for the retrieval of functional dependencies between proteins that have pleiotropic effects on mRNA transcription. MC EMiNEM is based on Nested Effects Models (NEMs), a class of probabilistic graphical models that extends the idea of hierarchical clustering. It combines mode-hopping Monte Carlo (MC) sampling with an Expectation-Maximization (EM) algorithm for NEMs to increase sensitivity compared to existing methods. A meta-analysis of four Mediator perturbation studies in Saccharomyces cerevisiae, three of which are unpublished, provides new insight into the Mediator signaling network. In addition to the known modular organization of the Mediator subunits, MC EMiNEM reveals a hierarchical ordering of its internal information flow, which is putatively transmitted through structural changes within the complex. We identify the N-terminus of Med7 as a peripheral entity, entailing only local structural changes upon perturbation, while the C-terminus of Med7 and Med19 appear to play a central role. MC EMiNEM associates Mediator subunits to most directly affected genes, which, in conjunction with gene set enrichment analysis, allows us to construct an interaction map of Mediator subunits and transcription factors.

Hormonally-mediated Epigenetic Changes to Steroid Receptors in the Developing Brain: Implications for Sexual Differentiation

Science.gov (United States)

Nugent, Bridget M.; Schwarz, Jaclyn M.; McCarthy, Margaret M.

2010-01-01

The establishment of sex-specific neural morphology, which underlies sex-specific behaviors, occurs during a perinatal sensitive window in which brief exposure to gonadal steroid hormones produces permanent masculinization of the brain. In the rodent, estradiol derived from testicular androgens is a principle organizational hormone. The mechanism by which transient estradiol exposure induces permanent differences in neuronal anatomy has been widely investigated, but remains elusive. Epigenetic changes, such as DNA methylation, allow environmental influences to alter long-term gene expression patterns and therefore may be a potential mediator of estradiol-induced organization of the neonatal brain. Here we review data that demonstrate sex and estradiol-induced differences in DNA methylation on the estrogen receptor α (ERα), estrogen receptor β (ERβ), and progesterone receptor (PR) promoters in sexually dimorphic brain regions across development. Contrary to the overarching view of DNA methylation as a permanent modification directly tied to gene expression, these data demonstrate that methylation patterns on steroid hormone receptors change across the life span and do not necessarily predict expression. Although further exploration into the mechanism and significance of estradiol-induced alterations in DNA methylation patterns in the neonatal brain is necessary, these results provide preliminary evidence that epigenetic alterations can occur in response to early hormone exposure and may mediate estradiol-induced organization of sex differences in the neonatal brain. PMID:20800064

Interaction of the regulatory subunit of the cAMP-dependent protein kinase with PATZ1 (ZNF278)

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Yang, Weng-Lang [Long Island Jewish Medical Center, North Shore University Hospital, Manhasset, NY 11030 (United States); Ravatn, Roald [Department of Medicine, University of Toledo, College of Medicine, Toledo, OH 43614 (United States); Kudoh, Kazuya [Department of Medicine, University of Toledo, College of Medicine, Toledo, OH 43614 (United States); Department of Obstetrics and Gynecology, National Defense Medical College, Tokorozawa, Saitama (Japan); Alabanza, Leah [Department of Medicine, University of Toledo, College of Medicine, Toledo, OH 43614 (United States); Chin, Khew-Voon, E-mail: khew-voon.chin@utoledo.edu [Department of Medicine, University of Toledo, College of Medicine, Toledo, OH 43614 (United States)

2010-01-15

The effects of cAMP in cell are predominantly mediated by the cAMP-dependent protein kinase (PKA), which is composed of two genetically distinct subunits, catalytic (C) and regulatory (R), forming a tetrameric holoenzyme R{sub 2}C{sub 2}. The only known function for the R subunit is that of inhibiting the activity of the C subunit kinase. It has been shown that overexpression of RI{alpha}, but not the C subunit kinase, is associated with neoplastic transformation. In addition, it has also been demonstrated that mutation in the RI{alpha}, but not the C subunit is associated with increased resistance to the DNA-damaging anticancer drug cisplatin, thus suggesting that the RI{alpha} subunit of PKA may have functions independent of the kinase. We show here that the RI{alpha} subunit interacts with a BTB/POZ domain zinc-finger transcription factor, PATZ1 (ZNF278), and co-expression with RI{alpha} results in its sequestration in the cytoplasm. The cytoplasmic/nuclear translocation is inducible by cAMP. C-terminus deletion abolishes PATZ1 interaction with RI{alpha} and results in its localization in the nucleus. PATZ1 transactivates the cMyc promoter and the presence of cAMP and co-expression with RI{alpha} modulates its transactivation. Moreover, PATZ1 is aberrantly expressed in cancer. Taken together, our results showed a potentially novel mechanism of cAMP signaling mediated through the interaction of RI{alpha} with PATZ1 that is independent of the kinase activity of PKA, and the aberrant expression of PATZ1 in cancer point to its role in cell growth regulation.

The secreted form of the p40 subunit of interleukin (IL)-12 inhibits IL-23 functions and abrogates IL-23-mediated antitumour effects

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Shimozato, Osamu; Ugai, Shin-ichi; Chiyo, Masako; Takenobu, Hisanori; Nagakawa, Hiroyasu; Wada, Akihiko; Kawamura, Kiyoko; Yamamoto, Hiroshi; Tagawa, Masatoshi

2006-01-01

Interleukin (IL)-23 is a heterodimeric cytokine consisting of a novel p19 molecule and the p40 subunit of IL-12. Since secreted p40 can act as an antagonist for IL-12, we investigated whether p40 also inhibited IL-23-mediated immunological functions. p40 did not induce interferon (IFN)-γ or IL-17 production from splenocytes but impaired IL-23-induced cytokine production by competitive binding to the IL-23 receptors. Furthermore, a mixed population of murine colon carcinoma Colon 26 cells transduced with the p40 gene and those transduced with the IL-23 gene developed tumours in syngenic mice, whereas the IL-23-expressing Colon 26 cells were completely rejected. p40 also suppressed IFN-γ production of antigen-stimulated splenocytes and IL-23-mediated cytotoxic T-lymphocyte activities in the mice that rejected Colon 26 cells expressing IL-23. p40 can thereby antagonize IL-23 and is a possible therapeutic agent for suppression of IL-23 functions. PMID:16423037

Med5(Nut1) and Med17(Srb4) Are Direct Targets of Mediator Histone H4 Tail Interactions

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Liu, Zhongle; Myers, Lawrence C.

2012-01-01

The Mediator complex transmits activation signals from DNA bound transcription factors to the core transcription machinery. In addition to its canonical role in transcriptional activation, recent studies have demonstrated that S. cerevisiae Mediator can interact directly with nucleosomes, and their histone tails. Mutations in Mediator subunits have shown that Mediator and certain chromatin structures mutually impact each other structurally and functionally in vivo. We have taken a UV photo cross-linking approach to further delineate the molecular basis of Mediator chromatin interactions and help determine whether the impact of certain Mediator mutants on chromatin is direct. Specifically, by using histone tail peptides substituted with an amino acid analog that is a UV activatible crosslinker, we have identified specific subunits within Mediator that participate in histone tail interactions. Using Mediator purified from mutant yeast strains we have evaluated the impact of these subunits on histone tail binding. This analysis has identified the Med5 subunit of Mediator as a target for histone tail interactions and suggests that the previously observed effect of med5 mutations on telomeric heterochromatin and silencing is direct. PMID:22693636

GABAA receptor subunit expression changes in the human Alzheimer's disease hippocampus, subiculum, entorhinal cortex and superior temporal gyrus.

Science.gov (United States)

Kwakowsky, Andrea; Calvo-Flores Guzmán, Beatriz; Pandya, Madhavi; Turner, Clinton; Waldvogel, Henry J; Faull, Richard L

2018-02-27

Gamma-aminobutyric acid (GABA) is the primary inhibitory neurotransmitter in the central nervous system. GABA type A receptors (GABA A Rs) are severely affected in Alzheimer's disease (AD). However, the distribution and subunit composition of GABA A Rs in the AD brain are not well understood. This is the first comprehensive study to show brain region- and cell layer-specific alterations in the expression of the GABA A R subunits α1-3, α5, β1-3 and γ2 in the human AD hippocampus, entorhinal cortex and superior temporal gyrus (STG). In late-stage AD tissue samples using immunohistochemistry we found significant alteration of all investigated GABA A Rs subunits except for α3 and β1 that were well preserved. The most prominent changes include an increase in GABA A R α1 expression associated with AD in all layers of the CA3 region, in the stratum (str.) granulare and hilus of the dentate gyrus (DG). We found a significant increase in GABA A R α2 expression in the str. oriens of the CA1-3, str. radiatum of the CA2,3 and decrease in the str. pyramidale of the CA1 region in AD cases. In AD there was a significant increase in GABA A R α5 subunit expression in str. pyramidale, str. oriens of the CA1 region and decrease in the STG. We also found a significant decrease in the GABA A R β3 subunit immunoreactivity in the str. oriens of the CA2, str. granulare and str. moleculare of the DG. In conclusion, these findings indicate that the expression of the GABA A R subunits shows brain region- and layer-specific alterations in AD, and these changes could significantly influence and alter GABA A R function in the disease. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Identification of a conserved archaeal RNA polymerase subunit contacted by the basal transcription factor TFB.

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Magill, C P; Jackson, S P; Bell, S D

2001-12-14

Archaea possess two general transcription factors that are required to recruit RNA polymerase (RNAP) to promoters in vitro. These are TBP, the TATA-box-binding protein and TFB, the archaeal homologue of TFIIB. Thus, the archaeal and eucaryal transcription machineries are fundamentally related. In both RNAP II and archaeal transcription systems, direct contacts between TFB/TFIIB and the RNAP have been demonstrated to mediate recruitment of the polymerase to the promoter. However the subunit(s) directly contacted by these factors has not been identified. Using systematic yeast two-hybrid and biochemical analyses we have identified an interaction between the N-terminal domain of TFB and an evolutionarily conserved subunit of the RNA polymerase, RpoK. Intriguingly, homologues of RpoK are found in all three nuclear RNA polymerases (Rpb6) and also in the bacterial RNA polymerase (omega-subunit).

Nanoparticle-mediated transcriptional modification enhances neuronal differentiation of human neural stem cells following transplantation in rat brain.

Science.gov (United States)

Li, Xiaowei; Tzeng, Stephany Y; Liu, Xiaoyan; Tammia, Markus; Cheng, Yu-Hao; Rolfe, Andrew; Sun, Dong; Zhang, Ning; Green, Jordan J; Wen, Xuejun; Mao, Hai-Quan

2016-04-01

Strategies to enhance survival and direct the differentiation of stem cells in vivo following transplantation in tissue repair site are critical to realizing the potential of stem cell-based therapies. Here we demonstrated an effective approach to promote neuronal differentiation and maturation of human fetal tissue-derived neural stem cells (hNSCs) in a brain lesion site of a rat traumatic brain injury model using biodegradable nanoparticle-mediated transfection method to deliver key transcriptional factor neurogenin-2 to hNSCs when transplanted with a tailored hyaluronic acid (HA) hydrogel, generating larger number of more mature neurons engrafted to the host brain tissue than non-transfected cells. The nanoparticle-mediated transcription activation method together with an HA hydrogel delivery matrix provides a translatable approach for stem cell-based regenerative therapy. Copyright © 2016 Elsevier Ltd. All rights reserved.

Brain microvascular pericytes are immunoactive in culture: cytokine, chemokine, nitric oxide, and LRP-1 expression in response to lipopolysaccharide

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Erickson Michelle A

2011-10-01

Full Text Available Abstract Background Brain microvascular pericytes are important constituents of the neurovascular unit. These cells are physically the closest cells to the microvascular endothelial cells in brain capillaries. They significantly contribute to the induction and maintenance of the barrier functions of the blood-brain barrier. However, very little is known about their immune activities or their roles in neuroinflammation. Here, we focused on the immunological profile of brain pericytes in culture in the quiescent and immune-challenged state by studying their production of immune mediators such as nitric oxide (NO, cytokines, and chemokines. We also examined the effects of immune challenge on pericyte expression of low density lipoprotein receptor-related protein-1 (LRP-1, a protein involved in the processing of amyloid precursor protein and the brain-to-blood efflux of amyloid-β peptide. Methods Supernatants were collected from primary cultures of mouse brain pericytes. Release of nitric oxide (NO was measured by the Griess reaction and the level of S-nitrosylation of pericyte proteins measured with a modified "biotin-switch" method. Specific mitogen-activated protein kinase (MAPK pathway inhibitors were used to determine involvement of these pathways on NO production. Cytokines and chemokines were analyzed by multianalyte technology. The expression of both subunits of LRP-1 was analyzed by western blot. Results Lipopolysaccharide (LPS induced release of NO by pericytes in a dose-dependent manner that was mediated through MAPK pathways. Nitrative stress resulted in S-nitrosylation of cellular proteins. Eighteen of twenty-three cytokines measured were released constitutively by pericytes or with stimulation by LPS, including interleukin (IL-12, IL-13, IL-9, IL-10, granulocyte-colony stimulating factor, granulocyte macrophage-colony stimulating factor, eotaxin, chemokine (C-C motif ligand (CCL-3, and CCL-4. Pericyte expressions of both subunits of

The evolutionarily conserved mediator subunit MDT-15/MED15 links protective innate immune responses and xenobiotic detoxification.

Directory of Open Access Journals (Sweden)

Read Pukkila-Worley

2014-05-01

Full Text Available Metazoans protect themselves from environmental toxins and virulent pathogens through detoxification and immune responses. We previously identified a small molecule xenobiotic toxin that extends survival of Caenorhabditis elegans infected with human bacterial pathogens by activating the conserved p38 MAP kinase PMK-1 host defense pathway. Here we investigate the cellular mechanisms that couple activation of a detoxification response to innate immunity. From an RNAi screen of 1,420 genes expressed in the C. elegans intestine, we identified the conserved Mediator subunit MDT-15/MED15 and 28 other gene inactivations that abrogate the induction of PMK-1-dependent immune effectors by this small molecule. We demonstrate that MDT-15/MED15 is required for the xenobiotic-induced expression of p38 MAP kinase PMK-1-dependent immune genes and protection from Pseudomonas aeruginosa infection. We also show that MDT-15 controls the induction of detoxification genes and functions to protect the host from bacteria-derived phenazine toxins. These data define a central role for MDT-15/MED15 in the coordination of xenobiotic detoxification and innate immune responses.

The evolutionarily conserved mediator subunit MDT-15/MED15 links protective innate immune responses and xenobiotic detoxification.

Science.gov (United States)

Pukkila-Worley, Read; Feinbaum, Rhonda L; McEwan, Deborah L; Conery, Annie L; Ausubel, Frederick M

2014-05-01

Metazoans protect themselves from environmental toxins and virulent pathogens through detoxification and immune responses. We previously identified a small molecule xenobiotic toxin that extends survival of Caenorhabditis elegans infected with human bacterial pathogens by activating the conserved p38 MAP kinase PMK-1 host defense pathway. Here we investigate the cellular mechanisms that couple activation of a detoxification response to innate immunity. From an RNAi screen of 1,420 genes expressed in the C. elegans intestine, we identified the conserved Mediator subunit MDT-15/MED15 and 28 other gene inactivations that abrogate the induction of PMK-1-dependent immune effectors by this small molecule. We demonstrate that MDT-15/MED15 is required for the xenobiotic-induced expression of p38 MAP kinase PMK-1-dependent immune genes and protection from Pseudomonas aeruginosa infection. We also show that MDT-15 controls the induction of detoxification genes and functions to protect the host from bacteria-derived phenazine toxins. These data define a central role for MDT-15/MED15 in the coordination of xenobiotic detoxification and innate immune responses.

Importance of Mediator complex in the regulation and integration of diverse signaling pathways in plants.

Science.gov (United States)

Samanta, Subhasis; Thakur, Jitendra K

2015-01-01

Basic transcriptional machinery in eukaryotes is assisted by a number of cofactors, which either increase or decrease the rate of transcription. Mediator complex is one such cofactor, and recently has drawn a lot of interest because of its integrative power to converge different signaling pathways before channeling the transcription instructions to the RNA polymerase II machinery. Like yeast and metazoans, plants do possess the Mediator complex across the kingdom, and its isolation and subunit analyses have been reported from the model plant, Arabidopsis. Genetic, and molecular analyses have unraveled important regulatory roles of Mediator subunits at every stage of plant life cycle starting from flowering to embryo and organ development, to even size determination. It also contributes immensely to the survival of plants against different environmental vagaries by the timely activation of its resistance mechanisms. Here, we have provided an overview of plant Mediator complex starting from its discovery to regulation of stoichiometry of its subunits. We have also reviewed involvement of different Mediator subunits in different processes and pathways including defense response pathways evoked by diverse biotic cues. Wherever possible, attempts have been made to provide mechanistic insight of Mediator's involvement in these processes.

A Functional Switch of NuRD Chromatin Remodeling Complex Subunits Regulates Mouse Cortical Development

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Justyna Nitarska

2016-11-01

Full Text Available Histone modifications and chromatin remodeling represent universal mechanisms by which cells adapt their transcriptional response to rapidly changing environmental conditions. Extensive chromatin remodeling takes place during neuronal development, allowing the transition of pluripotent cells into differentiated neurons. Here, we report that the NuRD complex, which couples ATP-dependent chromatin remodeling with histone deacetylase activity, regulates mouse brain development. Subunit exchange of CHDs, the core ATPase subunits of the NuRD complex, is required for distinct aspects of cortical development. Whereas CHD4 promotes the early proliferation of progenitors, CHD5 facilitates neuronal migration and CHD3 ensures proper layer specification. Inhibition of each CHD leads to defects of neuronal differentiation and migration, which cannot be rescued by expressing heterologous CHDs. Finally, we demonstrate that NuRD complexes containing specific CHDs are recruited to regulatory elements and modulate the expression of genes essential for brain development.

Mediator: A key regulator of plant development.

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Buendía-Monreal, Manuel; Gillmor, C Stewart

2016-11-01

Mediator is a multiprotein complex that regulates transcription at the level of RNA pol II assembly, as well as through regulation of chromatin architecture, RNA processing and recruitment of epigenetic marks. Though its modular structure is conserved in eukaryotes, its subunit composition has diverged during evolution and varies in response to environmental and tissue-specific inputs, suggesting different functions for each subunit and/or Mediator conformation. In animals, Mediator has been implicated in the control of differentiation and morphogenesis through modulation of numerous signaling pathways. In plants, studies have revealed roles for Mediator in regulation of cell division, cell fate and organogenesis, as well as developmental timing and hormone responses. We begin this review with an overview of biochemical mechanisms of yeast and animal Mediator that are likely to be conserved in all eukaryotes, as well as a brief discussion of the role of Mediator in animal development. We then present a comprehensive review of studies of the role of Mediator in plant development. Finally, we point to important questions for future research on the role of Mediator as a master coordinator of development. Copyright © 2016 Elsevier Inc. All rights reserved.

Off-pathway assembly of fimbria subunits is prevented by chaperone CfaA of CFA/I fimbriae from enterotoxigenic E. coli.

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Bao, Rui; Liu, Yang; Savarino, Stephen J; Xia, Di

2016-12-01

The assembly of the class 5 colonization factor antigen I (CFA/I) fimbriae of enterotoxigenic E. coli was proposed to proceed via the alternate chaperone-usher pathway. Here, we show that in the absence of the chaperone CfaA, CfaB, the major pilin subunit of CFA/I fimbriae, is able to spontaneously refold and polymerize into cyclic trimers. CfaA kinetically traps CfaB to form a metastable complex that can be stabilized by mutations. Crystal structure of the stabilized complex reveals distinctive interactions provided by CfaA to trap CfaB in an assembly competent state through donor-strand complementation (DSC) and cleft-mediated anchorage. Mutagenesis indicated that DSC controls the stability of the chaperone-subunit complex and the cleft-mediated anchorage of the subunit C-terminus additionally assist in subunit refolding. Surprisingly, over-stabilization of the chaperone-subunit complex led to delayed fimbria assembly, whereas destabilizing the complex resulted in no fimbriation. Thus, CfaA acts predominantly as a kinetic trap by stabilizing subunit to avoid its off-pathway self-polymerization that results in energetically favorable trimers and could serve as a driving force for CFA/I pilus assembly, representing an energetic landscape unique to class 5 fimbria assembly. Published 2016. This article is a U.S. Government work and is in the public domain in the USA. Molecular Microbiology published by John Wiley & Sons Ltd.

Med5(Nut1 and Med17(Srb4 are direct targets of mediator histone H4 tail interactions.

Directory of Open Access Journals (Sweden)

Zhongle Liu

Full Text Available The Mediator complex transmits activation signals from DNA bound transcription factors to the core transcription machinery. In addition to its canonical role in transcriptional activation, recent studies have demonstrated that S. cerevisiae Mediator can interact directly with nucleosomes, and their histone tails. Mutations in Mediator subunits have shown that Mediator and certain chromatin structures mutually impact each other structurally and functionally in vivo. We have taken a UV photo cross-linking approach to further delineate the molecular basis of Mediator chromatin interactions and help determine whether the impact of certain Mediator mutants on chromatin is direct. Specifically, by using histone tail peptides substituted with an amino acid analog that is a UV activatible crosslinker, we have identified specific subunits within Mediator that participate in histone tail interactions. Using Mediator purified from mutant yeast strains we have evaluated the impact of these subunits on histone tail binding. This analysis has identified the Med5 subunit of Mediator as a target for histone tail interactions and suggests that the previously observed effect of med5 mutations on telomeric heterochromatin and silencing is direct.

Mediator phosphorylation prevents stress response transcription during non-stress conditions.

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Miller, Christian; Matic, Ivan; Maier, Kerstin C; Schwalb, Björn; Roether, Susanne; Strässer, Katja; Tresch, Achim; Mann, Matthias; Cramer, Patrick

2012-12-28

The multiprotein complex Mediator is a coactivator of RNA polymerase (Pol) II transcription that is required for the regulated expression of protein-coding genes. Mediator serves as an end point of signaling pathways and regulates Pol II transcription, but the mechanisms it uses are not well understood. Here, we used mass spectrometry and dynamic transcriptome analysis to investigate a functional role of Mediator phosphorylation in gene expression. Affinity purification and mass spectrometry revealed that Mediator from the yeast Saccharomyces cerevisiae is phosphorylated at multiple sites of 17 of its 25 subunits. Mediator phosphorylation levels change upon an external stimulus set by exposure of cells to high salt concentrations. Phosphorylated sites in the Mediator tail subunit Med15 are required for suppression of stress-induced changes in gene expression under non-stress conditions. Thus dynamic and differential Mediator phosphorylation contributes to gene regulation in eukaryotic cells.

Genome-wide association of mediator and RNA polymerase II in wild-type and mediator mutant yeast.

Science.gov (United States)

Paul, Emily; Zhu, Z Iris; Landsman, David; Morse, Randall H

2015-01-01

Mediator is a large, multisubunit complex that is required for essentially all mRNA transcription in eukaryotes. In spite of the importance of Mediator, the range of its targets and how it is recruited to these is not well understood. Previous work showed that in Saccharomyces cerevisiae, Mediator contributes to transcriptional activation by two distinct mechanisms, one depending on the tail module triad and favoring SAGA-regulated genes, and the second occurring independently of the tail module and favoring TFIID-regulated genes. Here, we use chromatin immunoprecipitation sequencing (ChIP-seq) to show that dependence on tail module subunits for Mediator recruitment and polymerase II (Pol II) association occurs preferentially at SAGA-regulated over TFIID-regulated genes on a genome-wide scale. We also show that recruitment of tail module subunits to active gene promoters continues genome-wide when Mediator integrity is compromised in med17 temperature-sensitive (ts) yeast, demonstrating the modular nature of the Mediator complex in vivo. In addition, our data indicate that promoters exhibiting strong and stable occupancy by Mediator have a wide range of activity and are enriched for targets of the Tup1-Cyc8 repressor complex. We also identify a number of strong Mediator occupancy peaks that overlap dubious open reading frames (ORFs) and are likely to include previously unrecognized upstream activator sequences. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

The mediator complex in genomic and non-genomic signaling in cancer.

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Weber, Hannah; Garabedian, Michael J

2018-05-01

Mediator is a conserved, multi-subunit macromolecular machine divided structurally into head, middle, and tail modules, along with a transiently associating kinase module. Mediator functions as an integrator of transcriptional regulatory activity by interacting with DNA-bound transcription factors and with RNA polymerase II (Pol II) to both activate and repress gene expression. Mediator has been shown to affect multiple steps in transcription, including chromatin looping between enhancers and promoters, pre-initiation complex formation, transcriptional elongation, and mRNA splicing. Individual Mediator subunits participate in regulation of gene expression by the estrogen and androgen receptors and are altered in a number of endocrine cancers, including breast and prostate cancer. In addition to its role in genomic signaling, MED12 has been implicated in non-genomic signaling by interacting with and activating TGF-beta receptor 2 in the cytoplasm. Recent structural studies have revealed extensive inter-domain interactions and complex architecture of the Mediator-Pol II complex, suggesting that Mediator is capable of reorganizing its conformation and composition to fit cellular needs. We propose that alterations in Mediator subunit expression that occur in various cancers could impact the organization and function of Mediator, resulting in changes in gene expression that promote malignancy. A better understanding of the role of Mediator in cancer could reveal new approaches to the diagnosis and treatment of Mediator-dependent endocrine cancers, especially in settings of therapy resistance. Copyright © 2017 Elsevier Inc. All rights reserved.

Attenuated traumatic axonal injury and improved functional outcome after traumatic brain injury in mice lacking Sarm1.

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Henninger, Nils; Bouley, James; Sikoglu, Elif M; An, Jiyan; Moore, Constance M; King, Jean A; Bowser, Robert; Freeman, Marc R; Brown, Robert H

2016-04-01

Axonal degeneration is a critical, early event in many acute and chronic neurological disorders. It has been consistently observed after traumatic brain injury, but whether axon degeneration is a driver of traumatic brain injury remains unclear. Molecular pathways underlying the pathology of traumatic brain injury have not been defined, and there is no efficacious treatment for traumatic brain injury. Here we show that mice lacking the mouse Toll receptor adaptor Sarm1 (sterile α/Armadillo/Toll-Interleukin receptor homology domain protein) gene, a key mediator of Wallerian degeneration, demonstrate multiple improved traumatic brain injury-associated phenotypes after injury in a closed-head mild traumatic brain injury model. Sarm1(-/-) mice developed fewer β-amyloid precursor protein aggregates in axons of the corpus callosum after traumatic brain injury as compared to Sarm1(+/+) mice. Furthermore, mice lacking Sarm1 had reduced plasma concentrations of the phophorylated axonal neurofilament subunit H, indicating that axonal integrity is maintained after traumatic brain injury. Strikingly, whereas wild-type mice exibited a number of behavioural deficits after traumatic brain injury, we observed a strong, early preservation of neurological function in Sarm1(-/-) animals. Finally, using in vivo proton magnetic resonance spectroscopy we found tissue signatures consistent with substantially preserved neuronal energy metabolism in Sarm1(-/-) mice compared to controls immediately following traumatic brain injury. Our results indicate that the SARM1-mediated prodegenerative pathway promotes pathogenesis in traumatic brain injury and suggest that anti-SARM1 therapeutics are a viable approach for preserving neurological function after traumatic brain injury. © The Author (2016). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Importance of Mediator complex in the regulation and integration of diverse signaling pathways in plants

Directory of Open Access Journals (Sweden)

Subhasis eSamanta

2015-09-01

Full Text Available Basic transcriptional machinery in eukaryotes is assisted by a number of cofactors, which either increase or decrease the rate of transcription. Mediator complex is one such cofactor, and recently has drawn a lot of interest because of its integrative power to converge different signaling pathways before channelling the transcription instructions to the RNA polymerase II machinery. Like yeast and metazoans, plants do possess the Mediator complex across the kingdom, and its isolation and subunit analyses have been reported from the model plant, Arabidopsis. Genetic and molecular analyses have unravelled important regulatory roles of Mediator subunits at every stage of plant life cycle starting from flowering to embryo and organ development, to even size determination. It also contributes immensely to the survival of plants against different environmental vagaries by the timely activation of its resistance mechanisms. Here, we have provided an overview of plant Mediator complex starting from its discovery to regulation of stoichiometry of its subunits. We have also reviewed involvement of different Mediator subunits in different processes and pathways including defense response pathways evoked by diverse biotic cues. Wherever possible, attempts have been made to provide mechanistic insight of Mediator’s involvement in these processes.

SAD1, an RNA polymerase I subunit A34.5 of rice, interacts with Mediator and controls various aspects of plant development.

Science.gov (United States)

Li, Weiqiang; Yoshida, Akiko; Takahashi, Megumu; Maekawa, Masahiko; Kojima, Mikiko; Sakakibara, Hitoshi; Kyozuka, Junko

2015-01-01

The DWARF14 (D14) gene of rice functions within the signaling pathway of strigolactones, a group of plant hormones that inhibits shoot branching. We isolated a recessive mutant named super apical dormant (sad1-1) from a suppressor screen of d14-1. The growth of tillers (vegetative shoot branches) is suppressed in both the d14-1 sad1-1 double mutant and the sad1-1 single mutant. In addition, the sad1-1 mutant shows pleiotropic defects throughout development. SAD1 encodes an ortholog of RPA34.5, a subunit of RNA polymerase I (Pol I). Consequently, the level of ribosomal RNA (rRNA) is severely reduced in the sad1-1 mutant. These results indicate that proper ribosome function is a prerequisite for normal development in plants. The Arabidopsis ortholog of SAD1 was previously isolated as a Mediator-interacting protein. Here we show that SAD1 interacts physically with the Mediator complex through direct binding with OsMED4, a component of the middle module of the Mediator complex in rice. It is known that Mediator interacts with Pol II, which transcribes mRNAs and functions as a central regulator of transcription. This study indicates a novel aspect of Mediator function in Pol I-controlled rRNA transcription. TFIIF2 and RPC53 are the counterparts of RPA34.5 in Pol II and Pol III, respectively. We demonstrate that the rice orthologs of these proteins also interact with OsMED4. Our results suggest that interaction with MED4 in the Mediator complex is a common feature of the three types of RNA polymerases. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

Editing modifies the GABA(A) receptor subunit alpha3

DEFF Research Database (Denmark)

Ohlson, Johan; Pedersen, Jakob Skou; Haussler, David

2007-01-01

Adenosine to inosine (A-to-I) pre-mRNA editing by the ADAR enzyme family has the potential to increase the variety of the proteome. This editing by adenosine deamination is essential in mammals for a functional brain. To detect novel substrates for A-to-I editing we have used an experimental method...... to find selectively edited sites and combined it with bioinformatic techniques that find stem-loop structures suitable for editing. We present here the first verified editing candidate detected by this screening procedure. We show that Gabra-3, which codes for the alpha3 subunit of the GABA(A) receptor......, is a substrate for editing by both ADAR1 and ADAR2. Editing of the Gabra-3 mRNA recodes an isoleucine to a methionine. The extent of editing is low at birth but increases with age, reaching close to 100% in the adult brain. We therefore propose that editing of the Gabra-3 mRNA is important for normal brain...

The Mediator complex and transcription regulation

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Poss, Zachary C.; Ebmeier, Christopher C.

2013-01-01

The Mediator complex is a multi-subunit assembly that appears to be required for regulating expression of most RNA polymerase II (pol II) transcripts, which include protein-coding and most non-coding RNA genes. Mediator and pol II function within the pre-initiation complex (PIC), which consists of Mediator, pol II, TFIIA, TFIIB, TFIID, TFIIE, TFIIF and TFIIH and is approximately 4.0 MDa in size. Mediator serves as a central scaffold within the PIC and helps regulate pol II activity in ways that remain poorly understood. Mediator is also generally targeted by sequence-specific, DNA-binding transcription factors (TFs) that work to control gene expression programs in response to developmental or environmental cues. At a basic level, Mediator functions by relaying signals from TFs directly to the pol II enzyme, thereby facilitating TF-dependent regulation of gene expression. Thus, Mediator is essential for converting biological inputs (communicated by TFs) to physiological responses (via changes in gene expression). In this review, we summarize an expansive body of research on the Mediator complex, with an emphasis on yeast and mammalian complexes. We focus on the basics that underlie Mediator function, such as its structure and subunit composition, and describe its broad regulatory influence on gene expression, ranging from chromatin architecture to transcription initiation and elongation, to mRNA processing. We also describe factors that influence Mediator structure and activity, including TFs, non-coding RNAs and the CDK8 module. PMID:24088064

Understanding large multiprotein complexes: applying a multiple allosteric networks model to explain the function of the Mediator transcription complex.

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Lewis, Brian A

2010-01-15

The regulation of transcription and of many other cellular processes involves large multi-subunit protein complexes. In the context of transcription, it is known that these complexes serve as regulatory platforms that connect activator DNA-binding proteins to a target promoter. However, there is still a lack of understanding regarding the function of these complexes. Why do multi-subunit complexes exist? What is the molecular basis of the function of their constituent subunits, and how are these subunits organized within a complex? What is the reason for physical connections between certain subunits and not others? In this article, I address these issues through a model of network allostery and its application to the eukaryotic RNA polymerase II Mediator transcription complex. The multiple allosteric networks model (MANM) suggests that protein complexes such as Mediator exist not only as physical but also as functional networks of interconnected proteins through which information is transferred from subunit to subunit by the propagation of an allosteric state known as conformational spread. Additionally, there are multiple distinct sub-networks within the Mediator complex that can be defined by their connections to different subunits; these sub-networks have discrete functions that are activated when specific subunits interact with other activator proteins.

The effects of poverty on childhood brain development: the mediating effect of caregiving and stressful life events.

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Luby, Joan; Belden, Andy; Botteron, Kelly; Marrus, Natasha; Harms, Michael P; Babb, Casey; Nishino, Tomoyuki; Barch, Deanna

2013-12-01

IMPORTANCE The study provides novel data to inform the mechanisms by which poverty negatively impacts childhood brain development. OBJECTIVE To investigate whether the income-to-needs ratio experienced in early childhood impacts brain development at school age and to explore the mediators of this effect. DESIGN, SETTING, AND PARTICIPANTS This study was conducted at an academic research unit at the Washington University School of Medicine in St Louis. Data from a prospective longitudinal study of emotion development in preschool children who participated in neuroimaging at school age were used to investigate the effects of poverty on brain development. Children were assessed annually for 3 to 6 years prior to the time of a magnetic resonance imaging scan, during which they were evaluated on psychosocial, behavioral, and other developmental dimensions. Preschoolers included in the study were 3 to 6 years of age and were recruited from primary care and day care sites in the St Louis metropolitan area; they were annually assessed behaviorally for 5 to 10 years. Healthy preschoolers and those with clinical symptoms of depression participated in neuroimaging at school age/early adolescence. EXPOSURE Household poverty as measured by the income-to-needs ratio. MAIN OUTCOMES AND MEASURES Brain volumes of children's white matter and cortical gray matter, as well as hippocampus and amygdala volumes, obtained using magnetic resonance imaging. Mediators of interest were caregiver support/hostility measured observationally during the preschool period and stressful life events measured prospectively. RESULTS Poverty was associated with smaller white and cortical gray matter and hippocampal and amygdala volumes. The effects of poverty on hippocampal volume were mediated by caregiving support/hostility on the left and right, as well as stressful life events on the left. CONCLUSIONS AND RELEVANCE The finding that exposure to poverty in early childhood materially impacts brain

Downstream targets of methyl CpG binding protein 2 and their abnormal expression in the frontal cortex of the human Rett syndrome brain

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Minchenko Dimitri

2010-04-01

Full Text Available Abstract Background The Rett Syndrome (RTT brain displays regional histopathology and volumetric reduction, with frontal cortex showing such abnormalities, whereas the occipital cortex is relatively less affected. Results Using microarrays and quantitative PCR, the mRNA expression profiles of these two neuroanatomical regions were compared in postmortem brain tissue from RTT patients and normal controls. A subset of genes was differentially expressed in the frontal cortex of RTT brains, some of which are known to be associated with neurological disorders (clusterin and cytochrome c oxidase subunit 1 or are involved in synaptic vesicle cycling (dynamin 1. RNAi-mediated knockdown of MeCP2 in vitro, followed by further expression analysis demonstrated that the same direction of abnormal expression was recapitulated with MeCP2 knockdown, which for cytochrome c oxidase subunit 1 was associated with a functional respiratory chain defect. Chromatin immunoprecipitation (ChIP analysis showed that MeCP2 associated with the promoter regions of some of these genes suggesting that loss of MeCP2 function may be responsible for their overexpression. Conclusions This study has shed more light on the subset of aberrantly expressed genes that result from MECP2 mutations. The mitochondrion has long been implicated in the pathogenesis of RTT, however it has not been at the forefront of RTT research interest since the discovery of MECP2 mutations. The functional consequence of the underexpression of cytochrome c oxidase subunit 1 indicates that this is an area that should be revisited.

Heteromeric α7β2 Nicotinic Acetylcholine Receptors in the Brain

DEFF Research Database (Denmark)

Wu, Jie; Liu, Qiang; Tang, Pei

2016-01-01

The α7 nicotinic acetylcholine receptor (α7 nAChR) is highly expressed in the brain, where it maintains various neuronal functions including (but not limited to) learning and memory. In addition, the protein expression levels of α7 nAChRs are altered in various brain disorders. The classic rule...... governing α7 nAChR assembly in the mammalian brain was that it was assembled from five α7 subunits to form a homomeric receptor pentamer. However, emerging evidence demonstrates the presence of heteromeric α7 nAChRs in heterologously expressed systems and naturally in brain neurons, where α7 subunits are co...... nAChR, which have provided new insights into the understanding of a novel target of cholinergic signaling....

Pharmacological isolation of postsynaptic currents mediated by NR2A- and NR2B-containing NMDA receptors in the anterior cingulate cortex

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Cao Xiaoyan

2007-04-01

Full Text Available Abstract NMDA receptors (NMDARs are involved in excitatory synaptic transmission and plasticity associated with a variety of brain functions, from memory formation to chronic pain. Subunit-selective antagonists for NMDARs provide powerful tools to dissect NMDAR functions in neuronal activities. Recently developed antagonist for NR2A-containing receptors, NVP-AAM007, triggered debates on its selectivity and involvement of the NMDAR subunits in bi-directional synaptic plasticity. Here, we re-examined the pharmacological properties of NMDARs in the anterior cingulate cortex (ACC using NVP-AAM007 as well as ifenprodil, a selective antagonist for NR2B-containing NMDARs. By alternating sequence of drug application and examining different concentrations of NVP-AAM007, we found that the presence of NVP-AAM007 did not significantly affect the effect of ifenprodil on NMDAR-mediated EPSCs. These results suggest that NVP-AAM007 shows great preference for NR2A subunit and could be used as a selective antagonist for NR2A-containing NMDARs in the ACC.

Molecular dynamics studies of the P pilus rod subunit PapA.

Science.gov (United States)

Vitagliano, Luigi; Ruggiero, Alessia; Pedone, Carlo; Berisio, Rita

2009-03-01

Adhesion of uropathogenic Escherichia coli to host tissues is mediated by pili, which extend from the outer cell membrane of the bacterium. Here we report molecular dynamics (MD) characterizations of the major constituent of P pili from the uropathogenic E. coli, PapA, in unliganded state and in complex with the G1 strand of the chaperone PapD. To mimic the PapA response to the gradual dissociation of the PapD G1 strand and to evaluate the role of PapA chaperone recognition sites, we also carried out MD simulations of complexes of PapA with fragments of PapD G1 strand, that leave either the P4 or both P3 and P4 sites unoccupied. Data on the unbound form of PapA indicate that, upon release of the chaperone, PapA evolves toward compact states that are likely not prone to subunit-subunit association. In line with recent experimental reports, this finding implies that chaperone release and subunit-subunit association must be concerted. Our data also indicated that the gradual unbinding of the chaperone from the PapA groove has increasingly strong structural consequences. Indeed, the release of the chaperone from the site P4, which is closest to the initiation site (P5), does not have dramatic effects on the domain structure, whereas its release from both the P4 and the adjacent P3 sites induces a quick structural transition toward a collapsed state, where the subunit groove is obstructed.

Direct evidence that FK506 inhibition of FcepsilonRI-mediated exocytosis from RBL mast cells involves calcineurin.

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Hultsch, T; Brand, P; Lohmann, S; Saloga, J; Kincaid, R L; Knop, J

1998-05-01

FcepsilonRI-mediated exocytosis of preformed mediators from mast cells and basophils (e.g. histamine, serotonin, beta-hexosaminidase) is sensitive to the immunosuppressants cyclosporin A and FK506 (IC50 200 and 4 nM, respectively) but not rapamycin. The mechanism of inhibition does not appear to involve tyrosine phosphorylation, hydrolysis of inositol phosphates or calcium flux. Here we report experiments using a molecular approach to assess the role of calcineurin, a serine/threonine phosphatase thought to be the primary pharmacological target of these drugs. Calcineurin's activity requires association of its catalytic (A) subunit with an intrinsic regulatory (B) subunit. We hypothesized that calcineurin-sensitive signalling events should be affected by the depletion of calcineurin B subunits, thereby reducing the number of active A:B complexes. We therefore transfected rat basophilic leukemia (RBL) cells with an inhibitory (dominant negative) form of the calcineurin A subunit, which binds the calcineurin B subunit with high affinity but does not possess catalytic activity (B subunit knock-out, BKO). In these transfected cells, the dose-response curve for the inhibition of FcepsilonRI-mediated exocytosis by FK506 was shifted to the left, indicating an increased drug sensitivity of BKO-transfected cells. We conclude that FK506 inhibition of FcepsilonRI-mediated exocytosis in mast cells specifically targets calcineurin activity.

Laccase/Mediator Systems: Their Reactivity toward Phenolic Lignin Structures.

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Hilgers, Roelant; Vincken, Jean-Paul; Gruppen, Harry; Kabel, Mirjam A

2018-02-05

Laccase-mediator systems (LMS) have been widely studied for their capacity to oxidize the nonphenolic subunits of lignin (70-90% of the polymer). The phenolic subunits (10-30% of the polymer), which can also be oxidized without mediators, have received considerably less attention. Consequently, it remains unclear to what extent the presence of a mediator influences the reactions of the phenolic subunits of lignin. To get more insight in this, UHPLC-MS was used to study the reactions of a phenolic lignin dimer (GBG), initiated by a laccase from Trametes versicolor , alone or in combination with the mediators HBT and ABTS. The role of HBT was negligible, as its oxidation by laccase occurred slowly in comparison to that of GBG. Laccase and laccase/HBT oxidized GBG at a comparable rate, resulting in extensive polymerization of GBG. In contrast, laccase/ABTS converted GBG at a higher rate, as GBG was oxidized both directly by laccase but also by ABTS radical cations, which were rapidly formed by laccase. The laccase/ABTS system resulted in Cα oxidation of GBG and coupling of ABTS to GBG, rather than polymerization of GBG. Based on these results, we propose reaction pathways of phenolic lignin model compounds with laccase/HBT and laccase/ABTS.

Construction of a hybrid β-hexosaminidase subunit capable of forming stable homodimers that hydrolyze GM2 ganglioside in vivo

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Michael B Tropak

2016-01-01

Full Text Available Tay-Sachs or Sandhoff disease result from mutations in either the evolutionarily related HEXA or HEXB genes encoding respectively, the α- or β-subunits of β-hexosaminidase A (HexA. Of the three Hex isozymes, only HexA can interact with its cofactor, the GM2 activator protein (GM2AP, and hydrolyze GM2 ganglioside. A major impediment to establishing gene or enzyme replacement therapy based on HexA is the need to synthesize both subunits. Thus, we combined the critical features of both α- and β-subunits into a single hybrid µ-subunit that contains the α-subunit active site, the stable β-subunit interface and unique areas in each subunit needed to interact with GM2AP. To facilitate intracellular analysis and the purification of the µ-homodimer (HexM, CRISPR-based genome editing was used to disrupt the HEXA and HEXB genes in a Human Embryonic Kidney 293 cell line stably expressing the µ-subunit. In association with GM2AP, HexM was shown to hydrolyze a fluorescent GM2 ganglioside derivative both in cellulo and in vitro. Gene transfer studies in both Tay-Sachs and Sandhoff mouse models demonstrated that HexM expression reduced brain GM2 ganglioside levels.

Construction of a hybrid β-hexosaminidase subunit capable of forming stable homodimers that hydrolyze GM2 ganglioside in vivo

Science.gov (United States)

Tropak, Michael B; Yonekawa, Sayuri; Karumuthil-Melethil, Subha; Thompson, Patrick; Wakarchuk, Warren; Gray, Steven J; Walia, Jagdeep S; Mark, Brian L; Mahuran, Don

2016-01-01

Tay-Sachs or Sandhoff disease result from mutations in either the evolutionarily related HEXA or HEXB genes encoding respectively, the α- or β-subunits of β-hexosaminidase A (HexA). Of the three Hex isozymes, only HexA can interact with its cofactor, the GM2 activator protein (GM2AP), and hydrolyze GM2 ganglioside. A major impediment to establishing gene or enzyme replacement therapy based on HexA is the need to synthesize both subunits. Thus, we combined the critical features of both α- and β-subunits into a single hybrid µ-subunit that contains the α-subunit active site, the stable β-subunit interface and unique areas in each subunit needed to interact with GM2AP. To facilitate intracellular analysis and the purification of the µ-homodimer (HexM), CRISPR-based genome editing was used to disrupt the HEXA and HEXB genes in a Human Embryonic Kidney 293 cell line stably expressing the µ-subunit. In association with GM2AP, HexM was shown to hydrolyze a fluorescent GM2 ganglioside derivative both in cellulo and in vitro. Gene transfer studies in both Tay-Sachs and Sandhoff mouse models demonstrated that HexM expression reduced brain GM2 ganglioside levels. PMID:26966698

Neural computations mediating one-shot learning in the human brain.

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Sang Wan Lee

2015-04-01

Full Text Available Incremental learning, in which new knowledge is acquired gradually through trial and error, can be distinguished from one-shot learning, in which the brain learns rapidly from only a single pairing of a stimulus and a consequence. Very little is known about how the brain transitions between these two fundamentally different forms of learning. Here we test a computational hypothesis that uncertainty about the causal relationship between a stimulus and an outcome induces rapid changes in the rate of learning, which in turn mediates the transition between incremental and one-shot learning. By using a novel behavioral task in combination with functional magnetic resonance imaging (fMRI data from human volunteers, we found evidence implicating the ventrolateral prefrontal cortex and hippocampus in this process. The hippocampus was selectively "switched" on when one-shot learning was predicted to occur, while the ventrolateral prefrontal cortex was found to encode uncertainty about the causal association, exhibiting increased coupling with the hippocampus for high-learning rates, suggesting this region may act as a "switch," turning on and off one-shot learning as required.

A Common Structural Component for β-Subunit Mediated Modulation of Slow Inactivation in Different KV Channels

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Nathalie Strutz-Seebohm

2013-06-01

Full Text Available Background/Aims: Potassium channels are tetrameric proteins providing potassium selective passage through lipid embedded proteinaceous pores with highest fidelity. The selectivity results from binding to discrete potassium binding sites and stabilization of a hydrated potassium ion in a central internal cavity. The four potassium binding sites, generated by the conserved TTxGYGD signature sequence are formed by the backbone carbonyls of the amino acids TXGYG. Residues KV1.5-Val481, KV4.3-Leu368 and KV7.1- Ile 313 represent the amino acids in the X position of the respective channels. Methods: Here, we study the impact of these residues on ion selectivity, permeation and inactivation kinetics as well as the modulation by β-subunits using site-specific mutagenesis, electrophysiological analyses and molecular dynamics simulations. Results: We identify this position as key in modulation of slow inactivation by structurally dissimilar β-subunits in different KV channels. Conclusion: We propose a model in which structural changes accompanying activation and β-subunit modulation allosterically constrain the backbone carbonyl oxygen atoms via the side chain of the respective X-residue in the signature sequence to reduce conductance during slow inactivation.

Roles of Neuroglobin Binding to Mitochondrial Complex III Subunit Cytochrome c1 in Oxygen-Glucose Deprivation-Induced Neurotoxicity in Primary Neurons.

Science.gov (United States)

Yu, Zhanyang; Zhang, Yu; Liu, Ning; Yuan, Jing; Lin, Li; Zhuge, Qichuan; Xiao, Jian; Wang, Xiaoying

2016-07-01

Neuroglobin (Ngb) is a tissue globin specifically expressed in brain neurons. Recent studies by our laboratory and others have demonstrated that Ngb is protective against stroke and related neurological disorders, but the mechanisms remain poorly understood. We previously identified cytochrome c1 (Cyc1) as an Ngb-interacting molecule by yeast two-hybrid screening. Cyc1 is a subunit of mitochondria complex III, which is a component of mitochondrial respiratory chain and a major source of reactive oxygen species (ROS) production under both physiological and pathological conditions. In this study, we for the first time defined Ngb-Cyc1 binding, and investigated its roles in oxygen-glucose deprivation (OGD)/reoxygenation-induced neurotoxicity and ROS production in primary neurons. Immunocytochemistry and co-immunoprecipitation validated Ngb-Cyc1 binding, which was significantly increased by OGD and Ngb overexpression. We found 4 h OGD with/without 4 h reoxygenation significantly increased complex III activity, but this activity elevation was significantly attenuated in three groups of neurons: Ngb overexpression, specific complex III inhibitor stigmatellin, or stigmatellin plus Ngb overexpression, whereas there was no significant differences between these three groups, suggesting Ngb-Cyc1 binding may function in suppressing OGD-mediated complex III activity elevation. Importantly, these three groups of neurons also showed significant decreases in OGD-induced superoxide anion generation and neurotoxicity. These results suggest that Ngb can bind to mitochondrial complex III subunit Cyc1, leading to suppression of OGD-mediated complex III activity and subsequent ROS production elevation, and eventually reduction of OGD-induced neurotoxicity. This molecular signaling cascade may be at least part of the mechanisms of Ngb neuroprotection against OGD-induced neurotoxicity.

Δ9-THC-caused synaptic and memory impairments are mediated through COX-2 signaling

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Yang, Hongwei; Tang, Ya-ping; Sun, Hao; Song, Yunping; Chen, Chu

2013-01-01

SUMMARY Marijuana has been used for thousands of years as a treatment for medical conditions. However, untoward side effects limit its medical value. Here we show that synaptic and cognitive impairments following repeated exposure to Δ9-tetrahydrocannabinol (Δ9-THC) are associated with the induction of cyclooxygenase-2 (COX-2), an inducible enzyme that converts arachidonic acid to prostanoids, in the brain. COX-2 induction by Δ9-THC is mediated via CB1 receptor-coupled G-protein βγ subunits. Pharmacological or genetic inhibition of COX-2 blocks down-regulation and internalization of glutamate receptor subunits and alterations of the dendritic spine density of hippocampal neurons induced by repeated Δ9-THC exposures. Ablation of COX-2 also eliminates Δ9-THC-impaired hippocampal long-term synaptic plasticity, spatial, and fear memories. Importantly, the beneficial effects of decreasing β-amyloid plaques and neurodegeneration by Δ9-THC in Alzheimer’s disease animals are retained in the presence of COX-2 inhibition. These results suggest that the applicability of medical marijuana would be broadened by concurrent inhibition of COX-2. PMID:24267894

Decreased surface expression of the δ subunit of the GABAA receptor contributes to reduced tonic inhibition in dentate granule cells in a mouse model of fragile X syndrome.

Science.gov (United States)

Zhang, Nianhui; Peng, Zechun; Tong, Xiaoping; Lindemeyer, A Kerstin; Cetina, Yliana; Huang, Christine S; Olsen, Richard W; Otis, Thomas S; Houser, Carolyn R

2017-11-01

While numerous changes in the GABA system have been identified in models of Fragile X Syndrome (FXS), alterations in subunits of the GABA A receptors (GABA A Rs) that mediate tonic inhibition are particularly intriguing. Considering the key role of tonic inhibition in controlling neuronal excitability, reduced tonic inhibition could contribute to FXS-associated disorders such as hyperactivity, hypersensitivity, and increased seizure susceptibility. The current study has focused on the expression and function of the δ subunit of the GABA A R, a major subunit involved in tonic inhibition, in granule cells of the dentate gyrus in the Fmr1 knockout (KO) mouse model of FXS. Electrophysiological studies of dentate granule cells revealed a marked, nearly four-fold, decrease in tonic inhibition in the Fmr1 KO mice, as well as reduced effects of two δ subunit-preferring pharmacological agents, THIP and DS2, supporting the suggestion that δ subunit-containing GABA A Rs are compromised in the Fmr1 KO mice. Immunohistochemistry demonstrated a small but statistically significant decrease in δ subunit labeling in the molecular layer of the dentate gyrus in Fmr1 KO mice compared to wildtype (WT) littermates. The discrepancy between the large deficits in GABA-mediated tonic inhibition in granule cells in the Fmr1 KO mice and only modest reductions in immunolabeling of the δ subunit led to studies of surface expression of the δ subunit. Cross-linking experiments followed by Western blot analysis demonstrated a small, non-significant decrease in total δ subunit protein in the hippocampus of Fmr1 KO mice, but a four-fold decrease in surface expression of the δ subunit in these mice. No significant changes were observed in total or surface expression of the α4 subunit protein, a major partner of the δ subunit in the forebrain. Postembedding immunogold labeling for the δ subunit demonstrated a large, three-fold, decrease in the number of symmetric synapses with

The Schizosaccharomyces pombe Mediator

DEFF Research Database (Denmark)

Venturi, Michela

, Schizosaccharomyces pombe and mammalian Mediator. In our study, we have taken the S. pombe Mediator into consideration and characterized genetically and biochemically two subunits already know in S. cerevisiae, Med9 and Med11, but still not identified in the S. pombe Mediator. Genetic analysis has shown that med9......In the past several years great attention has been dedicated to the characterization of the Mediator complex in a different range of model organisms. Mediator is a conserved co-activator complex involved in transcriptional regulation and it conveys signals from regulatory transcription factors...... to the basal transcription machinery. Mediator was initially isolated from Saccharomyces cerevisiae based on its ability to render a RNA polymerase II in vitro transcription system responsive to activators. Additionally, structural studies have revealed striking structural similarities between S. cerevisiae...

Receptor-mediated transcytosis of cyclophilin B through the blood-brain barrier.

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Carpentier, M; Descamps, L; Allain, F; Denys, A; Durieux, S; Fenart, L; Kieda, C; Cecchelli, R; Spik, G

1999-07-01

Cyclophilin B (CyPB) is a cyclosporin A (CsA)-binding protein mainly located in intracellular vesicles and secreted in biological fluids. In previous works, we demonstrated that CyPB interacts with T lymphocytes and enhances in vitro cellular incorporation and activity of CsA. In addition to its immunosuppressive activity, CsA is able to promote regeneration of damaged peripheral nerves. However, the crossing of the drug from plasma to neural tissue is restricted by the relative impermeability of the blood-brain barrier. To know whether CyPB might also participate in the delivery of CsA into the brain, we have analyzed the interactions of CyPB with brain capillary endothelial cells. First, we demonstrated that CyPB binds to two types of binding sites present at the surface of capillary endothelial cells from various species of tissues. The first type of binding sites (K(D) = 300 nM; number of sites = 3 x 10(6)) is related to interactions with negatively charged compounds such as proteoglycans. The second type of binding sites, approximately 50,000 per cell, exhibits a higher affinity for CyPB (K(D) = 15 nM) and is involved in an endocytosis process, indicating it might correspond to a functional receptor. Finally, the use of an in vitro model of blood-brain barrier allowed us to demonstrate that CyPB is transcytosed by a receptor-mediated pathway (flux = 16.5 fmol/cm2/h). In these conditions, CyPB did not significantly modify the passage of CsA, indicating that it is unlikely to provide a pathway for CsA brain delivery.

Expression of Shiga toxin 2e glycosphingolipid receptors of primary porcine brain endothelial cells and toxin-mediated breakdown of the blood-brain barrier.

Science.gov (United States)

Meisen, Iris; Rosenbrück, Regina; Galla, Hans-Joachim; Hüwel, Sabine; Kouzel, Ivan U; Mormann, Michael; Karch, Helge; Müthing, Johannes

2013-06-01

Shiga toxin (Stx) 2e, released by certain Stx-producing Escherichia coli, is presently the best characterized virulence factor responsible for pig edema disease, which is characterized by hemorrhagic lesions, neurological disorders and often fatal outcomes. Although Stx2e-mediated brain vascular injury is the key event in development of neurologic signs, the glycosphingolipid (GSL) receptors of Stx2e and toxin-mediated impairment of pig brain endothelial cells have not been investigated so far. Here, we report on the detailed structural characterization of Stx2e receptors globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer), which make up the major neutral GSLs in primary porcine brain capillary endothelial cells (PBCECs). Various Gb3Cer and Gb4Cer lipoforms harboring sphingenine (d18:1) or sphinganine (d18:0) and mostly a long-chain fatty acid (C20-C24) were detected. A notable batch-to-batch heterogeneity of primary endothelial cells was observed regarding the extent of ceramide hydroxylation of Gb3Cer or Gb4Cer species. Gb3Cer, Gb4Cer and sphingomyelin preferentially distribute to detergent-resistant membrane fractions and can be considered lipid raft markers in PBCECs. Moreover, we employed an in vitro model of the blood-brain barrier (BBB), which exhibited strong cytotoxic effects of Stx2e on the endothelial monolayer and a rapid collapse of the BBB. These data strongly suggest the involvement of Stx2e in cerebral vascular damage with resultant neurological disturbance characteristic of edema disease.

Decreased α1-adrenergic receptor-mediated inositide hydrolysis in neurons from hypertensive rat brain

International Nuclear Information System (INIS)

Feldstein, J.B.; Gonzales, R.A.; Baker, S.P.; Sumners, C.; Crews, F.T.; Raizada, M.K.

1986-01-01

The expression of α 1 -adrenergic receptors and norepinephrine (NE)-stimulated hydrolysis of inositol phospholipid has been studied in neuronal cultures from the brains of normotensive (Wistar-Kyoto, WKY) and spontaneously hypertensive (SH) rats. Binding of 125 I-1-[β-(4-hydroxyphenyl)-ethyl-aminomethyl] tetralone (HEAT) to neuronal membranes was 68-85% specific and was rapid. Competition-inhibition experiments with various agonists and antagonists suggested that 125 I-HEAT bound selectively to α 1 -adrenergic receptors. Specific binding of 125 I-HEAT to neuronal membranes from SH rat brain cultures was 30-45% higher compared with binding in WKY normotensive controls. This increase was attributed to an increase in the number of α 1 -adrenergic receptors on SH rat brain neurons. Incubation of neuronal cultures of rat brain from both strains with NE resulted in a concentration-dependent stimulation of release of inositol phosphates, although neurons from SH rat brains were 40% less responsive compared with WKY controls. The decrease in responsiveness of SH rat brain neurons to NE, even though the α 1 -adrenergic receptors are increased, does not appear to be due to a general defect in membrane receptors and postreceptor signal transduction mechanisms. This is because neither the number of muscarinic-cholinergic receptors nor the carbachol-stimulated release of inositol phosphates is different in neuronal cultures from the brains of SH rats compared with neuronal cultures from the brains of WKY rats. These observations suggest that the increased expression of α 1 -adrenergic receptors does not parallel the receptor-mediated inositol phosphate hydrolysis in neuronal cultures from SH rat brain

Redefining the modular organization of the core Mediator complex.

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Wang, Xuejuan; Sun, Qianqian; Ding, Zhenrui; Ji, Jinhua; Wang, Jianye; Kong, Xiao; Yang, Jianghong; Cai, Gang

2014-07-01

The Mediator complex plays an essential role in the regulation of eukaryotic transcription. The Saccharomyces cerevisiae core Mediator comprises 21 subunits, which are organized into Head, Middle and Tail modules. Previously, the Head module was assigned to a distinct dense domain at the base, and the Middle and Tail modules were identified to form a tight structure above the Head module, which apparently contradicted findings from many biochemical and functional studies. Here, we compared the structures of the core Mediator and its subcomplexes, especially the first 3D structure of the Head + Middle modules, which permitted an unambiguous assignment of the three modules. Furthermore, nanogold labeling pinpointing four Mediator subunits from different modules conclusively validated the modular assignment, in which the Head and Middle modules fold back on one another and form the upper portion of the core Mediator, while the Tail module forms a distinct dense domain at the base. The new modular model of the core Mediator has reconciled the previous inconsistencies between the structurally and functionally defined Mediator modules. Collectively, these analyses completely redefine the modular organization of the core Mediator, which allow us to integrate the structural and functional information into a coherent mechanism for the Mediator's modularity and regulation in transcription initiation.

Anti-Amyloid-?-Mediated Positron Emission Tomography Imaging in Alzheimer's Disease Mouse Brains

OpenAIRE

McLean, Daniel; Cooke, Michael J.; Wang, Yuanfei; Green, David; Fraser, Paul E.; George-Hyslop, Peter St; Shoichet, Molly S.

2012-01-01

Antibody-mediated imaging of amyloid β (Aβ) in Alzheimer's disease (AD) offers a promising strategy to detect and monitor specific Aβ species, such as oligomers, that have important pathological and therapeutic relevance. The major current limitation of antibodies as a diagnostic and imaging device is poor blood-brain-barrier permeability. A classical anti-Aβ antibody, 6E10, is modified with 10 kDa polyethylene glycol (PEG) and a positron emitting isotope, Copper-64 (t(½) = 12.7 h), and intra...

Differential distribution of calcineurin Aα isoenzyme mRNA's in rat brain

NARCIS (Netherlands)

Buttini, M.; Limonta, S.; Luyten, M.; Boddeke, H.

1993-01-01

Specific antisense oligonucleotide probes for the α isoforms of the catalytic subunit (A-subunit) of calcineurin were prepared and the distribution of Aα1 and Aα2 mRNA's has been studied in rat brain using in situ hybridization histochemistry. Clear regional differences have been observed for the

Selective increases of AMPA, NMDA and kainate receptor subunit mRNAs in the hippocampus and orbitofrontal cortex but not in prefrontal cortex of human alcoholics

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Zhe eJin

2014-01-01

Full Text Available Glutamate is the main excitatory transmitter in the human brain. Drugs that affect the glutamatergic signaling will alter neuronal excitability. Ethanol inhibits glutamate receptors. We examined the expression level of glutamate receptor subunit mRNAs in human post-mortem samples from alcoholics and compared the results to brain samples from control subjects. RNA from hippocampal dentate gyrus (HP-DG, orbitofrontal cortex (OFC, and dorso-lateral prefrontal cortex (DL-PFC samples from 21 controls and 19 individuals with chronic alcohol dependence were included in the study. Total RNA was assayed using quantitative RT-PCR. Out of the 16 glutamate receptor subunits, mRNAs encoding two AMPA (2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-ylpropanoic acid receptor subunits GluA2 and GluA3; three kainate receptor subunits GluK2, GluK3 and GluK5 and five NMDA (N-methyl-D-aspartate receptor subunits GluN1, GluN2A, GluN2C, GluN2D and GluN3A were significantly increased in the HP-DG region in alcoholics. In the OFC, mRNA encoding the NMDA receptor subunit GluN3A was increased, whereas in the DL-PFC, no differences in mRNA levels were observed. Our laboratory has previously shown that the expression of genes encoding inhibitory GABA-A receptors is altered in the HP-DG and OFC of alcoholics (Jin et al., 2011. Whether the changes in one neurotransmitter system drives changes in the other or if they change independently is currently not known. The results demonstrate that excessive long-term alcohol consumption is associated with altered expression of genes encoding glutamate receptors in a brain region-specific manner. It is an intriguing possibility that genetic predisposition to alcoholism may contribute to these gene expression changes.

Detection of constitutive heterodimerization of the integrin Mac-1 subunits by fluorescence resonance energy transfer in living cells

International Nuclear Information System (INIS)

Fu Guo; Yang Huayan; Wang Chen; Zhang Feng; You Zhendong; Wang Guiying; He Cheng; Chen Yizhang; Xu Zhihan

2006-01-01

Macrophage differentiation antigen associated with complement three receptor function (Mac-1) belongs to β 2 subfamily of integrins that mediate important cell-cell and cell-extracellular matrix interactions. Biochemical studies have indicated that Mac-1 is a constitutive heterodimer in vitro. Here, we detected the heterodimerization of Mac-1 subunits in living cells by means of two fluorescence resonance energy transfer (FRET) techniques (fluorescence microscopy and fluorescence spectroscopy) and our results demonstrated that there is constitutive heterodimerization of the Mac-1 subunits and this constitutive heterodimerization of the Mac-1 subunits is cell-type independent. Through FRET imaging, we found that heterodimers of Mac-1 mainly localized in plasma membrane, perinuclear, and Golgi area in living cells. Furthermore, through analysis of the estimated physical distances between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) fused to Mac-1 subunits, we suggested that the conformation of Mac-1 subunits is not affected by the fusion of CFP or YFP and inferred that Mac-1 subunits take different conformation when expressed in Chinese hamster ovary (CHO) and human embryonic kidney (HEK) 293T cells, respectively

G-protein signaling leverages subunit-dependent membrane affinity to differentially control βγ translocation to intracellular membranes.

Science.gov (United States)

O'Neill, Patrick R; Karunarathne, W K Ajith; Kalyanaraman, Vani; Silvius, John R; Gautam, N

2012-12-18

Activation of G-protein heterotrimers by receptors at the plasma membrane stimulates βγ-complex dissociation from the α-subunit and translocation to internal membranes. This intermembrane movement of lipid-modified proteins is a fundamental but poorly understood feature of cell signaling. The differential translocation of G-protein βγ-subunit types provides a valuable experimental model to examine the movement of signaling proteins between membranes in a living cell. We used live cell imaging, mathematical modeling, and in vitro measurements of lipidated fluorescent peptide dissociation from vesicles to determine the mechanistic basis of the intermembrane movement and identify the interactions responsible for differential translocation kinetics in this family of evolutionarily conserved proteins. We found that the reversible translocation is mediated by the limited affinity of the βγ-subunits for membranes. The differential kinetics of the βγ-subunit types are determined by variations among a set of basic and hydrophobic residues in the γ-subunit types. G-protein signaling thus leverages the wide variation in membrane dissociation rates among different γ-subunit types to differentially control βγ-translocation kinetics in response to receptor activation. The conservation of primary structures of γ-subunits across mammalian species suggests that there can be evolutionary selection for primary structures that confer specific membrane-binding affinities and consequent rates of intermembrane movement.

The U-box E3 ubiquitin ligase TUD1 functions with a heterotrimeric G α subunit to regulate Brassinosteroid-mediated growth in rice.

Directory of Open Access Journals (Sweden)

Xingming Hu

Full Text Available Heterotrimeric G proteins are an important group of signaling molecules found in eukaryotes. They function with G-protein-coupled-receptors (GPCRs to transduce various signals such as steroid hormones in animals. Nevertheless, their functions in plants are not well-defined. Previous studies suggested that the heterotrimeric G protein α subunit known as D1/RGA1 in rice is involved in a phytohormone gibberellin-mediated signaling pathway. Evidence also implicates D1 in the action of a second phytohormone Brassinosteroid (BR and its pathway. However, it is unclear how D1 functions in this pathway, because so far no partner has been identified to act with D1. In this study, we report a D1 genetic interactor Taihu Dwarf1 (TUD1 that encodes a functional U-box E3 ubiquitin ligase. Genetic, phenotypic, and physiological analyses have shown that tud1 is epistatic to d1 and is less sensitive to BR treatment. Histological observations showed that the dwarf phenotype of tud1 is mainly due to decreased cell proliferation and disorganized cell files in aerial organs. Furthermore, we found that D1 directly interacts with TUD1. Taken together, these results demonstrate that D1 and TUD1 act together to mediate a BR-signaling pathway. This supports the idea that a D1-mediated BR signaling pathway occurs in rice to affect plant growth and development.

The U-Box E3 Ubiquitin Ligase TUD1 Functions with a Heterotrimeric G α Subunit to Regulate Brassinosteroid-Mediated Growth in Rice

Science.gov (United States)

Hu, Xingming; Qian, Qian; Xu, Ting; Zhang, Yu'e; Dong, Guojun; Gao, Ting; Xie, Qi; Xue, Yongbiao

2013-01-01

Heterotrimeric G proteins are an important group of signaling molecules found in eukaryotes. They function with G-protein-coupled-receptors (GPCRs) to transduce various signals such as steroid hormones in animals. Nevertheless, their functions in plants are not well-defined. Previous studies suggested that the heterotrimeric G protein α subunit known as D1/RGA1 in rice is involved in a phytohormone gibberellin-mediated signaling pathway. Evidence also implicates D1 in the action of a second phytohormone Brassinosteroid (BR) and its pathway. However, it is unclear how D1 functions in this pathway, because so far no partner has been identified to act with D1. In this study, we report a D1 genetic interactor Taihu Dwarf1 (TUD1) that encodes a functional U-box E3 ubiquitin ligase. Genetic, phenotypic, and physiological analyses have shown that tud1 is epistatic to d1 and is less sensitive to BR treatment. Histological observations showed that the dwarf phenotype of tud1 is mainly due to decreased cell proliferation and disorganized cell files in aerial organs. Furthermore, we found that D1 directly interacts with TUD1. Taken together, these results demonstrate that D1 and TUD1 act together to mediate a BR-signaling pathway. This supports the idea that a D1-mediated BR signaling pathway occurs in rice to affect plant growth and development. PMID:23526892

KCNE4 is an inhibitory subunit to Kv1.1 and Kv1.3 potassium channels

DEFF Research Database (Denmark)

Grunnet, Morten; Rasmussen, Hannne B; Hay-Schmidt, Anders

2003-01-01

is detected in the heart and in five different parts of the brain. Having the broad distribution of Kv1 channels in mind, the demonstrated inhibitory property of KCNE4-subunits could locally and/or transiently have a dramatic influence on cellular excitability and on setting resting membrane potentials....

Downregulation of GABA[Subscript A] Receptor Protein Subunits a6, ß2, d, e, ?2, ?, and ?2 in Superior Frontal Cortex of Subjects with Autism

Science.gov (United States)

Fatemi, S. Hossein; Reutiman, Teri J.; Folsom, Timothy D.; Rustan, Oyvind G.; Rooney, Robert J.; Thuras, Paul D.

2014-01-01

We measured protein and mRNA levels for nine gamma-aminobutyric acid A (GABA[subscript A]) receptor subunits in three brain regions (cerebellum, superior frontal cortex, and parietal cortex) in subjects with autism versus matched controls. We observed changes in mRNA for a number of GABA[subscript A] and GABA[subscript B] subunits and overall…

Progesterone modulation of alpha5 nAChR subunits influences anxiety-related behavior during estrus cycle.

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Gangitano, D; Salas, R; Teng, Y; Perez, E; De Biasi, M

2009-06-01

Smokers often report an anxiolytic effect of cigarettes. In addition, stress-related disorders such as anxiety, post-traumatic stress syndrome and depression are often associated with chronic nicotine use. To study the role of the alpha5 nicotinic acetylcholine receptor subunit in anxiety-related responses, control and alpha5 subunit null mice (alpha5(-/-)) were subjected to the open field activity (OFA), light-dark box (LDB) and elevated plus maze (EPM) tests. In the OFA and LDB, alpha5(-/-) behaved like wild-type controls. In the EPM, female alpha5(-/-) mice displayed an anxiolytic-like phenotype, while male alpha5(-/-) mice were undistinguishable from littermate controls. We studied the hypothalamus-pituitary-adrenal axis by measuring plasma corticosterone and hypothalamic corticotropin-releasing factor. Consistent with an anxiolytic-like phenotype, female alpha5(-/-) mice displayed lower basal corticosterone levels. To test whether gonadal steroids regulate the expression of alpha5, we treated cultured NTera 2 cells with progesterone and found that alpha5 protein levels were upregulated. In addition, brain levels of alpha5 mRNA increased upon progesterone injection into ovariectomized wild-type females. Finally, we tested anxiety levels in the EPM during the estrous cycle. The estrus phase (when progesterone levels are low) is anxiolytic-like in wild-type mice, but no cycle-dependent fluctuations in anxiety levels were found in alpha5(-/-) females. Thus, alpha5-containing neuronal nicotinic acetylcholine receptors may be mediators of anxiogenic responses, and progesterone-dependent modulation of alpha5 expression may contribute to fluctuations in anxiety levels during the ovarian cycle.

Activity-dependent control of NMDA receptor subunit composition at hippocampal mossy fibre synapses.

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Carta, Mario; Srikumar, Bettadapura N; Gorlewicz, Adam; Rebola, Nelson; Mulle, Christophe

2018-02-15

CA3 pyramidal cells display input-specific differences in the subunit composition of synaptic NMDA receptors (NMDARs). Although at low density, GluN2B contributes significantly to NMDAR-mediated EPSCs at mossy fibre synapses. Long-term potentiation (LTP) of NMDARs triggers a modification in the subunit composition of synaptic NMDARs by insertion of GluN2B. GluN2B subunits are essential for the expression of LTP of NMDARs at mossy fibre synapses. Single neurons express NMDA receptors (NMDARs) with distinct subunit composition and biophysical properties that can be segregated in an input-specific manner. The dynamic control of the heterogeneous distribution of synaptic NMDARs is crucial to control input-dependent synaptic integration and plasticity. In hippocampal CA3 pyramidal cells from mice of both sexes, we found that mossy fibre (MF) synapses display a markedly lower proportion of GluN2B-containing NMDARs than associative/commissural synapses. The mechanism involved in such heterogeneous distribution of GluN2B subunits is not known. Here we show that long-term potentiation (LTP) of NMDARs, which is selectively expressed at MF-CA3 pyramidal cell synapses, triggers a modification in the subunit composition of synaptic NMDARs by insertion of GluN2B. This activity-dependent recruitment of GluN2B at mature MF-CA3 pyramidal cell synapses contrasts with the removal of GluN2B subunits at other glutamatergic synapses during development and in response to activity. Furthermore, although expressed at low levels, GluN2B is necessary for the expression of LTP of NMDARs at MF-CA3 pyramidal cell synapses. Altogether, we reveal a previously unknown activity-dependent regulation and function of GluN2B subunits that may contribute to the heterogeneous plasticity induction rules in CA3 pyramidal cells. © 2017 Centre Nationnal de la Recherche Scientifique. The Journal of Physiology © 2017 The Physiological Society.

Fto colocalizes with a satiety mediator oxytocin in the brain and upregulates oxytocin gene expression

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Olszewski, Pawel K., E-mail: olsze005@umn.edu [Department of Neuroscience, Functional Pharmacology, Uppsala University, 75124 Uppsala (Sweden); Minnesota Obesity Center, Saint Paul, MN 55108 (United States); Fredriksson, Robert; Eriksson, Jenny D. [Department of Neuroscience, Functional Pharmacology, Uppsala University, 75124 Uppsala (Sweden); Mitra, Anaya [Department of Food Science and Nutrition, Saint Paul, MN 55108 (United States); Radomska, Katarzyna J. [Department of Neuroscience, Functional Pharmacology, Uppsala University, 75124 Uppsala (Sweden); Gosnell, Blake A. [Department of Food Science and Nutrition, Saint Paul, MN 55108 (United States); Solvang, Maria N. [Department of Neuroscience, Functional Pharmacology, Uppsala University, 75124 Uppsala (Sweden); Levine, Allen S. [Minnesota Obesity Center, Saint Paul, MN 55108 (United States); Department of Food Science and Nutrition, Saint Paul, MN 55108 (United States); Schioeth, Helgi B. [Department of Neuroscience, Functional Pharmacology, Uppsala University, 75124 Uppsala (Sweden)

2011-05-13

Highlights: {yields} The majority of neurons synthesizing a satiety mediator, oxytocin, coexpress Fto. {yields} The level of colocalization is similar in the male and female brain. {yields} Fto overexpression in hypothalamic neurons increases oxytocin mRNA levels by 50%. {yields} Oxytocin does not affect Fto expression through negative feedback mechanisms. -- Abstract: Single nucleotide polymorphisms in the fat mass and obesity-associated (FTO) gene have been associated with obesity in humans. Alterations in Fto expression in transgenic animals affect body weight, energy expenditure and food intake. Fto, a nuclear protein and proposed transcription co-factor, has been speculated to affect energy balance through a functional relationship with specific genes encoding feeding-related peptides. Herein, we employed double immunohistochemistry and showed that the majority of neurons synthesizing a satiety mediator, oxytocin, coexpress Fto in the brain of male and female mice. We then overexpressed Fto in a murine hypothalamic cell line and, using qPCR, detected a 50% increase in the level of oxytocin mRNA. Expression levels of several other feeding-related genes, including neuropeptide Y (NPY) and Agouti-related protein (AgRP), were unaffected by the FTO transfection. Addition of 10 and 100 nmol oxytocin to the cell culture medium did not affect Fto expression in hypothalamic cells. We conclude that Fto, a proposed transcription co-factor, influences expression of the gene encoding a satiety mediator, oxytocin.

Fto colocalizes with a satiety mediator oxytocin in the brain and upregulates oxytocin gene expression

International Nuclear Information System (INIS)

Olszewski, Pawel K.; Fredriksson, Robert; Eriksson, Jenny D.; Mitra, Anaya; Radomska, Katarzyna J.; Gosnell, Blake A.; Solvang, Maria N.; Levine, Allen S.; Schioeth, Helgi B.

2011-01-01

Highlights: → The majority of neurons synthesizing a satiety mediator, oxytocin, coexpress Fto. → The level of colocalization is similar in the male and female brain. → Fto overexpression in hypothalamic neurons increases oxytocin mRNA levels by 50%. → Oxytocin does not affect Fto expression through negative feedback mechanisms. -- Abstract: Single nucleotide polymorphisms in the fat mass and obesity-associated (FTO) gene have been associated with obesity in humans. Alterations in Fto expression in transgenic animals affect body weight, energy expenditure and food intake. Fto, a nuclear protein and proposed transcription co-factor, has been speculated to affect energy balance through a functional relationship with specific genes encoding feeding-related peptides. Herein, we employed double immunohistochemistry and showed that the majority of neurons synthesizing a satiety mediator, oxytocin, coexpress Fto in the brain of male and female mice. We then overexpressed Fto in a murine hypothalamic cell line and, using qPCR, detected a 50% increase in the level of oxytocin mRNA. Expression levels of several other feeding-related genes, including neuropeptide Y (NPY) and Agouti-related protein (AgRP), were unaffected by the FTO transfection. Addition of 10 and 100 nmol oxytocin to the cell culture medium did not affect Fto expression in hypothalamic cells. We conclude that Fto, a proposed transcription co-factor, influences expression of the gene encoding a satiety mediator, oxytocin.

Migraine with aura and silent brain infarcts lack of mediation of patent foramen ovale.

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Calviere, L; Tall, P; Massabuau, P; Bonneville, F; Larrue, V

2013-12-01

Population-based studies have shown a heightened prevalence of clinically silent brain infarcts in subjects who have migraine with aura (MA). We sought to determine whether this association could be confirmed in young patients with cryptogenic ischemic stroke, and explored the role of patent foramen ovale (PFO) as a potential underlying mechanism. Patients were selected from a registry of young patients consecutively treated for ischemic stroke in a tertiary university hospital among those without definite cause of stroke. Patients with PFO were matched for age and gender with patients with normal atrial septum. Migraine and MA were evaluated after patient selection and matching. Silent brain infarcts were independently evaluated on MRI. We included 100 patients [60 men; mean age (SD), 44.8 years (8.3)], 50 patients with PFO. We found silent brain infarcts in 36 patients and MA in 13 patients. MA was more frequent in patients with silent brain infarcts than in patients without silent brain infarcts (25.0% vs. 6.3%; OR, 5; 95% CI, 1.4-17.6; P = 0.01). Traditional cardiovascular risk factors were not associated with silent brain infarcts. PFO was neither associated with MA (OR, 1.7; 95% CI, 0.5-5.3) nor silent brain infarcts (OR, 0.7; 95% CI, 0.3-1.5). The association of MA with silent brain infarcts was not altered after adjustment for PFO. Findings suggest that silent brain infarcts in young patients with cryptogenic stroke is associated with MA. We found no evidence for a mediating effect of PFO on this association. © 2013 The Author(s) European Journal of Neurology © 2013 EFNS.

AKAP18:PKA-RIIα structure reveals crucial anchor points for recognition of regulatory subunits of PKA.

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Götz, Frank; Roske, Yvette; Schulz, Maike Svenja; Autenrieth, Karolin; Bertinetti, Daniela; Faelber, Katja; Zühlke, Kerstin; Kreuchwig, Annika; Kennedy, Eileen J; Krause, Gerd; Daumke, Oliver; Herberg, Friedrich W; Heinemann, Udo; Klussmann, Enno

2016-07-01

A-kinase anchoring proteins (AKAPs) interact with the dimerization/docking (D/D) domains of regulatory subunits of the ubiquitous protein kinase A (PKA). AKAPs tether PKA to defined cellular compartments establishing distinct pools to increase the specificity of PKA signalling. Here, we elucidated the structure of an extended PKA-binding domain of AKAP18β bound to the D/D domain of the regulatory RIIα subunits of PKA. We identified three hydrophilic anchor points in AKAP18β outside the core PKA-binding domain, which mediate contacts with the D/D domain. Such anchor points are conserved within AKAPs that bind regulatory RII subunits of PKA. We derived a different set of anchor points in AKAPs binding regulatory RI subunits of PKA. In vitro and cell-based experiments confirm the relevance of these sites for the interaction of RII subunits with AKAP18 and of RI subunits with the RI-specific smAKAP. Thus we report a novel mechanism governing interactions of AKAPs with PKA. The sequence specificity of each AKAP around the anchor points and the requirement of these points for the tight binding of PKA allow the development of selective inhibitors to unequivocally ascribe cellular functions to the AKAP18-PKA and other AKAP-PKA interactions. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

Role of the Rubisco Small Subunit

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Spreitzer, Robert Joseph [Univ. of Nebraska, Lincoln, NE (United States)

2016-11-05

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the rate-limiting step of CO₂ fixation in photosynthesis. However, it is a slow enzyme, and O₂ competes with CO₂ at the active site. Oxygenation initiates the photorespiratory pathway, which also results in the loss of CO₂. If carboxylation could be increased or oxygenation decreased, an increase in net CO₂ fixation would be realized. Because Rubisco provides the primary means by which carbon enters all life on earth, there is much interest in engineering Rubisco to increase the production of food and renewable energy. Rubisco is located in the chloroplasts of plants, and it is comprised of two subunits. Much is known about the chloroplast-gene-encoded large subunit (rbcL gene), which contains the active site, but much less is known about the role of the nuclear-gene-encoded small subunit in Rubisco function (rbcS gene). Both subunits are coded by multiple genes in plants, which makes genetic engineering difficult. In the eukaryotic, green alga Chlamydomonas reinhardtii, it has been possible to eliminate all the Rubisco genes. These Rubisco-less mutants can be maintained by providing acetate as an alternative carbon source. In this project, focus has been placed on determining whether the small subunit might be a better genetic-engineering target for improving Rubisco. Analysis of a variable-loop structure (βA-βB loop) of the small subunit by genetic selection, directed mutagenesis, and construction of chimeras has shown that the small subunit can influence CO₂/O₂ specificity. X-ray crystal structures of engineered chimeric-loop enzymes have indicated that additional residues and regions of the small subunit may also contribute to Rubisco function. Structural dynamics of the small-subunit carboxyl terminus was also investigated. Alanine-scanning mutagenesis of the most-conserved small-subunit residues has identified a

Neural Computations Mediating One-Shot Learning in the Human Brain

Science.gov (United States)

Lee, Sang Wan; O’Doherty, John P.; Shimojo, Shinsuke

2015-01-01

Incremental learning, in which new knowledge is acquired gradually through trial and error, can be distinguished from one-shot learning, in which the brain learns rapidly from only a single pairing of a stimulus and a consequence. Very little is known about how the brain transitions between these two fundamentally different forms of learning. Here we test a computational hypothesis that uncertainty about the causal relationship between a stimulus and an outcome induces rapid changes in the rate of learning, which in turn mediates the transition between incremental and one-shot learning. By using a novel behavioral task in combination with functional magnetic resonance imaging (fMRI) data from human volunteers, we found evidence implicating the ventrolateral prefrontal cortex and hippocampus in this process. The hippocampus was selectively “switched” on when one-shot learning was predicted to occur, while the ventrolateral prefrontal cortex was found to encode uncertainty about the causal association, exhibiting increased coupling with the hippocampus for high-learning rates, suggesting this region may act as a “switch,” turning on and off one-shot learning as required. PMID:25919291

Two conserved modules of Schizosaccharomyces pombe Mediator regulate distinct cellular pathways

DEFF Research Database (Denmark)

Linder, Tomas; Rasmussen, Nina; Samuelsen, Camilla O

2008-01-01

Mediator is an evolutionary conserved coregulator complex required for transcription of almost all RNA polymerase II-dependent genes. The Schizosaccharomyces pombe Mediator consists of two dissociable components-a core complex organized into a head and middle domain as well as the Cdk8 regulatory...... subcomplex. In this work we describe a functional characterization of the S. pombe Mediator. We report the identification of the S. pombe Med20 head subunit and the isolation of ts alleles of the core head subunit encoding med17+. Biochemical analysis of med8(ts), med17(ts), Deltamed18, Deltamed20...... and Deltamed27 alleles revealed a stepwise head domain molecular architecture. Phenotypical analysis of Cdk8 and head module alleles including expression profiling classified the Mediator mutant alleles into one of two groups. Cdk8 module mutants flocculate due to overexpression of adhesive cell...

The Cac2 subunit is essential for productive histone binding and nucleosome assembly in CAF-1

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Mattiroli, Francesca; Gu, Yajie; Balsbaugh, Jeremy L.; Ahn, Natalie G.; Luger, Karolin

2017-04-18

Nucleosome assembly following DNA replication controls epigenome maintenance and genome integrity. Chromatin assembly factor 1 (CAF-1) is the histone chaperone responsible for histone (H3-H4)2 deposition following DNA synthesis. Structural and functional details for this chaperone complex and its interaction with histones are slowly emerging. Using hydrogen-deuterium exchange coupled to mass spectrometry, combined with in vitro and in vivo mutagenesis studies, we identified the regions involved in the direct interaction between the yeast CAF-1 subunits, and mapped the CAF-1 domains responsible for H3-H4 binding. The large subunit, Cac1 organizes the assembly of CAF-1. Strikingly, H3-H4 binding is mediated by a composite interface, shaped by Cac1-bound Cac2 and the Cac1 acidic region. Cac2 is indispensable for productive histone binding, while deletion of Cac3 has only moderate effects on H3-H4 binding and nucleosome assembly. These results define direct structural roles for yeast CAF-1 subunits and uncover a previously unknown critical function of the middle subunit in CAF-1.

Posttraumatic Propofol Neurotoxicity Is Mediated via the Pro-Brain-Derived Neurotrophic Factor-p75 Neurotrophin Receptor Pathway in Adult Mice.

Science.gov (United States)

Sebastiani, Anne; Granold, Matthias; Ditter, Anja; Sebastiani, Philipp; Gölz, Christina; Pöttker, Bruno; Luh, Clara; Schaible, Eva-Verena; Radyushkin, Konstantin; Timaru-Kast, Ralph; Werner, Christian; Schäfer, Michael K; Engelhard, Kristin; Moosmann, Bernd; Thal, Serge C

2016-02-01

The gamma-aminobutyric acid modulator propofol induces neuronal cell death in healthy immature brains by unbalancing neurotrophin homeostasis via p75 neurotrophin receptor signaling. In adulthood, p75 neurotrophin receptor becomes down-regulated and propofol loses its neurotoxic effect. However, acute brain lesions, such as traumatic brain injury, reactivate developmental-like programs and increase p75 neurotrophin receptor expression, probably to foster reparative processes, which in turn could render the brain sensitive to propofol-mediated neurotoxicity. This study investigates the influence of delayed single-bolus propofol applications at the peak of p75 neurotrophin receptor expression after experimental traumatic brain injury in adult mice. Randomized laboratory animal study. University research laboratory. Adult C57BL/6N and nerve growth factor receptor-deficient mice. Sedation by IV propofol bolus application delayed after controlled cortical impact injury. Propofol sedation at 24 hours after traumatic brain injury increased lesion volume, enhanced calpain-induced αII-spectrin cleavage, and increased cell death in perilesional tissue. Thirty-day postinjury motor function determined by CatWalk (Noldus Information Technology, Wageningen, The Netherlands) gait analysis was significantly impaired in propofol-sedated animals. Propofol enhanced pro-brain-derived neurotrophic factor/brain-derived neurotrophic factor ratio, which aggravates p75 neurotrophin receptor-mediated cell death. Propofol toxicity was abolished both by pharmacologic inhibition of the cell death domain of the p75 neurotrophin receptor (TAT-Pep5) and in mice lacking the extracellular neurotrophin binding site of p75 neurotrophin receptor. This study provides first evidence that propofol sedation after acute brain lesions can have a deleterious impact and implicates a role for the pro-brain-derived neurotrophic factor-p75 neurotrophin receptor pathway. This observation is important as sedation

FXYD proteins reverse inhibition of the Na+-K+ pump mediated by glutathionylation of its beta1 subunit.

Science.gov (United States)

Bibert, Stéphanie; Liu, Chia-Chi; Figtree, Gemma A; Garcia, Alvaro; Hamilton, Elisha J; Marassi, Francesca M; Sweadner, Kathleen J; Cornelius, Flemming; Geering, Käthi; Rasmussen, Helge H

2011-05-27

The seven members of the FXYD protein family associate with the Na(+)-K(+) pump and modulate its activity. We investigated whether conserved cysteines in FXYD proteins are susceptible to glutathionylation and whether such reactivity affects Na(+)-K(+) pump function in cardiac myocytes and Xenopus oocytes. Glutathionylation was detected by immunoblotting streptavidin precipitate from biotin-GSH loaded cells or by a GSH antibody. Incubation of myocytes with recombinant FXYD proteins resulted in competitive displacement of native FXYD1. Myocyte and Xenopus oocyte pump currents were measured with whole-cell and two-electrode voltage clamp techniques, respectively. Native FXYD1 in myocytes and FXYD1 expressed in oocytes were susceptible to glutathionylation. Mutagenesis identified the specific cysteine in the cytoplasmic terminal that was reactive. Its reactivity was dependent on flanking basic amino acids. We have reported that Na(+)-K(+) pump β(1) subunit glutathionylation induced by oxidative signals causes pump inhibition in a previous study. In the present study, we found that β(1) subunit glutathionylation and pump inhibition could be reversed by exposing myocytes to exogenous wild-type FXYD3. A cysteine-free FXYD3 derivative had no effect. Similar results were obtained with wild-type and mutant FXYD proteins expressed in oocytes. Glutathionylation of the β(1) subunit was increased in myocardium from FXYD1(-/-) mice. In conclusion, there is a dependence of Na(+)-K(+) pump regulation on reactivity of two specifically identified cysteines on separate components of the multimeric Na(+)-K(+) pump complex. By facilitating deglutathionylation of the β(1) subunit, FXYD proteins reverse oxidative inhibition of the Na(+)-K(+) pump and play a dynamic role in its regulation.

Brain's reward circuits mediate itch relief. a functional MRI study of active scratching.

Directory of Open Access Journals (Sweden)

Alexandru D P Papoiu

Full Text Available Previous brain imaging studies investigating the brain processing of scratching used an exogenous intervention mimicking scratching, performed not by the subjects themselves, but delivered by an investigator. In real life, scratching is a conscious, voluntary, controlled motor response to itching, which is directed to the perceived site of distress. In this study we aimed to visualize in real-time by brain imaging the core mechanisms of the itch-scratch cycle when scratching was performed by subjects themselves. Secondly, we aimed to assess the correlations between brain patterns of activation and psychophysical ratings of itch relief or pleasurability of scratching. We also compared the patterns of brain activity evoked by self-scratching vs. passive scratching. We used a robust tridimensional Arterial Spin Labeling fMRI technique that is less sensitive to motion artifacts: 3D gradient echo and spin echo (GRASE--Propeller. Active scratching was accompanied by a higher pleasurability and induced a more pronounced deactivation of the anterior cingulate cortex and insula, in comparison with passive scratching. A significant involvement of the reward system including the ventral tegmentum of the midbrain, coupled with a mechanism deactivating the periaqueductal gray matter (PAG, suggests that itch modulation operates in reverse to the mechanism known to suppress pain. Our findings not only confirm a role for the central networks processing reward in the pleasurable aspects of scratching, but also suggest they play a role in mediating itch relief.

Neuropeptides as mediators of the early-life impact on the brain; implications for alcohol use disorders

Directory of Open Access Journals (Sweden)

Ingrid eNylander

2012-07-01

Full Text Available The brain is constantly exposed to external and internal input and to function in an ever-changing environment we are dependent on processes that enable the brain to adapt to new stimuli. Exposure to postnatal environmental stimuli can interfere with vital adaption processes and cause long-term changes in physiological function and behaviour. Early-life alterations in brain function may result in impaired ability to adapt to new situations, in altered sensitivity to challenges later in life and thereby mediate risk or protection for psychopathology such as alcohol use disorders (AUD. In clinical research the studies of mechanisms, mediators and causal relation between early environmental factors and vulnerability to AUD are restricted and attempts are made to find valid animal models for studies of the early-life influence on the brain. This review focuses on rodent models and the effects of adverse and naturalistic conditions on peptide networks within the brain and pituitary gland. Importantly, the consequences of alcohol addiction are not discussed but rather neurobiological alterations that can cause risk consumption and vulnerability to addiction. The article reviews earlier results and includes new data with emphasis on endogenous opioid peptides but also oxytocin and vasopressin. These peptides are vital for developmental processes and it is hypothesized that early-life changes in peptide networks may interfere with neuronal processes and thereby contribute the individual vulnerability for AUD. The summarized results indicate a link between early-life rearing conditions, opioids and ethanol consumption and that the ethanol-induced effects and the treatment with opioid antagonists later in life are dependent on early-life experiences. Endogenous opioids are therefore of interest to further study in the early-life impact on individual differences in vulnerability to AUD and treatment outcome.

Omega-3 polyunsaturated fatty acids promote amyloid-β clearance from the brain through mediating the function of the glymphatic system.

Science.gov (United States)

Ren, Huixia; Luo, Chuanming; Feng, Yanqing; Yao, Xiaoli; Shi, Zhe; Liang, Fengyin; Kang, Jing X; Wan, Jian-Bo; Pei, Zhong; Su, Huanxing

2017-01-01

Impairment of amyloid-β (Aβ) clearance leads to Aβ accumulation in the brain during the development of Alzheimer's disease (AD). Strategies that can restore or improve the clearance function hold great promise in delaying or preventing the onset of AD. Here, we show that n-3 polyunsaturated fatty acids (PUFAs), by use of fat-1 transgenic mice and oral administration of fish oil, significantly promote interstitial Aβ clearance from the brain and resist Aβ injury. Such beneficial effects were abolished in Aqp4-knockout mice, suggesting that the AQP4-dependent glymphatic system is actively involved in the promoting the effects of n-3 PUFAs on the clearance of extracellular Aβ. Imaging on clarified brain tissues clearly displayed that n-3 PUFAs markedly inhibit the activation of astrocytes and protect the AQP4 polarization in the affected brain region after Aβ injection. The results of the present study prove a novel mechanism by which n-3 PUFAs exert protective roles in reducing Aβ accumulation via mediating the glymphatic system function.-Ren, H., Luo, C., Feng, Y., Yao, X., Shi, Z., Liang, F., Kang, J. X., Wan, J.-B., Pei, Z., Su, H. Omega-3 polyunsaturated fatty acids promote amyloid-β clearance from the brain through mediating the function of the glymphatic system. © FASEB.

PKC/CREB pathway mediates the expressions of GABAA receptor subunits in cultured hippocampal neurons after low-Mg2+ solution treatment.

Science.gov (United States)

Wu, Guofeng; Yu, Jinpeng; Wang, Likun; Ren, Siying; Zhang, Yixia

2018-02-01

To investigate the potential effects of the PKC/CREB pathway on the expressions of GABA A receptor subunits α1, γ2, and δ in cultured hippocampal neurons using a model of epilepsy that employed conditions of low magnesium (Mg 2+ ). A total of 108 embryonic rats at the age of 18 embryonic days (E18)prepared from adult female SD rats were used as experimental subjects. Primary rat hippocampal cultures were prepared from the embryonic 18 days rats. The cultured hippocampal neurons were then treated with artificial cerebrospinal fluid containing low Mg 2+ solutions to generate a low Mg 2+ model of epilepsy. The low Mg 2+ stimulation lasted for 3 h and then returned to in maintenance medium for 20 h. The changes of the GABA A receptor subunit α1, γ2, δ were observed by blocking or activating the function of the CREB. The quantification of the GABA A receptor subunit α1, γ2, δ and the CREB were determined by a qRT-PCR and a Western blot method. After the neurons were exposed to a low-Mg 2+ solution for 3 h, GABA A receptor mRNA expression markedly increased compared to the control, and then gradually decreased. In contrast, CREB mRNA levels exhibited a dramatic down-regulation 3 h after terminating low-Mg 2+ treatment, and then peaked at 9 h. Western blot analyses verified that staurosporine suppressed CREB phosphorylation (p-CREB). The mRNA expression of GABA A receptor subunit α1 increased only in the presence of staurosporine, whereas the expressions of subunits γ2 and δ significantly increased in the presence of either KG-501 or staurosporine. Furthermore, phorbol 12-myristate 13-acetate (PMA) decreased the expressions of GABA A subunits α1, γ2, and δ when administered alone. However, the administration of either KG-501 or staurosporine reversed the inhibitory effects of PMA. The PKC/CREB pathway may negatively regulate the expressions of GABA A receptor subunits α1, γ2, and δ in cultured hippocampal neurons in low Mg 2+ model of

Neuroprotection by Caffeine in Hyperoxia-Induced Neonatal Brain Injury

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Stefanie Endesfelder

2017-01-01

Full Text Available Sequelae of prematurity triggered by oxidative stress and free radical-mediated tissue damage have coined the term “oxygen radical disease of prematurity”. Caffeine, a potent free radical scavenger and adenosine receptor antagonist, reduces rates of brain damage in preterm infants. In the present study, we investigated the effects of caffeine on oxidative stress markers, anti-oxidative response, inflammation, redox-sensitive transcription factors, apoptosis, and extracellular matrix following the induction of hyperoxia in neonatal rats. The brain of a rat pups at postnatal Day 6 (P6 corresponds to that of a human fetal brain at 28–32 weeks gestation and the neonatal rat is an ideal model in which to investigate effects of oxidative stress and neuroprotection of caffeine on the developing brain. Six-day-old Wistar rats were pre-treated with caffeine and exposed to 80% oxygen for 24 and 48 h. Caffeine reduced oxidative stress marker (heme oxygenase-1, lipid peroxidation, hydrogen peroxide, and glutamate-cysteine ligase catalytic subunit (GCLC, promoted anti-oxidative response (superoxide dismutase, peroxiredoxin 1, and sulfiredoxin 1, down-regulated pro-inflammatory cytokines, modulated redox-sensitive transcription factor expression (Nrf2/Keap1, and NFκB, reduced pro-apoptotic effectors (poly (ADP-ribose polymerase-1 (PARP-1, apoptosis inducing factor (AIF, and caspase-3, and diminished extracellular matrix degeneration (matrix metalloproteinases (MMP 2, and inhibitor of metalloproteinase (TIMP 1/2. Our study affirms that caffeine is a pleiotropic neuroprotective drug in the developing brain due to its anti-oxidant, anti-inflammatory, and anti-apoptotic properties.

Altered organization of GABAA receptor mRNA expression in the depressed suicide brain

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Michael O Poulter

2010-03-01

Full Text Available Inter-relationships ordinarily exist between mRNA expression of GABA-A subunits in the frontopolar cortex (FPC of individuals that had died suddenly from causes other than suicide. However, these correlations were largely absent in persons that had died by suicide. In the present investigation, these findings were extended by examining GABA-A receptor expression patterns (of controls and depressed individuals that died by suicide in the orbital frontal cortex (OFC, hippocampus, amygdala. locus coeruleus (LC,and paraventricular nucleus (PVN, all of which have been implicated in either depression, anxiety or stress responsivity. Results Using QPCR analysis, we found that in controls the inter-relations between GABA-A subunits varied across brain regions, being high in the hippocampus and amygdala, intermediate in the LC, and low in the OFC and PVN. The GABA-A subunit inter-relations were markedly different in persons that died by suicide, being reduced in hippocampus and amygdala, stable in the LC, but more coordinated in the OFC and to some extent in the PVN. Conclusions It seems that altered brain region-specific inhibitory signaling, stemming from altered GABA-A subunit coordination, are associated with depression/suicide. Although, it is unknown whether GABA-A subunit re-organization was specifically tied to depression, suicide, or the accompanying distress, these data show that the co-ordinate expression of this transcriptome does vary depending on brain region and is plastic.

Mammalian mediator 19 mediates H1299 lung adenocarcinoma cell clone conformation, growth, and metastasis.

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Xu, Lu-Lu; Guo, Shu-Liang; Ma, Su-Ren; Luo, Yong-Ai

2012-01-01

Mammalian mediator (MED) is a multi-protein coactivator that has been identified by several research groups. The involvement of the MED complex subunit 19 (MED 19) in the metastasis of lung adenocarcinoma cell line (H1299), which expresses the MED 19 subunit, was here investigated. When MED 19 expression was decreased by RNA interference H1299 cells demonstrated reduced clone formation, arrest in the S phase of the cell cycle, and lowered metastatic capacity. Thus, MED 19 appears to play important roles in the biological behavior of non-small cell lung carcinoma cells. These findings may be important for the development of novel lung carcinoma treatments.

Disruption of Mediator rescues the stunted growth of a lignin-deficient Arabidopsis mutant.

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Bonawitz, Nicholas D; Kim, Jeong Im; Tobimatsu, Yuki; Ciesielski, Peter N; Anderson, Nickolas A; Ximenes, Eduardo; Maeda, Junko; Ralph, John; Donohoe, Bryon S; Ladisch, Michael; Chapple, Clint

2014-05-15

Lignin is a phenylpropanoid-derived heteropolymer important for the strength and rigidity of the plant secondary cell wall. Genetic disruption of lignin biosynthesis has been proposed as a means to improve forage and bioenergy crops, but frequently results in stunted growth and developmental abnormalities, the mechanisms of which are poorly understood. Here we show that the phenotype of a lignin-deficient Arabidopsis mutant is dependent on the transcriptional co-regulatory complex, Mediator. Disruption of the Mediator complex subunits MED5a (also known as REF4) and MED5b (also known as RFR1) rescues the stunted growth, lignin deficiency and widespread changes in gene expression seen in the phenylpropanoid pathway mutant ref8, without restoring the synthesis of guaiacyl and syringyl lignin subunits. Cell walls of rescued med5a/5b ref8 plants instead contain a novel lignin consisting almost exclusively of p-hydroxyphenyl lignin subunits, and moreover exhibit substantially facilitated polysaccharide saccharification. These results demonstrate that guaiacyl and syringyl lignin subunits are largely dispensable for normal growth and development, implicate Mediator in an active transcriptional process responsible for dwarfing and inhibition of lignin biosynthesis, and suggest that the transcription machinery and signalling pathways responding to cell wall defects may be important targets to include in efforts to reduce biomass recalcitrance.

Mediator subunit MED1 is a T3-dependent and T3-independent coactivator on the thyrotropin β gene promoter

Energy Technology Data Exchange (ETDEWEB)

Matsui, Keiji; Oda, Kasumi; Mizuta, Shumpei; Ishino, Ruri; Urahama, Norinaga; Hasegawa, Natsumi [Laboratory of Hematology, Division of Medical Biophysics, Kobe University Graduate School of Health Sciences, 7-10-2 Tomogaoka, Suma-ku, Kobe 654-0142 (Japan); Roeder, Robert G. [Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10065 (United States); Ito, Mitsuhiro, E-mail: itomi@med.kobe-u.ac.jp [Laboratory of Hematology, Division of Medical Biophysics, Kobe University Graduate School of Health Sciences, 7-10-2 Tomogaoka, Suma-ku, Kobe 654-0142 (Japan); Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10065 (United States); Department of Family and Community Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 654-0142 (Japan); Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 159-8555 (Japan)

2013-10-11

Highlights: •MED1 is a bona fide T3-dependent coactivator on TSHB promoter. •Mice with LxxLL-mutant MED1 have attenuated TSHβ mRNA and thyroid hormone levels. •MED1 activates TSHB promoter T3-dependently in cultured cells. •T3-dependent MED1 action is enhanced when SRC1/SRC2 or HDAC2 is downregulated. •MED1 is also a T3-independent GATA2/Pit1 coactivator on TSHB promoter. -- Abstract: The MED1 subunit of the Mediator transcriptional coregulator complex is a nuclear receptor-specific coactivator. A negative feedback mechanism of thyroid-stimulating hormone (TSH, or thyrotropin) expression in the thyrotroph in the presence of triiodothyronine (T3) is employed by liganded thyroid hormone receptor β (TRβ) on the TSHβ gene promoter, where conventional histone-modifying coactivators act as corepressors. We now provide evidence that MED1 is a ligand-dependent positive cofactor on this promoter. TSHβ gene transcription was attenuated in MED1 mutant mice in which the nuclear receptor-binding ability of MED1 was specifically disrupted. MED1 stimulated GATA2- and Pit1-mediated TSHβ gene promoter activity in a ligand-independent manner in cultured cells. MED1 also stimulated transcription from the TSHβ gene promoter in a T3-dependent manner. The transcription was further enhanced when the T3-dependent corepressors SRC1, SRC2, and HDAC2 were downregulated. Hence, MED1 is a T3-dependent and -independent coactivator on the TSHβ gene promoter.

Evidence that Mediator is essential for Pol II transcription, but is not a required component of the preinitiation complex in vivo.

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Petrenko, Natalia; Jin, Yi; Wong, Koon Ho; Struhl, Kevin

2017-07-12

The Mediator complex has been described as a general transcription factor, but it is unclear if it is essential for Pol II transcription and/or is a required component of the preinitiation complex (PIC) in vivo. Here, we show that depletion of individual subunits, even those essential for cell growth, causes a general but only modest decrease in transcription. In contrast, simultaneous depletion of all Mediator modules causes a drastic decrease in transcription. Depletion of head or middle subunits, but not tail subunits, causes a downstream shift in the Pol II occupancy profile, suggesting that Mediator at the core promoter inhibits promoter escape. Interestingly, a functional PIC and Pol II transcription can occur when Mediator is not detected at core promoters. These results provide strong evidence that Mediator is essential for Pol II transcription and stimulates PIC formation, but it is not a required component of the PIC in vivo.

Δ9-THC-caused synaptic and memory impairments are mediated through COX-2 signaling.

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Chen, Rongqing; Zhang, Jian; Fan, Ni; Teng, Zhao-Qian; Wu, Yan; Yang, Hongwei; Tang, Ya-Ping; Sun, Hao; Song, Yunping; Chen, Chu

2013-11-21

Marijuana has been used for thousands of years as a treatment for medical conditions. However, untoward side effects limit its medical value. Here, we show that synaptic and cognitive impairments following repeated exposure to Δ(9)-tetrahydrocannabinol (Δ(9)-THC) are associated with the induction of cyclooxygenase-2 (COX-2), an inducible enzyme that converts arachidonic acid to prostanoids in the brain. COX-2 induction by Δ(9)-THC is mediated via CB1 receptor-coupled G protein βγ subunits. Pharmacological or genetic inhibition of COX-2 blocks downregulation and internalization of glutamate receptor subunits and alterations of the dendritic spine density of hippocampal neurons induced by repeated Δ(9)-THC exposures. Ablation of COX-2 also eliminates Δ(9)-THC-impaired hippocampal long-term synaptic plasticity, working, and fear memories. Importantly, the beneficial effects of decreasing β-amyloid plaques and neurodegeneration by Δ(9)-THC in Alzheimer's disease animals are retained in the presence of COX-2 inhibition. These results suggest that the applicability of medical marijuana would be broadened by concurrent inhibition of COX-2. Copyright © 2013 Elsevier Inc. All rights reserved.

Alzheimer's Disease Brain-Derived Amyloid-{beta}-Mediated Inhibition of LTP In Vivo Is Prevented by Immunotargeting Cellular Prion Protein.

LENUS (Irish Health Repository)

Barry, Andrew E

2011-05-18

Synthetic amyloid-β protein (Aβ) oligomers bind with high affinity to cellular prion protein (PrP(C)), but the role of this interaction in mediating the disruption of synaptic plasticity by such soluble Aβ in vitro is controversial. Here we report that intracerebroventricular injection of Aβ-containing aqueous extracts of Alzheimer\\'s disease (AD) brain robustly inhibits long-term potentiation (LTP) without significantly affecting baseline excitatory synaptic transmission in the rat hippocampus in vivo. Moreover, the disruption of LTP was abrogated by immunodepletion of Aβ. Importantly, intracerebroventricular administration of antigen-binding antibody fragment D13, directed to a putative Aβ-binding site on PrP(C), prevented the inhibition of LTP by AD brain-derived Aβ. In contrast, R1, a Fab directed to the C terminus of PrP(C), a region not implicated in binding of Aβ, did not significantly affect the Aβ-mediated inhibition of LTP. These data support the pathophysiological significance of SDS-stable Aβ dimer and the role of PrP(C) in mediating synaptic plasticity disruption by soluble Aβ.

Evidence for a role of transporter-mediated currents in the depletion of brain serotonin induced by serotonin transporter substrates.

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Baumann, Michael H; Bulling, Simon; Benaderet, Tova S; Saha, Kusumika; Ayestas, Mario A; Partilla, John S; Ali, Syed F; Stockner, Thomas; Rothman, Richard B; Sandtner, Walter; Sitte, Harald H

2014-05-01

Serotonin (5-HT) transporter (SERT) substrates like fenfluramine and 3,4-methylenedioxymethamphetamine cause long-term depletion of brain 5-HT, while certain other substrates do not. The 5-HT deficits produced by SERT substrates are dependent upon transporter proteins, but the exact mechanisms responsible are unclear. Here, we compared the pharmacology of several SERT substrates: fenfluramine, d-fenfluramine, 1-(m-chlorophenyl)piperazine (mCPP) and 1-(m-trifluoromethylphenyl)piperainze (TFMPP), to establish relationships between acute drug mechanisms and the propensity for long-term 5-HT depletions. In vivo microdialysis was carried out in rat nucleus accumbens to examine acute 5-HT release and long-term depletion in the same subjects. In vitro assays were performed to measure efflux of [(3)H]5-HT in rat brain synaptosomes and transporter-mediated ionic currents in SERT-expressing Xenopus oocytes. When administered repeatedly to rats (6 mg/kg, i.p., four doses), all drugs produce large sustained elevations in extracellular 5-HT (>5-fold) with minimal effects on dopamine. Importantly, 2 weeks after dosing, only rats exposed to fenfluramine and d-fenfluramine display depletion of brain 5-HT. All test drugs evoke fluoxetine-sensitive efflux of [(3)H]5-HT from synaptosomes, but d-fenfluramine and its bioactive metabolite d-norfenfluramine induce significantly greater SERT-mediated currents than phenylpiperazines. Our data confirm that drug-induced 5-HT release probably does not mediate 5-HT depletion. However, the magnitude of transporter-mediated inward current may be a critical factor in the cascade of events leading to 5-HT deficits. This hypothesis warrants further study, especially given the growing popularity of designer drugs that target SERT.

Leptin and insulin engage specific PI3K subunits in hypothalamic SF1 neurons

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Jong-Woo Sohn

2016-08-01

Full Text Available Objective: The ventromedial hypothalamic nucleus (VMH regulates energy balance and glucose homeostasis. Leptin and insulin exert metabolic effects via their cognate receptors expressed by the steroidogenic factor 1 (SF1 neurons within the VMH. However, detailed cellular mechanisms involved in the regulation of these neurons by leptin and insulin remain to be identified. Methods: We utilized genetically-modified mouse models and performed patch-clamp electrophysiology experiments to resolve this issue. Results: We identified distinct populations of leptin-activated and leptin-inhibited SF1 neurons. In contrast, insulin uniformly inhibited SF1 neurons. Notably, we found that leptin-activated, leptin-inhibited, and insulin-inhibited SF1 neurons are distinct subpopulations within the VMH. Leptin depolarization of SF1 neuron also required the PI3K p110β catalytic subunit. This effect was mediated by the putative transient receptor potential C (TRPC channel. On the other hand, hyperpolarizing responses of SF1 neurons by leptin and insulin required either of the p110α or p110β catalytic subunits, and were mediated by the putative ATP-sensitive K+ (KATP channel. Conclusions: Our results demonstrate that specific PI3K catalytic subunits are responsible for the acute effects of leptin and insulin on VMH SF1 neurons, and provide insights into the cellular mechanisms of leptin and insulin action on VMH SF1 neurons that regulate energy balance and glucose homeostasis. Author Video: Author Video Watch what authors say about their articles Keywords: Cellular mechanism, Conditional knockout mouse, Patch clamp technique, Functional heterogeneity, Homeostasis

Mediator MED23 regulates basal transcription in vivo via an interaction with P-TEFb.

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Wang, Wei; Yao, Xiao; Huang, Yan; Hu, Xiangming; Liu, Runzhong; Hou, Dongming; Chen, Ruichuan; Wang, Gang

2013-01-01

The Mediator is a multi-subunit complex that transduces regulatory information from transcription regulators to the RNA polymerase II apparatus. Growing evidence suggests that Mediator plays roles in multiple stages of eukaryotic transcription, including elongation. However, the detailed mechanism by which Mediator regulates elongation remains elusive. In this study, we demonstrate that Mediator MED23 subunit controls a basal level of transcription by recruiting elongation factor P-TEFb, via an interaction with its CDK9 subunit. The mRNA level of Egr1, a MED23-controlled model gene, is reduced 4-5 fold in Med23 (-/-) ES cells under an unstimulated condition, but Med23-deficiency does not alter the occupancies of RNAP II, GTFs, Mediator complex, or activator ELK1 at the Egr1 promoter. Instead, Med23 depletion results in a significant decrease in P-TEFb and RNAP II (Ser2P) binding at the coding region, but no changes for several other elongation regulators, such as DSIF and NELF. ChIP-seq revealed that Med23-deficiency partially reduced the P-TEFb occupancy at a set of MED23-regulated gene promoters. Further, we demonstrate that MED23 interacts with CDK9 in vivo and in vitro. Collectively, these results provide the mechanistic insight into how Mediator promotes RNAP II into transcription elongation.

Reflections on: "A general role for adaptations in G-Proteins and the cyclic AMP system in mediating the chronic actions of morphine and cocaine on neuronal function".

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Nestler, Eric J

2016-08-15

In 1991 we demonstrated that chronic morphine exposure increased levels of adenylyl cyclase and protein kinase A (PKA) in several regions of the rat central nervous system as inferred from measures of enzyme activity in crude extracts (Terwilliger et al., 1991). These findings led us to hypothesize that a concerted upregulation of the cAMP pathway is a general mechanism of opiate tolerance and dependence. Moreover, in the same study we showed similar induction of adenylyl cyclase and PKA activity in nucleus accumbens (NAc) in response to chronic administration of cocaine, but not of several non-abused psychoactive drugs. Morphine and cocaine also induced equivalent changes in inhibitory G protein subunits in this brain region. We thus extended our hypothesis to suggest that, particularly within brain reward regions such as NAc, cAMP pathway upregulation represents a common mechanism of reward tolerance and dependence shared by several classes of drugs of abuse. Research since that time, by many laboratories, has provided substantial support for these hypotheses. Specifically, opiates in several CNS regions including NAc, and cocaine more selectively in NAc, induce expression of certain adenylyl cyclase isoforms and PKA subunits via the transcription factor, CREB, and these transcriptional adaptations serve a homeostatic function to oppose drug action. In certain brain regions, such as locus coeruleus, these adaptations mediate aspects of physical opiate dependence and withdrawal, whereas in NAc they mediate reward tolerance and dependence that drives increased drug self-administration. This work has had important implications for understanding the molecular basis of addiction. "A general role for adaptations in G-proteins and the cyclic AMP system in mediating the chronic actions of morphine and cocaine on neuronal function". Previous studies have shown that chronic morphine increases levels of the G-protein subunits Giα and Goα, adenylate cyclase, cyclic AMP

A functional portrait of Med7 and the mediator complex in Candida albicans.

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Tebbji, Faiza; Chen, Yaolin; Richard Albert, Julien; Gunsalus, Kearney T W; Kumamoto, Carol A; Nantel, André; Sellam, Adnane; Whiteway, Malcolm

2014-11-01

Mediator is a multi-subunit protein complex that regulates gene expression in eukaryotes by integrating physiological and developmental signals and transmitting them to the general RNA polymerase II machinery. We examined, in the fungal pathogen Candida albicans, a set of conditional alleles of genes encoding Mediator subunits of the head, middle, and tail modules that were found to be essential in the related ascomycete Saccharomyces cerevisiae. Intriguingly, while the Med4, 8, 10, 11, 14, 17, 21 and 22 subunits were essential in both fungi, the structurally highly conserved Med7 subunit was apparently non-essential in C. albicans. While loss of CaMed7 did not lead to loss of viability under normal growth conditions, it dramatically influenced the pathogen's ability to grow in different carbon sources, to form hyphae and biofilms, and to colonize the gastrointestinal tracts of mice. We used epitope tagging and location profiling of the Med7 subunit to examine the distribution of the DNA sites bound by Mediator during growth in either the yeast or the hyphal form, two distinct morphologies characterized by different transcription profiles. We observed a core set of 200 genes bound by Med7 under both conditions; this core set is expanded moderately during yeast growth, but is expanded considerably during hyphal growth, supporting the idea that Mediator binding correlates with changes in transcriptional activity and that this binding is condition specific. Med7 bound not only in the promoter regions of active genes but also within coding regions and at the 3' ends of genes. By combining genome-wide location profiling, expression analyses and phenotyping, we have identified different Med7p-influenced regulons including genes related to glycolysis and the Filamentous Growth Regulator family. In the absence of Med7, the ribosomal regulon is de-repressed, suggesting Med7 is involved in central aspects of growth control.

Divergence of RNA polymerase ? subunits in angiosperm plastid genomes is mediated by genomic rearrangement

OpenAIRE

Blazier, J. Chris; Ruhlman, Tracey A.; Weng, Mao-Lun; Rehman, Sumaiyah K.; Sabir, Jamal S. M.; Jansen, Robert K.

2016-01-01

Genes for the plastid-encoded RNA polymerase (PEP) persist in the plastid genomes of all photosynthetic angiosperms. However, three unrelated lineages (Annonaceae, Passifloraceae and Geraniaceae) have been identified with unusually divergent open reading frames (ORFs) in the conserved region of rpoA, the gene encoding the PEP ? subunit. We used sequence-based approaches to evaluate whether these genes retain function. Both gene sequences and complete plastid genome sequences were assembled an...

Permeability transition in human mitochondria persists in the absence of peripheral stalk subunits of ATP synthase.

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He, Jiuya; Carroll, Joe; Ding, Shujing; Fearnley, Ian M; Walker, John E

2017-08-22

The opening of a nonspecific channel, known as the permeability transition pore (PTP), in the inner membranes of mitochondria can be triggered by calcium ions, leading to swelling of the organelle, disruption of the inner membrane and ATP synthesis, and cell death. Pore opening can be inhibited by cyclosporin A mediated via cyclophilin D. It has been proposed that the pore is associated with the dimeric ATP synthase and the oligomycin sensitivity conferral protein (OSCP), a component of the enzyme's peripheral stalk, provides the site at which cyclophilin D interacts. Subunit b contributes a central α-helical structure to the peripheral stalk, extending from near the top of the enzyme's catalytic domain and crossing the membrane domain of the enzyme via two α-helices. We investigated the possible involvement of the subunit b and the OSCP in the PTP by generating clonal cells, HAP1-Δb and HAP1-ΔOSCP, lacking the membrane domain of subunit b or the OSCP, respectively, in which the corresponding genes, ATP5F1 and ATP5O , had been disrupted. Both cell lines preserve the characteristic properties of the PTP; therefore, the membrane domain of subunit b does not contribute to the PTP, and the OSCP does not provide the site of interaction with cyclophilin D. The membrane subunits ATP6, ATP8, and subunit c have been eliminated previously from possible participation in the PTP; thus, the only subunits of ATP synthase that could participate in pore formation are e, f, g, diabetes-associated protein in insulin-sensitive tissues (DAPIT), and the 6.8-kDa proteolipid.

Molecular cloning of the human casein kinase II α subunit

International Nuclear Information System (INIS)

Meisner, H.; Heller-Harrison, R.; Buxton, J.; Czech, M.P.

1989-01-01

A human cDNA encoding the α subunit of casein kinase II and a partial cDNA encoding the rat homologue were isolated by using a Drosophila casein kinase II cDNA probe. The 2.2-kb human cDNA contains a 1.2-kb open reading frame, 150 nucleotides of 5' leader, and 850 nucleotides of 3' noncoding region. Except for the first 7 deduced amino acids that are missing in the rat cDNA, the 328 amino acids beginning with the amino terminus are identical between human and rat. The Drosophila enzyme sequence is 90% identical with the human casein kinase II sequence, and there is only a single amino acid difference between the published partial bovine sequence and the human sequence. In addition, the C-terminus of the human cDNA has an extra 53 amino acids not present in Drosophila. Northern analysis of rat and human RNA showed predominant bands of 5.5, 3.1, and 1.8 kb. In rat tissues, brain and spleen had the highest levels of casein kinase II α subunit specific RNA, while skeletal muscle showed the lowest. Southern analysis of human cultured cell and tissue genomic DNA using the full-length cDNA probe revealed two bands with restriction enzymes that have no recognition sites within the cDNA and three to six bands with enzymes having single internal sites. These results are consistent with the possibility that two genes encode the α subunits

Brain acetylcholinesterase activity controls systemic cytokine levels through the cholinergic anti-inflammatory pathway

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Pavlov, Valentin A.; Parrish, William R.; Rosas-Ballina, Mauricio; Ochani, Mahendar; Puerta, Margot; Ochani, Kanta; Chavan, Sangeeta; Al-Abed, Yousef; Tracey, Kevin J.

2015-01-01

The excessive release of cytokines by the immune system contributes importantly to the pathogenesis of inflammatory diseases. Recent advances in understanding the biology of cytokine toxicity led to the discovery of the “cholinergic anti-inflammatory pathway,” defined as neural signals transmitted via the vagus nerve that inhibit cytokine release through a mechanism that requires the alpha7 subunit-containing nicotinic acetylcholine receptor (α7nAChR). Vagus nerve regulation of peripheral functions is controlled by brain nuclei and neural networks, but despite considerable importance, little is known about the molecular basis for central regulation of the vagus nerve-based cholinergic anti-inflammatory pathway. Here we report that brain acetylcholinesterase activity controls systemic and organ specific TNF production during endotoxemia. Peripheral administration of the acetylcholinesterase inhibitor galantamine significantly reduced serum TNF levels through vagus nerve signaling, and protected against lethality during murine endotoxemia. Administration of a centrally-acting muscarinic receptor antagonist abolished the suppression of TNF by galantamine, indicating that suppressing acetylcholinesterase activity, coupled with central muscarinic receptors, controls peripheral cytokine responses. Administration of galantamine to α7nAChR knockout mice failed to suppress TNF levels, indicating that the α7nAChR-mediated cholinergic anti-inflammatory pathway is required for the anti-inflammatory effect of galantamine. These findings show that inhibition of brain acetylcholinesterase suppresses systemic inflammation through a central muscarinic receptor-mediated and vagal- and α7nAChR-dependent mechanism. Our data also indicate that a clinically used centrally-acting acetylcholinesterase inhibitor can be utilized to suppress abnormal inflammation to therapeutic advantage. PMID:18639629

The brain cytoplasmic RNA BC1 regulates dopamine D2 receptor-mediated transmission in the striatum.

Science.gov (United States)

Centonze, Diego; Rossi, Silvia; Napoli, Ilaria; Mercaldo, Valentina; Lacoux, Caroline; Ferrari, Francesca; Ciotti, Maria Teresa; De Chiara, Valentina; Prosperetti, Chiara; Maccarrone, Mauro; Fezza, Filomena; Calabresi, Paolo; Bernardi, Giorgio; Bagni, Claudia

2007-08-15

Dopamine D(2) receptor (D(2)DR)-mediated transmission in the striatum is remarkably flexible, and changes in its efficacy have been heavily implicated in a variety of physiological and pathological conditions. Although receptor-associated proteins are clearly involved in specific forms of synaptic plasticity, the molecular mechanisms regulating the sensitivity of D(2) receptors in this brain area are essentially obscure. We have studied the physiological responses of the D(2)DR stimulations in mice lacking the brain cytoplasmic RNA BC1, a small noncoding dendritically localized RNA that is supposed to play a role in mRNA translation. We show that the efficiency of D(2)-mediated transmission regulating striatal GABA synapses is under the control of BC1 RNA, through a negative influence on D(2) receptor protein level affecting the functional pool of receptors. Ablation of the BC1 gene did not result in widespread dysregulation of synaptic transmission, because the sensitivity of cannabinoid CB(1) receptors was intact in the striatum of BC1 knock-out (KO) mice despite D(2) and CB(1) receptors mediated similar electrophysiological actions. Interestingly, the fragile X mental retardation protein FMRP, one of the multiple BC1 partners, is not involved in the BC1 effects on the D(2)-mediated transmission. Because D(2)DR mRNA is apparently equally translated in the BC1-KO and wild-type mice, whereas the protein level is higher in BC1-KO mice, we suggest that BC1 RNA controls D(2)DR indirectly, probably regulating translation of molecules involved in D(2)DR turnover and/or stability.

Functional characterization of the mammalian iAAA protease subunit, YME1L

OpenAIRE

Majczak, Joanna

2008-01-01

The iAAA protease is an ATP-dependent proteolytic complex in the mitochondrial inner membrane and belongs to the highly conserved family of AAA proteins. In the yeast Saccharomyces cerevisiae, the iAAA protease is a homo-oligomeric complex composed of Yme1p subunits which are active in the intermembrane space and mediate protein quality control. Yeast cells lacking Yme1p are characterized by pleiotropic phenotypes including a respiratory deficiency at elevated temperature and an aberrant mito...

Mice Lacking the Alpha9 Subunit of the Nicotinic Acetylcholine Receptor Exhibit Deficits in Frequency Difference Limens and Sound Localization

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Amanda Clause

2017-06-01

Full Text Available Sound processing in the cochlea is modulated by cholinergic efferent axons arising from medial olivocochlear neurons in the brainstem. These axons contact outer hair cells in the mature cochlea and inner hair cells during development and activate nicotinic acetylcholine receptors composed of α9 and α10 subunits. The α9 subunit is necessary for mediating the effects of acetylcholine on hair cells as genetic deletion of the α9 subunit results in functional cholinergic de-efferentation of the cochlea. Cholinergic modulation of spontaneous cochlear activity before hearing onset is important for the maturation of central auditory circuits. In α9KO mice, the developmental refinement of inhibitory afferents to the lateral superior olive is disturbed, resulting in decreased tonotopic organization of this sound localization nucleus. In this study, we used behavioral tests to investigate whether the circuit anomalies in α9KO mice correlate with sound localization or sound frequency processing. Using a conditioned lick suppression task to measure sound localization, we found that three out of four α9KO mice showed impaired minimum audible angles. Using a prepulse inhibition of the acoustic startle response paradigm, we found that the ability of α9KO mice to detect sound frequency changes was impaired, whereas their ability to detect sound intensity changes was not. These results demonstrate that cholinergic, nicotinic α9 subunit mediated transmission in the developing cochlear plays an important role in the maturation of hearing.

β2-Adrenergic Receptor-Mediated HIF-1α Upregulation Mediates Blood Brain Barrier Damage in Acute Cerebral Ischemia

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Yanyun Sun

2017-08-01

Full Text Available Disruption of the blood brain barrier (BBB within the thrombolytic time window is an antecedent event to intracerebral hemorrhage in ischemic stroke. Our recent studies showed that 2-h cerebral ischemia induced BBB damage in non-infarcted area and secreted matrix metalloproteinase-2 (MMP-2 accounted for this disruption. However, the factors that affect MMP-2 secretion and regulate BBB damage remains unknown. Since hypoxia-inducible factor-1 alpha (HIF-1α was discovered as a mater regulator in hypoxia, we sought to investigate the roles of HIF-1α in BBB damage as well as the factors regulating HIF-1α expression in the ischemic brain. in vivo rat middle cerebral artery occlusion (MCAO and in vitro oxygen glucose deprivation (OGD models were used to mimic ischemia. Pretreatment with HIF-1α inhibitor YC-1 significantly inhibited 2-h MCAO-induced BBB damage, which was accompanied by suppressed occludin degradation and vascular endothelial growth factor (VEGF mRNA upregulation. Interestingly, β2-adrenergic receptor (β2-AR antagonist ICI 118551 attenuated ischemia-induced BBB damage by regulating HIF-1α expression. Double immunostaining showed that HIF-1α was upregulated in ischemic neurons but not in astrocytes andendothelial cells. Of note, HIF-1α inhibition with inhibitor YC-1 or siRNA significantly prevented OGD-induced VEGF upregulation as well as the secretion of VEGF and MMP-2 in neurons. More importantly, blocking β2-AR with ICI 118551 suppressedHIF-1α upregulation in ischemic neurons and attenuated occludin degradation induced by the conditioned media of OGD-treatedneurons. Taken together, blockade of β2-AR-mediated HIF-1α upregulation mediates BBB damage during acute cerebral ischemia. These findings provide new mechanistic understanding of early BBB damage in ischemic stroke and may help reduce thrombolysis-related hemorrhagic complications.

Cyclic AMP regulation of the human glycoprotein hormone α-subunit gene is mediated by an 18-base-pair element

International Nuclear Information System (INIS)

Silver, B.J.; Bokar, J.A.; Virgin, J.B.; Vallen, E.A.; Milsted, A.; Nilson, J.H.

1987-01-01

cAMP regulates transcription of the gene encoding the α-subunit of human chorionic gonadotropin (hCG) in the choriocarcinoma cells (BeWo). To define the sequences required for regulation by cAMP, the authors inserted fragments from the 5' flanking region of the α-subunit gene into a test vector containing the simian virus 40 early promoter (devoid of its enhancer) linked to the bacterial chloramphenicol acetyltransferase (CAT) gene. Results from transient expression assays in BeWo cells indicated that a 1500-base-pair (bp) fragment conferred cAMP responsiveness on the CAT gene regardless of position or orientation of the insert relative to the viral promoter. A subfragment extending from position -169 to position -100 had the same effect on cAMP-induced expression. Furthermore, the entire stimulatory effect could be achieved with an 18-bp synthetic oligodeoxynucleotide corresponding to a direct repeat between position -146 and -111. In the absence of cAMP, the α-subunit 5' flanking sequence also enhanced transcription from the simian virus 40 early promoter. They localized this enhancer activity to the same -169/-100 fragment containing the cAMP response element. The 18-bp element alone, however, had no effect on basal expression. Thus, this short DNA sequence serves as a cAMP response element and also functions independently of other promoter-regulatory elements located in the 5' flanking sequence of the α-subunit gene

Alternative Splicing of AMPA subunits in Prefrontal Cortical Fields of Cynomolgus Monkeys following Chronic Ethanol Self-Administration

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Glen eAcosta

2012-01-01

Full Text Available Functional impairment of the orbital and medial prefrontal cortex underlies deficits in executive control that characterize addictive disorders, including alcohol addiction. Previous studies indicate that alcohol alters glutamate neurotransmission and one substrate of these effects may be through the reconfiguration of the subunits constituting ionotropic glutamate receptor (iGluR complexes. Glutamatergic transmission is integral to cortico-cortical and cortico-subcortical communication and alcohol-induced changes in the abundance of the receptor subunits and/or their splice variants may result in critical functional impairments of prefrontal cortex in alcohol dependence. To this end, the effects of chronic ethanol self-administration on glutamate receptor ionotropic AMPA (GRIA subunit variant and kainate (GRIK subunit mRNA expression were studied in the orbitofrontal cortex (OFC, dorsolateral prefrontal cortex (DLPFC and anterior cingulate cortex (ACC of male cynomolgus monkeys. In DLPFC, total AMPA splice variant expression and total kainate receptor subunit expression were significantly decreased in alcohol drinking monkeys. Expression levels of GRIA3 flip and flop and GRIA4 flop mRNAs in this region were positively correlated with daily ethanol intake and blood ethanol concentrations averaged over the six months prior to necropsy. In OFC, AMPA subunit splice variant expression was reduced in the alcohol treated group. GRIA2 flop mRNA levels in this region were positively correlated with daily ethanol intake and blood ethanol concentrations averaged over the six months prior to necropsy. Results from these studies provide further evidence of transcriptional regulation of iGluR subunits in the primate brain following chronic alcohol self-administration. Additional studies examining the cellular localization of such effects in the framework of primate prefrontal cortical circuitry are warranted.

The MDT-15 subunit of mediator interacts with dietary restriction to modulate longevity and fluoranthene toxicity in Caenorhabditis elegans.

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Jennifer Schleit

Full Text Available Dietary restriction (DR, the limitation of calorie intake while maintaining proper nutrition, has been found to extend life span and delay the onset of age-associated disease in a wide range of species. Previous studies have suggested that DR can reduce the lethality of environmental toxins. To further examine the role of DR in toxin response, we measured life spans of the nematode Caenorhabditis elegans treated with the mutagenic polyaromatic hydrocarbon, fluoranthene (FLA. FLA is a direct byproduct of combustion, and is one of U.S. Environmental Protection Agency's sixteen priority environmental toxins. Treatment with 5 µg/ml FLA shortened the life spans of ad libitum fed nematodes, and DR resulted in increased sensitivity to FLA. To determine the role of detoxifying enzymes in the toxicity of FLA, we tested nematodes with mutations in the gene encoding the MDT-15 subunit of mediator, a transcriptional coactivator that regulates genes involved in fatty acid metabolism and detoxification. Mutation of mdt-15 increased the life span of FLA treated animals compared to wild-type animals with no difference observed between DR and ad libitum fed mdt-15 animals. We also examined mutants with altered insulin-IGF-1-like signaling (IIS, which is known to modulate life span and stress resistance in C. elegans independently of DR. Mutation of the genes coding for the insulin-like receptor DAF-2 or the FOXO-family transcription factor DAF16 did not alter the animals' susceptibility to FLA compared to wild type. Taken together, our results suggest that certain compounds have increased toxicity when combined with a DR regimen through increased metabolic activation. This increased metabolic activation appears to be mediated through the MDT-15 transcription factor and is independent of the IIS pathway.

Structures of transcription pre-initiation complex with TFIIH and Mediator.

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Schilbach, S; Hantsche, M; Tegunov, D; Dienemann, C; Wigge, C; Urlaub, H; Cramer, P

2017-11-09

For the initiation of transcription, RNA polymerase II (Pol II) assembles with general transcription factors on promoter DNA to form the pre-initiation complex (PIC). Here we report cryo-electron microscopy structures of the Saccharomyces cerevisiae PIC and PIC-core Mediator complex at nominal resolutions of 4.7 Å and 5.8 Å, respectively. The structures reveal transcription factor IIH (TFIIH), and suggest how the core and kinase TFIIH modules function in the opening of promoter DNA and the phosphorylation of Pol II, respectively. The TFIIH core subunit Ssl2 (a homologue of human XPB) is positioned on downstream DNA by the 'E-bridge' helix in TFIIE, consistent with TFIIE-stimulated DNA opening. The TFIIH kinase module subunit Tfb3 (MAT1 in human) anchors the kinase Kin28 (CDK7), which is mobile in the PIC but preferentially located between the Mediator hook and shoulder in the PIC-core Mediator complex. Open spaces between the Mediator head and middle modules may allow access of the kinase to its substrate, the C-terminal domain of Pol II.

Role for the MED21-MED7 Hinge in Assembly of the Mediator-RNA Polymerase II Holoenzyme*

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Sato, Shigeo; Tomomori-Sato, Chieri; Tsai, Kuang-Lei; Yu, Xiaodi; Sardiu, Mihaela; Saraf, Anita; Washburn, Michael P.; Florens, Laurence; Asturias, Francisco J.; Conaway, Ronald C.

2016-01-01

Mediator plays an integral role in activation of RNA polymerase II (Pol II) transcription. A key step in activation is binding of Mediator to Pol II to form the Mediator-Pol II holoenzyme. Here, we exploit a combination of biochemistry and macromolecular EM to investigate holoenzyme assembly. We identify a subset of human Mediator head module subunits that bind Pol II independent of other subunits and thus probably contribute to a major Pol II binding site. In addition, we show that binding of human Mediator to Pol II depends on the integrity of a conserved “hinge” in the middle module MED21-MED7 heterodimer. Point mutations in the hinge region leave core Mediator intact but lead to increased disorder of the middle module and markedly reduced affinity for Pol II. These findings highlight the importance of Mediator conformation for holoenzyme assembly. PMID:27821593

The regulatory beta-subunit of protein kinase CK2 regulates cell-cycle progression at the onset of mitosis

DEFF Research Database (Denmark)

Yde, C W; Olsen, B B; Meek, D

2008-01-01

25 dual-specificity phosphatase family members. In somatic cells, Wee1 is downregulated by phosphorylation and ubiquitin-mediated degradation to ensure rapid activation of CDK1 at the beginning of M phase. Here, we show that downregulation of the regulatory beta-subunit of protein kinase CK2 by RNA...

Anti-amyloid-β-mediated positron emission tomography imaging in Alzheimer's disease mouse brains.

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Daniel McLean

Full Text Available Antibody-mediated imaging of amyloid β (Aβ in Alzheimer's disease (AD offers a promising strategy to detect and monitor specific Aβ species, such as oligomers, that have important pathological and therapeutic relevance. The major current limitation of antibodies as a diagnostic and imaging device is poor blood-brain-barrier permeability. A classical anti-Aβ antibody, 6E10, is modified with 10 kDa polyethylene glycol (PEG and a positron emitting isotope, Copper-64 (t(½ = 12.7 h, and intravenously delivered to the TgCRND8 mouse model of Alzheimer's disease. Modification of 6E10 with PEG (6E10-PEG increases accumulation of 6E10 in brain tissue in both TgCRND8 and wild type control animals. 6E10-PEG differentiates TgCRND8 animals from wild type controls using positron emission tomography (PET and provides a framework for using antibodies to detect pathology using non-invasive medical imaging techniques.

Identification of a GTP-binding protein α subunit that lacks an apparent ADP-ribosylation site for pertussis toxin

International Nuclear Information System (INIS)

Fong, H.K.W.; Yoshimoto, K.K.; Eversole-Cire, P.; Simon, M.I.

1988-01-01

Recent molecular cloning of cDNA for the α subunit of bovine transducin (a guanine nucleotide-binding regulatory protein, or G protein) has revealed the presence of two retinal-specific transducins, called T/sub r/ and T/sub c/, which are expressed in rod or cone photoreceptor cells. In a further study of G-protein diversity and signal transduction in the retina, the authors have identified a G-protein α subunit, which they refer to as G/sub z/α, by isolating a human retinal cDNA clone that cross-hybridizes at reduced stringency with bovine T/sub r/ α-subunit cDNA. The deduced amino acid sequence of G/sub z/α is 41-67% identical with those of other known G-protein α subunits. However, the 355-residue G/sub z/α lacks a consensus site for ADP-ribosylation by pertussis toxin, and its amino acid sequence varies within a number of regions that are strongly conserved among all of the other G-protein α subunits. They suggest that G/sub z/α, which appears to be highly expressed in neural tissues, represents a member of a subfamily of G proteins that mediate signal transduction in pertussis toxin-insensitive systems

Ethanol activation of protein kinase A regulates GABA-A receptor subunit expression in the cerebral cortex and contributes to ethanol-induced hypnosis

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Sandeep eKumar

2012-04-01

Full Text Available Protein kinases are implicated in neuronal cell functions such as modulation of ion channel function, trafficking and synaptic excitability. Both protein kinase C (PKC and A (PKA are involved in regulation of γ-aminobutyric acid type A (GABA-A receptors through phosphorylation. However, the role of PKA in regulating GABA-A receptors following acute ethanol exposure is not known. The present study investigated the role of PKA in ethanol effects on GABA-A receptor α1 subunit expression in the P2 synaptosomal fraction of the rat cerebral cortex. Additionally, GABA-related behaviors were also examined. Rats were administered ethanol (2.0 – 3.5 g/kg or saline and PKC, PKA and GABA-A receptor α1 subunit levels were measured by Western blot analysis. Ethanol (3.5 g/kg transiently increased GABA-A receptor α1 subunit expression and PKA RIIβ subunit expression at similar time points whereas PKA RIIα was increased at later time points. In contrast, PKC isoform expression remained unchanged. Notably, the moderate ethanol dose (2.0g/kg had no effect on GABA-A α1 subunit levels although PKA RIIα and RIIβ were increased at 10 and 60 minutes, when PKC isozymes are also known to be elevated. To determine if PKA activation was responsible for the ethanol-induced elevation of GABA-A α1 subunits, the PKA antagonist H89 was administered to rats prior to ethanol exposure. H89 administration prevented ethanol-induced increases in GABA-A receptor α1 subunit expression. Moreover, increasing PKA activity intracerebroventricularly with Sp-cAMP prior to a hypnotic dose of ethanol increased ethanol-induced loss of righting reflex duration. This effect appears to be mediated in part by GABA-A receptors as increasing PKA activity also increased the duration of muscimol-induced loss of righting reflex. Overall these data suggest that PKA mediates ethanol-induced GABA-A receptor expression and contributes to ethanol behavioral effects involving GABA-A receptors.

Vagally mediated effects of brain stem dopamine on gastric tone and phasic contractions of the rat.

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Anselmi, L; Toti, L; Bove, C; Travagli, R A

2017-11-01

Dopamine (DA)-containing fibers and neurons are embedded within the brain stem dorsal vagal complex (DVC); we have shown previously that DA modulates the membrane properties of neurons of the dorsal motor nucleus of the vagus (DMV) via DA1 and DA2 receptors. The vagally dependent modulation of gastric tone and phasic contractions, i.e., motility, by DA, however, has not been characterized. With the use of microinjections of DA in the DVC while recording gastric tone and motility, the aims of the present study were 1 ) assess the gastric effects of brain stem DA application, 2 ) identify the DA receptor subtype, and, 3 ) identify the postganglionic pathway(s) activated. Dopamine microinjection in the DVC decreased gastric tone and motility in both corpus and antrum in 29 of 34 rats, and the effects were abolished by ipsilateral vagotomy and fourth ventricular treatment with the selective DA2 receptor antagonist L741,626 but not by application of the selective DA1 receptor antagonist SCH 23390. Systemic administration of the cholinergic antagonist atropine attenuated the inhibition of corpus and antrum tone in response to DA microinjection in the DVC. Conversely, systemic administration of the nitric oxide synthase inhibitor nitro-l-arginine methyl ester did not alter the DA-induced decrease in gastric tone and motility. Our data provide evidence of a dopaminergic modulation of a brain stem vagal neurocircuit that controls gastric tone and motility. NEW & NOTEWORTHY Dopamine administration in the brain stem decreases gastric tone and phasic contractions. The gastric effects of dopamine are mediated via dopamine 2 receptors on neurons of the dorsal motor nucleus of the vagus. The inhibitory effects of dopamine are mediated via inhibition of the postganglionic cholinergic pathway. Copyright © 2017 the American Physiological Society.

[Paradoxical brain embolism mediated through a pulmonary arteriovenous malformation with hereditary hemorrhagic telangiectasia in a Japanese patient].

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Takeda, June; Todo, Kenichi; Yamamoto, Shiro; Yamagami, Hiroshi; Kawamoto, Michi; Kohara, Nobuo

2012-01-01

We report a case of paradoxical brain embolism mediated through a pulmonary arteriovenous malformation (PAVM) with hereditary hemorrhagic telangiectasia (HHT). A 25-year-old right handed man was admitted to our hospital after sudden headache and visual field abnormality. In neurologic examinations, he had left superior-quadrantanopsia. Laboratory findings showed iron deficiency anemia. Diffusion weighted images disclosed a high-signal-intensity area in the right occipito-temporal lobe, and intraarterial digital subtraction cerebral angiography revealed occlusion of the right posterior cerebral artery. Transesophageal echocardiography revealed continuous right-to-left shunt. We confirmed his history of spontaneous recurrent epistaxis and the first-degree relatives with epistaxis or PAVM. A contrast enhanced CT scan of the chest revealed a PAVM. The diagnosis of paradoxical brain embolism mediated through the PAVM with HHT was, thus, established. The PAVM was occluded by using embolization coils successfully. In Asian countries, the prevalence of PAVM with HHT is thought to be lower than in European countries. We should carefully take medical and family histories, especially epistaxis, in a young stroke patient.

Antioxidant mediated response of Scoparia dulcis in noise-induced redox imbalance and immunohistochemical changes in rat brain.

Science.gov (United States)

Wankhar, Wankupar; Srinivasan, Sakthivel; Rajan, Ravindran; Sheeladevi, Rathinasamy

2017-01-19

Noise has been regarded as an environmental/occupational stressor that causes damages to both auditory and non-auditory organs. Prolonged exposure to these mediators of stress has often resulted in detrimental effect, where oxidative/nitrosative stress plays a major role. Hence, it would be appropriate to examine the possible role of free radicals in brain discrete regions and the "antioxidants" mediated response of S. dulcis. Animals were subjected to noise stress for 15 days (100 dB/4 hours/day) and estimation of endogenous free radical and antioxidant activity were carried out on brain discrete regions (the cerebral cortex, cerebellum, brainstem, striatum, hippocampus and hypothalamus). The result showed that exposure to noise could alleviate endogenous free radical generation and altered antioxidant status in brain discrete regions when compared to that of the control groups. This alleviated free radical generation (H 2 O 2 and NO) is well supported by an upregulated protein expression on immunohistochemistry of both iNOS and nNOS in the cerebral cortex on exposure to noise stress. These findings suggest that increased free radical generation and altered anti-oxidative status can cause redox imbalance in the brain discrete regions. However, free radical scavenging activity of the plant was evident as the noise exposed group treated with S. dulcis[200 mg/(kg·b·w)] displayed a therapeutic effect by decreasing the free radical level and regulate the anti-oxidative status to that of control animals. Hence, it can be concluded that the efficacy of S. dulcis could be attributed to its free radical scavenging activity and anti-oxidative property.

Changes in Glutamate/NMDA Receptor Subunit 1 Expression in Rat Brain after Acute and Subacute Exposure to Methamphetamine

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Walailuk Kerdsan

2009-01-01

Full Text Available Methamphetamine (METH is a psychostimulant drug of abuse that produces long-term behavioral changes including behavioral sensitization, tolerance, and dependence. METH has been reported to induce neurotoxic effects in several areas of the brain via the dopaminergic system. Changes of dopamine function can induce malfunction of the glutamatergic system. Therefore, the aim of the present study was to examine the effects of METH administration on the expression of glutamate N-methyl-D-aspartate receptor subunit 1 (NMDAR1 in frontal cortex, striatum, and hippocampal formation after acute and subacute exposure to METH by western blotting. Male Sprague-Dawley rats were injected intraperitoneally with a single dose of 8 mg/kg METH, 4 mg/kg/day METH for 14 days and saline in acute, subacute, and control groups, respectively. A significant increase in NMDAR1 immunoreactive protein was found in frontal cortex in the subacute group (P=.036 but not in the acute group (P=.580. Moreover, a significant increase in NMDAR1 was also observed in striatum in both acute (P=.025 and subacute groups (P=.023. However, no significant differences in NMDAR1 in hippocampal formation were observed in either acute or subacute group. The results suggest that an upregulation of NMDA receptor expression may be a consequence of glutamatergic dysfunction induced by METH.

Alternate day fasting impacts the brain insulin-signaling pathway of young adult male C57BL/6 mice.

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Lu, Jianghua; E, Lezi; Wang, Wenfang; Frontera, Jennifer; Zhu, Hao; Wang, Wen-Tung; Lee, Phil; Choi, In Young; Brooks, William M; Burns, Jeffrey M; Aires, Daniel; Swerdlow, Russell H

2011-04-01

Dietary restriction (DR) has recognized health benefits that may extend to brain. We examined how DR affects bioenergetics-relevant enzymes and signaling pathways in the brains of C57BL/6 mice. Five-month-old male mice were placed in ad libitum or one of two repeated fasting and refeeding (RFR) groups, an alternate day (intermittent fed; IF) or alternate day plus antioxidants (blueberry, pomegranate, and green tea extracts) (IF + AO) fed group. During the 24-h fast blood glucose levels initially fell but stabilized within 6 h of starting the fast, thus avoiding frank hypoglycemia. DR in general appeared to enhance insulin sensitivity. After six weeks brain AKT and glycogen synthase kinase 3 beta phosphorylation were lower in the RFR mice, suggesting RFR reduced brain insulin-signaling pathway activity. Pathways that mediate mitochondrial biogenesis were not activated; AMP kinase phosphorylation, silent information regulator 2 phosphorylation, peroxisomal proliferator-activated receptor-gamma coactivator 1 alpha levels, and cytochrome oxidase subunit 4 levels did not change. ATP levels also did not decline, which suggests the RFR protocols did not directly impact brain bioenergetics. Antioxidant supplementation did not affect the brain parameters we evaluated. Our data indicate in young adult male C57BL/6 mice, RFR primarily affects brain energy metabolism by reducing brain insulin signaling, which potentially results indirectly as a consequence of reduced peripheral insulin production. © 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.

LRP1 in brain vascular smooth muscle cells mediates local clearance of Alzheimer's amyloid-β.

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Kanekiyo, Takahisa; Liu, Chia-Chen; Shinohara, Mitsuru; Li, Jie; Bu, Guojun

2012-11-14

Impaired clearance of amyloid-β (Aβ) is a major pathogenic event for Alzheimer's disease (AD). Aβ depositions in brain parenchyma as senile plaques and along cerebrovasculature as cerebral amyloid angiopathy (CAA) are hallmarks of AD. A major pathway that mediates brain Aβ clearance is the cerebrovascular system where Aβ is eliminated through the blood-brain barrier (BBB) and/or degraded by cerebrovascular cells along the interstitial fluid drainage pathway. An Aβ clearance receptor, the low-density lipoprotein receptor-related protein 1 (LRP1), is abundantly expressed in cerebrovasculature, in particular in vascular smooth muscle cells. Previous studies have indicated a role of LRP1 in endothelial cells in transcytosing Aβ out of the brain across the BBB; however, whether this represents a significant pathway for brain Aβ clearance remains controversial. Here, we demonstrate that Aβ can be cleared locally in the cerebrovasculature by an LRP1-dependent endocytic pathway in smooth muscle cells. The uptake and degradation of both endogenous and exogenous Aβ were significantly reduced in LRP1-suppressed human brain vascular smooth muscle cells. Conditional deletion of Lrp1 in vascular smooth muscle cell in amyloid model APP/PS1 mice accelerated brain Aβ accumulation and exacerbated Aβ deposition as amyloid plaques and CAA without affecting Aβ production. Our results demonstrate that LRP1 is a major Aβ clearance receptor in cerebral vascular smooth muscle cell and a disturbance of this pathway contributes to Aβ accumulation. These studies establish critical functions of the cerebrovasculature system in Aβ metabolism and identify a new pathway involved in the pathogenesis of both AD and CAA.

Serotonin: A mediator of the gut-brain axis in multiple sclerosis.

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Malinova, Tsveta S; Dijkstra, Christine D; de Vries, Helga E

2017-11-01

The significance of the gut microbiome for the pathogenesis of multiple sclerosis (MS) has been established, although the underlying signaling mechanisms of this interaction have not been sufficiently explored. We address this point and use serotonin (5-hydroxytryptamine (5-HT))-a microbial-modulated neurotransmitter (NT) as a showcase to demonstrate that NTs regulated by the gut microbiome are potent candidates for mediators of the gut-brain axis in demyelinating disorders. Methods, Results, and Conclusion: Our comprehensive overview of literature provides evidence that 5-HT levels in the gut are controlled by the microbiome, both via secretion and through regulation of metabolites. In addition, we demonstrate that the gut microbiome can influence the formation of the serotonergic system (SS) in the brain. We also show that SS alterations have been related to MS directly-altered expression of 5-HT transporters in central nervous system (CNS) and indirectly-beneficial effects of 5-HT modulating drugs on the course of the disease and higher prevalence of depression in patients with MS. Finally, we discuss briefly the role of other microbiome-modulated NTs such as γ-aminobutyric acid and dopamine in MS to highlight a new direction for future research aiming to relate microbiome-regulated NTs to demyelinating disorders.

Crystal structure and pharmacological characterization of a novel N-methyl-D-aspartate (NMDA) receptor antagonist at the GluN1 glycine binding site

DEFF Research Database (Denmark)

Kvist, Trine; Steffensen, Thomas Bielefeldt; Greenwood, Jeremy R

2013-01-01

NMDA receptors are ligand-gated ion channels that mediate excitatory neurotransmission in the brain. They are tetrameric complexes composed of glycine-binding GluN1 and GluN3 subunits together with glutamate-binding GluN2 subunits. Subunit-selective antagonists that discriminate between the glyci...... screening. Furthermore, the structure reveals that the imino acetamido group of TK40 acts as an α-amino acid bioisostere, which could be of importance in bioisosteric replacement strategies for future ligand design....

Flexible Connectors between Capsomer Subunits that Regulate Capsid Assembly.

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Hasek, Mary L; Maurer, Joshua B; Hendrix, Roger W; Duda, Robert L

2017-08-04

Viruses build icosahedral capsids of specific size and shape by regulating the spatial arrangement of the hexameric and pentameric protein capsomers in the growing shell during assembly. In the T=7 capsids of Escherichia coli bacteriophage HK97 and other phages, 60 capsomers are hexons, while the rest are pentons that are correctly positioned during assembly. Assembly of the HK97 capsid to the correct size and shape has been shown to depend on specific ionic contacts between capsomers. We now describe additional ionic interactions within capsomers that also regulate assembly. Each is between the long hairpin, the "E-loop," that extends from one subunit to the adjacent subunit within the same capsomer. Glutamate E153 on the E-loop and arginine R210 on the adjacent subunit's backbone alpha-helix form salt bridges in hexamers and pentamers. Mutations that disrupt these salt bridges were lethal for virus production, because the mutant proteins assembled into tubes or sheets instead of capsids. X-ray structures show that the E153-R210 links are flexible and maintained during maturation despite radical changes in capsomer shape. The E153-R210 links appear to form early in assembly to enable capsomers to make programmed changes in their shape during assembly. The links also prevent flattening of capsomers and premature maturation. Mutant phenotypes and modeling support an assembly model in which flexible E153-R210 links mediate capsomer shape changes that control where pentons are placed to create normal-sized capsids. The E-loop may be conserved in other systems in order to play similar roles in regulating assembly. Copyright © 2017 Elsevier Ltd. All rights reserved.

Synaptic Changes in AMPA Receptor Subunit Expression in Cortical Parvalbumin Interneurons in the Stargazer Model of Absence Epilepsy

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Nadia K. Adotevi

2017-12-01

Full Text Available Feedforward inhibition is essential to prevent run away excitation within the brain. Recent evidence suggests that a loss of feed-forward inhibition in the corticothalamocortical circuitry may underlie some absence seizures. However, it is unclear if this aberration is specifically linked to loss of synaptic excitation onto local fast-spiking parvalbumin-containing (PV+ inhibitory interneurons, which are responsible for mediating feedforward inhibition within cortical networks. We recently reported a global tissue loss of AMPA receptors (AMPARs, and a specific mistrafficking of these AMPARs in PV+ interneurons in the stargazer somatosensory cortex. The current study was aimed at investigating if cellular changes in AMPAR expression were translated into deficits in receptors at specific synapses in the feedforward inhibitory microcircuit. Using western blot immunolabeling on biochemically isolated synaptic fractions, we demonstrate a loss of AMPAR GluA1–4 subunits in the somatosensory cortex of stargazers compared to non-epileptic control mice. Furthermore, using double post-embedding immunogold-cytochemistry, we show a loss of GluA1–4-AMPARs at excitatory synapses onto cortical PV+ interneurons. Altogether, these data indicate a loss of synaptic AMPAR-mediated excitation of cortical PV+ inhibitory neurons. As the cortex is considered the site of initiation of spike wave discharges (SWDs within the corticothalamocortical circuitry, loss of AMPARs at cortical PV+ interneurons likely impairs feed-forward inhibitory output, and contributes to the generation of SWDs and absence seizures in stargazers.

Genome-wide occupancy profile of mediator and the Srb8-11 module reveals interactions with coding regions

DEFF Research Database (Denmark)

Zhu, Xuefeng; Wirén, Marianna; Sinha, Indranil

2006-01-01

Mediator exists in a free form containing the Med12, Med13, CDK8, and CycC subunits (the Srb8-11 module) and a smaller form, which lacks these four subunits and associates with RNA polymerase II (Pol II), forming a holoenzyme. We use chromatin immunoprecipitation (ChIP) and DNA microarrays...... to investigate genome-wide localization of Mediator and the Srb8-11 module in fission yeast. Mediator and the Srb8-11 module display similar binding patterns, and interactions with promoters and upstream activating sequences correlate with increased transcription activity. Unexpectedly, Mediator also interacts...... with the downstream coding region of many genes. These interactions display a negative bias for positions closer to the 5' ends of open reading frames (ORFs) and appear functionally important, because downregulation of transcription in a temperature-sensitive med17 mutant strain correlates with increased Mediator...

Role of regulatory subunits and protein kinase inhibitor (PKI) in determining nuclear localization and activity of the catalytic subunit of protein kinase A.

Science.gov (United States)

Wiley, J C; Wailes, L A; Idzerda, R L; McKnight, G S

1999-03-05

Regulation of protein kinase A by subcellular localization may be critical to target catalytic subunits to specific substrates. We employed epitope-tagged catalytic subunit to correlate subcellular localization and gene-inducing activity in the presence of regulatory subunit or protein kinase inhibitor (PKI). Transiently expressed catalytic subunit distributed throughout the cell and induced gene expression. Co-expression of regulatory subunit or PKI blocked gene induction and prevented nuclear accumulation. A mutant PKI lacking the nuclear export signal blocked gene induction but not nuclear accumulation, demonstrating that nuclear export is not essential to inhibit gene induction. When the catalytic subunit was targeted to the nucleus with a nuclear localization signal, it was not sequestered in the cytoplasm by regulatory subunit, although its activity was completely inhibited. PKI redistributed the nuclear catalytic subunit to the cytoplasm and blocked gene induction, demonstrating that the nuclear export signal of PKI can override a strong nuclear localization signal. With increasing PKI, the export process appeared to saturate, resulting in the return of catalytic subunit to the nucleus. These results demonstrate that both the regulatory subunit and PKI are able to completely inhibit the gene-inducing activity of the catalytic subunit even when the catalytic subunit is forced to concentrate in the nuclear compartment.

Progesterone mediates brain functional connectivity changes during the menstrual cycle - A pilot resting state MRI study

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Katrin eArelin

2015-02-01

Full Text Available The growing interest in intrinsic brain organization has sparked various innovative approaches to generating comprehensive connectivity-based maps of the human brain. Prior reports point to a sexual dimorphism of the structural and functional human connectome. However, it is uncertain whether subtle changes in sex hormones, as occur during the monthly menstrual cycle, substantially impact the functional architecture of the female brain. Here, we performed eigenvector centrality (EC mapping in 32 longitudinal resting state fMRI scans of a single healthy subject without oral contraceptive use, across four menstrual cycles, and assessed estrogen and progesterone levels. To investigate associations between cycle-dependent hormones and brain connectivity, we performed correlation analyses between the EC maps and the respective hormone levels. On the whole brain level, we found a significant positive correlation between progesterone and EC in the bilateral DLPFC and bilateral sensorimotor cortex. In a secondary region-of-interest analysis, we detected a progesterone-modulated increase in functional connectivity of both bilateral DLPFC and bilateral sensorimotor cortex with the hippocampus. Our results suggest that the menstrual cycle substantially impacts intrinsic functional connectivity, particularly in brain areas associated with contextual memory-regulation, such as the hippocampus. These findings are the first to link the subtle hormonal fluctuations that occur during the menstrual cycle, to significant changes in regional functional connectivity in the hippocampus in a longitudinal design, given the limitation of data acquisition in a single subject. Our study demonstrates the feasibility of such a longitudinal rs-fMRI design and illustrates a means of creating a personalized map of the human brain by integrating potential mediators of brain states, such as menstrual cycle phase.

The δ subunit of RNA polymerase guides promoter selectivity and virulence in Staphylococcus aureus.

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Weiss, Andy; Ibarra, J Antonio; Paoletti, Jessica; Carroll, Ronan K; Shaw, Lindsey N

2014-04-01

In Gram-positive bacteria, and particularly the Firmicutes, the DNA-dependent RNA polymerase (RNAP) complex contains an additional subunit, termed the δ factor, or RpoE. This enigmatic protein has been studied for more than 30 years for various organisms, but its function is still not well understood. In this study, we investigated its role in the major human pathogen Staphylococcus aureus. We showed conservation of important structural regions of RpoE in S. aureus and other species and demonstrated binding to core RNAP that is mediated by the β and/or β' subunits. To identify the impact of the δ subunit on transcription, we performed transcriptome sequencing (RNA-seq) analysis and observed 191 differentially expressed genes in the rpoE mutant. Ontological analysis revealed, quite strikingly, that many of the downregulated genes were known virulence factors, while several mobile genetic elements (SaPI5 and prophage SA3usa) were strongly upregulated. Phenotypically, the rpoE mutant had decreased accumulation and/or activity of a number of key virulence factors, including alpha toxin, secreted proteases, and Panton-Valentine leukocidin (PVL). We further observed significantly decreased survival of the mutant in whole human blood, increased phagocytosis by human leukocytes, and impaired virulence in a murine model of infection. Collectively, our results demonstrate that the δ subunit of RNAP is a critical component of the S. aureus transcription machinery and plays an important role during infection.

Acetylcholine Receptor: Complex of Homologous Subunits

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Raftery, Michael A.; Hunkapiller, Michael W.; Strader, Catherine D.; Hood, Leroy E.

1980-06-01

The acetylcholine receptor from the electric ray Torpedo californica is composed of five subunits; two are identical and the other three are structurally related to them. Microsequence analysis of the four polypeptides demonstrates amino acid homology among the subunits. Further sequence analysis of both membrane-bound and Triton-solubilized, chromatographically purified receptor gave the stoichiometry of the four subunits (40,000:50,000:60,000:65,000 daltons) as 2:1:1:1, indicating that this protein is a pentameric complex with a molecular weight of 255,000 daltons. Genealogical analysis suggests that divergence from a common ancestral gene occurred early in the evolution of the receptor. This shared ancestry argues that each of the four subunits plays a functional role in the receptor's physiological action.

The structure of the TFIIH p34 subunit reveals a von Willebrand factor A like fold.

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Dominik R Schmitt

Full Text Available RNA polymerase II dependent transcription and nucleotide excision repair are mediated by a multifaceted interplay of subunits within the general transcription factor II H (TFIIH. A better understanding of the molecular structure of TFIIH is the key to unravel the mechanism of action of this versatile protein complex within these vital cellular processes. The importance of this complex becomes further evident in the context of severe diseases like xeroderma pigmentosum, Cockayne's syndrome and trichothiodystrophy, that arise from single point mutations in TFIIH subunits. Here we describe the structure of the p34 subunit of the TFIIH complex from the eukaryotic thermophilic fungus Chaetomium thermophilum. The structure revealed that p34 contains a von Willebrand Factor A (vWA like domain, a fold which is generally known to be involved in protein-protein interactions. Within TFIIH p34 strongly interacts with p44, a positive regulator of the helicase XPD. Putative protein-protein interfaces are analyzed and possible binding sites for the p34-p44 interaction suggested.

Ghrelin upregulates the phosphorylation of the GluN2B subunit of the NMDA receptor by activating GHSR1a and Fyn in the rat hippocampus.

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Berrout, Liza; Isokawa, Masako

2018-01-01

Ghrelin and its receptor GHSR1a have been shown to exert numerous physiological functions in the brain, in addition to the well-established orexigenic role in the hypothalamus. Earlier work indicated that ghrelin stimulated the phosphorylation of the GluN1 subunit of the NMDA receptor (NMDAR) and enhanced synaptic transmission in the hippocampus. In the present study, we report that the exogenous application of ghrelin increased GluN2B phosphorylation. This increase was independent of GluN2B subunit activity or NMDAR channel activity. However, it depended on the activation of GHSR1a and Fyn as it was blocked by D-Lys3-GHRP-6 and PP2, respectively. Inhibitors for G-protein-regulated second messengers, such as Rp-cAMP, H89, TBB, ryanodine, and thapsigargin, unexpectedly enhanced GluN2B phosphorylation, suggesting that cAMP, PKA, casein kinase II, and cytosolic calcium signaling may oppose to the effect of ghrelin on the phosphorylation of GluN2B. Our findings suggest that 1) GluN2B is likely a molecular target of ghrelin and GHSR1a-driven signaling cascades, and 2) the ghrelin-mediated phosphorylation of GluN2B depends on Fyn activation under complex negative regulation by other second messengers. Copyright © 2017 Elsevier B.V. All rights reserved.

Ferroxidase-Mediated Iron Oxide Biomineralization

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Zeth, Kornelius; Hoiczyk, Egbert; Okuda, Mitsuhiro

2016-01-01

Iron oxide biomineralization occurs in all living organisms and typically involves protein compartments ranging from 5 to 100nm in size. The smallest iron-oxo particles are formed inside dodecameric Dps protein cages, while the structurally related ferritin compartments consist of twice as many......, translocation, oxidation, nucleation, and storage, that are mediated by ferroxidase centers. Thus, compartmentalized iron oxide biomineralization yields uniform nanoparticles strictly determined by the sizes of the compartments, allowing customization for highly diverse nanotechnological applications....... identical protein subunits. The largest known compartments are encapsulins, icosahedra made of up to 180 protein subunits that harbor additional ferritin-like proteins in their interior. The formation of iron-oxo particles in all these compartments requires a series of steps including recruitment of iron...

Distribution of the branched-chain α-ketoacid dehydrogenase complex E1α subunit and glutamate dehydrogenase in the human brain and their role in neuro-metabolism.

Science.gov (United States)

Hull, Jonathon; Usmari Moraes, Marcela; Brookes, Emma; Love, Seth; Conway, Myra E

2018-01-01

Glutamate is the major excitatory neurotransmitter of the central nervous system, with the branched-chain amino acids (BCAAs) acting as key nitrogen donors for de novo glutamate synthesis. Despite the importance of these major metabolites, their metabolic pathway in the human brain is still not well characterised. The metabolic pathways that influence the metabolism of BCAAs have been well characterised in rat models. However, the expression of key proteins such as the branched-chain α-ketoacid dehydrogenase (BCKD) complex and glutamate dehydrogenase isozymes (GDH) in the human brain is still not well characterised. We have used specific antibodies to these proteins to analyse their distribution within the human brain and report, for the first time, that the E1α subunit of the BCKD is located in both neurons and vascular endothelial cells. We also demonstrate that GDH is localised to astrocytes, although vascular immunolabelling does occur. The labelling of GDH was most intense in astrocytes adjacent to the hippocampus, in keeping with glutamatergic neurotransmission in this region. GDH was also present in astrocyte processes abutting vascular endothelial cells. Previously, we demonstrated that the branched-chain aminotransferase (hBCAT) proteins were most abundant in vascular cells (hBCATm) and neurons (hBCATc). Present findings are further evidence that BCAAs are metabolised within both the vasculature and neurons in the human brain. We suggest that GDH, hBCAT and the BCKD proteins operate in conjunction with astrocytic glutamate transporters and glutamine synthetase to regulate the availability of glutamate. This has important implications given that the dysregulation of glutamate metabolism, leading to glutamate excitotoxicity, is an important contributor to the pathogenesis of several neurodegenerative conditions such as Alzheimer's disease. Crown Copyright © 2017. Published by Elsevier Ltd. All rights reserved.

Absorptive-mediated endocytosis of cationized albumin and a beta-endorphin-cationized albumin chimeric peptide by isolated brain capillaries. Model system of blood-brain barrier transport

International Nuclear Information System (INIS)

Kumagai, A.K.; Eisenberg, J.B.; Pardridge, W.M.

1987-01-01

Cationized albumin (pI greater than 8), unlike native albumin (pI approximately 4), enters cerebrospinal fluid (CSF) rapidly from blood. This suggests that a specific uptake mechanism for cationized albumin may exist at the brain capillary wall, i.e. the blood-brain barrier. Isolated bovine brain capillaries rapidly bound cationized [ 3 H]albumin and approximately 70% of the bound radioactivity was resistant to mild acid wash, which is assumed to represent internalized peptide. Binding was saturable and a Scatchard plot gave a maximal binding capacity (Ro) = 5.5 +/- 0.7 micrograms/mgp (79 +/- 10 pmol/mgp), and a half-saturation constant (KD) = 55 +/- 8 micrograms/ml (0.8 +/- 0.1 microM). The binding of cationized [ 3 H]albumin (pI = 8.5-9) was inhibited by protamine, protamine sulfate, and polylysine (molecular weight = 70,000) with a Ki of approximately 3 micrograms/ml for all three proteins. The use of cationized albumin in directed delivery of peptides through the blood-brain barrier was examined by coupling [ 3 H]beta-endorphin to unlabeled cationized albumin (pI = 8.5-9) using the bifunctional reagent, N-succinimidyl 3-(2-pyridyldithio)proprionate. The [ 3 H]beta-endorphin-cationized albumin chimeric peptide was rapidly bound and endocytosed by isolated bovine brain capillaries, and this was inhibited by unlabeled cationized albumin but not by unconjugated beta-endorphin or native bovine albumin. Cationized albumin provides a new tool for studying absorptive-mediated endocytosis at the brain capillary and may also provide a vehicle for directed drug delivery through the blood-brain barrier

Absorptive-mediated endocytosis of cationized albumin and a beta-endorphin-cationized albumin chimeric peptide by isolated brain capillaries. Model system of blood-brain barrier transport

Energy Technology Data Exchange (ETDEWEB)

Kumagai, A.K.; Eisenberg, J.B.; Pardridge, W.M.

1987-11-05

Cationized albumin (pI greater than 8), unlike native albumin (pI approximately 4), enters cerebrospinal fluid (CSF) rapidly from blood. This suggests that a specific uptake mechanism for cationized albumin may exist at the brain capillary wall, i.e. the blood-brain barrier. Isolated bovine brain capillaries rapidly bound cationized (/sup 3/H)albumin and approximately 70% of the bound radioactivity was resistant to mild acid wash, which is assumed to represent internalized peptide. Binding was saturable and a Scatchard plot gave a maximal binding capacity (Ro) = 5.5 +/- 0.7 micrograms/mgp (79 +/- 10 pmol/mgp), and a half-saturation constant (KD) = 55 +/- 8 micrograms/ml (0.8 +/- 0.1 microM). The binding of cationized (/sup 3/H)albumin (pI = 8.5-9) was inhibited by protamine, protamine sulfate, and polylysine (molecular weight = 70,000) with a Ki of approximately 3 micrograms/ml for all three proteins. The use of cationized albumin in directed delivery of peptides through the blood-brain barrier was examined by coupling (/sup 3/H)beta-endorphin to unlabeled cationized albumin (pI = 8.5-9) using the bifunctional reagent, N-succinimidyl 3-(2-pyridyldithio)proprionate. The (/sup 3/H)beta-endorphin-cationized albumin chimeric peptide was rapidly bound and endocytosed by isolated bovine brain capillaries, and this was inhibited by unlabeled cationized albumin but not by unconjugated beta-endorphin or native bovine albumin. Cationized albumin provides a new tool for studying absorptive-mediated endocytosis at the brain capillary and may also provide a vehicle for directed drug delivery through the blood-brain barrier.

Developmental aspects of the rat brain insulin receptor: loss of sialic acid and fluctuation in number characterize fetal development

International Nuclear Information System (INIS)

Brennan, W.A. Jr.

1988-01-01

In this study, I have investigated the structure of the rat brain insulin receptor during fetal development. There is a progressive decrease in the apparent molecular size of the brain alpha-subunit during development: 130K on day 16 of gestation, 126K at birth, and 120K in the adult. Glycosylation was investigated as a possible reason for the observed differences in the alpha-subunit molecular size. The results show that the developmental decrease in the brain alpha-subunit apparent molecular size is due to a parallel decrease in sialic acid content. This was further confirmed by measuring the retention of autophosphorylated insulin receptors on wheat germ agglutinin (WGA)-Sepharose. An inverse correlation between developmental age and retention of 32 P-labeled insulin receptors on the lectin column was observed. Insulin binding increases 6-fold between 16 and 20 days of gestation [61 +/- 25 (+/- SE) fmol/mg protein and 364 +/- 42 fmol/mg, respectively]. Thereafter, binding in brain membranes decreases to 150 +/- 20 fmol/mg by 2 days after birth, then reaches the adult level of 63 +/- 15 fmol/mg. In addition, the degree of insulin-stimulated autophosphorylation closely parallels the developmental changes in insulin binding. Between 16 and 20 days of fetal life, insulin-stimulated phosphorylation of the beta-subunit increases 6-fold. Thereafter, the extent of phosphorylation decreases rapidly, reaching adult values identical with those in 16-day-old fetal brain. These results suggest that the embryonic brain possesses competent insulin receptors whose expression changes markedly during fetal development. This information should be important in defining the role of insulin in the developing nervous system

Regulation of metabolism by the Mediator complex.

Science.gov (United States)

Youn, Dou Yeon; Xiaoli, Alus M; Pessin, Jeffrey E; Yang, Fajun

2016-01-01

The Mediator complex was originally discovered in yeast, but it is conserved in all eukaryotes. Its best-known function is to regulate RNA polymerase II-dependent gene transcription. Although the mechanisms by which the Mediator complex regulates transcription are often complicated by the context-dependent regulation, this transcription cofactor complex plays a pivotal role in numerous biological pathways. Biochemical, molecular, and physiological studies using cancer cell lines or model organisms have established the current paradigm of the Mediator functions. However, the physiological roles of the mammalian Mediator complex remain poorly defined, but have attracted a great interest in recent years. In this short review, we will summarize some of the reported functions of selective Mediator subunits in the regulation of metabolism. These intriguing findings suggest that the Mediator complex may be an important player in nutrient sensing and energy balance in mammals.

Human placental Na+, K+-ATPase α subunit: cDNA cloning, tissue expression, DNA polymorphism, and chromosomal localization

International Nuclear Information System (INIS)

Chehab, F.F.; Kan, Y.W.; Law, M.L.; Hartz, J.; Kao, F.T.; Blostein, R.

1987-01-01

A 2.2-kilobase clone comprising a major portion of the coding sequence of the Na + , K + -ATPase α subunit was cloned from human placenta and its sequence was identical to that encoding the α subunit of human kidney and HeLa cells. Transfer blot analysis of the mRNA products of the Na + , K + -ATPase gene from various human tissues and cell lines revealed only one band (≅ 4.7 kilobases) under low and high stringency washing conditions. The levels of expression in the tissues were intestine > placenta > liver > pancreas, and in the cell lines the levels were human erythroleukemia > butyrate-induced colon > colon > brain > HeLa cells. mRNA was undetectable in reticulocytes, consistent with the authors failure to detect positive clones in a size-selected ( > 2 kilobases) λgt11 reticulocyte cDNA library. DNA analysis revealed by a polymorphic EcoRI band and chromosome localization by flow sorting and in situ hybridization showed that the α subunit is on the short is on the short arm (band p11-p13) of chromosome 1

Mechanistic Exploration of Cancer Stem Cell Marker Voltage-Dependent Calcium Channel α2δ1 Subunit-mediated Chemotherapy Resistance in Small-Cell Lung Cancer.

Science.gov (United States)

Yu, Jiangyong; Wang, Shuhang; Zhao, Wei; Duan, Jianchun; Wang, Zhijie; Chen, Hanxiao; Tian, Yanhua; Wang, Di; Zhao, Jun; An, Tongtong; Bai, Hua; Wu, Meina; Wang, Jie

2018-05-01

Purpose: Chemoresistance in small-cell lung cancer (SCLC) is reportedly attributed to the existence of resistant cancer stem cells (CSC). Studies involving CSC-specific markers and related mechanisms in SCLC remain limited. This study explored the role of the voltage-dependent calcium channel α2δ1 subunit as a CSC marker in chemoresistance of SCLC, and explored the potential mechanisms of α2δ1-mediated chemoresistance and strategies of overcoming the resistance. Experimental Design: α2δ1-positive cells were identified and isolated from SCLC cell lines and patient-derived xenograft (PDX) models, and CSC-like properties were subsequently verified. Transcriptome sequencing and Western blotting were carried out to identify pathways involved in α2δ1-mediated chemoresistance in SCLC. In addition, possible interventions to overcome α2δ1-mediated chemoresistance were examined. Results: Different proportions of α2δ1 + cells were identified in SCLC cell lines and PDX models. α2δ1 + cells exhibited CSC-like properties (self-renewal, tumorigenic, differentiation potential, and high expression of genes related to CSCs and drug resistance). Chemotherapy induced the enrichment of α2δ1 + cells instead of CD133 + cells in PDXs, and an increased proportion of α2δ1 + cells corresponded to increased chemoresistance. Activation and overexpression of ERK in the α2δ1-positive H1048 cell line was identified at the protein level. mAb 1B50-1 was observed to improve the efficacy of chemotherapy and delay relapse as maintenance therapy in PDX models. Conclusions: SCLC cells expressing α2δ1 demonstrated CSC-like properties, and may contribute to chemoresistance. ERK may play a key role in α2δ1-mediated chemoresistance. mAb 1B50-1 may serve as a potential anti-SCLC drug. Clin Cancer Res; 24(9); 2148-58. ©2018 AACR . ©2018 American Association for Cancer Research.

Subunit stoichiometry of the chloroplast photosystem I complex

International Nuclear Information System (INIS)

Bruce, B.D.; Malkin, R.

1988-01-01

A native photosystem I (PS I) complex and a PS I core complex depleted of antenna subunits has been isolated from the uniformly 14 C-labeled aquatic higher plant, Lemna. These complexes have been analyzed for their subunit stoichiometry by quantitative sodium dodecyl sulfate-polyacrylamide gel electrophoresis methods. The results for both preparations indicate that one copy of each high molecular mass subunit is present per PS I complex and that a single copy of most low molecular mass subunits is also present. These results suggest that iron-sulfur center X, an early PS I electron acceptor proposed to bind to the high molecular mass subunits, contains a single [4Fe-4S] cluster which is bound to a dimeric structure of high molecular mass subunits, each providing 2 cysteine residues to coordinate this cluster

miR-339-5p inhibits alcohol-induced brain inflammation through regulating NF-κB pathway

International Nuclear Information System (INIS)

Zhang, Yu; Wei, Guangkuan; Di, Zhiyong; Zhao, Qingjie

2014-01-01

Graphical abstract: - Highlights: • Alcohol upregulates miR-339-5p expression. • miR-339-5p inhibits the NF-kB pathway. • miR-339-5p interacts with and blocks activity of IKK-beat and IKK-epsilon. • miR-339-5p modulates IL-1β, IL-6 and TNF-α. - Abstract: Alcohol-induced neuroinflammation is mediated by the innate immunesystem. Pro-inflammatory responses to alcohol are modulated by miRNAs. The miRNA miR-339-5p has previously been found to be upregulated in alcohol-induced neuroinflammation. However, little has been elucidated on the regulatory functions of this miRNA in alcohol-induced neuroinflammation. We investigated the function of miR-339-5p in alcohol exposed brain tissue and isolated microglial cells using ex vivo and in vitro techniques. Our results show that alcohol induces transcription of miR 339-5p, IL-6, IL-1β and TNF-α in mouse brain tissue and isolated microglial cells by activating NF-κB. Alcohol activation of NF-κB allows for nuclear translocation of the NF-κB subunit p65 and expression of pro-inflammatory mediators. miR-339-5p inhibited expression of these pro-inflammatory factors through the NF-κB pathway by abolishing IKK-β and IKK-ε activity

miR-339-5p inhibits alcohol-induced brain inflammation through regulating NF-κB pathway

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Zhang, Yu [Department of Neurology, The First Affiliated School of Harbin Medical University, Harbin 150001 (China); Wei, Guangkuan [Department of Neurology, Heilongjiang Provincial Hospital, Harbin 150036 (China); Di, Zhiyong [Department of Laboratory, Heilongjiang Provincial Hospital, Harbin 150036 (China); Zhao, Qingjie, E-mail: zhaoqingjie2013@163.com [Department of Neurology, The First Affiliated School of Harbin Medical University, Harbin 150001 (China)

2014-09-26

Graphical abstract: - Highlights: • Alcohol upregulates miR-339-5p expression. • miR-339-5p inhibits the NF-kB pathway. • miR-339-5p interacts with and blocks activity of IKK-beat and IKK-epsilon. • miR-339-5p modulates IL-1β, IL-6 and TNF-α. - Abstract: Alcohol-induced neuroinflammation is mediated by the innate immunesystem. Pro-inflammatory responses to alcohol are modulated by miRNAs. The miRNA miR-339-5p has previously been found to be upregulated in alcohol-induced neuroinflammation. However, little has been elucidated on the regulatory functions of this miRNA in alcohol-induced neuroinflammation. We investigated the function of miR-339-5p in alcohol exposed brain tissue and isolated microglial cells using ex vivo and in vitro techniques. Our results show that alcohol induces transcription of miR 339-5p, IL-6, IL-1β and TNF-α in mouse brain tissue and isolated microglial cells by activating NF-κB. Alcohol activation of NF-κB allows for nuclear translocation of the NF-κB subunit p65 and expression of pro-inflammatory mediators. miR-339-5p inhibited expression of these pro-inflammatory factors through the NF-κB pathway by abolishing IKK-β and IKK-ε activity.

SAHA (Vorinostat Corrects Inhibitory Synaptic Deficits Caused by Missense Epilepsy Mutations to the GABAA Receptor γ2 Subunit

Directory of Open Access Journals (Sweden)

Nela Durisic

2018-03-01

Full Text Available The GABAA receptor (GABAAR α1 subunit A295D epilepsy mutation reduces the surface expression of α1A295Dβ2γ2 GABAARs via ER-associated protein degradation. Suberanilohydroxamic acid (SAHA, also known as Vorinostat was recently shown to correct the misfolding of α1A295D subunits and thereby enhance the functional surface expression of α1A295Dβ2γ2 GABAARs. Here we investigated whether SAHA can also restore the surface expression of γ2 GABAAR subunits that incorporate epilepsy mutations (N40S, R43Q, P44S, R138G known to reduce surface expression via ER-associated protein degradation. As a control, we also investigated the γ2K289M epilepsy mutation that impairs gating without reducing surface expression. Effects of mutations were evaluated on inhibitory postsynaptic currents (IPSCs mediated by the major synaptic α1β2γ2 GABAAR isoform. Recordings were performed in neuron-HEK293 cell artificial synapses to minimise contamination by GABAARs of undefined subunit composition. Transfection with α1β2γ2N40S, α1β2γ2R43Q, α1β2γ2P44S and α1β2γ2R138G subunits produced IPSCs with decay times slower than those of unmutated α1β2γ2 GABAARs due to the low expression of mutant γ2 subunits and the correspondingly high expression of slow-decaying α1β2 GABAARs. SAHA pre-treatment significantly accelerated the decay time constants of IPSCs consistent with the upregulation of mutant γ2 subunit expression. This increase in surface expression was confirmed by immunohistochemistry. SAHA had no effect on either the IPSC kinetics or surface expression levels of α1β2γ2K289M GABAARs, confirming its specificity for ER-retained mutant γ2 subunits. We also found that α1β2γ2K289M GABAARs and SAHA-treated α1β2γ2R43Q, α1β2γ2P44S and α1β2γ2R138G GABAARs all mediated IPSCs that decayed at significantly faster rates than wild type receptors as temperature was increased from 22 to 40°C. This may help explain why these mutations cause febrile

Aggravation of brain infarction through an increase in acrolein production and a decrease in glutathione with aging.

Science.gov (United States)

Uemura, Takeshi; Watanabe, Kenta; Ishibashi, Misaki; Saiki, Ryotaro; Kuni, Kyoshiro; Nishimura, Kazuhiro; Toida, Toshihiko; Kashiwagi, Keiko; Igarashi, Kazuei

2016-04-29

We previously reported that tissue damage during brain infarction was mainly caused by inactivation of proteins by acrolein. This time, it was tested why brain infarction increases in parallel with aging. A mouse model of photochemically induced thrombosis (PIT) was studied using 2, 6, and 12 month-old female C57BL/6 mice. The size of brain infarction in the mouse PIT model increased with aging. The volume of brain infarction in 12 month-old mice was approximately 2-fold larger than that in 2 month-old mice. The larger brain infarction in 12 month-old mice was due to an increase in acrolein based on an increase in the activity of spermine oxidase, together with a decrease in glutathione (GSH), a major acrolein-detoxifying compound in cells, based on the decrease in one of the subunits of glutathione biosynthesizing enzymes, γ-glutamylcysteine ligase modifier subunit, with aging. The results indicate that aggravation of brain infarction with aging was mainly due to the increase in acrolein production and the decrease in GSH in brain. Copyright © 2016 Elsevier Inc. All rights reserved.

Pharmacological and biochemical properties of the benzodiazepine-GABA receptor in codfish brain in comparison with mammalian brain

International Nuclear Information System (INIS)

Deng, L.

1989-01-01

The GABA receptor of codfish brain is encoded by an ancestral gene of the mammalian GABA receptor based on phylogenetic studies. The mammalian GABA receptor consists of at least two subunits (β and α) which could be photoaffinity labeled by the GABA agonist [ 3 H]muscimol (57 kDa) and the benzodiazepine (BZ) agonist [ 3 H]flunitrazepam (52 kDa), respectively. In contrast, electrophoresis of codfish GABA receptor photoaffinity labeled by the same ligands showed a single radioactive peak on sodium dodecyl surface polyarcylamide gel, giving rise to a relative molecular weight of 56-57 kDa equivalent to the β subunit of 57 kDa in mammals. The homogeneity of purified receptor using benzodiazepine (Ro 7-1986/1) affinity chromatography was further verified by two-dimensional gel electrophoresis based on isoelectric point and molecular weight, in addition to a single band on a silver stained gel and specific activity. The receptor density and affinity constant for [ 3 H]muscimol and [ 3 H]flunitrazepam are comparable to those in bovine, rate, and human brain

Role of adenosine 5'-monophosphate-activated protein kinase subunits in skeletal muscle mammalian target of rapamycin signaling

DEFF Research Database (Denmark)

Deshmukh, Atul S.; Treebak, Jonas Thue; Long, Yun Chau

2008-01-01

AMP-activated protein kinase (AMPK) is an important energy-sensing protein in skeletal muscle. Mammalian target of rapamycin (mTOR) mediates translation initiation and protein synthesis through ribosomal S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). AMPK...... activation reduces muscle protein synthesis by down-regulating mTOR signaling, whereas insulin mediates mTOR signaling via Akt activation. We hypothesized that AMPK-mediated inhibitory effects on mTOR signaling depend on catalytic alpha2 and regulatory gamma3 subunits. Extensor digitorum longus muscle from...... (Thr37/46) (P mTOR targets, suggesting mTOR signaling is blocked by prior AMPK activation. The AICAR-induced inhibition was partly rescued...

The NDUFB6 subunit of the mitochondrial respiratory chain complex I is required for electron transfer activity: A proof of principle study on stable and controlled RNA interference in human cell lines

International Nuclear Information System (INIS)

Loublier, Sandrine; Bayot, Aurelien; Rak, Malgorzata; El-Khoury, Riyad; Benit, Paule; Rustin, Pierre

2011-01-01

Highlights: → NDUFB6 is required for activity of mitochondrial complex I in human cell lines. → Lentivirus based RNA interference results in frequent off target insertions. → Flp-In recombinase mediated miRNA insertion allows gene-specific extinction. -- Abstract: Molecular bases of inherited deficiencies of mitochondrial respiratory chain complex I are still unknown in a high proportion of patients. Among 45 subunits making up this large complex, more than half has unknown function(s). Understanding the function of these subunits would contribute to our knowledge on mitochondrial physiology but might also reveal that some of these subunits are not required for the catalytic activity of the complex. A direct consequence of this finding would be the reduction of the number of candidate genes to be sequenced in patients with decreased complex I activity. In this study, we tested two different methods to stably extinct complex I subunits in cultured cells. We first found that lentivirus-mediated shRNA expression frequently resulted in the unpredicted extinction of additional gene(s) beside targeted ones. This can be ascribed to uncontrolled genetic material insertions in the genome of the host cell. This approach thus appeared inappropriate to study unknown functions of a gene. Next, we found it possible to specifically extinct a CI subunit gene by direct insertion of a miR targeting CI subunits in a Flp site (HEK293 Flp-In cells). By using this strategy we unambiguously demonstrated that the NDUFB6 subunit is required for complex I activity, and defined conditions suitable to undertake a systematic and stable extinction of the different supernumerary subunits in human cells.

The NDUFB6 subunit of the mitochondrial respiratory chain complex I is required for electron transfer activity: A proof of principle study on stable and controlled RNA interference in human cell lines

Energy Technology Data Exchange (ETDEWEB)

Loublier, Sandrine; Bayot, Aurelien; Rak, Malgorzata; El-Khoury, Riyad; Benit, Paule [Inserm U676, Hopital Robert Debre, F-75019 Paris (France); Universite Paris 7, Faculte de medecine Denis Diderot, IFR02 Paris (France); Rustin, Pierre, E-mail: pierre.rustin@inserm.fr [Inserm U676, Hopital Robert Debre, F-75019 Paris (France); Universite Paris 7, Faculte de medecine Denis Diderot, IFR02 Paris (France)

2011-10-22

Highlights: {yields} NDUFB6 is required for activity of mitochondrial complex I in human cell lines. {yields} Lentivirus based RNA interference results in frequent off target insertions. {yields} Flp-In recombinase mediated miRNA insertion allows gene-specific extinction. -- Abstract: Molecular bases of inherited deficiencies of mitochondrial respiratory chain complex I are still unknown in a high proportion of patients. Among 45 subunits making up this large complex, more than half has unknown function(s). Understanding the function of these subunits would contribute to our knowledge on mitochondrial physiology but might also reveal that some of these subunits are not required for the catalytic activity of the complex. A direct consequence of this finding would be the reduction of the number of candidate genes to be sequenced in patients with decreased complex I activity. In this study, we tested two different methods to stably extinct complex I subunits in cultured cells. We first found that lentivirus-mediated shRNA expression frequently resulted in the unpredicted extinction of additional gene(s) beside targeted ones. This can be ascribed to uncontrolled genetic material insertions in the genome of the host cell. This approach thus appeared inappropriate to study unknown functions of a gene. Next, we found it possible to specifically extinct a CI subunit gene by direct insertion of a miR targeting CI subunits in a Flp site (HEK293 Flp-In cells). By using this strategy we unambiguously demonstrated that the NDUFB6 subunit is required for complex I activity, and defined conditions suitable to undertake a systematic and stable extinction of the different supernumerary subunits in human cells.

Identification of the large subunit of Ribulose 1,5-bisphosphate carboxylase/oxygenase as a substrate for transglutaminase in Medicageo sativa L. (alfalfa)

International Nuclear Information System (INIS)

Margosiak, S.A.; Dharma, A.; Carver, M.R.B.; Gonzales, A.P.; Louie, D.; Kuehn, G.D.

1990-01-01

Extract prepared from floral meristematic tissue of alfalfa (Medicago sativa L.) were investigated for expression of the enzyme transglutaminase in order to identify the major protein substrate for transglutaminase-directed modifications among plant proteins. The large polymorphic subunits of ribulose 1,5-bisphosphate carboxylase/oxygenase in alfalfa, with molecular weights of 52,700 and 57,600, are major substrates for transglutaminase in these extracts. This was established by: (a) covalent conjugation of monodansylcadaverine to the large subunit followed by fluorescent detection in SDS-polyacrylamide gels; (b) covalent conjugation of [ 14 C]putrescine to the large subunit with detection by autoradiography; (c) covalent conjugation of monodansylcadaverine to the large subunit and demonstration of immunocross-reactivity on nitrocellulose transblot of the modified large subunit with antibody prepared in rabbits against dansylated-ovalbumin; (d) demonstration of a direct dependence of the rate of transglutaminase-mediated, [ 14 C]putresciene incorporation upon the concentration of ribulose, 1,5-bisphosphate carboxylase/oxygenase from alfalfa or spinach; and (e) presumptive evidence from size exclusion chromatography that transglutaminase may cofractionate with native molecules of ribulose 1,5-bisphosphate carboxylase/oxygenase in crude extracts

Effect of sodium-glucose cotransporter 2 (SGLT2) inhibition on weight loss is partly mediated by liver-brain-adipose neurocircuitry.

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Sawada, Yoshikazu; Izumida, Yoshihiko; Takeuchi, Yoshinori; Aita, Yuichi; Wada, Nobuhiro; Li, EnXu; Murayama, Yuki; Piao, Xianying; Shikama, Akito; Masuda, Yukari; Nishi-Tatsumi, Makiko; Kubota, Midori; Sekiya, Motohiro; Matsuzaka, Takashi; Nakagawa, Yoshimi; Sugano, Yoko; Iwasaki, Hitoshi; Kobayashi, Kazuto; Yatoh, Shigeru; Suzuki, Hiroaki; Yagyu, Hiroaki; Kawakami, Yasushi; Kadowaki, Takashi; Shimano, Hitoshi; Yahagi, Naoya

2017-11-04

Sodium-glucose cotransporter 2 (SGLT2) inhibitors have both anti-diabetic and anti-obesity effects. However, the precise mechanism of the anti-obesity effect remains unclear. We previously demonstrated that the glycogen depletion signal triggers lipolysis in adipose tissue via liver-brain-adipose neurocircuitry. In this study, therefore, we investigated whether the anti-obesity mechanism of SGLT2 inhibitor is mediated by this mechanism. Diet-induced obese mice were subjected to hepatic vagotomy (HVx) or sham operation and loaded with high fat diet containing 0.015% tofogliflozin (TOFO), a highly selective SGLT2 inhibitor, for 3 weeks. TOFO-treated mice showed a decrease in fat mass and the effect of TOFO was attenuated in HVx group. Although both HVx and sham mice showed a similar level of reduction in hepatic glycogen by TOFO treatment, HVx mice exhibited an attenuated response in protein phosphorylation by protein kinase A (PKA) in white adipose tissue compared with the sham group. As PKA pathway is known to act as an effector of the liver-brain-adipose axis and activate triglyceride lipases in adipocytes, these results indicated that SGLT2 inhibition triggered glycogen depletion signal and actuated liver-brain-adipose axis, resulting in PKA activation in adipocytes. Taken together, it was concluded that the effect of SGLT2 inhibition on weight loss is in part mediated via the liver-brain-adipose neurocircuitry. Copyright © 2017 Elsevier Inc. All rights reserved.

Soybean glycinin subunits: Characterization of physicochemical and adhesion properties.

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Mo, Xiaoqun; Zhong, Zhikai; Wang, Donghai; Sun, Xiuzhi

2006-10-04

Soybean proteins have shown great potential for applications as renewable and environmentally friendly adhesives. The objective of this work was to study physicochemical and adhesion properties of soy glycinin subunits. Soybean glycinin was extracted from soybean flour and then fractionated into acidic and basic subunits with an estimated purity of 90 and 85%, respectively. Amino acid composition of glycinin subunits was determined. The high hydrophobic amino acid content is a major contributor to the solubility behavior and water resistance of the basic subunits. Acidic subunits and glycinin had similar solubility profiles, showing more than 80% solubility at pH 2.0-4.0 or 6.5-12.0, whereas basic subunits had considerably lower solubility with the minimum at pH 4.5-8.0. Thermal analysis using a differential scanning calorimeter suggested that basic subunits form new oligomeric structures with higher thermal stability than glycinin but no highly ordered structures present in isolated acidic subunits. The wet strength of basic subunits was 160% more than that of acidic subunits prepared at their respective isoelectric points (pI) and cured at 130 degrees C. Both pH and the curing temperature significantly affected adhesive performance. High-adhesion water resistance was usually observed for adhesives from protein prepared at their pI values and cured at elevated temperatures. Basic subunits are responsible for the water resistance of glycinin and are a good starting material for the development of water-resistant adhesives.

Solution Structure of the N-Terminal Domain of Mediator Subunit MED26 and Molecular Characterization of Its Interaction with EAF1 and TAF7.

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Lens, Zoé; Cantrelle, François-Xavier; Peruzzini, Riccardo; Hanoulle, Xavier; Dewitte, Frédérique; Ferreira, Elisabeth; Baert, Jean-Luc; Monté, Didier; Aumercier, Marc; Villeret, Vincent; Verger, Alexis; Landrieu, Isabelle

2017-10-13

MED26 is a subunit of Mediator, a large complex central to the regulation of gene transcription by RNA Polymerase II. MED26 plays a role in the switch between the initiation and elongation phases of RNA Polymerase II-mediated transcription process. Regulation of these steps requires successive binding of MED26 N-terminal domain (NTD) to TATA-binding protein-associated factor 7 (TAF7) and Eleven-nineteen lysine-rich in leukemia-Associated Factor 1 (EAF1). In order to investigate the mechanism of regulation by MED26, MED26-NTD structure was solved by NMR, revealing a 4-helix bundle. EAF1 (239-268) and TAF7 (205-235) peptide interactions were both mapped to the same groove formed by H3 and H4 helices of MED26-NTD. Both interactions are characterized by dissociation constants in the 10-μM range. Further experiments revealed a folding-upon-binding mechanism that leads to the formation of EAF1 (N247-S260) and TAF7 (L214-S227) helices. Chemical shift perturbations and nuclear Overhauser enhancement contacts support the involvement of residues I222/F223 in anchoring TAF7 helix to a hydrophobic pocket of MED26-NTD, including residues L48, W80 and I84. In addition, Ala mutations of charged residues located in the C-terminal disordered part of TAF7 and EAF1 peptides affected the binding, with a loss of affinity characterized by a 10-time increase of dissociation constants. A structural model of MED26-NTD/TAF7 complex shows bi-partite components, combining ordered and disordered segments, as well as hydrophobic and electrostatic contributions to the binding. This study provides molecular detail that will help to decipher the mechanistic basis for the initiation to elongation switch-function mediated by MED26-NTD. Copyright © 2017 Elsevier Ltd. All rights reserved.

Characterisation of 5-HT3C, 5-HT3D and 5-HT3E receptor subunits: evolution, distribution and function.

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Holbrook, Joanna D; Gill, Catherine H; Zebda, Noureddine; Spencer, Jon P; Leyland, Rebecca; Rance, Kim H; Trinh, Han; Balmer, Gemma; Kelly, Fiona M; Yusaf, Shahnaz P; Courtenay, Nicola; Luck, Jane; Rhodes, Andrew; Modha, Sundip; Moore, Stephen E; Sanger, Gareth J; Gunthorpe, Martin J

2009-01-01

The 5-HT(3) receptor is a member of the 'Cys-loop' family of ligand-gated ion channels that mediate fast excitatory and inhibitory transmission in the nervous system. Current evidence points towards native 5-HT(3) receptors originating from homomeric assemblies of 5-HT(3A) or heteromeric assembly of 5-HT(3A) and 5-HT(3B). Novel genes encoding 5-HT(3C), 5-HT(3D), and 5-HT(3E) have recently been described but the functional importance of these proteins is unknown. In the present study, in silico analysis (confirmed by partial cloning) indicated that 5-HT(3C), 5-HT(3D), and 5-HT(3E) are not human-specific as previously reported: they are conserved in multiple mammalian species but are absent in rodents. Expression profiles of the novel human genes indicated high levels in the gastrointestinal tract but also in the brain, Dorsal Root Ganglion (DRG) and other tissues. Following the demonstration that these subunits are expressed at the cell membrane, the functional properties of the recombinant human subunits were investigated using patch clamp electrophysiology. 5-HT(3C), 5-HT(3D), and 5-HT(3E) were all non-functional when expressed alone. Co-transfection studies to determine potential novel heteromeric receptor interactions with 5-HT(3A) demonstrated that the expression or function of the receptor was modified by 5-HT(3C) and 5-HT(3E), but not 5-HT(3D). The lack of distinct effects on current rectification, kinetics or pharmacology of 5-HT(3A) receptors does not however provide unequivocal evidence to support a direct contribution of 5-HT(3C) or 5-HT(3E) to the lining of the ion channel pore of novel heteromeric receptors. The functional and pharmacological contributions of these novel subunits to human biology and diseases such as irritable bowel syndrome for which 5-HT(3) receptor antagonists have major clinical usage, therefore remains to be fully determined.

Expression of NMDA receptor subunits in human blood lymphocytes: A peripheral biomarker in online computer game addiction.

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Sadat-Shirazi, Mitra-Sadat; Vousooghi, Nasim; Alizadeh, Bentolhoda; Makki, Seyed Mohammad; Zarei, Seyed Zeinolabedin; Nazari, Shahrzad; Zarrindast, Mohammad Reza

2018-05-23

Background and aims Repeated performance of some behaviors such as playing computer games could result in addiction. The NMDA receptor is critically involved in the development of behavioral and drug addictions. It has been claimed that the expression level of neurotransmitter receptors in the brain may be reflected in peripheral blood lymphocytes (PBLs). Methods Here, using a real-time PCR method, we have investigated the mRNA expression of GluN2A, GluN2D, GluN3A, and GluN3B subunits of the NMDA receptor in PBLs of male online computer game addicts (n = 25) in comparison with normal subjects (n = 26). Results Expression levels of GluN2A, GluN2D, and GluN3B subunits were not statistically different between game addicts and the control group. However, the mRNA expression of the GluN3A subunit was downregulated in PBLs of game addicts. Discussion and conclusions Transcriptional levels of GluN2A and GluN2D subunits in online computer game addicts are similar to our previously reported data of opioid addiction and are not different from the control group. However, unlike our earlier finding of drug addiction, the mRNA expression levels of GluN3A and GluN3B subunits in PBLs of game addicts are reduced and unchanged, respectively, compared with control subjects. It seems that the downregulated state of the GluN3A subunit of NMDA receptor in online computer game addicts is a finding that deserves more studies in the future to see whether it can serve as a peripheral biomarker in addiction studies, where the researcher wants to rule out the confusing effects of abused drugs.

Intrasteric control of AMPK via the gamma1 subunit AMP allosteric regulatory site.

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Adams, Julian; Chen, Zhi-Ping; Van Denderen, Bryce J W; Morton, Craig J; Parker, Michael W; Witters, Lee A; Stapleton, David; Kemp, Bruce E

2004-01-01

AMP-activated protein kinase (AMPK) is a alphabetagamma heterotrimer that is activated in response to both hormones and intracellular metabolic stress signals. AMPK is regulated by phosphorylation on the alpha subunit and by AMP allosteric control previously thought to be mediated by both alpha and gamma subunits. Here we present evidence that adjacent gamma subunit pairs of CBS repeat sequences (after Cystathionine Beta Synthase) form an AMP binding site related to, but distinct from the classical AMP binding site in phosphorylase, that can also bind ATP. The AMP binding site of the gamma(1) CBS1/CBS2 pair, modeled on the structures of the CBS sequences present in the inosine monophosphate dehydrogenase crystal structure, contains three arginine residues 70, 152, and 171 and His151. The yeast gamma homolog, snf4 contains a His151Gly substitution, and when this is introduced into gamma(1), AMP allosteric control is substantially lost and explains why the yeast snf1p/snf4p complex is insensitive to AMP. Arg70 in gamma(1) corresponds to the site of mutation in human gamma(2) and pig gamma(3) genes previously identified to cause an unusual cardiac phenotype and glycogen storage disease, respectively. Mutation of any of AMP binding site Arg residues to Gln substantially abolishes AMP allosteric control in expressed AMPK holoenzyme. The Arg/Gln mutations also suppress the previously described inhibitory properties of ATP and render the enzyme constitutively active. We propose that ATP acts as an intrasteric inhibitor by bridging the alpha and gamma subunits and that AMP functions to derepress AMPK activity.

Brain activation to negative stimuli mediates a relationship between adolescent marijuana use and later emotional functioning

OpenAIRE

Heitzeg, Mary M.; Cope, Lora M.; Martz, Meghan E.; Hardee, Jillian E.; Zucker, Robert A.

2015-01-01

This work investigated the impact of heavy marijuana use during adolescence on emotional functioning, as well as the brain functional mediators of this effect. Participants (n = 40) were recruited from the Michigan Longitudinal Study (MLS). Data on marijuana use were collected prospectively beginning in childhood as part of the MLS. Participants were classified as heavy marijuana users (n = 20) or controls with minimal marijuana use. Two facets of emotional functioning—negative emotionality a...

The brain cytoplasmic RNA BC1 regulates dopamine D-2 receptor-mediated transmission in the striatum

OpenAIRE

Centonze, Diego; Rossi, Silvia; Napoli, Ilaria; Mercaldo, Valentina; Lacoux, Caroline; Ferrari, Francesca; Ciotti, Maria Teresa; De Chiara, Valentina; Prosperetti, Chiara; Maccarrone, Mauro; Fezza, Filomena; Calabresi, Paolo; Bernardi, Giorgio; Bagni, Claudia

2007-01-01

Dopamine D-2 receptor (D2DR)-mediated transmission in the striatum is remarkably flexible, and changes in its efficacy have been heavily implicated in a variety of physiological and pathological conditions. Although receptor-associated proteins are clearly involved in specific forms of synaptic plasticity, the molecular mechanisms regulating the sensitivity of D-2 receptors in this brain area are essentially obscure. We have studied the physiological responses of the D2DR stimulations in mice...

High αv Integrin Level of Cancer Cells Is Associated with Development of Brain Metastasis in Athymic Rats.

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Wu, Yingjen Jeffrey; Pagel, Michael A; Muldoon, Leslie L; Fu, Rongwei; Neuwelt, Edward A

2017-08-01

Brain metastases commonly occur in patients with malignant skin, lung and breast cancers resulting in high morbidity and poor prognosis. Integrins containing an αv subunit are cell adhesion proteins that contribute to cancer cell migration and cancer progression. We hypothesized that high expression of αv integrin cell adhesion protein promoted metastatic phenotypes in cancer cells. Cancer cells from different origins were used and studied regarding their metastatic ability and intetumumab, anti-αv integrin mAb, sensitivity using in vitro cell migration assay and in vivo brain metastases animal models. The number of brain metastases and the rate of occurrence were positively correlated with cancer cell αv integrin levels. High αv integrin-expressing cancer cells showed significantly faster cell migration rate in vitro than low αv integrin-expressing cells. Intetumumab significantly inhibited cancer cell migration in vitro regardless of αv integrin expression level. Overexpression of αv integrin in cancer cells with low αv integrin level accelerated cell migration in vitro and increased the occurrence of brain metastases in vivo. αv integrin promotes brain metastases in cancer cells and may mediate early steps in the metastatic cascade, such as adhesion to brain vasculature. Targeting αv integrin with intetumumab could provide clinical benefit in treating cancer patients who develop metastases. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Toll-like receptor 4 mediates microglial activation and production of inflammatory mediators in neonatal rat brain following hypoxia: role of TLR4 in hypoxic microglia

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2013-01-01

Background Hypoxia induces microglial activation which causes damage to the developing brain. Microglia derived inflammatory mediators may contribute to this process. Toll-like receptor 4 (TLR4) has been reported to induce microglial activation and cytokines production in brain injuries; however, its role in hypoxic injury remains uncertain. We investigate here TLR4 expression and its roles in neuroinflammation in neonatal rats following hypoxic injury. Methods One day old Wistar rats were subjected to hypoxia for 2 h. Primary cultured microglia and BV-2 cells were subjected to hypoxia for different durations. TLR4 expression in microglia was determined by RT-PCR, western blot and immunofluorescence staining. Small interfering RNA (siRNA) transfection and antibody neutralization were employed to downregulate TLR4 in BV-2 and primary culture. mRNA and protein expression of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β) and inducible nitric oxide synthase (iNOS) was assessed. Reactive oxygen species (ROS), nitric oxide (NO) and NF-κB levels were determined by flow cytometry, colorimetric and ELISA assays respectively. Hypoxia-inducible factor-1 alpha (HIF-1α) mRNA and protein expression was quantified and where necessary, the protein expression was depleted by antibody neutralization. In vivo inhibition of TLR4 with CLI-095 injection was carried out followed by investigation of inflammatory mediators expression via double immunofluorescence staining. Results TLR4 immunofluorescence and protein expression in the corpus callosum and cerebellum in neonatal microglia were markedly enhanced post-hypoxia. In vitro, TLR4 protein expression was significantly increased in both primary microglia and BV-2 cells post-hypoxia. TLR4 neutralization in primary cultured microglia attenuated the hypoxia-induced expression of TNF-α, IL-1β and iNOS. siRNA knockdown of TLR4 reduced hypoxia-induced upregulation of TNF-α, IL-1β, iNOS, ROS and NO in BV-2 cells. TLR4

Function and regulation of the Mediator complex.

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Conaway, Ronald C; Conaway, Joan Weliky

2011-04-01

Over the past few years, advances in biochemical and genetic studies of the structure and function of the Mediator complex have shed new light on its subunit architecture and its mechanism of action in transcription by RNA polymerase II (pol II). The development of improved methods for reconstitution of recombinant Mediator subassemblies is enabling more in-depth analyses of basic features of the mechanisms by which Mediator interacts with and controls the activity of pol II and the general initiation factors. The discovery and characterization of multiple, functionally distinct forms of Mediator characterized by the presence or absence of the Cdk8 kinase module have led to new insights into how Mediator functions in both Pol II transcription activation and repression. Finally, progress in studies of the mechanisms by which the transcriptional activation domains (ADs) of DNA binding transcription factors target Mediator have brought to light unexpected complexities in the way Mediator participates in signal transduction. Copyright © 2011 Elsevier Ltd. All rights reserved.

Neuroprotection by biodegradable PAMAM ester (e-PAM-R)-mediated HMGB1 siRNA delivery in primary cortical cultures and in the postischemic brain.

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Kim, Il-Doo; Lim, Chae-Moon; Kim, Jung-Bin; Nam, Hye Yeong; Nam, Kihoon; Kim, Seung-Woo; Park, Jong-Sang; Lee, Ja-Kyeong

2010-03-19

Although RNA interference (RNAi)-mediated gene silencing provides a powerful strategy for modulating specific gene functions, difficulties associated with siRNA delivery have impeded the development of efficient therapeutic applications. In particular, the efficacy of siRNA delivery into neurons has been limited by extremely low transfection efficiencies. e-PAM-R is a biodegradable arginine ester of PAMAM dendrimer, which is readily degradable under physiological conditions (pH 7.4, 37 degrees C). In the present study, we investigated the efficiency of siRNA delivery by e-PAM-R in primary cortical cultures and in rat brain. e-PAM-R/siRNA complexes showed high transfection efficiencies and low cytotoxicities in primary cortical cultures. Localization of fluorescence-tagged siRNA revealed that siRNA was delivered not only into the nucleus and cytoplasm, but also along the processes of the neuron. e-PAM-R/siRNA complex-mediated target gene reduction was observed in over 40% of cells and it was persistent for over 48 h. The potential use of e-PAM-R was demonstrated by gene knockdown after transfecting High mobility group box-1 (HMGB1, a novel cytokine-like molecule) siRNA into H(2)O(2)- or NMDA-treated primary cortical cultures. In these cells, HMGB1 siRNA delivery successfully reduced both basal and H(2)O(2)- or NMDA-induced HMGB1 levels, and as a result of that, neuronal cell death was significantly suppressed in both cases. Furthermore, we showed that e-PAM-R successfully delivered HMGB1 siRNA into the rat brain, wherein HMGB1 expression was depleted in over 40% of neurons and astrocytes of the normal brain. Moreover, e-PAM-R-mediated HMGB1 siRNA delivery notably reduced infarct volume in the postischemic rat brain, which is generated by occluding the middle cerebral artery for 60 min. These results indicate that e-PAM-R, a novel biodegradable nonviral gene carrier, offers an efficient means of transfecting siRNA into primary neuronal cells and in the brain and of

Mediator Undergoes a Compositional Change during Transcriptional Activation.

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Petrenko, Natalia; Jin, Yi; Wong, Koon Ho; Struhl, Kevin

2016-11-03

Mediator is a transcriptional co-activator recruited to enhancers by DNA-binding activators, and it also interacts with RNA polymerase (Pol) II as part of the preinitiation complex (PIC). We demonstrate that a single Mediator complex associates with the enhancer and core promoter in vivo, indicating that it can physically bridge these transcriptional elements. However, the Mediator kinase module associates strongly with the enhancer, but not with the core promoter, and it dissociates from the enhancer upon depletion of the TFIIH kinase. Severing the kinase module from Mediator by removing the connecting subunit Med13 does not affect Mediator association at the core promoter but increases occupancy at enhancers. Thus, Mediator undergoes a compositional change in which the kinase module, recruited via Mediator to the enhancer, dissociates from Mediator to permit association with Pol II and the PIC. As such, Mediator acts as a dynamic bridge between the enhancer and core promoter. Copyright © 2016 Elsevier Inc. All rights reserved.

Ectopic Expression of α6 and δ GABAA Receptor Subunits in Hilar Somatostatin Neurons Increases Tonic Inhibition and Alters Network Activity in the Dentate Gyrus

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Tong, Xiaoping; Peng, Zechun; Zhang, Nianhui; Cetina, Yliana; Huang, Christine S.; Wallner, Martin; Otis, Thomas S.

2015-01-01

The role of GABAA receptor (GABAAR)-mediated tonic inhibition in interneurons remains unclear and may vary among subgroups. Somatostatin (SOM) interneurons in the hilus of the dentate gyrus show negligible expression of nonsynaptic GABAAR subunits and very low tonic inhibition. To determine the effects of ectopic expression of tonic GABAAR subtypes in these neurons, Cre-dependent viral vectors were used to express GFP-tagged GABAAR subunits (α6 and δ) selectively in hilar SOM neurons in SOM-Cre mice. In single-transfected animals, immunohistochemistry demonstrated strong expression of either the α6 or δ subunit; in cotransfected animals, both subunits were consistently expressed in the same neurons. Electrophysiology revealed a robust increase of tonic current, with progressively larger increases following transfection of δ, α6, and α6/δ subunits, respectively, indicating formation of functional receptors in all conditions and likely coassembly of the subunits in the same receptor following cotransfection. An in vitro model of repetitive bursting was used to determine the effects of increased tonic inhibition in hilar SOM interneurons on circuit activity in the dentate gyrus. Upon cotransfection, the frequency of GABAAR-mediated bursting in granule cells was reduced, consistent with a reduction in synchronous firing among hilar SOM interneurons. Moreover, in vivo studies of Fos expression demonstrated reduced activation of α6/δ-cotransfected neurons following acute seizure induction by pentylenetetrazole. The findings demonstrate that increasing tonic inhibition in hilar SOM interneurons can alter dentate gyrus circuit activity during strong stimulation and suggest that tonic inhibition of interneurons could play a role in regulating excessive synchrony within the network. SIGNIFICANCE STATEMENT In contrast to many hippocampal interneurons, somatostatin (SOM) neurons in the hilus of the dentate gyrus have very low levels of nonsynaptic GABAARs and exhibit

Transcriptional regulators of Na, K-ATPase subunits

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Zhiqin eLi

2015-10-01

Full Text Available The Na,K-ATPase classically serves as an ion pump creating an electrochemical gradient across the plasma membrane that is essential for transepithelial transport, nutrient uptake and membrane potential. In addition, Na,K-ATPase also functions as a receptor, a signal transducer and a cell adhesion molecule. With such diverse roles, it is understandable that the Na,K-ATPase subunits, the catalytic alpha-subunit, the beta-subunit and the FXYD proteins, are controlled extensively during development and to accommodate physiological needs. The spatial and temporal expression of Na,K-ATPase is partially regulated at the transcriptional level. Numerous transcription factors, hormones, growth factors, lipids and extracellular stimuli modulate the transcription of the Na,K-ATPase subunits. Moreover, epigenetic mechanisms also contribute to the regulation of Na,K-ATPase expression. With the ever growing knowledge about diseases associated with the malfunction of Na,K-ATPase, this review aims at summarizing the best-characterized transcription regulators that modulate Na,K-ATPase subunit levels. As abnormal expression of Na,K-ATPase subunits have been observed in many carcinoma, we will also discuss transcription factors that are associated with epithelial-to-mesenchymal transition, a crucial step in the progression of many tumors to malignant disease.

Analysis of lipid raft molecules in the living brain slices.

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Kotani, Norihiro; Nakano, Takanari; Ida, Yui; Ito, Rina; Hashizume, Miki; Yamaguchi, Arisa; Seo, Makoto; Araki, Tomoyuki; Hojo, Yasushi; Honke, Koichi; Murakoshi, Takayuki

2017-08-24

Neuronal plasma membrane has been thought to retain a lot of lipid raft components which play important roles in the neural function. Although the biochemical analyses of lipid raft using brain tissues have been extensively carried out in the past 20 years, many of their experimental conditions do not coincide with those of standard neuroscience researches such as neurophysiology and neuropharmacology. Hence, the physiological methods for lipid raft analysis that can be compatible with general neuroscience have been required. Herein, we developed a system to physiologically analyze ganglioside GM1-enriched lipid rafts in brain tissues using the "Enzyme-Mediated Activation of Radical Sources (EMARS)" method that we reported (Kotani N. et al. Proc. Natl. Acad. Sci. U S A 105, 7405-7409 (2008)). The EMARS method was applied to acute brain slices prepared from mouse brains in aCSF solution using the EMARS probe, HRP-conjugated cholera toxin subunit B, which recognizes ganglioside GM1. The membrane molecules present in the GM1-enriched lipid rafts were then labeled with fluorescein under the physiological condition. The fluorescein-tagged lipid raft molecules called "EMARS products" distributed differentially among various parts of the brain. On the other hand, appreciable differences were not detected among segments along the longitudinal axis of the hippocampus. We further developed a device to label the lipid raft molecules in acute hippocampal slices under two different physiological conditions to detect dynamics of the lipid raft molecules during neural excitation. Using this device, several cell membrane molecules including Thy1, known as a lipid raft resident molecule in neurons, were confirmed by the EMARS method in living hippocampal slices. Copyright © 2017 Elsevier Ltd. All rights reserved.

BIASED AGONISM OF THREE DIFFERENT CANNABINOID RECEPTOR AGONISTS IN MOUSE BRAIN CORTEX

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Rebeca Diez-Alarcia

2016-11-01

Full Text Available Cannabinoid receptors are able to couple to different families of G-proteins when activated by an agonist drug. It has been suggested that different intracellular responses may be activated depending on the ligand. The goal of the present study was to characterize the pattern of G protein subunit stimulation triggered by three different cannabinoid ligands, THC, WIN55212-2 and ACEA in mouse brain cortex.Stimulation of the [35S]GTPS binding coupled to specific immunoprecipitation with antibodies against different subtypes of G proteins (Gαi1, Gαi2, Gαi3, Gαo, Gαz, Gαs, Gαq/11, and Gα12/13, in the presence of Δ9-THC, WIN55212-2 and ACEA (submaximal concentration 10 µM was determined by Scintillation Proximity Assay (SPA technique in mouse cortex of wild type, CB1 knock-out, CB2 knock-out and CB1/CB2 double knock-out mice. Results show that, in mouse brain cortex, cannabinoid agonists are able to significantly stimulate not only the classical inhibitory Gαi/o subunits but also other G subunits like Gαz, Gαq/11, and Gα12/13. Moreover, the specific pattern of G protein subunit activation is different depending on the ligand. In conclusion, our results demonstrate that, in mice brain native tissue, different exogenous cannabinoid ligands are able to selectively activate different inhibitory and non-inhibitory Gα protein subtypes, through the activation of CB1 and/or CB2 receptors. Results of the present study may help to understand the specific molecular pathways involved in the pharmacological effects of cannabinoid-derived drugs.

The Mediator Kinase Module Restrains Epidermal Growth Factor Receptor Signaling and Represses Vulval Cell Fate Specification in Caenorhabditis elegans.

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Grants, Jennifer M; Ying, Lisa T L; Yoda, Akinori; You, Charlotte C; Okano, Hideyuki; Sawa, Hitoshi; Taubert, Stefan

2016-02-01

Cell signaling pathways that control proliferation and determine cell fates are tightly regulated to prevent developmental anomalies and cancer. Transcription factors and coregulators are important effectors of signaling pathway output, as they regulate downstream gene programs. In Caenorhabditis elegans, several subunits of the Mediator transcriptional coregulator complex promote or inhibit vulva development, but pertinent mechanisms are poorly defined. Here, we show that Mediator's dissociable cyclin dependent kinase 8 (CDK8) module (CKM), consisting of cdk-8, cic-1/Cyclin C, mdt-12/dpy-22, and mdt-13/let-19, is required to inhibit ectopic vulval cell fates downstream of the epidermal growth factor receptor (EGFR)-Ras-extracellular signal-regulated kinase (ERK) pathway. cdk-8 inhibits ectopic vulva formation by acting downstream of mpk-1/ERK, cell autonomously in vulval cells, and in a kinase-dependent manner. We also provide evidence that the CKM acts as a corepressor for the Ets-family transcription factor LIN-1, as cdk-8 promotes transcriptional repression by LIN-1. In addition, we find that CKM mutation alters Mediator subunit requirements in vulva development: the mdt-23/sur-2 subunit, which is required for vulva development in wild-type worms, is dispensable for ectopic vulva formation in CKM mutants, which instead display hallmarks of unrestrained Mediator tail module activity. We propose a model whereby the CKM controls EGFR-Ras-ERK transcriptional output by corepressing LIN-1 and by fine tuning Mediator specificity, thus balancing transcriptional repression vs. activation in a critical developmental signaling pathway. Collectively, these data offer an explanation for CKM repression of EGFR signaling output and ectopic vulva formation and provide the first evidence of Mediator CKM-tail module subunit crosstalk in animals. Copyright © 2016 by the Genetics Society of America.

The first transmembrane domain (TM1) of β2-subunit binds to the transmembrane domain S1 of α-subunit in BK potassium channels

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Morera, Francisco J.; Alioua, Abderrahmane; Kundu, Pallob; Salazar, Marcelo; Gonzalez, Carlos; Martinez, Agustin D.; Stefani, Enrico; Toro, Ligia; Latorre, Ramon

2012-01-01

The BK channel is one of the most broadly expressed ion channels in mammals. In many tissues, the BK channel pore-forming α-subunit is associated to an auxiliary β-subunit that modulates the voltage- and Ca2+-dependent activation of the channel. Structural components present in β-subunits that are important for the physical association with the α-subunit are yet unknown. Here, we show through co-immunoprecipitation that the intracellular C-terminus, the second transmembrane domain (TM2) and the extracellular loop of the β2-subunit are dispensable for association with the α-subunit pointing transmembrane domain 1 (TM1) as responsible for the interaction. Indeed, the TOXCAT assay for transmembrane protein–protein interactions demonstrated for the first time that TM1 of the β2-subunit physically binds to the transmembrane S1 domain of the α-subunit. PMID:22710124

Recruitment of Mediator Complex by Cell Type and Stage-Specific Factors Required for Tissue-Specific TAF Dependent Gene Activation in an Adult Stem Cell Lineage.

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Lu, Chenggang; Fuller, Margaret T

2015-12-01

Onset of terminal differentiation in adult stem cell lineages is commonly marked by robust activation of new transcriptional programs required to make the appropriate differentiated cell type(s). In the Drosophila male germ line stem cell lineage, the switch from proliferating spermatogonia to spermatocyte is accompanied by one of the most dramatic transcriptional changes in the fly, as over 1000 new transcripts turn on in preparation for meiosis and spermatid differentiation. Here we show that function of the coactivator complex Mediator is required for activation of hundreds of new transcripts in the spermatocyte program. Mediator appears to act in a sequential hierarchy, with the testis activating Complex (tMAC), a cell type specific form of the Mip/dREAM general repressor, required to recruit Mediator subunits to the chromatin, and Mediator function required to recruit the testis TAFs (tTAFs), spermatocyte specific homologs of subunits of TFIID. Mediator, tMAC and the tTAFs co-regulate expression of a major set of spermatid differentiation genes. The Mediator subunit Med22 binds the tMAC component Topi when the two are coexpressed in S2 cells, suggesting direct recruitment. Loss of Med22 function in spermatocytes causes meiosis I maturation arrest male infertility, similar to loss of function of the tMAC subunits or the tTAFs. Our results illuminate how cell type specific versions of the Mip/dREAM complex and the general transcription machinery cooperate to drive selective gene activation during differentiation in stem cell lineages.

Alteration of skin wound healing in keratinocyte-specific mediator complex subunit 1 null mice.

Science.gov (United States)

Noguchi, Fumihito; Nakajima, Takeshi; Inui, Shigeki; Reddy, Janardan K; Itami, Satoshi

2014-01-01

MED1 (Mediator complex subunit 1) is a co-activator of various transcription factors that function in multiple transcriptional pathways. We have already established keratinocyte-specific MED1 null mice (Med1(epi-/-)) that develop epidermal hyperplasia. Herein, to investigate the function(s) of MED1 in skin wound healing, full-thickness skin wounds were generated in Med1(epi-/-) and age-matched wild-type mice and the healing process was analyzed. Macroscopic wound closure and the re-epithelialization rate were accelerated in 8-week-old Med1(epi-/-) mice compared with age-matched wild-type mice. Increased lengths of migrating epithelial tongues and numbers of Ki67-positive cells at the wounded epidermis were observed in 8-week-old Med1(epi-/-) mice, whereas wound contraction and the area of α-SMA-positive myofibroblasts in the granulation tissue were unaffected. Migration was enhanced in Med1(epi-/-) keratinocytes compared with wild-type keratinocytes in vitro. Immunoblotting revealed that the expression of follistatin was significantly decreased in Med1(epi-/-) keratinocytes. Moreover, the mitogen-activated protein kinase pathway was enhanced before and after treatment of Med1(epi-/-) keratinocytes with activin A in vitro. Cell-cycle analysis showed an increased ratio of S phase cells after activin A treatment of Med1(epi-/-) keratinocytes compared with wild-type keratinocytes. These findings indicate that the activin-follistatin system is involved in this acceleration of skin wound healing in 8-week-old Med1(epi-/-) mice. On the other hand, skin wound healing in 6-month-old Med1(epi-/-) mice was significantly delayed with decreased numbers of Ki67-positive cells at the wounded epidermis as well as BrdU-positive label retaining cells in hair follicles compared with age-matched wild-type mice. These results agree with our previous observation that hair follicle bulge stem cells are reduced in older Med1(epi-/-) mice, indicating a decreased contribution of hair

Alteration of skin wound healing in keratinocyte-specific mediator complex subunit 1 null mice.

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Fumihito Noguchi

Full Text Available MED1 (Mediator complex subunit 1 is a co-activator of various transcription factors that function in multiple transcriptional pathways. We have already established keratinocyte-specific MED1 null mice (Med1(epi-/- that develop epidermal hyperplasia. Herein, to investigate the function(s of MED1 in skin wound healing, full-thickness skin wounds were generated in Med1(epi-/- and age-matched wild-type mice and the healing process was analyzed. Macroscopic wound closure and the re-epithelialization rate were accelerated in 8-week-old Med1(epi-/- mice compared with age-matched wild-type mice. Increased lengths of migrating epithelial tongues and numbers of Ki67-positive cells at the wounded epidermis were observed in 8-week-old Med1(epi-/- mice, whereas wound contraction and the area of α-SMA-positive myofibroblasts in the granulation tissue were unaffected. Migration was enhanced in Med1(epi-/- keratinocytes compared with wild-type keratinocytes in vitro. Immunoblotting revealed that the expression of follistatin was significantly decreased in Med1(epi-/- keratinocytes. Moreover, the mitogen-activated protein kinase pathway was enhanced before and after treatment of Med1(epi-/- keratinocytes with activin A in vitro. Cell-cycle analysis showed an increased ratio of S phase cells after activin A treatment of Med1(epi-/- keratinocytes compared with wild-type keratinocytes. These findings indicate that the activin-follistatin system is involved in this acceleration of skin wound healing in 8-week-old Med1(epi-/- mice. On the other hand, skin wound healing in 6-month-old Med1(epi-/- mice was significantly delayed with decreased numbers of Ki67-positive cells at the wounded epidermis as well as BrdU-positive label retaining cells in hair follicles compared with age-matched wild-type mice. These results agree with our previous observation that hair follicle bulge stem cells are reduced in older Med1(epi-/- mice, indicating a decreased contribution of hair

Generation of recombinant antibodies to rat GABAA receptor subunits by affinity selection on synthetic peptides.

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Sujatha P Koduvayur

Full Text Available The abundance and physiological importance of GABAA receptors in the central nervous system make this neurotransmitter receptor an attractive target for localizing diagnostic and therapeutic biomolecules. GABAA receptors are expressed within the retina and mediate synaptic signaling at multiple stages of the visual process. To generate monoclonal affinity reagents that can specifically recognize GABAA receptor subunits, we screened two bacteriophage M13 libraries, which displayed human scFvs, by affinity selection with synthetic peptides predicted to correspond to extracellular regions of the rat α1 and β2 GABAA subunits. We isolated three anti-β2 and one anti-α1 subunit specific scFvs. Fluorescence polarization measurements revealed all four scFvs to have low micromolar affinities with their cognate peptide targets. The scFvs were capable of detecting fully folded GABAA receptors heterologously expressed by Xenopus laevis oocytes, while preserving ligand-gated channel activity. Moreover, A10, the anti-α1 subunit-specific scFv, was capable of detecting native GABAA receptors in the mouse retina, as observed by immunofluorescence staining. In order to improve their apparent affinity via avidity, we dimerized the A10 scFv by fusing it to the Fc portion of the IgG. The resulting scFv-Fc construct had a Kd of ∼26 nM, which corresponds to an approximately 135-fold improvement in binding, and a lower detection limit in dot blots, compared to the monomeric scFv. These results strongly support the use of peptides as targets for generating affinity reagents to membrane proteins and encourage investigation of molecular conjugates that use scFvs as anchoring components to localize reagents of interest at GABAA receptors of retina and other neural tissues, for studies of receptor activation and subunit structure.

Salt-Induced Hypertension in a Mouse Model of Liddle's Syndrome is Mediated by Epithelial Sodium Channels in the Brain

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Van Huysse, James W.; Amin, Md. Shahrier; Yang, Baoli; Leenen, Frans H. H.

2012-01-01

Neural precursor cell expressed and developmentally downregulated 4-2 protein (Nedd4-2) facilitates the endocytosis of epithelial Na channels (ENaC). Both mice and humans with a loss of regulation of ENaC by Nedd4-2 have salt-induced hypertension. ENaC is also expressed in the brain, where it is critical for hypertension on high salt diet in salt-sensitive rats. In the present studies we assessed whether Nedd4-2 knockout (−/−) mice have: 1) increased brain ENaC; 2) elevated CSF sodium on high salt diet; and 3) enhanced pressor responses to CSF sodium and hypertension on high salt diet, both mediated by brain ENaC. Prominent choroid plexus and neuronal ENaC staining was present in −/− but not in wild-type (W/T) mice. In chronically instrumented mice, intracerebroventricular (icv) infusion of Na-rich aCSF increased MAP 3-fold higher in −/− than W/T. Icv infusion of the ENaC blocker benzamil abolished this enhancement. In telemetered −/− mice on high salt diet (8% NaCl), CSF [Na+], MAP and HR increased significantly, MAP by 30-35 mmHg. These MAP and HR responses were largely prevented by icv benzamil, but only to a minor extent by sc benzamil at the icv rate. We conclude that increased ENaC expression in the brain of Nedd 4-2 −/− mice mediates their hypertensive response to high salt diet, by causing increased sodium levels in the CSF as well as hyper-responsiveness to CSF sodium. These findings highlight the possible causative contribution of CNS ENaC in the etiology of salt-induced hypertension. PMID:22802227

28 CFR 51.6 - Political subunits.

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2010-07-01

... 28 Judicial Administration 2 2010-07-01 2010-07-01 false Political subunits. 51.6 Section 51.6 Judicial Administration DEPARTMENT OF JUSTICE (CONTINUED) PROCEDURES FOR THE ADMINISTRATION OF SECTION 5 OF THE VOTING RIGHTS ACT OF 1965, AS AMENDED General Provisions § 51.6 Political subunits. All political...

The Mediator Complex and Lipid Metabolism.

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Zhang, Yi; Xiaoli; Zhao, Xiaoping; Yang, Fajun

2013-03-01

The precise control of gene expression is essential for all biological processes. In addition to DNA-binding transcription factors, numerous transcription cofactors contribute another layer of regulation of gene transcription in eukaryotic cells. One of such transcription cofactors is the highly conserved Mediator complex, which has multiple subunits and is involved in various biological processes through directly interacting with relevant transcription factors. Although the current understanding on the biological functions of Mediator remains incomplete, research in the past decade has revealed an important role of Mediator in regulating lipid metabolism. Such function of Mediator is dependent on specific transcription factors, including peroxisome proliferator-activated receptor-gamma (PPARγ) and sterol regulatory element-binding proteins (SREBPs), which represent the master regulators of lipid metabolism. The medical significance of these findings is apparent, as aberrant lipid metabolism is intimately linked to major human diseases, such as type 2 diabetes and cardiovascular disease. Here, we briefly review the functions and molecular mechanisms of Mediator in regulation of lipid metabolism.

Plant Mediator complex and its critical functions in transcription regulation.

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Yang, Yan; Li, Ling; Qu, Li-Jia

2016-02-01

The Mediator complex is an important component of the eukaryotic transcriptional machinery. As an essential link between transcription factors and RNA polymerase II, the Mediator complex transduces diverse signals to genes involved in different pathways. The plant Mediator complex was recently purified and comprises conserved and specific subunits. It functions in concert with transcription factors to modulate various responses. In this review, we summarize the recent advances in understanding the plant Mediator complex and its diverse roles in plant growth, development, defense, non-coding RNA production, response to abiotic stresses, flowering, genomic stability and metabolic homeostasis. In addition, the transcription factors interacting with the Mediator complex are also highlighted. © 2015 Institute of Botany, Chinese Academy of Sciences.

Involvement of proteasomal subunits zeta and iota in RNA degradation.

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Petit, F; Jarrousse, A S; Dahlmann, B; Sobek, A; Hendil, K B; Buri, J; Briand, Y; Schmid, H P

1997-01-01

We have identified two distinct subunits of 20 S proteasomes that are associated with RNase activity. Proteasome subunits zeta and iota, eluted from two-dimensional Western blots, hydrolysed tobacco mosaic virus RNA, whereas none of the other subunits degraded this substrate under the same conditions. Additionally, proteasomes were dissociated by 6 M urea, and subunit zeta, containing the highest RNase activity, was isolated by anion-exchange chromatography and gel filtration. Purified subunit zeta migrated as a single spot on two-dimensional PAGE with a molecular mass of approx. 28 kDa. Addition of anti-(subunit zeta) antibodies led to the co-precipitation of this proteasome subunit and nuclease activity. This is the first evidence that proteasomal alpha-type subunits are associated with an enzymic activity, and our results provide further evidence that proteasomes may be involved in cellular RNA metabolism. PMID:9337855

Subunit Stoichiometry of Human Muscle Chloride Channels

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Fahlke, Christoph; Knittle, Timothy; Gurnett, Christina A.; Campbell, Kevin P.; George, Alfred L.

1997-01-01

Voltage-gated Cl? channels belonging to the ClC family appear to function as homomultimers, but the number of subunits needed to form a functional channel is controversial. To determine subunit stoichiometry, we constructed dimeric human skeletal muscle Cl? channels in which one subunit was tagged by a mutation (D136G) that causes profound changes in voltage-dependent gating. Sucrose-density gradient centrifugation experiments indicate that both monomeric and dimeric hClC-1 channels in their ...

Transcriptional regulators of Na, K-ATPase subunits

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Zhiqin eLi; Sigrid A Langhans

2015-01-01

The Na,K-ATPase classically serves as an ion pump creating an electrochemical gradient across the plasma membrane that is essential for transepithelial transport, nutrient uptake and membrane potential. In addition, Na,K-ATPase also functions as a receptor, a signal transducer and a cell adhesion molecule. With such diverse roles, it is understandable that the Na,K-ATPase subunits, the catalytic alpha-subunit, the beta-subunit and the FXYD proteins, are controlled extensively during developme...

The metazoan Mediator co-activator complex as an integrative hub for transcriptional regulation

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Malik, Sohail; Roeder, Robert G.

2010-01-01

The Mediator is an evolutionarily conserved, multiprotein complex that is a key regulator of protein-coding genes. In metazoan cells, multiple pathways that are responsible for homeostasis, cell growth and differentiation converge on the Mediator through transcriptional activators and repressors that target one or more of the almost 30 subunits of this complex. Besides interacting directly with RNA polymerase II, Mediator has multiple functions and can interact with and coordinate the action ...

HMGB1 mediates endogenous TLR2 activation and brain tumor regression.

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James F Curtin

2009-01-01

Full Text Available Glioblastoma multiforme (GBM is the most aggressive primary brain tumor that carries a 5-y survival rate of 5%. Attempts at eliciting a clinically relevant anti-GBM immune response in brain tumor patients have met with limited success, which is due to brain immune privilege, tumor immune evasion, and a paucity of dendritic cells (DCs within the central nervous system. Herein we uncovered a novel pathway for the activation of an effective anti-GBM immune response mediated by high-mobility-group box 1 (HMGB1, an alarmin protein released from dying tumor cells, which acts as an endogenous ligand for Toll-like receptor 2 (TLR2 signaling on bone marrow-derived GBM-infiltrating DCs.Using a combined immunotherapy/conditional cytotoxic approach that utilizes adenoviral vectors (Ad expressing Fms-like tyrosine kinase 3 ligand (Flt3L and thymidine kinase (TK delivered into the tumor mass, we demonstrated that CD4(+ and CD8(+ T cells were required for tumor regression and immunological memory. Increased numbers of bone marrow-derived, tumor-infiltrating myeloid DCs (mDCs were observed in response to the therapy. Infiltration of mDCs into the GBM, clonal expansion of antitumor T cells, and induction of an effective anti-GBM immune response were TLR2 dependent. We then proceeded to identify the endogenous ligand responsible for TLR2 signaling on tumor-infiltrating mDCs. We demonstrated that HMGB1 was released from dying tumor cells, in response to Ad-TK (+ gancyclovir [GCV] treatment. Increased levels of HMGB1 were also detected in the serum of tumor-bearing Ad-Flt3L/Ad-TK (+GCV-treated mice. Specific activation of TLR2 signaling was induced by supernatants from Ad-TK (+GCV-treated GBM cells; this activation was blocked by glycyrrhizin (a specific HMGB1 inhibitor or with antibodies to HMGB1. HMGB1 was also released from melanoma, small cell lung carcinoma, and glioma cells treated with radiation or temozolomide. Administration of either glycyrrhizin or anti

Dissociable brain systems mediate vicarious learning of stimulus-response and action-outcome contingencies.

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Liljeholm, Mimi; Molloy, Ciara J; O'Doherty, John P

2012-07-18

Two distinct strategies have been suggested to support action selection in humans and other animals on the basis of experiential learning: a goal-directed strategy that generates decisions based on the value and causal antecedents of action outcomes, and a habitual strategy that relies on the automatic elicitation of actions by environmental stimuli. In the present study, we investigated whether a similar dichotomy exists for actions that are acquired vicariously, through observation of other individuals rather than through direct experience, and assessed whether these strategies are mediated by distinct brain regions. We scanned participants with functional magnetic resonance imaging while they performed an observational learning task designed to encourage either goal-directed encoding of the consequences of observed actions, or a mapping of observed actions to conditional discriminative cues. Activity in different parts of the action observation network discriminated between the two conditions during observational learning and correlated with the degree of insensitivity to outcome devaluation in subsequent performance. Our findings suggest that, in striking parallel to experiential learning, neural systems mediating the observational acquisition of actions may be dissociated into distinct components: a goal-directed, outcome-sensitive component and a less flexible stimulus-response component.

Deletion of P2X2 and P2X3 receptor subunits does not alter motility of the mouse colon

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Matthew DeVries

2010-03-01

Full Text Available Purinergic P2X receptors contribute to neurotransmission in the gut. P2X receptors are ligand-gated cation channels that mediate synaptic excitation in subsets of enteric neurons. The present study evaluated colonic motility in vitro and in vivo in wild type (WT and P2X2 and P2X3 subunit knockout (KO mice. The muscarinic receptor agonist, bethanechol (0.3-3 micromolar, caused similar contractions of the longitudinal muscle in colon segments from WT, P2X2 and P2X3 subunit KO mice. Nicotine (1-300 micromolar, acting at neuronal nicotinic receptors, caused similar longitudinal muscle relaxations in colonic segments from WT and P2X2 and P2X3 subunit KO mice. Nicotine-induced relaxations were inhibited by nitro-L-arginine (NLA, 100 micromolar and apamin (0.1 micromolar which block inhibitory neuromuscular transmission. ATP (1-1000 micromolar caused contractions only in the presence of NLA and apamin. ATP-induced contractions were similar in colon segments from WT, P2X2 and P2X3 KO mice. The mouse colon generates spontaneous migrating motor complexes (MMCs in vitro. The MMC frequency was higher in P2X2 KO compared to WT tissues; other parameters of the MMC were similar in colon segments from WT, P2X2 and P2X3 KO mice. 5-Hydroxytryptophan-induced fecal output was similar in WT, P2X2 and P2X3 KO mice. These data indicate that nicotinic receptors are located predominately on inhibitory motor neurons supplying the longitudinal muscle in the mouse colon. P2X2 or P2X3 subunit containing receptors are not localized to motorneurons supplying the longitudinal muscle. Synaptic transmission mediated by P2X2 or P2X3 subunit containing receptors is not required for propulsive motility in the mouse colon.

MC EMiNEM maps the interaction landscape of the Mediator

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Niederberger, Theresa; Etzold, Stefanie; Lidschreiber, Michael; Maier, Kerstin C.; Martin, Dietmar E.; Fröhlich, Holger; Cramer, Patrick; Tresch, Achim

2012-01-01

The Mediator is a highly conserved, large multiprotein complex that is involved essentially in the regulation of eukaryotic mRNA transcription. It acts as a general transcription factor by integrating regulatory signals from gene-specific activators or repressors to the RNA Polymerase II. The internal network of interactions between Mediator subunits that conveys these signals is largely unknown. Here, we introduce MC EMiNEM, a novel method for the retrieval of functional dependencies between...

Three alpha-subunits of heterotrimeric G proteins and an adenylyl cyclase have distinct roles in fruiting body development in the homothallic fungus Sordaria macrospora.

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Kamerewerd, Jens; Jansson, Malin; Nowrousian, Minou; Pöggeler, Stefanie; Kück, Ulrich

2008-09-01

Sordaria macrospora, a self-fertile filamentous ascomycete, carries genes encoding three different alpha-subunits of heterotrimeric G proteins (gsa, G protein Sordaria alpha subunit). We generated knockout strains for all three gsa genes (Deltagsa1, Deltagsa2, and Deltagsa3) as well as all combinations of double mutants. Phenotypic analysis of single and double mutants showed that the genes for Galpha-subunits have distinct roles in the sexual life cycle. While single mutants show some reduction of fertility, double mutants Deltagsa1Deltagsa2 and Deltagsa1Deltagsa3 are completely sterile. To test whether the pheromone receptors PRE1 and PRE2 mediate signaling via distinct Galpha-subunits, two recently generated Deltapre strains were crossed with all Deltagsa strains. Analyses of the corresponding double mutants revealed that compared to GSA2, GSA1 is a more predominant regulator of a signal transduction cascade downstream of the pheromone receptors and that GSA3 is involved in another signaling pathway that also contributes to fruiting body development and fertility. We further isolated the gene encoding adenylyl cyclase (AC) (sac1) for construction of a knockout strain. Analyses of the three DeltagsaDeltasac1 double mutants and one Deltagsa2Deltagsa3Deltasac1 triple mutant indicate that SAC1 acts downstream of GSA3, parallel to a GSA1-GSA2-mediated signaling pathway. In addition, the function of STE12 and PRO41, two presumptive signaling components, was investigated in diverse double mutants lacking those developmental genes in combination with the gsa genes. This analysis was further completed by expression studies of the ste12 and pro41 transcripts in wild-type and mutant strains. From the sum of all our data, we propose a model for how different Galpha-subunits interact with pheromone receptors, adenylyl cyclase, and STE12 and thus cooperatively regulate sexual development in S. macrospora.

Assembly of the Arp5 (Actin-related Protein) Subunit Involved in Distinct INO80 Chromatin Remodeling Activities*

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Yao, Wei; Beckwith, Sean L.; Zheng, Tina; Young, Thomas; Dinh, Van T.; Ranjan, Anand; Morrison, Ashby J.

2015-01-01

ATP-dependent chromatin remodeling, which repositions and restructures nucleosomes, is essential to all DNA-templated processes. The INO80 chromatin remodeling complex is an evolutionarily conserved complex involved in diverse cellular processes, including transcription, DNA repair, and replication. The functional diversity of the INO80 complex can, in part, be attributed to specialized activities of distinct subunits that compose the complex. Furthermore, structural analyses have identified biochemically discrete subunit modules that assemble along the Ino80 ATPase scaffold. Of particular interest is the Saccharomyces cerevisiae Arp5-Ies6 module located proximal to the Ino80 ATPase and the Rvb1-Rvb2 helicase module needed for INO80-mediated in vitro activity. In this study we demonstrate that the previously uncharacterized Ies2 subunit is required for Arp5-Ies6 association with the catalytic components of the INO80 complex. In addition, Arp5-Ies6 module assembly with the INO80 complex is dependent on distinct conserved domains within Arp5, Ies6, and Ino80, including the spacer region within the Ino80 ATPase domain. Arp5-Ies6 interacts with chromatin via assembly with the INO80 complex, as IES2 and INO80 deletion results in loss of Arp5-Ies6 chromatin association. Interestingly, ectopic addition of the wild-type Arp5-Ies6 module stimulates INO80-mediated ATP hydrolysis and nucleosome sliding in vitro. However, the addition of mutant Arp5 lacking unique insertion domains facilitates ATP hydrolysis in the absence of nucleosome sliding. Collectively, these results define the requirements of Arp5-Ies6 assembly, which are needed to couple ATP hydrolysis to productive nucleosome movement. PMID:26306040

The head module of Mediator directs activation of preloaded RNAPII in vivo.

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Lee, Sarah K; Chen, Xu; Huang, Liangqun; Stargell, Laurie A

2013-12-01

The successful synthesis of a transcript by RNA polymerase II (RNAPII) is a multistage process with distinct rate-limiting steps that can vary depending on the particular gene. A growing number of genes in a variety of organisms are regulated at steps after the recruitment of RNAPII. The best-characterized Saccharomyces cerevisiae gene regulated in this manner is CYC1. This gene has high occupancy of RNAPII under non-inducing conditions, defining it as a poised gene. Here, we find that subunits of the head module of Mediator, Med18 and Med20, and Med19 are required for activation of transcription at the CYC1 promoter in response to environmental cues. These subunits of Mediator are required at the preloaded promoter for normal levels of recruitment and activity of the general transcription factor TFIIH. Strikingly, these Mediator components are dispensable for activation by the same activator at a different gene, which lacks a preloaded polymerase in the promoter region. Based on these results and other studies, we speculate that Mediator plays an essential role in triggering an inactive polymerase at CYC1 into a productively elongating form.

The Mediator Complex and Lipid Metabolism

OpenAIRE

Zhang, Yi; Xiaoli,; Zhao, Xiaoping; Yang, Fajun

2013-01-01

The precise control of gene expression is essential for all biological processes. In addition to DNA-binding transcription factors, numerous transcription cofactors contribute another layer of regulation of gene transcription in eukaryotic cells. One of such transcription cofactors is the highly conserved Mediator complex, which has multiple subunits and is involved in various biological processes through directly interacting with relevant transcription factors. Although the current understan...

Molecular and functional characterization of seven Na+/K+-ATPase β subunit paralogs in Senegalese sole (Solea senegalensis Kaup, 1858).

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Armesto, Paula; Infante, Carlos; Cousin, Xavier; Ponce, Marian; Manchado, Manuel

2015-04-01

In the present work, seven genes encoding Na(+),K(+)-ATPase (NKA) β-subunits in the teleost Solea senegalensis are described for the first time. Sequence analysis of the predicted polypeptides revealed a high degree of conservation with those of other vertebrate species and maintenance of important motifs involved in structure and function. Phylogenetic analysis clustered the seven genes into four main clades: β1 (atp1b1a and atp1b1b), β2 (atp1b2a and atp1b2b), β3 (atp1b3a and atp1b3b) and β4 (atp1b4). In juveniles, all paralogous transcripts were detected in the nine tissues examined albeit with different expression patterns. The most ubiquitous expressed gene was atp1b1a whereas atp1b1b was mainly detected in osmoregulatory organs (gill, kidney and intestine), and atp1b2a, atp1b2b, atp1b3a, atp1b3b and atp1b4 in brain. An expression analysis in three brain regions and pituitary revealed that β1-type transcripts were more abundant in pituitary than the other β paralogs with slight differences between brain regions. Quantification of mRNA abundance in gills after a salinity challenge showed an activation of atp1b1a and atp1b1b at high salinity water (60 ppt) and atp1b3a and atp1b3b in response to low salinity (5 ppt). Transcriptional analysis during larval development showed specific expression patterns for each paralog. Moreover, no differences in the expression profiles between larvae cultivated at 10 and 35 ppt were observed except for atp1b4 with higher mRNA levels at 10 than 35 ppt at 18 days post hatch. Whole-mount in situ hybridization analysis revealed that atp1b1b was mainly localized in gut, pronephric tubule, gill, otic vesicle, and chordacentrum of newly hatched larvae. All these data suggest distinct roles of NKA β subunits in tissues, during development and osmoregulation with β1 subunits involved in the adaptation to hyperosmotic conditions and β3 subunits to hypoosmotic environments. Copyright © 2014 Elsevier Inc. All rights reserved.

Bacopa monnieri Extract (CDRI-08 Modulates the NMDA Receptor Subunits and nNOS-Apoptosis Axis in Cerebellum of Hepatic Encephalopathy Rats

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Papia Mondal

2015-01-01

Full Text Available Hepatic encephalopathy (HE, characterized by impaired cerebellar functions during chronic liver failure (CLF, involves N-methyl-D-aspartate receptor (NMDAR overactivation in the brain cells. Bacopa monnieri (BM extract is a known neuroprotectant. The present paper evaluates whether BM extract is able to modulate the two NMDAR subunits (NR2A and NR2B and its downstream mediators in cerebellum of rats with chronic liver failure (CLF, induced by administration of 50 mg/kg bw thioacetamide (TAA i.p. for 14 days, and in the TAA group rats orally treated with 200 mg/kg bw BM extract from days 8 to 14. NR2A is known to impart neuroprotection and that of NR2B induces neuronal death during NMDAR activation. Neuronal nitric oxide synthase- (nNOS- apoptosis pathway is known to mediate NMDAR led excitotoxicity. The level of NR2A was found to be significantly reduced with a concomitant increase of NR2B in cerebellum of the CLF rats. This was consistent with significantly enhanced nNOS expression, nitric oxide level, and reduced Bcl2/Bax ratio. Moreover, treatment with BM extract reversed the NR2A/NR2B ratio and also normalized the levels of nNOS-apoptotic factors in cerebellum of those rats. The findings suggest modulation of NR2A and NR2B expression by BM extract to prevent neurochemical alterations associated with HE.

Antagonism at the NR2B subunit of NMDA receptors induces increased connectivity of the prefrontal and subcortical regions regulating reward behavior.

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Gass, Natalia; Becker, Robert; Sack, Markus; Schwarz, Adam J; Reinwald, Jonathan; Cosa-Linan, Alejandro; Zheng, Lei; von Hohenberg, Christian Clemm; Inta, Dragos; Meyer-Lindenberg, Andreas; Weber-Fahr, Wolfgang; Gass, Peter; Sartorius, Alexander

2018-04-01

Evidence indicates that ketamine's rapid antidepressant efficacy likely results from its antagonism of NR2B-subunit-containing NMDA receptors (NMDAR). Since ketamine equally blocks NR2A- and NR2B-containing NMDAR, and has affinity to other receptors, NR2B-selective drugs might have improved therapeutic efficiency and side effect profile. We aimed to compare the effects of (S)-ketamine and two different types of NR2B-selective antagonists on functional brain networks in rats, in order to find common circuits, where their effects intersect, and that might explain their antidepressant action. The experimental design comprised four parallel groups of rats (N = 37), each receiving (S)-Ketamine, CP-101,606, Ro 25-6981 or saline. After compound injection, we acquired resting-state functional magnetic resonance imaging time series. We used graph theoretical approach to calculate brain network properties. Ketamine and CP-101,606 diminished the global clustering coefficient and small-worldness index. At the nodal level, all compounds induced increased connectivity of the regions mediating reward and cognitive aspects of emotional processing, such as ventromedial prefrontal cortex, septal nuclei, and nucleus accumbens. The dorsal hippocampus and regions involved in sensory processing and aversion, such as superior and inferior colliculi, exhibited an opposite effect. The effects common to ketamine and NR2B-selective compounds were localized to the same brain regions as those reported in depression, but in the opposite direction. The upregulation of the reward circuitry might partially underlie the antidepressant and anti-anhedonic effects of the antagonists and could potentially serve as a translational imaging phenotype for testing putative antidepressants, especially those targeting the NR2B receptor subtype.

Reorganization of functional brain networks mediates the improvement of cognitive performance following real-time neurofeedback training of working memory.

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Zhang, Gaoyan; Yao, Li; Shen, Jiahui; Yang, Yihong; Zhao, Xiaojie

2015-05-01

Working memory (WM) is essential for individuals' cognitive functions. Neuroimaging studies indicated that WM fundamentally relied on a frontoparietal working memory network (WMN) and a cinguloparietal default mode network (DMN). Behavioral training studies demonstrated that the two networks can be modulated by WM training. Different from the behavioral training, our recent study used a real-time functional MRI (rtfMRI)-based neurofeedback method to conduct WM training, demonstrating that WM performance can be significantly improved after successfully upregulating the activity of the target region of interest (ROI) in the left dorsolateral prefrontal cortex (Zhang et al., [2013]: PloS One 8:e73735); however, the neural substrate of rtfMRI-based WM training remains unclear. In this work, we assessed the intranetwork and internetwork connectivity changes of WMN and DMN during the training, and their correlations with the change of brain activity in the target ROI as well as with the improvement of post-training behavior. Our analysis revealed an "ROI-network-behavior" correlation relationship underlying the rtfMRI training. Further mediation analysis indicated that the reorganization of functional brain networks mediated the effect of self-regulation of the target brain activity on the improvement of cognitive performance following the neurofeedback training. The results of this study enhance our understanding of the neural basis of real-time neurofeedback and suggest a new direction to improve WM performance by regulating the functional connectivity in the WM related networks. © 2014 Wiley Periodicals, Inc.

Detachment of Chain-Forming Neuroblasts by Fyn-Mediated Control of cell-cell Adhesion in the Postnatal Brain.

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Fujikake, Kazuma; Sawada, Masato; Hikita, Takao; Seto, Yayoi; Kaneko, Naoko; Herranz-Pérez, Vicente; Dohi, Natsuki; Homma, Natsumi; Osaga, Satoshi; Yanagawa, Yuchio; Akaike, Toshihiro; García-Verdugo, Jose Manuel; Hattori, Mitsuharu; Sobue, Kazuya; Sawamoto, Kazunobu

2018-05-09

In the rodent olfactory system, neuroblasts produced in the ventricular-subventricular zone of the postnatal brain migrate tangentially in chain-like cell aggregates toward the olfactory bulb (OB) through the rostral migratory stream (RMS). After reaching the OB, the chains are dissociated and the neuroblasts migrate individually and radially toward their final destination. The cellular and molecular mechanisms controlling cell-cell adhesion during this detachment remain unclear. Here we report that Fyn, a nonreceptor tyrosine kinase, regulates the detachment of neuroblasts from chains in the male and female mouse OB. By performing chemical screening and in vivo loss-of-function and gain-of-function experiments, we found that Fyn promotes somal disengagement from the chains and is involved in neuronal migration from the RMS into the granule cell layer of the OB. Fyn knockdown or Dab1 (disabled-1) deficiency caused p120-catenin to accumulate and adherens junction-like structures to be sustained at the contact sites between neuroblasts. Moreover, a Fyn and N-cadherin double-knockdown experiment indicated that Fyn regulates the N-cadherin-mediated cell adhesion between neuroblasts. These results suggest that the Fyn-mediated control of cell-cell adhesion is critical for the detachment of chain-forming neuroblasts in the postnatal OB. SIGNIFICANCE STATEMENT In the postnatal brain, newly born neurons (neuroblasts) migrate in chain-like cell aggregates toward their destination, where they are dissociated into individual cells and mature. The cellular and molecular mechanisms controlling the detachment of neuroblasts from chains are not understood. Here we show that Fyn, a nonreceptor tyrosine kinase, promotes the somal detachment of neuroblasts from chains, and that this regulation is critical for the efficient migration of neuroblasts to their destination. We further show that Fyn and Dab1 (disabled-1) decrease the cell-cell adhesion between chain-forming neuroblasts

Comprehensive analysis of the transcriptional profile of the Mediator complex across human cancer types.

Science.gov (United States)

Syring, Isabella; Klümper, Niklas; Offermann, Anne; Braun, Martin; Deng, Mario; Boehm, Diana; Queisser, Angela; von Mässenhausen, Anne; Brägelmann, Johannes; Vogel, Wenzel; Schmidt, Doris; Majores, Michael; Schindler, Anne; Kristiansen, Glen; Müller, Stefan C; Ellinger, Jörg; Shaikhibrahim, Zaki; Perner, Sven

2016-04-26

The Mediator complex is a key regulator of gene transcription and several studies demonstrated altered expressions of particular subunits in diverse human diseases, especially cancer. However a systematic study deciphering the transcriptional expression of the Mediator across different cancer entities is still lacking.We therefore performed a comprehensive in silico cancer vs. benign analysis of the Mediator complex subunits (MEDs) for 20 tumor entities using Oncomine datasets. The transcriptional expression profiles across almost all cancer entities showed differentially expressed MEDs as compared to benign tissue. Differential expression of MED8 in renal cell carcinoma (RCC) and MED12 in lung cancer (LCa) were validated and further investigated by immunohistochemical staining on tissue microarrays containing large numbers of specimen. MED8 in clear cell RCC (ccRCC) associated with shorter survival and advanced TNM stage and showed higher expression in metastatic than primary tumors. In vitro, siRNA mediated MED8 knockdown significantly impaired proliferation and motility in ccRCC cell lines, hinting at a role for MED8 to serve as a novel therapeutic target in ccRCC. Taken together, our Mediator complex transcriptome proved to be a valid tool for identifying cancer-related shifts in Mediator complex composition, revealing that MEDs do exhibit cancer specific transcriptional expression profiles.

Glymphatic system disruption as a mediator of brain trauma and chronic traumatic encephalopathy.

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Sullan, Molly J; Asken, Breton M; Jaffee, Michael S; DeKosky, Steven T; Bauer, Russell M

2018-01-01

Traumatic brain injury (TBI) is an increasingly important issue among veterans, athletes and the general public. Difficulties with sleep onset and maintenance are among the most commonly reported symptoms following injury, and sleep debt is associated with increased accumulation of beta amyloid (Aβ) and phosphorylated tau (p-tau) in the interstitial space. Recent research into the glymphatic system, a lymphatic-like metabolic clearance mechanism in the central nervous system (CNS) which relies on cerebrospinal fluid (CSF), interstitial fluid (ISF), and astrocytic processes, shows that clearance is potentiated during sleep. This system is damaged in the acute phase following mTBI, in part due to re-localization of aquaporin-4 channels away from astrocytic end feet, resulting in reduced potential for waste removal. Long-term consequences of chronic dysfunction within this system in the context of repetitive brain trauma and insomnia have not been established, but potentially provide one link in the explanatory chain connecting repetitive TBI with later neurodegeneration. Current research has shown p-tau deposition in perivascular spaces and along interstitial pathways in chronic traumatic encephalopathy (CTE), pathways related to glymphatic flow; these are the main channels by which metabolic waste is cleared. This review addresses possible links between mTBI-related damage to glymphatic functioning and physiological changes found in CTE, and proposes a model for the mediating role of sleep disruption in increasing the risk for developing CTE-related pathology and subsequent clinical symptoms following repetitive brain trauma. Copyright © 2017 Elsevier Ltd. All rights reserved.

Extracellular vesicle-mediated transfer of genetic information between the hematopoietic system and the brain in response to inflammation.

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Kirsten Ridder

2014-06-01

Full Text Available Mechanisms behind how the immune system signals to the brain in response to systemic inflammation are not fully understood. Transgenic mice expressing Cre recombinase specifically in the hematopoietic lineage in a Cre reporter background display recombination and marker gene expression in Purkinje neurons. Here we show that reportergene expression in neurons is caused by intercellular transfer of functional Cre recombinase messenger RNA from immune cells into neurons in the absence of cell fusion. In vitro purified secreted extracellular vesicles (EVs from blood cells contain Cre mRNA, which induces recombination in neurons when injected into the brain. Although Cre-mediated recombination events in the brain occur very rarely in healthy animals, their number increases considerably in different injury models, particularly under inflammatory conditions, and extend beyond Purkinje neurons to other neuronal populations in cortex, hippocampus, and substantia nigra. Recombined Purkinje neurons differ in their miRNA profile from their nonrecombined counterparts, indicating physiological significance. These observations reveal the existence of a previously unrecognized mechanism to communicate RNA-based signals between the hematopoietic system and various organs, including the brain, in response to inflammation.

Chemotaxis to cyclic AMP and folic acid is mediated by different G proteins in Dictyostelium discoideum

NARCIS (Netherlands)

Kesbeke, Fanja; Haastert, Peter J.M. van; Wit, René J.W. de; Snaar-Jagalska, B. Ewa

1990-01-01

Mutant Frigid A (fgdA) of Dictyostelium discoideum is defective in a functional Gα2 subunit of a G protein and is characterized by a complete blockade of the cyclic AMP-mediated sensory transduction steps, including cyclic AMP relay, chemotaxis and the cyclic GMP response. Folic acid-mediated

Cloning of the γ-aminobutyric acid (GABA) ρ1 cDNA: A GABA receptor subunit highly expressed in the retina

International Nuclear Information System (INIS)

Cutting, G.R.; Lu, Luo; Kasch, L.M.; Montrose-Rafizadeh, C.; Antonarakis, S.E.; Guggino, W.B.; Kazazian, H.H. Jr.; O'Hara, B.F.; Donovan, D.M.; Shimada, Shoichi; Uhl, G.R.

1991-01-01

Type A γ-aminobutyric acid (GABA A ) receptors are a family of ligand-gated chloride channels that are the major inhibitory neurotransmitter receptors in the nervous system. Molecular cloning has revealed diversity in the subunits that compose this heterooligomeric receptor, but each previously elucidated subunit displays amino acid similarity in conserved structural elements. The authors have used these highly conserved regions to identify additional members of this family by using the polymerase chain reaction (PCR). One PCR product was used to isolate a full-length cDNA from a human retina cDNA library. The mature protein predicted from this cDNA sequence is 458 amino acids long and displays between 30 and 38% amino acid similarity to the previously identified GABA A subunits. This gene is expressed primarily in the retina but transcripts are also detected in the brain, lung, and thymus. Injection of Xenopus oocytes with RNA transcribed in vitro produces a GABA-responsive chloride conductance and expression of the cDNA in COS cells yields GABA-displaceable muscimol binding. These features are consistent with our identification of a GABA subunit, GABA ρ 1 , with prominent retinal expression that increases the diversity and tissue specificity of this ligand-gated ion-channel receptor family

Brain docosahexaenoic acid uptake and metabolism.

Science.gov (United States)

Lacombe, R J Scott; Chouinard-Watkins, Raphaël; Bazinet, Richard P

2018-02-08

Docosahexaenoic acid (DHA) is the most abundant n-3 polyunsaturated fatty acid in the brain where it serves to regulate several important processes and, in addition, serves as a precursor to bioactive mediators. Given that the capacity of the brain to synthesize DHA locally is appreciably low, the uptake of DHA from circulating lipid pools is essential to maintaining homeostatic levels. Although, several plasma pools have been proposed to supply the brain with DHA, recent evidence suggests non-esterified-DHA and lysophosphatidylcholine-DHA are the primary sources. The uptake of DHA into the brain appears to be regulated by a number of complementary pathways associated with the activation and metabolism of DHA, and may provide mechanisms for enrichment of DHA within the brain. Following entry into the brain, DHA is esterified into and recycled amongst membrane phospholipids contributing the distribution of DHA in brain phospholipids. During neurotransmission and following brain injury, DHA is released from membrane phospholipids and converted to bioactive mediators which regulate signaling pathways important to synaptogenesis, cell survival, and neuroinflammation, and may be relevant to treating neurological diseases. In the present review, we provide a comprehensive overview of brain DHA metabolism, encompassing many of the pathways and key enzymatic regulators governing brain DHA uptake and metabolism. In addition, we focus on the release of non-esterified DHA and subsequent production of bioactive mediators and the evidence of their proposed activity within the brain. We also provide a brief review of the evidence from post-mortem brain analyses investigating DHA levels in the context of neurological disease and mood disorder, highlighting the current disparities within the field. Copyright © 2017. Published by Elsevier Ltd.

The beta subunit of casein kinase II

DEFF Research Database (Denmark)

Boldyreff, B; Piontek, K; Schmidt-Spaniol, I

1991-01-01

cDNAs encoding the beta subunit of pig and mouse CKII were isolated. The porcine cDNA was expressed as a fusion protein in Escherichia coli and used for the production of anti-CKII-beta subunit specific antibodies....

Characterization of fimbrial subunits from Bordetella species

NARCIS (Netherlands)

Mooi, F.R.; Heide, H.G.J. van der; Avest, A.R. ter; Welinder, K.G.; Livey, I.; Zeijst, B.A.M. van der; Gaastra, W.

Using antisera raised against serotype 2 and 3 fimbrial subunits from Bordetella pertussis, serologically related polypeptides were detected in Bordetella bronchiseptica, Bordetella parapertussis and Bordetella avium strains. The two B. pertussis fimbrial subunits, and three of the serologically

GABAA receptor B subunit expression in the superior frontal cortex of human alcoholics

International Nuclear Information System (INIS)

Buckley, S.T.; Dodd, P.R.

2001-01-01

Full text: Changes in GABA A receptor pharmacology can be ascribed to alterations in expression of specific GABA A receptor subunits. Ethanol is known to be a potent agonist of the GABA A receptor. Chronic abuse of alcohol in humans results in damage of selective brain regions such as the superior frontal cortex (SFC), leading to neuronal cell loss. Studies in our laboratory 1 and elsewhere 2 have shown differences in expression of a number of GABA A receptor subunits in chronic human alcoholism. This suggests that alterations in GABA A receptor composition may be involved in the pathogenesis of alcoholic brain damage. We analysed the expression of the β 1 ,β 2 and β 3 isoforms of the GABA A receptor by a competitive reverse transcription polymerase chain reaction (RT-PCR) technique, which utilised an internal standard (IS) for quantitation. 35 S-dATP was incorporated to enable visualisation of the PCR products. Human brain tissue was obtained at autopsy and stored in 0.32 M sucrose at -80 deg C. Total RNA was extracted from pathologically susceptible and spared regions, SFC and motor cortex respectively,of 22 control and 22 alcoholic patients. 1 μg of total RNA from each sample was co-amplified with 0.5 pg of IS and a ratio determined. A standard consisting of known amounts of β 1 cRNA titrated against 0.5 pg of IS enabled a standard curve to be generated for quantitation of each unknown sample. The samples were subjected to polyacrylamide gel electrophoresis and the dried gel exposed to a phosphorimager screen. Data analysis was performed using the ImageQuant program. Initial results indicate that there is a reduction in expression of all the β transcripts in alcoholics when compared with controls, which supports the hypothesis that the GABA A receptor is altered by alcohol abuse. Supported by NHMRC. Copyright (2001) Australian Neuroscience Society

Recruitment of Mediator Complex by Cell Type and Stage-Specific Factors Required for Tissue-Specific TAF Dependent Gene Activation in an Adult Stem Cell Lineage.

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Chenggang Lu

2015-12-01

Full Text Available Onset of terminal differentiation in adult stem cell lineages is commonly marked by robust activation of new transcriptional programs required to make the appropriate differentiated cell type(s. In the Drosophila male germ line stem cell lineage, the switch from proliferating spermatogonia to spermatocyte is accompanied by one of the most dramatic transcriptional changes in the fly, as over 1000 new transcripts turn on in preparation for meiosis and spermatid differentiation. Here we show that function of the coactivator complex Mediator is required for activation of hundreds of new transcripts in the spermatocyte program. Mediator appears to act in a sequential hierarchy, with the testis activating Complex (tMAC, a cell type specific form of the Mip/dREAM general repressor, required to recruit Mediator subunits to the chromatin, and Mediator function required to recruit the testis TAFs (tTAFs, spermatocyte specific homologs of subunits of TFIID. Mediator, tMAC and the tTAFs co-regulate expression of a major set of spermatid differentiation genes. The Mediator subunit Med22 binds the tMAC component Topi when the two are coexpressed in S2 cells, suggesting direct recruitment. Loss of Med22 function in spermatocytes causes meiosis I maturation arrest male infertility, similar to loss of function of the tMAC subunits or the tTAFs. Our results illuminate how cell type specific versions of the Mip/dREAM complex and the general transcription machinery cooperate to drive selective gene activation during differentiation in stem cell lineages.

(-)1-(Benzofuran-2-yl)-2-propylaminopentane, [(-)BPAP], a selective enhancer of the impulse propagation mediated release of catecholamines and serotonin in the brain.

Science.gov (United States)

Knoll, J; Yoneda, F; Knoll, B; Ohde, H; Miklya, I

1999-12-01

1. The brain constituents beta-phenylethylamine (PEA) and tryptamine enhance the impulse propagation mediated transmitter release (exocytosis) from the catecholaminergic and serotoninergic neurons in the brain ('catecholaminergic/serotoninergic activity enhancer, CAE/SAE, effect'). (-)Deprenyl (Selegiline) and (-)1-phenyl-2-propylaminopentane [(-)PPAP] are amphetamine derived CAE substances devoid of the catecholamine releasing property. 2. By changing the aromatic ring in PPAP we developed highly potent and selective CAE/SAE substances, structurally unrelated to the amphetamines. Out of 65 newly synthetized compounds, a tryptamine derived structure, (-)1-(benzofuran-2-yl)-2-propylaminopentane [(-)BPAP] was selected as a potential follower of (-)deprenyl in the clinic and as a reference compound for further analysis of the CAE/SAE mechanism in the mammalian brain. 3. (-)BPAP significantly enhanced in 0.18 micromol 1(-1) concentration the impulse propagation mediated release of [(3)H]-noradrenaline and [(3)H]-dopamine and in 36 nmol 1(-1) concentration the release of [(3)H]-serotonin from the isolated brain stem of rats. The amount of catecholamines and serotonin released from isolated discrete rat brain regions (dopamine from the striatum, substantia nigra and tuberculum olfactorium, noradrenaline from the locus coeruleus and serotonin from the raphe) enhanced significantly in the presence of 10(-12) - 10(-14) M (-)BPAP. BPAP protected cultured hippocampal neurons from the neurotoxic effect of beta-amyloid in 10(-14) M concentration. In rats (-)BPAP significantly enhanced the activity of the catecholaminergic and serotoninergic neurons in the brain 30 min after acute injection of 0.1 microg kg(-1) s.c. In the shuttle box, (-)BPAP in rats was about 130 times more potent than (-)deprenyl in antagonizing tetrabenazine induced inhibition of performance.

The PKA-C3 catalytic subunit is required in two pairs of interneurons for successful mating of Drosophila.

Science.gov (United States)

Cassar, Marlène; Sunderhaus, Elizabeth; Wentzell, Jill S; Kuntz, Sara; Strauss, Roland; Kretzschmar, Doris

2018-02-06

Protein kinase A (PKA) has been shown to play a role in a plethora of cellular processes ranging from development to memory formation. Its activity is mediated by the catalytic subunits whereby many species express several paralogs. Drosophila encodes three catalytic subunits (PKA-C1-3) and whereas PKA-C1 has been well studied, the functions of the other two subunits were unknown. PKA-C3 is the orthologue of mammalian PRKX/Pkare and they are structurally more closely related to each other than to other catalytic subunits within their species. PRKX is expressed in the nervous system in mice but its function is also unknown. We now show that the loss of PKA-C3 in Drosophila causes copulation defects, though the flies are active and show no defects in other courtship behaviours. This phenotype is specifically due to the loss of PKA-C3 because PKA-C1 cannot replace PKA-C3. PKA-C3 is expressed in two pairs of interneurons that send projections to the ventro-lateral protocerebrum and the mushroom bodies and that synapse onto motor neurons in the ventral nerve cord. Rescue experiments show that expression of PKA-C3 in these interneurons is sufficient for copulation, suggesting a role in relaying information from the sensory system to motor neurons to initiate copulation.

Differences in activity of cytochrome C oxidase in brain between sleep and wakefulness.

Science.gov (United States)

Nikonova, Elena V; Vijayasarathy, Camasamudram; Zhang, Lin; Cater, Jacqueline R; Galante, Raymond J; Ward, Stephen E; Avadhani, Narayan G; Pack, Allan I

2005-01-01

Increased mRNA level of subunit 1 cytochrome c oxidase (COXI) during wakefulness and after short-term sleep deprivation has been described in brain. We hypothesized that this might contribute to increased activity of cytochrome oxidase (COX) enzyme during wakefulness, as part of the mechanisms to provide sufficient amounts of adenosine triphosphate to meet increased neuronal energy demands. COX activity was measured in isolated mitochondria from different brain regions in groups of rats with 3 hours of spontaneous sleep, 3 hours of spontaneous wake, and 3 hours of sleep deprivation. The group with 3 hours of spontaneous wake was added to delineate the circadian component of changes in the enzyme activity. Northern blot analysis was performed to examine the mRNA levels of 2 subunits of the enzyme COXI and COXIV, encoded by mitochondrial and nuclear DNA, respectively. Laboratory of Biochemistry, Department of Animal Biology, and Center for Sleep and Respiratory Neurobiology, University of Pennsylvania. 2-month-old male Fischer rats (N = 21) implanted for polygraphic recording. For COX activity, there was a main effect by analysis of variance of experimental group (P sleep-deprived groups as compared to the sleep group. A main effect of brain region was also significant (P sleep. There is an increase in COX activity after both 3 hours of spontaneous wake and 3 hours of sleep deprivation as compared with 3 hours of spontaneous sleep in diverse brain regions, which could be, in part, explained by the increased levels of bigenomic transcripts of the enzyme. This likely contributes to increased adenosine triphosphate production during wakefulness. ADP, adenosine diphosphate; ATP, adenosine triphosphate; COXI, cytochrome c oxidase subunit 1 mRNA; COX, cytochrome c oxidase (protein); CREB, cyclic AMP response element binding protein; DNA, deoxyribonucleic acid; EDTA, ethylenediaminetetraacetic acid; EEG, electroencephalography; EMG, electromyography; GABP, GA binding

A multi-subunit Chlamydia vaccine inducing neutralizing antibodies and strong IFN-γ(+) CMI responses protects against a genital infection in minipigs

DEFF Research Database (Denmark)

Bøje, Sarah; Olsen, Anja Weinreich; Erneholm, Karin

2016-01-01

Chlamydia is the most widespread sexually transmitted bacterial disease and a prophylactic vaccine is highly needed. Ideally, this vaccine is required to induce a combined response of Th1 cell-mediated immune (CMI) response in concert with neutralizing antibodies. Using a novel Göttingen minipig...... animal model, we evaluated the immunogenicity and efficacy of a multi-subunit vaccine formulated in the strong Th1-inducing adjuvant CAF01. We evaluated a mixture of two fusion proteins (Hirep1 and CTH93) designed to promote either neutralizing antibodies or cell-mediated immunity, respectively. Hirep1...

Crossing the Blood-Brain Barrier: Recent Advances in Drug Delivery to the Brain.

Science.gov (United States)

Patel, Mayur M; Patel, Bhoomika M

2017-02-01

CNS disorders are on the rise despite advancements in our understanding of their pathophysiological mechanisms. A major hurdle to the treatment of these disorders is the blood-brain barrier (BBB), which serves as an arduous janitor to protect the brain. Many drugs are being discovered for CNS disorders, which, however fail to enter the market because of their inability to cross the BBB. This is a pronounced challenge for the pharmaceutical fraternity. Hence, in addition to the discovery of novel entities and drug candidates, scientists are also developing new formulations of existing drugs for brain targeting. Several approaches have been investigated to allow therapeutics to cross the BBB. As the molecular structure of the BBB is better elucidated, several key approaches for brain targeting include physiological transport mechanisms such as adsorptive-mediated transcytosis, inhibition of active efflux pumps, receptor-mediated transport, cell-mediated endocytosis, and the use of peptide vectors. Drug-delivery approaches comprise delivery from microspheres, biodegradable wafers, and colloidal drug-carrier systems (e.g., liposomes, nanoparticles, nanogels, dendrimers, micelles, nanoemulsions, polymersomes, exosomes, and quantum dots). The current review discusses the latest advancements in these approaches, with a major focus on articles published in 2015 and 2016. In addition, we also cover the alternative delivery routes, such as intranasal and convection-enhanced diffusion methods, and disruption of the BBB for brain targeting.

Malleable machines in transcription regulation: the mediator complex.

Directory of Open Access Journals (Sweden)

Agnes Tóth-Petróczy

2008-12-01

Full Text Available The Mediator complex provides an interface between gene-specific regulatory proteins and the general transcription machinery including RNA polymerase II (RNAP II. The complex has a modular architecture (Head, Middle, and Tail and cryoelectron microscopy analysis suggested that it undergoes dramatic conformational changes upon interactions with activators and RNAP II. These rearrangements have been proposed to play a role in the assembly of the preinitiation complex and also to contribute to the regulatory mechanism of Mediator. In analogy to many regulatory and transcriptional proteins, we reasoned that Mediator might also utilize intrinsically disordered regions (IDRs to facilitate structural transitions and transmit transcriptional signals. Indeed, a high prevalence of IDRs was found in various subunits of Mediator from both Saccharomyces cerevisiae and Homo sapiens, especially in the Tail and the Middle modules. The level of disorder increases from yeast to man, although in both organisms it significantly exceeds that of multiprotein complexes of a similar size. IDRs can contribute to Mediator's function in three different ways: they can individually serve as target sites for multiple partners having distinctive structures; they can act as malleable linkers connecting globular domains that impart modular functionality on the complex; and they can also facilitate assembly and disassembly of complexes in response to regulatory signals. Short segments of IDRs, termed molecular recognition features (MoRFs distinguished by a high protein-protein interaction propensity, were identified in 16 and 19 subunits of the yeast and human Mediator, respectively. In Saccharomyces cerevisiae, the functional roles of 11 MoRFs have been experimentally verified, and those in the Med8/Med18/Med20 and Med7/Med21 complexes were structurally confirmed. Although the Saccharomyces cerevisiae and Homo sapiens Mediator sequences are only weakly conserved, the

Kin28 regulates the transient association of Mediator with core promoters.

Science.gov (United States)

Jeronimo, Célia; Robert, François

2014-05-01

Mediator is an essential, broadly used eukaryotic transcriptional coactivator. How and what Mediator communicates from activators to RNA polymerase II (RNAPII) remains an open question. Here we performed genome-wide location profiling of Saccharomyces cerevisiae Mediator subunits. Mediator is not found at core promoters but rather occupies the upstream activating sequence, upstream of the pre-initiation complex. In the absence of Kin28 (CDK7) kinase activity or in cells in which the RNAPII C-terminal domain is mutated to replace Ser5 with alanine, however, Mediator accumulates at core promoters together with RNAPII. We propose that Mediator is released quickly from promoters after phosphorylation of Ser5 by Kin28 (CDK7), which also allows for RNAPII to escape from the promoter.

The functions of Mediator in Candida albicans support a role in shaping species-specific gene expression.

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Nathalie Uwamahoro

Full Text Available The Mediator complex is an essential co-regulator of RNA polymerase II that is conserved throughout eukaryotes. Here we present the first study of Mediator in the pathogenic fungus Candida albicans. We focused on the Middle domain subunit Med31, the Head domain subunit Med20, and Srb9/Med13 from the Kinase domain. The C. albicans Mediator shares some roles with model yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, such as functions in the response to certain stresses and the role of Med31 in the expression of genes regulated by the activator Ace2. The C. albicans Mediator also has additional roles in the transcription of genes associated with virulence, for example genes related to morphogenesis and gene families enriched in pathogens, such as the ALS adhesins. Consistently, Med31, Med20, and Srb9/Med13 contribute to key virulence attributes of C. albicans, filamentation, and biofilm formation; and ALS1 is a biologically relevant target of Med31 for development of biofilms. Furthermore, Med31 affects virulence of C. albicans in the worm infection model. We present evidence that the roles of Med31 and Srb9/Med13 in the expression of the genes encoding cell wall adhesins are different between S. cerevisiae and C. albicans: they are repressors of the FLO genes in S. cerevisiae and are activators of the ALS genes in C. albicans. This suggests that Mediator subunits regulate adhesion in a distinct manner between these two distantly related fungal species.

Transcription regulation by the Mediator complex.

Science.gov (United States)

Soutourina, Julie

2018-04-01

Alterations in the regulation of gene expression are frequently associated with developmental diseases or cancer. Transcription activation is a key phenomenon in the regulation of gene expression. In all eukaryotes, mediator of RNA polymerase II transcription (Mediator), a large complex with modular organization, is generally required for transcription by RNA polymerase II, and it regulates various steps of this process. The main function of Mediator is to transduce signals from the transcription activators bound to enhancer regions to the transcription machinery, which is assembled at promoters as the preinitiation complex (PIC) to control transcription initiation. Recent functional studies of Mediator with the use of structural biology approaches and functional genomics have revealed new insights into Mediator activity and its regulation during transcription initiation, including how Mediator is recruited to transcription regulatory regions and how it interacts and cooperates with PIC components to assist in PIC assembly. Novel roles of Mediator in the control of gene expression have also been revealed by showing its connection to the nuclear pore and linking Mediator to the regulation of gene positioning in the nuclear space. Clear links between Mediator subunits and disease have also encouraged studies to explore targeting of this complex as a potential therapeutic approach in cancer and fungal infections.

The cAMP-induced G protein subunits dissociation monitored in live Dictyostelium cells by BRET reveals two activation rates, a positive effect of caffeine and potential role of microtubules.

Science.gov (United States)

Tariqul Islam, A F M; Yue, Haicen; Scavello, Margarethakay; Haldeman, Pearce; Rappel, Wouter-Jan; Charest, Pascale G

2018-08-01

To study the dynamics and mechanisms controlling activation of the heterotrimeric G protein Gα2βγ in Dictyostelium in response to stimulation by the chemoattractant cyclic AMP (cAMP), we monitored the G protein subunit interaction in live cells using bioluminescence resonance energy transfer (BRET). We found that cAMP induces the cAR1-mediated dissociation of the G protein subunits to a similar extent in both undifferentiated and differentiated cells, suggesting that only a small number of cAR1 (as expressed in undifferentiated cells) is necessary to induce the full activation of Gα2βγ. In addition, we found that treating cells with caffeine increases the potency of cAMP-induced Gα2βγ activation; and that disrupting the microtubule network but not F-actin inhibits the cAMP-induced dissociation of Gα2βγ. Thus, microtubules are necessary for efficient cAR1-mediated activation of the heterotrimeric G protein. Finally, kinetics analyses of Gα2βγ subunit dissociation induced by different cAMP concentrations indicate that there are two distinct rates at which the heterotrimeric G protein subunits dissociate when cells are stimulated with cAMP concentrations above 500 nM versus only one rate at lower cAMP concentrations. Quantitative modeling suggests that the kinetics profile of Gα2βγ subunit dissociation results from the presence of both uncoupled and G protein pre-coupled cAR1 that have differential affinities for cAMP and, consequently, induce G protein subunit dissociation through different rates. We suggest that these different signaling kinetic profiles may play an important role in initial chemoattractant gradient sensing. Copyright © 2018 Elsevier Inc. All rights reserved.

The beta1 subunit of the Na,K-ATPase pump interacts with megalencephalic leucoencephalopathy with subcortical cysts protein 1 (MLC1) in brain astrocytes: new insights into MLC pathogenesis.

Science.gov (United States)

Brignone, Maria S; Lanciotti, Angela; Macioce, Pompeo; Macchia, Gianfranco; Gaetani, Matteo; Aloisi, Francesca; Petrucci, Tamara C; Ambrosini, Elena

2011-01-01

Megalencephalic leucoencephalopathy with subcortical cysts (MLC) is a rare congenital leucodystrophy caused by mutations in MLC1, a membrane protein of unknown function. MLC1 expression in astrocyte end-feet contacting blood vessels and meninges, along with brain swelling, fluid cysts and myelin vacuolation observed in MLC patients, suggests a possible role for MLC1 in the regulation of fluid and ion homeostasis and cellular volume changes. To identify MLC1 direct interactors and dissect the molecular pathways in which MLC1 is involved, we used NH2-MLC1 domain as a bait to screen a human brain library in a yeast two-hybrid assay. We identified the β1 subunit of the Na,K-ATPase pump as one of the interacting clones and confirmed it by pull-downs, co-fractionation assays and immunofluorescence stainings in human and rat astrocytes in vitro and in brain tissue. By performing ouabain-affinity chromatography on astrocyte and brain extracts, we isolated MLC1 and the whole Na,K-ATPase enzyme in a multiprotein complex that included Kir4.1, syntrophin and dystrobrevin. Because Na,K-ATPase is involved in intracellular osmotic control and volume regulation, we investigated the effect of hypo-osmotic stress on MLC1/Na,K-ATPase relationship in astrocytes. We found that hypo-osmotic conditions increased MLC1 membrane expression and favoured MLC1/Na,K-ATPase-β1 association. Moreover, hypo-osmosis induced astrocyte swelling and the reversible formation of endosome-derived vacuoles, where the two proteins co-localized. These data suggest that through its interaction with Na,K-ATPase, MLC1 is involved in the control of intracellular osmotic conditions and volume regulation in astrocytes, opening new perspectives for understanding the pathological mechanisms of MLC disease.

Distinct α subunit variations of the hypothalamic GABAA receptor triplets (αβγ are linked to hibernating state in hamsters

Directory of Open Access Journals (Sweden)

Alò Raffaella

2010-09-01

Full Text Available Abstract Background The structural arrangement of the γ-aminobutyric acid type A receptor (GABAAR is known to be crucial for the maintenance of cerebral-dependent homeostatic mechanisms during the promotion of highly adaptive neurophysiological events of the permissive hibernating rodent, i.e the Syrian golden hamster. In this study, in vitro quantitative autoradiography and in situ hybridization were assessed in major hypothalamic nuclei. Reverse Transcription Reaction-Polymerase chain reaction (RT-PCR tests were performed for specific GABAAR receptor subunit gene primers synthases of non-hibernating (NHIB and hibernating (HIB hamsters. Attempts were made to identify the type of αβγ subunit combinations operating during the switching ON/OFF of neuronal activities in some hypothalamic nuclei of hibernators. Results Both autoradiography and molecular analysis supplied distinct expression patterns of all α subunits considered as shown by a strong (p 1 ratio (over total α subunits considered in the present study in the medial preoptic area (MPOA and arcuate nucleus (Arc of NHIBs with respect to HIBs. At the same time α2 subunit levels proved to be typical of periventricular nucleus (Pe and Arc of HIB, while strong α4 expression levels were detected during awakening state in the key circadian hypothalamic station, i.e. the suprachiasmatic nucleus (Sch; 60%. Regarding the other two subunits (β and γ, elevated β3 and γ3 mRNAs levels mostly characterized MPOA of HIBs, while prevalently elevated expression concentrations of the same subunits were also typical of Sch, even though this time during the awakening state. In the case of Arc, notably elevated levels were obtained for β3 and γ2 during hibernating conditions. Conclusion We conclude that different αβγ subunits are operating as major elements either at the onset of torpor or during induction of the arousal state in the Syrian golden hamster. The identification of a brain regional

Peptide carrier-mediated non-covalent delivery of unmodified cisplatin, methotrexate and other agents via intravenous route to the brain.

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Gobinda Sarkar

Full Text Available BACKGROUND: Rapid pre-clinical evaluation of chemotherapeutic agents against brain cancers and other neurological disorders remains largely unattained due to the presence of the blood-brain barrier (BBB, which limits transport of most therapeutic compounds to the brain. A synthetic peptide carrier, K16ApoE, was previously developed that enabled transport of target proteins to the brain by mimicking a ligand-receptor system. The peptide carrier was found to generate transient BBB permeability, which was utilized for non-covalent delivery of cisplatin, methotrexate and other compounds to the brain. APPROACH: Brain delivery of the chemotherapeutics and other agents was achieved either by injecting the carrier peptide and the drugs separately or as a mixture, to the femoral vein. A modification of the method comprised injection of K16ApoE pre-mixed with cetuximab, followed by injection of a 'small-molecule' drug. PRINCIPAL FINDINGS: Seven-of-seven different small molecules were successfully delivered to the brain via K16ApoE. Depending on the method, brain uptake with K16ApoE was 0.72-1.1% for cisplatin and 0.58-0.92% for methotrexate (34-50-fold and 54-92 fold greater for cisplatin and methotrexate, respectively, with K16ApoE than without. Visually intense brain-uptake of Evans Blue, Light Green SF and Crocein scarlet was also achieved. Direct intracranial injection of EB show locally restricted distribution of the dye in the brain, whereas K16ApoE-mediated intravenous injection of EB resulted in the distribution of the dye throughout the brain. Experiments with insulin suggest that ligand-receptor signaling intrinsic to the BBB provides a natural means for passive transport of some molecules across the BBB. SIGNIFICANCE: The results suggest that the carrier peptide can non-covalently transport various chemotherapeutic agents to the brain. Thus, the method offers an avenue for pre-clinical evaluation of various small and large therapeutic molecules

Neonatal seizures alter NMDA glutamate receptor GluN2A and 3A subunit expression and function in hippocampal CA1 neurons

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Zhou, Chengwen; Sun, Hongyu; Klein, Peter M.; Jensen, Frances E.

2015-01-01

Neonatal seizures are commonly caused by hypoxic and/or ischemic injury during birth and can lead to long-term epilepsy and cognitive deficits. In a rodent hypoxic seizure (HS) model, we have previously demonstrated a critical role for seizure-induced enhancement of the AMPA subtype of glutamate receptor (GluA) in epileptogenesis and cognitive consequences, in part due to GluA maturational upregulation of expression. Similarly, as the expression and function of the N-Methyl-D-aspartate (NMDA) subtype of glutamate receptor (GluN) is also developmentally controlled, we examined how early life seizures during the critical period of synaptogenesis could modify GluN development and function. In a postnatal day (P)10 rat model of neonatal seizures, we found that seizures could alter GluN2/3 subunit composition of GluNs and physiological function of synaptic GluNs. In hippocampal slices removed from rats within 48–96 h following seizures, the amplitudes of synaptic GluN-mediated evoked excitatory postsynaptic currents (eEPSCs) were elevated in CA1 pyramidal neurons. Moreover, GluN eEPSCs showed a decreased sensitivity to GluN2B selective antagonists and decreased Mg2+ sensitivity at negative holding potentials, indicating a higher proportion of GluN2A and GluN3A subunit function, respectively. These physiological findings were accompanied by a concurrent increase in GluN2A phosphorylation and GluN3A protein. These results suggest that altered GluN function and expression could potentially contribute to future epileptogenesis following neonatal seizures, and may represent potential therapeutic targets for the blockade of future epileptogenesis in the developing brain. PMID:26441533

Implications of astrocytes in mediating the protective effects of Selective Estrogen Receptor Modulators upon brain damage

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George E. Barreto

2015-04-01

Full Text Available Selective Estrogen Receptor Modulators (SERMs are steroidal or non-steroidal compounds that are already used in clinical practice for the treatment of breast cancer, osteoporosis and menopausal symptoms. While SERMs actions in the breast, bone, and uterus have been well characterized, their actions in the brain are less well understood. Previous works have demonstrated the beneficial effects of SERMs in different chronic neurodegenerative diseases like Alzheimer, Parkinson’s disease and Multiple sclerosis, as well as acute degeneration as stroke and traumatic brain injury. Moreover, these compounds exhibit similar protective actions as those of estradiol in the Central Nervous System, overt any secondary effect. For these reasons, in the past few years, there has been a growing interest in the neuroprotective effects exerted directly or indirectly by SERMs in the SNC. In this context, astrocytes play an important role in the maintenance of brain metabolism, and antioxidant support to neurons, thus indicating that better protection of astrocytes are an important asset targeting neuronal protection. Moreover, various clinical and experimental studies have reported that astrocytes are essential for the neuroprotective effects of SERMs during neuronal injuries, as these cells express different estrogen receptors in cell membrane, demonstrating that part of SERMs effects upon injury may be mediated by astrocytes. The present work highlights the current evidence on the protective mechanisms of SERMs, such as tamoxifen and raloxifene, in the SNC, and their modulation of astrocytic properties as promising therapeutic targets during brain damage.

Immunopathogenesis of brain abscess

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Kielian Tammy

2004-08-01

Full Text Available Abstract Brain abscess represents a significant medical problem despite recent advances made in detection and therapy. Due to the emergence of multi-drug resistant strains and the ubiquitous nature of bacteria, the occurrence of brain abscess is likely to persist. Our laboratory has developed a mouse experimental brain abscess model allowing for the identification of key mediators in the CNS anti-bacterial immune response through the use of cytokine and chemokine knockout mice. Studies of primary microglia and astrocytes from neonatal mice have revealed that S. aureus, one of the main etiologic agents of brain abscess in humans, is a potent stimulus for proinflammatory mediator production. Recent evidence from our laboratory indicates that Toll-like receptor 2 plays a pivotal role in the recognition of S. aureus and its cell wall product peptidoglycan by glia, although other receptors also participate in the recognition event. This review will summarize the consequences of S. aureus on CNS glial activation and the resultant neuroinflammatory response in the experimental brain abscess model.

The Subunit Principle in Scar Face Revision.

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Elshahat, Ahmed; Lashin, Riham

2017-06-01

Facial scaring is considered one of the most difficult cosmetic problems for any plastic surgeon to solve. The condition is more difficult if the direction of the scar is not parallel to relaxed skin tension lines. Attempts to manage this difficult situation included revisions using geometric designs, Z plasties or W plasties to camouflage the straight line visible scaring. The use of long-lasting resorbable sutures was tried too. Recently, the use of botulinum toxin during revision improved the results. Fractional CO2 lasers, microfat grafts, and platelet-rich plasma were added to the armamentarium. The scar is least visible if placed in the junction between the facial subunits. The aim of this study is to investigate the use of the subunit principle to improve the results of scar revision. Four patients were included in this study. Tissue expansion of the intact part of the subunit allowed shifting the scar to the junction between the affected subunit and the adjacent one. Tissue expansion, delivery of the expanders, and advancement of the flaps were successful in all patients. The fact that this is a 2-stage procedure and sacrifices some of the intact skin from the affected facial subunit, makes this technique reserved to patients with ugly facial scars who are ambitious to improve their appearance.

Brain function in carriers of a genome-wide supported bipolar disorder variant.

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Erk, Susanne; Meyer-Lindenberg, Andreas; Schnell, Knut; Opitz von Boberfeld, Carola; Esslinger, Christine; Kirsch, Peter; Grimm, Oliver; Arnold, Claudia; Haddad, Leila; Witt, Stephanie H; Cichon, Sven; Nöthen, Markus M; Rietschel, Marcella; Walter, Henrik

2010-08-01

The neural abnormalities underlying genetic risk for bipolar disorder, a severe, common, and highly heritable psychiatric condition, are largely unknown. An opportunity to define these mechanisms is provided by the recent discovery, through genome-wide association, of a single-nucleotide polymorphism (rs1006737) strongly associated with bipolar disorder within the CACNA1C gene, encoding the alpha subunit of the L-type voltage-dependent calcium channel Ca(v)1.2. To determine whether the genetic risk associated with rs1006737 is mediated through hippocampal function. Functional magnetic resonance imaging study. University hospital. A total of 110 healthy volunteers of both sexes and of German descent in the Hardy-Weinberg equilibrium for rs1006737. Blood oxygen level-dependent signal during an episodic memory task and behavioral and psychopathological measures. Using an intermediate phenotype approach, we show that healthy carriers of the CACNA1C risk variant exhibit a pronounced reduction of bilateral hippocampal activation during episodic memory recall and diminished functional coupling between left and right hippocampal regions. Furthermore, risk allele carriers exhibit activation deficits of the subgenual anterior cingulate cortex, a region repeatedly associated with affective disorders and the mediation of adaptive stress-related responses. The relevance of these findings for affective disorders is supported by significantly higher psychopathology scores for depression, anxiety, obsessive-compulsive thoughts, interpersonal sensitivity, and neuroticism in risk allele carriers, correlating negatively with the observed regional brain activation. Our data demonstrate that rs1006737 or genetic variants in linkage disequilibrium with it are functional in the human brain and provide a neurogenetic risk mechanism for bipolar disorder backed by genome-wide evidence.

Theory of mind mediates the prospective relationship between abnormal social brain network morphology and chronic behavior problems after pediatric traumatic brain injury.

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Ryan, Nicholas P; Catroppa, Cathy; Beare, Richard; Silk, Timothy J; Crossley, Louise; Beauchamp, Miriam H; Yeates, Keith Owen; Anderson, Vicki A

2016-04-01

Childhood and adolescence coincide with rapid maturation and synaptic reorganization of distributed neural networks that underlie complex cognitive-affective behaviors. These regions, referred to collectively as the 'social brain network' (SBN) are commonly vulnerable to disruption from pediatric traumatic brain injury (TBI); however, the mechanisms that link morphological changes in the SBN to behavior problems in this population remain unclear. In 98 children and adolescents with mild to severe TBI, we acquired 3D T1-weighted MRIs at 2-8 weeks post-injury. For comparison, 33 typically developing controls of similar age, sex and education were scanned. All participants were assessed on measures of Theory of Mind (ToM) at 6 months post-injury and parents provided ratings of behavior problems at 24-months post-injury. Severe TBI was associated with volumetric reductions in the overall SBN package, as well as regional gray matter structural change in multiple component regions of the SBN. When compared with TD controls and children with milder injuries, the severe TBI group had significantly poorer ToM, which was associated with more frequent behavior problems and abnormal SBN morphology. Mediation analysis indicated that impaired theory of mind mediated the prospective relationship between abnormal SBN morphology and more frequent chronic behavior problems. Our findings suggest that sub-acute alterations in SBN morphology indirectly contribute to long-term behavior problems via their influence on ToM. Volumetric change in the SBN and its putative hub regions may represent useful imaging biomarkers for prediction of post-acute social cognitive impairment, which may in turn elevate risk for chronic behavior problems. © The Author (2016). Published by Oxford University Press. For Permissions, please email: journals.permissions@oup.com.

The GABAA receptor α and β subunits but not the density of muscimol binding sites are altered in the auditory-linguistic association cortex of subjects with schizophrenia

International Nuclear Information System (INIS)

Farnbach-Pralong, D.; Bradbury, R.; Tomaskovic, E.; Copolov, D.; Dean, B.

1998-01-01

Full text: An increase in the density of postsynaptic GABA A receptors has recently been reported in the prefrontal cortex of subjects with schizophrenia. This increase has been hypothesised to represent an up-regulation in response a decrease in the density of GABAergic interneurons. In order to determine whether the GABA A receptor is also altered in the auditory-linguistic association cortex of the schizophrenic brain, we used quantitative autoradiography to measure the density of that receptor in tissue obtained at autopsy from 20 control subjects and 20 subjects with schizophrenia matched for sex and age. The density of GABA A receptors was measured as the difference in the binding of the specific ligand [ 3 H]muscimol (100 nM) in the presence or in the absence of 10 5 M SR95531. There was no significant difference in the density of [ 3 H]muscimol binding between tissue from schizophrenic (554.9±20,5 fmol/mg TE) and non-schizophrenic (580.1±26.2 fmol/mg TE) subjects. The abundance of the α and β subunits of the GABA A receptor was also measured in particulate membranes prepared from tissue from 6 control and 6 schizophrenic subjects using Western blots. Detection with monoclonal antibodies and chemiluminescence showed that in tissue from control subjects, there was a significant correlation between the levels of α and β subunits (r=0.817, p=0.047). However, there was no such correlation in tissue from schizophrenic subjects (r=0.265, p=0.61), where in 2 subjects large levels of β-subunit were not matched by similar levels of α subunit. These preliminary results suggest mat there may be a failure for up-regulated GABA A receptor subunits to assemble into functional receptors in this brain region for some subjects with schizophrenia. Copyright (1998) Australian Neuroscience Society

Mapping glucose-mediated gut-to-brain signalling pathways in humans.

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Little, Tanya J; McKie, Shane; Jones, Richard B; D'Amato, Massimo; Smith, Craig; Kiss, Orsolya; Thompson, David G; McLaughlin, John T

2014-08-01

Previous fMRI studies have demonstrated that glucose decreases the hypothalamic BOLD response in humans. However, the mechanisms underlying the CNS response to glucose have not been defined. We recently demonstrated that the slowing of gastric emptying by glucose is dependent on activation of the gut peptide cholecystokinin (CCK1) receptor. Using physiological functional magnetic resonance imaging this study aimed to determine the whole brain response to glucose, and whether CCK plays a central role. Changes in blood oxygenation level-dependent (BOLD) signal were monitored using fMRI in 12 healthy subjects following intragastric infusion (250ml) of: 1M glucose+predosing with dexloxiglumide (CCK1 receptor antagonist), 1M glucose+placebo, or 0.9% saline (control)+placebo, in a single-blind, randomised fashion. Gallbladder volume, blood glucose, insulin, and GLP-1 and CCK concentrations were determined. Hunger, fullness and nausea scores were also recorded. Intragastric glucose elevated plasma glucose, insulin, and GLP-1, and reduced gall bladder volume (an in vivo assay for CCK secretion). Glucose decreased BOLD signal, relative to saline, in the brainstem and hypothalamus as well as the cerebellum, right occipital cortex, putamen and thalamus. The timing of the BOLD signal decrease was negatively correlated with the rise in blood glucose and insulin levels. The glucose+dex arm highlighted a CCK1-receptor dependent increase in BOLD signal only in the motor cortex. Glucose induces site-specific differences in BOLD response in the human brain; the brainstem and hypothalamus show a CCK1 receptor-independent reduction which is likely to be mediated by a circulatory effect of glucose and insulin, whereas the motor cortex shows an early dexloxiglumide-reversible increase in signal, suggesting a CCK1 receptor-dependent neural pathway. Copyright © 2014. Published by Elsevier Inc.

Mapping glucose-mediated gut-to-brain signalling pathways in humans☆

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Little, Tanya J.; McKie, Shane; Jones, Richard B.; D'Amato, Massimo; Smith, Craig; Kiss, Orsolya; Thompson, David G.; McLaughlin, John T.

2014-01-01

Objectives Previous fMRI studies have demonstrated that glucose decreases the hypothalamic BOLD response in humans. However, the mechanisms underlying the CNS response to glucose have not been defined. We recently demonstrated that the slowing of gastric emptying by glucose is dependent on activation of the gut peptide cholecystokinin (CCK1) receptor. Using physiological functional magnetic resonance imaging this study aimed to determine the whole brain response to glucose, and whether CCK plays a central role. Experimental design Changes in blood oxygenation level-dependent (BOLD) signal were monitored using fMRI in 12 healthy subjects following intragastric infusion (250 ml) of: 1 M glucose + predosing with dexloxiglumide (CCK1 receptor antagonist), 1 M glucose + placebo, or 0.9% saline (control) + placebo, in a single-blind, randomised fashion. Gallbladder volume, blood glucose, insulin, and GLP-1 and CCK concentrations were determined. Hunger, fullness and nausea scores were also recorded. Principal observations Intragastric glucose elevated plasma glucose, insulin, and GLP-1, and reduced gall bladder volume (an in vivo assay for CCK secretion). Glucose decreased BOLD signal, relative to saline, in the brainstem and hypothalamus as well as the cerebellum, right occipital cortex, putamen and thalamus. The timing of the BOLD signal decrease was negatively correlated with the rise in blood glucose and insulin levels. The glucose + dex arm highlighted a CCK1-receptor dependent increase in BOLD signal only in the motor cortex. Conclusions Glucose induces site-specific differences in BOLD response in the human brain; the brainstem and hypothalamus show a CCK1 receptor-independent reduction which is likely to be mediated by a circulatory effect of glucose and insulin, whereas the motor cortex shows an early dexloxiglumide-reversible increase in signal, suggesting a CCK1 receptor-dependent neural pathway. PMID:24685436

Double dissociation of spike timing-dependent potentiation and depression by subunit-preferring NMDA receptor antagonists in mouse barrel cortex.

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Banerjee, Abhishek; Meredith, Rhiannon M; Rodríguez-Moreno, Antonio; Mierau, Susanna B; Auberson, Yves P; Paulsen, Ole

2009-12-01

Spike timing-dependent plasticity (STDP) is a strong candidate for an N-methyl-D-aspartate (NMDA) receptor-dependent form of synaptic plasticity that could underlie the development of receptive field properties in sensory neocortices. Whilst induction of timing-dependent long-term potentiation (t-LTP) requires postsynaptic NMDA receptors, timing-dependent long-term depression (t-LTD) requires the activation of presynaptic NMDA receptors at layer 4-to-layer 2/3 synapses in barrel cortex. Here we investigated the developmental profile of t-LTD at layer 4-to-layer 2/3 synapses of mouse barrel cortex and studied their NMDA receptor subunit dependence. Timing-dependent LTD emerged in the first postnatal week, was present during the second week and disappeared in the adult, whereas t-LTP persisted in adulthood. An antagonist at GluN2C/D subunit-containing NMDA receptors blocked t-LTD but not t-LTP. Conversely, a GluN2A subunit-preferring antagonist blocked t-LTP but not t-LTD. The GluN2C/D subunit requirement for t-LTD appears to be synapse specific, as GluN2C/D antagonists did not block t-LTD at horizontal cross-columnar layer 2/3-to-layer 2/3 synapses, which was blocked by a GluN2B antagonist instead. These data demonstrate an NMDA receptor subunit-dependent double dissociation of t-LTD and t-LTP mechanisms at layer 4-to-layer 2/3 synapses, and suggest that t-LTD is mediated by distinct molecular mechanisms at different synapses on the same postsynaptic neuron.

Epac Signaling Is Required for Cocaine-Induced Change in AMPA Receptor Subunit Composition in the Ventral Tegmental Area.

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Liu, Xiaojie; Chen, Yao; Tong, Jiaqing; Reynolds, Ashley M; Proudfoot, Sarah C; Qi, Jinshun; Penzes, Peter; Lu, Youming; Liu, Qing-Song

2016-04-27

Exchange protein directly activated by cAMP (Epac) and protein kinase A (PKA) are intracellular receptors for cAMP. Although PKA and its downstream effectors have been studied extensively in the context of drug addiction, whether and how Epac regulates cellular and behavioral effects of drugs of abuse remain essentially unknown. Epac is known to regulate AMPA receptor (AMPAR) trafficking. Previous studies have shown that a single cocaine exposure in vivo leads to an increase in GluA2-lacking AMPARs in dopamine neurons of the ventral tegmental area (VTA). We tested the hypothesis that Epac mediates cocaine-induced changes in AMPAR subunit composition in the VTA. We report that a single cocaine injection in vivo in wild-type mice leads to inward rectification of EPSCs and renders EPSCs sensitive to a GluA2-lacking AMPAR blocker in VTA dopamine neurons. The cocaine-induced increase in GluA2-lacking AMPARs was absent in Epac2-deficient mice but not in Epac1-deficient mice. In addition, activation of Epac with the selective Epac agonist 8-CPT-2Me-cAMP (8-CPT) recapitulated the cocaine-induced increase in GluA2-lacking AMPARs, and the effects of 8-CPT were mediated by Epac2. We also show that conditioned place preference to cocaine was impaired in Epac2-deficient mice and in mice in which Epac2 was knocked down in the VTA but was not significantly altered in Epac1-deficient mice. Together, these results suggest that Epac2 is critically involved in the cocaine-induced change in AMPAR subunit composition and drug-cue associative learning. Addictive drugs, such as cocaine, induce long-lasting adaptions in the reward circuits of the brain. A single intraperitoneal injection of cocaine leads to changes in the composition and property of the AMPAR that carries excitatory inputs to dopamine neurons. Here, we provide evidence that exchange protein directly activated by cAMP (Epac), a cAMP sensor protein, is required for the cocaine-induced changes of the AMPAR. We found that the

What will this do to me and my brain? Ethical issues in brain-to-brain interfacing

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Elisabeth eHildt

2015-02-01

Full Text Available For several years now, brain-computer interfaces (BCIs in which brain signals are used to navigate a computer, a prostheses or a technical device, have been developed in various experimental contexts (Wolpaw & Wolpaw 2012; Grübler & Hildt 2014. Researchers have recently taken the next step and run experiments based on connections between two brains. These so-called brain-to-brain interfaces (abbreviation: BBIs or BTBIs involve not only a BCI component deriving information from a brain and sending it to a computer, but also a computer-brain interface (CBI component delivering information to another brain. What results is technology-mediated brain-to-brain communication (B2B communication, i.e. direct communication between two brains that does not involve any activity of the peripheral nervous system. In what follows, ethical issues that arise in neural interfacing will be discussed after a short introduction to recent BBI experiments. In this, the focus will be on the implications BBIs may have on the individual at the CBI side of the BBI, i.e. on the recipient.

Conscious brain-to-brain communication in humans using non-invasive technologies.

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Grau, Carles; Ginhoux, Romuald; Riera, Alejandro; Nguyen, Thanh Lam; Chauvat, Hubert; Berg, Michel; Amengual, Julià L; Pascual-Leone, Alvaro; Ruffini, Giulio

2014-01-01

Human sensory and motor systems provide the natural means for the exchange of information between individuals, and, hence, the basis for human civilization. The recent development of brain-computer interfaces (BCI) has provided an important element for the creation of brain-to-brain communication systems, and precise brain stimulation techniques are now available for the realization of non-invasive computer-brain interfaces (CBI). These technologies, BCI and CBI, can be combined to realize the vision of non-invasive, computer-mediated brain-to-brain (B2B) communication between subjects (hyperinteraction). Here we demonstrate the conscious transmission of information between human brains through the intact scalp and without intervention of motor or peripheral sensory systems. Pseudo-random binary streams encoding words were transmitted between the minds of emitter and receiver subjects separated by great distances, representing the realization of the first human brain-to-brain interface. In a series of experiments, we established internet-mediated B2B communication by combining a BCI based on voluntary motor imagery-controlled electroencephalographic (EEG) changes with a CBI inducing the conscious perception of phosphenes (light flashes) through neuronavigated, robotized transcranial magnetic stimulation (TMS), with special care taken to block sensory (tactile, visual or auditory) cues. Our results provide a critical proof-of-principle demonstration for the development of conscious B2B communication technologies. More fully developed, related implementations will open new research venues in cognitive, social and clinical neuroscience and the scientific study of consciousness. We envision that hyperinteraction technologies will eventually have a profound impact on the social structure of our civilization and raise important ethical issues.

Conscious brain-to-brain communication in humans using non-invasive technologies.

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Carles Grau

Full Text Available Human sensory and motor systems provide the natural means for the exchange of information between individuals, and, hence, the basis for human civilization. The recent development of brain-computer interfaces (BCI has provided an important element for the creation of brain-to-brain communication systems, and precise brain stimulation techniques are now available for the realization of non-invasive computer-brain interfaces (CBI. These technologies, BCI and CBI, can be combined to realize the vision of non-invasive, computer-mediated brain-to-brain (B2B communication between subjects (hyperinteraction. Here we demonstrate the conscious transmission of information between human brains through the intact scalp and without intervention of motor or peripheral sensory systems. Pseudo-random binary streams encoding words were transmitted between the minds of emitter and receiver subjects separated by great distances, representing the realization of the first human brain-to-brain interface. In a series of experiments, we established internet-mediated B2B communication by combining a BCI based on voluntary motor imagery-controlled electroencephalographic (EEG changes with a CBI inducing the conscious perception of phosphenes (light flashes through neuronavigated, robotized transcranial magnetic stimulation (TMS, with special care taken to block sensory (tactile, visual or auditory cues. Our results provide a critical proof-of-principle demonstration for the development of conscious B2B communication technologies. More fully developed, related implementations will open new research venues in cognitive, social and clinical neuroscience and the scientific study of consciousness. We envision that hyperinteraction technologies will eventually have a profound impact on the social structure of our civilization and raise important ethical issues.

The Mediator co-activator complex regulates Ty1 retromobility by controlling the balance between Ty1i and Ty1 promoters.

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Salinero, Alicia C; Knoll, Elisabeth R; Zhu, Z Iris; Landsman, David; Curcio, M Joan; Morse, Randall H

2018-02-01

The Ty1 retrotransposons present in the genome of Saccharomyces cerevisiae belong to the large class of mobile genetic elements that replicate via an RNA intermediary and constitute a significant portion of most eukaryotic genomes. The retromobility of Ty1 is regulated by numerous host factors, including several subunits of the Mediator transcriptional co-activator complex. In spite of its known function in the nucleus, previous studies have implicated Mediator in the regulation of post-translational steps in Ty1 retromobility. To resolve this paradox, we systematically examined the effects of deleting non-essential Mediator subunits on the frequency of Ty1 retromobility and levels of retromobility intermediates. Our findings reveal that loss of distinct Mediator subunits alters Ty1 retromobility positively or negatively over a >10,000-fold range by regulating the ratio of an internal transcript, Ty1i, to the genomic Ty1 transcript. Ty1i RNA encodes a dominant negative inhibitor of Ty1 retromobility that blocks virus-like particle maturation and cDNA synthesis. These results resolve the conundrum of Mediator exerting sweeping control of Ty1 retromobility with only minor effects on the levels of Ty1 genomic RNA and the capsid protein, Gag. Since the majority of characterized intrinsic and extrinsic regulators of Ty1 retromobility do not appear to effect genomic Ty1 RNA levels, Mediator could play a central role in integrating signals that influence Ty1i expression to modulate retromobility.

Efficient expression of functional (α6β22β3 AChRs in Xenopus oocytes from free subunits using slightly modified α6 subunits.

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Carson Kai-Kwong Ley

Full Text Available Human (α6β2(α4β2β3 nicotinic acetylcholine receptors (AChRs are essential for addiction to nicotine and a target for drug development for smoking cessation. Expressing this complex AChR is difficult, but has been achieved using subunit concatamers. In order to determine what limits expression of α6* AChRs and to efficiently express α6* AChRs using free subunits, we investigated expression of the simpler (α6β22β3 AChR. The concatameric form of this AChR assembles well, but is transported to the cell surface inefficiently. Various chimeras of α6 with the closely related α3 subunit increased expression efficiency with free subunits and produced pharmacologically equivalent functional AChRs. A chimera in which the large cytoplasmic domain of α6 was replaced with that of α3 increased assembly with β2 subunits and transport of AChRs to the oocyte surface. Another chimera replacing the unique methionine 211 of α6 with leucine found at this position in transmembrane domain 1 of α3 and other α subunits increased assembly of mature subunits containing β3 subunits within oocytes. Combining both α3 sequences in an α6 chimera increased expression of functional (α6β22β3 AChRs to 12-fold more than with concatamers. This is pragmatically useful, and provides insights on features of α6 subunit structure that limit its expression in transfected cells.

V-ATPase-mediated granular acidification is regulated by the V-ATPase accessory subunit Ac45 in POMC-producing cells.

NARCIS (Netherlands)

Jansen, E.J.S.; Hafmans, T.G.M.; Martens, G.J.

2010-01-01

The vacuolar (H(+))-ATPase (V-ATPase) is an important proton pump, and multiple critical cell-biological processes depend on the proton gradient provided by the pump. Yet, the mechanism underlying the control of the V-ATPase is still elusive but has been hypothesized to involve an accessory subunit

Age-dependent modifications of AMPA receptor subunit expression levels and related cognitive effects in 3xTg-AD mice

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Pamela eCantanelli

2014-08-01

Full Text Available GluA1, GluA2, GluA3, and GluA4 are the constitutive subunits of AMPA receptors (AMPARs, the major mediators of fast excitatory transmission in the mammalian central nervous system. Most AMPARs are Ca2+-impermeable because of the presence of the GluA2 subunit. GluA2 mRNA undergoes an editing process that results in a Q to R substitution, a key factor in the regulation of AMPAR Ca2+-permeability. AMPARs lacking GluA2 or containing the unedited subunit are permeable to Ca2+ and Zn2+. The phenomenon physiologically modulates synaptic plasticity while, in pathologic conditions, leads to increased vulnerability to excitotoxic neuronal death. Given the importance of these subunits, we have therefore evaluated possible associations between changes in expression levels of AMPAR subunits and development of cognitive deficits in 3xTg-AD mice, a widely investigated transgenic mouse model of Alzheimer’s disease. With qRT-PCR, we assayed hippocampal mRNA expression levels of GluA1-4 subunits occurring in young [3 months of age (m.o.a.] and old (12 m.o.a Tg-AD mice and made comparisons with levels found in age-matched wild type (WT mice. Efficiency of GluA2 RNA editing was also analyzed. All animals were cognitively tested for short- and long-term spatial memory with the Morris Water Maze (MWM navigation task. 3xTg-AD mice showed age-dependent decreases of mRNA levels for all the AMPAR subunits, with the exception of GluA2. Editing remained fully efficient with aging in 3xTg-AD and WT mice. A one-to-one correlation analysis between MWM performances and GluA1-4 mRNA expression profiles showed negative correlations between GluA2 levels and MWM performances in young 3xTg-AD mice. On the contrary, positive correlations between GluA2 mRNA and MWM performances were found in young WT mice. Our data suggest that increases of AMPARs that contain GluA1, GluA3, and GluA4 subunits may help in maintaining cognition in pre-symptomatic 3xTg-AD mice.

Brain activation to negative stimuli mediates a relationship between adolescent marijuana use and later emotional functioning

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Mary M. Heitzeg

2015-12-01

Full Text Available This work investigated the impact of heavy marijuana use during adolescence on emotional functioning, as well as the brain functional mediators of this effect. Participants (n = 40 were recruited from the Michigan Longitudinal Study (MLS. Data on marijuana use were collected prospectively beginning in childhood as part of the MLS. Participants were classified as heavy marijuana users (n = 20 or controls with minimal marijuana use. Two facets of emotional functioning—negative emotionality and resiliency (a self-regulatory mechanism—were assessed as part of the MLS at three time points: mean age 13.4, mean age 19.6, and mean age 23.1. Functional neuroimaging data during an emotion-arousal word task were collected at mean age 20.2. Negative emotionality decreased and resiliency increased across the three time points in controls but not heavy marijuana users. Compared with controls, heavy marijuana users had less activation to negative words in temporal, prefrontal, and occipital cortices, insula, and amygdala. Activation of dorsolateral prefrontal cortex to negative words mediated an association between marijuana group and later negative emotionality. Activation of the cuneus/lingual gyrus mediated an association between marijuana group and later resiliency. Results support growing evidence that heavy marijuana use during adolescence affects later emotional outcomes.

Sorting Tubules Regulate Blood-Brain Barrier Transcytosis

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Roberto Villaseñor

2017-12-01

Full Text Available Transcytosis across the blood-brain barrier (BBB regulates key processes of the brain, but the intracellular sorting mechanisms that determine successful receptor-mediated transcytosis in brain endothelial cells (BECs remain unidentified. Here, we used Transferrin receptor-based Brain Shuttle constructs to investigate intracellular transport in BECs, and we uncovered a pathway for the regulation of receptor-mediated transcytosis. By combining live-cell imaging and mathematical modeling in vitro with super-resolution microscopy of the BBB, we show that intracellular tubules promote transcytosis across the BBB. A monovalent construct (sFab sorted for transcytosis was localized to intracellular tubules, whereas a bivalent construct (dFab sorted for degradation formed clusters with impaired transport along tubules. Manipulating tubule biogenesis by overexpressing the small GTPase Rab17 increased dFab transport into tubules and induced its transcytosis in BECs. We propose that sorting tubules regulate transcytosis in BECs and may be a general mechanism for receptor-mediated transport across the BBB.

Genetic analysis of the cytoplasmic dynein subunit families.

Science.gov (United States)

Pfister, K Kevin; Shah, Paresh R; Hummerich, Holger; Russ, Andreas; Cotton, James; Annuar, Azlina Ahmad; King, Stephen M; Fisher, Elizabeth M C

2006-01-01

Cytoplasmic dyneins, the principal microtubule minus-end-directed motor proteins of the cell, are involved in many essential cellular processes. The major form of this enzyme is a complex of at least six protein subunits, and in mammals all but one of the subunits are encoded by at least two genes. Here we review current knowledge concerning the subunits, their interactions, and their functional roles as derived from biochemical and genetic analyses. We also carried out extensive database searches to look for new genes and to clarify anomalies in the databases. Our analysis documents evolutionary relationships among the dynein subunits of mammals and other model organisms, and sheds new light on the role of this diverse group of proteins, highlighting the existence of two cytoplasmic dynein complexes with distinct cellular roles.

Genetic analysis of the cytoplasmic dynein subunit families.

Directory of Open Access Journals (Sweden)

K Kevin Pfister

2006-01-01

Full Text Available Cytoplasmic dyneins, the principal microtubule minus-end-directed motor proteins of the cell, are involved in many essential cellular processes. The major form of this enzyme is a complex of at least six protein subunits, and in mammals all but one of the subunits are encoded by at least two genes. Here we review current knowledge concerning the subunits, their interactions, and their functional roles as derived from biochemical and genetic analyses. We also carried out extensive database searches to look for new genes and to clarify anomalies in the databases. Our analysis documents evolutionary relationships among the dynein subunits of mammals and other model organisms, and sheds new light on the role of this diverse group of proteins, highlighting the existence of two cytoplasmic dynein complexes with distinct cellular roles.

Connexin 43-Mediated Astroglial Metabolic Networks Contribute to the Regulation of the Sleep-Wake Cycle.

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Clasadonte, Jerome; Scemes, Eliana; Wang, Zhongya; Boison, Detlev; Haydon, Philip G

2017-09-13

Astrocytes produce and supply metabolic substrates to neurons through gap junction-mediated astroglial networks. However, the role of astroglial metabolic networks in behavior is unclear. Here, we demonstrate that perturbation of astroglial networks impairs the sleep-wake cycle. Using a conditional Cre-Lox system in mice, we show that knockout of the gap junction subunit connexin 43 in astrocytes throughout the brain causes excessive sleepiness and fragmented wakefulness during the nocturnal active phase. This astrocyte-specific genetic manipulation silenced the wake-promoting orexin neurons located in the lateral hypothalamic area (LHA) by impairing glucose and lactate trafficking through astrocytic networks. This global wakefulness instability was mimicked with viral delivery of Cre recombinase to astrocytes in the LHA and rescued by in vivo injections of lactate. Our findings propose a novel regulatory mechanism critical for maintaining normal daily cycle of wakefulness and involving astrocyte-neuron metabolic interactions. Copyright © 2017 Elsevier Inc. All rights reserved.

Structural insights into RipC, a putative citrate lyase β subunit from a Yersinia pestis virulence operon

International Nuclear Information System (INIS)

Torres, Rodrigo; Chim, Nicholas; Sankaran, Banumathi; Pujol, Céline; Bliska, James B.; Goulding, Celia W.

2011-01-01

Comparison of the 2.45 Å resolution crystal structure of homotrimeric RipC, a putative citrate lyase β subunit from Y. pestis, with structural homologs reveals conserved RipC residues that are implicated in CoA binding. Yersinia pestis remains a threat, with outbreaks of plague occurring in rural areas and its emergence as a weapon of bioterrorism; thus, an improved understanding of its various pathogenicity pathways is warranted. The rip (required for intracellular proliferation) virulence operon is required for Y. pestis survival in interferon-γ-treated macrophages and has been implicated in lowering macrophage-produced nitric oxide levels. RipC, one of three gene products from the rip operon, is annotated as a citrate lyase β subunit. Furthermore, the Y. pestis genome lacks genes that encode citrate lyase α and γ subunits, suggesting a unique functional role of RipC in the Y. pestisrip-mediated survival pathway. Here, the 2.45 Å resolution crystal structure of RipC revealed a homotrimer in which each monomer consists of a (β/α) 8 TIM-barrel fold. Furthermore, the trimeric state was confirmed in solution by size-exclusion chromatography. Through sequence and structure comparisons with homologous proteins, it is proposed that RipC is a putative CoA- or CoA-derivative binding protein

GABA-A Receptors Mediate Tonic Inhibition and Neurosteroid Sensitivity in the Brain.

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Reddy, Doodipala Samba

2018-01-01

Neurosteroids like allopregnanolone (AP) are positive allosteric modulators of synaptic and extrasynaptic GABA-A receptors. AP and related neurosteroids exhibit a greater potency for δ-containing extrasynaptic receptors. The δGABA-A receptors, which are expressed extrasynaptically in the dentate gyrus and other regions, contribute to tonic inhibition, promoting network shunting as well as reducing seizure susceptibility. Levels of endogenous neurosteroids fluctuate with ovarian cycle. Natural and synthetic neurosteroids maximally potentiate tonic inhibition in the hippocampus and provide robust protection against a variety of limbic seizures and status epilepticus. Recently, a consensus neurosteroid pharmacophore model has been proposed at extrasynaptic δGABA-A receptors based on structure-activity relationship for functional activation of tonic currents and seizure protection. Aside from anticonvulsant actions, neurosteroids have been found to be powerful anxiolytic and anesthetic agents. Neurosteroids and Zn 2+ have preferential affinity for δ-containing receptors. Thus, Zn 2+ can prevent neurosteroid activation of extrasynaptic δGABA-A receptor-mediated tonic inhibition. Recently, we demonstrated that Zn 2+ selectively inhibits extrasynaptic δGABA-A receptors and thereby fully prevents AP activation of tonic inhibition and seizure protection. We confirmed that neurosteroids exhibit greater sensitivity at extrasynaptic δGABA-A receptors. Overall, extrasynaptic GABA-A receptors are primary mediators of tonic inhibition in the brain and play a key role in the pathophysiology of epilepsy and other neurological disorders. © 2018 Elsevier Inc. All rights reserved.

Oxcarbazepine causes neurocyte apoptosis and developing brain damage by triggering Bax/Bcl-2 signaling pathway mediated caspase 3 activation in neonatal rats.

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Song, Y; Zhong, M; Cai, F-C

2018-01-01

Anti-epileptic drugs (AEDs) are the main methods for treatment of neonatal seizures; however, a few AEDs may cause developing brain damage of neonate. This study aims to investigate effects of oxcarbazepine (OXC) on developing brain damage of neonatal rats. Both of neonatal and adult rats were divided into 6 groups, including Control, OXC 187.5 mg/kg, OXC 281.25 mg/kg, OXC 375 mg/kg group, LEV and PHT group. Body weight and brain weight were evaluated. Hematoxylin and eosin (HE) and Nissl staining were used to observe neurocyte morphology and Nissl bodies, respectively. Apoptosis was examined using TUNEL assay, and caspase 8 activity was evaluated using spectrophotometer method. Cytochrome C-release was evaluated using flow cytometry. Western blot was used to examine Bax and Bcl-2 expression. OXC 375 mg/kg treatment significantly decreased brain weight compared to Control group in neonatal rats (P5 rats) (pOxcarbazepine at a concentration of 281.25 mg/kg or more causes neurocyte apoptosis and developing brain damage by triggering Bax/Bcl-2 signaling pathway mediated caspase 3 activation in neonatal rats.

Anxiogenic properties of an inverse agonist selective for α3 subunit-containing GABAA receptors

OpenAIRE

Atack, John R; Hutson, Peter H; Collinson, Neil; Marshall, George; Bentley, Graham; Moyes, Christopher; Cook, Susan M; Collins, Ian; Wafford, Keith; McKernan, Ruth M; Dawson, Gerard R

2005-01-01

α3IA (6-(4-pyridyl)-5-(4-methoxyphenyl)-3-carbomethoxy-1-methyl-1H-pyridin-2-one) is a pyridone with higher binding and functional affinity and greater inverse agonist efficacy for GABAA receptors containing an α3 rather than an α1, α2 or α5 subunit. If doses are selected that minimise the occupancy at these latter subtypes, then the in vivo effects of α3IA are most probably mediated by the α3 subtype.α3IA has good CNS penetration in rats and mice as measured using a [3H]Ro 15-1788 in vivo bi...

Three α-Subunits of Heterotrimeric G Proteins and an Adenylyl Cyclase Have Distinct Roles in Fruiting Body Development in the Homothallic Fungus Sordaria macrospora

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Kamerewerd, Jens; Jansson, Malin; Nowrousian, Minou; Pöggeler, Stefanie; Kück, Ulrich

2008-01-01

Sordaria macrospora, a self-fertile filamentous ascomycete, carries genes encoding three different α-subunits of heterotrimeric G proteins (gsa, G protein Sordaria alpha subunit). We generated knockout strains for all three gsa genes (Δgsa1, Δgsa2, and Δgsa3) as well as all combinations of double mutants. Phenotypic analysis of single and double mutants showed that the genes for Gα-subunits have distinct roles in the sexual life cycle. While single mutants show some reduction of fertility, double mutants Δgsa1Δgsa2 and Δgsa1Δgsa3 are completely sterile. To test whether the pheromone receptors PRE1 and PRE2 mediate signaling via distinct Gα-subunits, two recently generated Δpre strains were crossed with all Δgsa strains. Analyses of the corresponding double mutants revealed that compared to GSA2, GSA1 is a more predominant regulator of a signal transduction cascade downstream of the pheromone receptors and that GSA3 is involved in another signaling pathway that also contributes to fruiting body development and fertility. We further isolated the gene encoding adenylyl cyclase (AC) (sac1) for construction of a knockout strain. Analyses of the three ΔgsaΔsac1 double mutants and one Δgsa2Δgsa3Δsac1 triple mutant indicate that SAC1 acts downstream of GSA3, parallel to a GSA1–GSA2-mediated signaling pathway. In addition, the function of STE12 and PRO41, two presumptive signaling components, was investigated in diverse double mutants lacking those developmental genes in combination with the gsa genes. This analysis was further completed by expression studies of the ste12 and pro41 transcripts in wild-type and mutant strains. From the sum of all our data, we propose a model for how different Gα-subunits interact with pheromone receptors, adenylyl cyclase, and STE12 and thus cooperatively regulate sexual development in S. macrospora. PMID:18723884

Roles of the β subunit hinge domain in ATP synthase F1 sector: Hydrophobic network formed by introduced βPhe174 inhibits subunit rotation

International Nuclear Information System (INIS)

Nakanishi-Matsui, Mayumi; Kashiwagi, Sachiko; Kojima, Masaki; Nonaka, Takamasa; Futai, Masamitsu

2010-01-01

The ATP synthase β subunit hinge domain (βPhe148 ∼ βGly186, P-loop/α-helixB/loop/β-sheet4, Escherichia coli residue numbering) dramatically changes in conformation upon nucleotide binding. We previously reported that F 1 with the βSer174 to Phe mutation in the domain lowered the γ subunit rotation speed, and thus decreased the ATPase activity [M. Nakanishi-Matsui, S. Kashiwagi, T. Ubukata, A. Iwamoto-Kihara, Y. Wada, M. Futai, Rotational catalysis of Escherichia coli ATP synthase F 1 sector. Stochastic fluctuation and a key domain of the β subunit, J. Biol. Chem. 282 (2007) 20698-20704.]. Homology modeling indicates that the amino acid replacement induces a hydrophobic network, in which the βMet159, βIle163, and βAla167 residues of the β subunit are involved together with the mutant βPhe174. The network is expected to stabilize the conformation of β DP (nucleotide-bound form of the β subunit), resulting in increased activation energy for transition to β E (empty β subunit). The modeling further predicts that replacement of βMet159 with Ala or Ile weakens the hydrophobic network. As expected, these two mutations experimentally suppressed the ATPase activities as well as subunit rotation of βS174F. Furthermore, the rotation rate decreased with the increase of the strength in the hydrophobic network. These results indicate that the smooth conformational change of the β subunit hinge domain is pertinent for the rotational catalysis.

The autophosphorylation and p34cdc2 phosphorylation sites of casein kinase-2 beta-subunit are not essential for reconstituting the fully-active heterotetrameric holoenzyme

DEFF Research Database (Denmark)

Meggio, F; Boldyreff, B; Issinger, O G

1993-01-01

Two mutants of human casein kinase-2 beta-subunit with short deletions at either their amino (delta 1-4) or carboxy (delta 209-215) terminal side have been created that have lost the capability to undergo autophosphorylation and p34cdc2 mediated phosphorylation, respectively. Both mutants give rise...

Studies on the subunits of human glycoprotein hormones in relation to reproduction

International Nuclear Information System (INIS)

Hagen, C.

1977-01-01

In this review summarising present knowledge of the biological and immunological activity of the subunits of human glycoprotein hormones, the specificity of the α-subunit and β-subunit radioimmunoassays are discussed. The crossreaction studies performed with the α-subunit radioimmunoassays are aummarised in one table while those with the β-subunit radioimmunoassays are presented in a second table. (JIW)

Augmenting brain metabolism to increase macro- and chaperone-mediated autophagy for decreasing neuronal proteotoxicity and aging.

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Loos, Ben; Klionsky, Daniel J; Wong, Esther

2017-09-01

Accumulation of toxic protein aggregates in the nerve cells is a hallmark of neuronal diseases and brain aging. Mechanisms to enhance neuronal surveillance to improve neuronal proteostasis have a direct impact on promoting neuronal health and forestalling age-related decline in brain function. Autophagy is a lysosomal degradative pathway pivotal for neuronal protein quality control. Different types of autophagic mechanisms participate in protein handling in neurons. Macroautophagy targets misfolded and aggregated proteins in autophagic vesicles to the lysosomes for destruction, while chaperone-mediated autophagy (CMA) degrades specific soluble cytosolic proteins delivered to the lysosomes by chaperones. Dysfunctions in macroautophagy and CMA contribute to proteo- and neuro-toxicity associated with neurodegeneration and aging. Thus, augmenting or preserving both autophagic mechanisms pose significant benefits in delaying physiological and pathological neuronal demises. Recently, life-style interventions that modulate metabolite ketone bodies, energy intake by caloric restriction and energy expenditure by exercise have shown to enhance both autophagy and brain health. However, to what extent these interventions affect neuronal autophagy to promote brain fitness remains largely unclear. Here, we review the functional connections of how macroautophagy and CMA are affected by ketone bodies, caloric restriction and exercise in the context of neurodegeneration. A concomitant assessment of yeast Saccharomyces cerevisiae is performed to reveal the conserved nature of such autophagic responses to substrate perturbations. In doing so, we provide novel insights and integrated evidence for a potential adjuvant therapeutic strategy to intervene in the neuronal decline in neurodegenerative diseases by controlling both macroautophagy and CMA fluxes favorably. Copyright © 2017 Elsevier Ltd. All rights reserved.

Regulation of KV channel voltage-dependent activation by transmembrane β subunits

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Xiaohui eSun

2012-04-01

Full Text Available Voltage-activated K+ (KV channels are important for shaping action potentials and maintaining resting membrane potential in excitable cells. KV channels contain a central pore-gate domain (PGD surrounded by four voltage-sensing domains (VSD. The VSDs will change conformation in response to alterations of the membrane potential thereby inducing the opening of the PGD. Many KV channels are heteromeric protein complexes containing auxiliary β subunits. These β subunits modulate channel expression and activity to increase functional diversity and render tissue specific phenotypes. This review focuses on the KV β subunits that contain transmembrane (TM segments including the KCNE family and the β subunits of large conductance, Ca2+- and voltage-activated K+ (BK channels. These TM β subunits affect the voltage-dependent activation of KV α subunits. Experimental and computational studies have described the structural location of these β subunits in the channel complexes and the biophysical effects on VSD activation, PGD opening and VSD-PGD coupling. These results reveal some common characteristics and mechanistic insights into KV channel modulation by TM β subunits.

Smuggling Drugs into the Brain: An Overview of Ligands Targeting Transcytosis for Drug Delivery across the Blood-Brain Barrier.

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Georgieva, Julia V; Hoekstra, Dick; Zuhorn, Inge S

2014-11-17

The blood-brain barrier acts as a physical barrier that prevents free entry of blood-derived substances, including those intended for therapeutic applications. The development of molecular Trojan horses is a promising drug targeting technology that allows for non-invasive delivery of therapeutics into the brain. This concept relies on the application of natural or genetically engineered proteins or small peptides, capable of specifically ferrying a drug-payload that is either directly coupled or encapsulated in an appropriate nanocarrier, across the blood-brain barrier via receptor-mediated transcytosis. Specifically, in this process the nanocarrier-drug system ("Trojan horse complex") is transported transcellularly across the brain endothelium, from the blood to the brain interface, essentially trailed by a native receptor. Naturally, only certain properties would favor a receptor to serve as a transporter for nanocarriers, coated with appropriate ligands. Here we briefly discuss brain microvascular endothelial receptors that have been explored until now, highlighting molecular features that govern the efficiency of nanocarrier-mediated drug delivery into the brain.

Role of desensitization and subunit expression for kainate receptor-mediated neurotoxicity in murine neocortical cultures

DEFF Research Database (Denmark)

Jensen, J B; Schousboe, A; Pickering, D S

1999-01-01

) toxicity mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors, and (3) toxicity that can be mediated by kainate receptors when desensitization of the receptors is blocked. The indirect action at NMDA receptors was discovered because (5R, 10S)-(+)-5-methyl-10,11-dihydro-5H...... nedioxy-5H-2,3-benzodiazepine (GYKI 53655), a selective AMPA receptor antagonist, abolished the remaining toxicity. These results indicated that kainate- and domoate-mediated toxicity involves both the NMDA and the AMPA receptors. Pretreatment of the cultures with concanavalin A to prevent desensitization...

Apoptotic role of TGF-β mediated by Smad4 mitochondria translocation and cytochrome c oxidase subunit II interaction.

Science.gov (United States)

Pang, Lijuan; Qiu, Tao; Cao, Xu; Wan, Mei

2011-07-01

Smad4, originally isolated from the human chromosome 18q21, is a key factor in transducing the signals of the TGF-β superfamily of growth hormones and plays a pivotal role in mediating antimitogenic and proapoptotic effects of TGF-β, but the mechanisms by which Smad4 induces apoptosis are elusive. Here we report that Smad4 directly translocates to the mitochondria of apoptotic cells. Smad4 gene silencing by siRNA inhibits TGF-β-induced apoptosis in Hep3B cells and UV-induced apoptosis in PANC-1 cells. Cell fractionation assays demonstrated that a fraction of Smad4 translocates to mitochondria after long time TGF-β treatment or UV exposure, during which the cells were under apoptosis. Smad4 mitochondria translocation during apoptosis was also confirmed by fluorescence observation of Smad4 colocalization with MitoTracker Red. We searched for mitochondria proteins that have physical interactions with Smad4 using yeast two-hybrid screening approach. DNA sequence analysis identified 34 positive clones, five of which encoded subunits in mitochondria complex IV, i.e., one clone encoded cytochrome c oxidase COXII, three clones encoded COXIII and one clone encoded COXVb. Strong interaction between Smad4 with COXII, an important apoptosis regulator, was verified in yeast by β-gal activity assays and in mammalian cells by immunoprecipitation assays. Further, mitochondrial portion of cells was isolated and the interaction between COXII and Smad4 in mitochondria upon TGF-β treatment or UV exposure was confirmed. Importantly, targeting Smad4 to mitochondria using import leader fusions enhanced TGF-β-induced apoptosis. Collectively, the results suggest that Smad4 promote apoptosis of the cells through its mitochondrial translocation and association with mitochondria protein COXII. Copyright © 2011 Elsevier Inc. All rights reserved.

Unc-51/ATG1 controls axonal and dendritic development via kinesin-mediated vesicle transport in the Drosophila brain.

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Hiroaki Mochizuki

2011-05-01

Full Text Available Members of the evolutionary conserved Ser/Thr kinase Unc-51 family are key regulatory proteins that control neural development in both vertebrates and invertebrates. Previous studies have suggested diverse functions for the Unc-51 protein, including axonal elongation, growth cone guidance, and synaptic vesicle transport.In this work, we have investigated the functional significance of Unc-51-mediated vesicle transport in the development of complex brain structures in Drosophila. We show that Unc-51 preferentially accumulates in newly elongating axons of the mushroom body, a center of olfactory learning in flies. Mutations in unc-51 cause disintegration of the core of the developing mushroom body, with mislocalization of Fasciclin II (Fas II, an IgG-family cell adhesion molecule important for axonal guidance and fasciculation. In unc-51 mutants, Fas II accumulates in the cell bodies, calyx, and the proximal peduncle. Furthermore, we show that mutations in unc-51 cause aberrant overshooting of dendrites in the mushroom body and the antennal lobe. Loss of unc-51 function leads to marked accumulation of Rab5 and Golgi components, whereas the localization of dendrite-specific proteins, such as Down syndrome cell adhesion molecule (DSCAM and No distributive disjunction (Nod, remains unaltered. Genetic analyses of kinesin light chain (Klc and unc-51 double heterozygotes suggest the importance of kinesin-mediated membrane transport for axonal and dendritic development. Moreover, our data demonstrate that loss of Klc activity causes similar axonal and dendritic defects in mushroom body neurons, recapitulating the salient feature of the developmental abnormalities caused by unc-51 mutations.Unc-51 plays pivotal roles in the axonal and dendritic development of the Drosophila brain. Unc-51-mediated membrane vesicle transport is important in targeted localization of guidance molecules and organelles that regulate elongation and compartmentalization of

Regulation of P450-mediated permethrin resistance in Culex quinquefasciatus by the GPCR/Gαs/AC/cAMP/PKA signaling cascade.

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Li, Ting; Liu, Nannan

2017-12-01

This study explores the role of G-protein-coupled receptor-intracellular signaling in the development of P450-mediated insecticide resistance in mosquitoes, Culex quinquefasciatus , focusing on the essential function of the GPCRs and their downstream effectors of Gs alpha subunit protein (Gαs) and adenylyl cyclase (ACs) in P450-mediated insecticide resistance of Culex mosquitoes. Our RNAi-mediated functional study showed that knockdown of Gαs caused the decreased expression of the downstream effectors of ACs and PKAs in the GPCR signaling pathway and resistance P450 genes, whereas knockdown of ACs decreased the expression of PKAs and resistance P450 genes. Knockdown of either Gαs or ACs resulted in an increased susceptibility of mosquitoes to permethrin. These results add significantly to our understanding of the molecular basis of resistance P450 gene regulation through GPCR/Gαs/AC/cAMP-PKA signaling pathways in the insecticide resistance of mosquitoes. The temporal and spatial dynamic analyses of GPCRs, Gαs, ACs, PKAs, and P450s in two insecticide resistant mosquito strains revealed that all the GPCR signaling pathway components tested, namely GPCRs, Gαs, ACs and PKAs, were most highly expressed in the brain for both resistant strains, suggesting the role played by these genes in signaling transduction and regulation. The resistance P450 genes were mainly expressed in the brain, midgut and malpighian tubules (MTs), suggesting their critical function in the central nervous system and importance for detoxification. The temporal dynamics analysis for the gene expression showed a diverse expression profile during mosquito development, indicating their initially functional importance in response to exposure to insecticides during their life stages.

The chemopreventive properties of chlorogenic acid reveal a potential new role for the microsomal glucose-6-phosphate translocase in brain tumor progression

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Desgagnés Julie

2006-03-01

Full Text Available Abstract Background Chlorogenic acid (CHL, the most potent functional inhibitor of the microsomal glucose-6-phosphate translocase (G6PT, is thought to possess cancer chemopreventive properties. It is not known, however, whether any G6PT functions are involved in tumorigenesis. We investigated the effects of CHL and the potential role of G6PT in regulating the invasive phenotype of brain tumor-derived glioma cells. Results RT-PCR was used to show that, among the adult and pediatric brain tumor-derived cells tested, U-87 glioma cells expressed the highest levels of G6PT mRNA. U-87 cells lacked the microsomal catalytic subunit glucose-6-phosphatase (G6Pase-α but expressed G6Pase-β which, when coupled to G6PT, allows G6P hydrolysis into glucose to occur in non-glyconeogenic tissues such as brain. CHL inhibited U-87 cell migration and matrix metalloproteinase (MMP-2 secretion, two prerequisites for tumor cell invasion. Moreover, CHL also inhibited cell migration induced by sphingosine-1-phosphate (S1P, a potent mitogen for glioblastoma multiform cells, as well as the rapid, S1P-induced extracellular signal-regulated protein kinase phosphorylation potentially mediated through intracellular calcium mobilization, suggesting that G6PT may also perform crucial functions in regulating intracellular signalling. Overexpression of the recombinant G6PT protein induced U-87 glioma cell migration that was, in turn, antagonized by CHL. MMP-2 secretion was also inhibited by the adenosine triphosphate (ATP-depleting agents 2-deoxyglucose and 5-thioglucose, a mechanism that may inhibit ATP-mediated calcium sequestration by G6PT. Conclusion We illustrate a new G6PT function in glioma cells that could regulate the intracellular signalling and invasive phenotype of brain tumor cells, and that can be targeted by the anticancer properties of CHL.

γ1-Containing GABA-A Receptors Cluster at Synapses Where they Mediate Slower Synaptic Currents than γ2-Containing GABA-A Receptors

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Christine L. Dixon

2017-06-01

Full Text Available GABA-A receptors (GABAARs are pentameric ligand-gated ion channels that are assembled mainly from α (α1–6, β (β1–3 and γ (γ1–3 subunits. Although GABAARs containing γ2L subunits mediate most of the inhibitory neurotransmission in the brain, significant expression of γ1 subunits is seen in the amygdala, pallidum and substantia nigra. However, the location and function of γ1-containing GABAARs in these regions remains unclear. In “artificial” synapses, where the subunit composition of postsynaptic receptors is specifically controlled, γ1 incorporation slows the synaptic current decay rate without affecting channel deactivation, suggesting that γ1-containing receptors are not clustered and therefore activated by diffuse neurotransmitter. However, we show that γ1-containing receptors are localized at neuronal synapses and form clusters in both synaptic and extrasynaptic regions. In addition, they exhibit rapid membrane diffusion and a higher frequency of exchange between synaptic and perisynaptic populations compared to γ2L-containing GABAARs. A point mutation in the large intracellular domain and a pharmacological analysis reveal that when a single non-conserved γ2L residue is mutated to its γ1 counterpart (T349L, the synaptic current decay is slowed from γ2L- to γ1-like without changing the clustering or diffusion properties of the receptors. In addition, previous fast perfusion and single channel kinetic experiments revealed no difference in the intrinsic closing rates of γ2L- and γ1-containing receptors when expressed in HEK293 cells. These observations together with Monte Carlo simulations of synaptic function confirm that decreased clustering does not control γ1-containing GABAAR kinetics. Rather, they suggest that γ1- and γ2L-containing receptors exhibit differential synaptic current decay rates due to differential gating dynamics when localized at the synapse.

Mechanisms of the gabapentinoids and α 2 δ-1 calcium channel subunit in neuropathic pain.

Science.gov (United States)

Patel, Ryan; Dickenson, Anthony H

2016-04-01

The gabapentinoid drugs gabapentin and pregabalin are key front-line therapies for various neuropathies of peripheral and central origin. Originally designed as analogs of GABA, the gabapentinoids bind to the α 2 δ-1 and α 2 δ-2 auxiliary subunits of calcium channels, though only the former has been implicated in the development of neuropathy in animal models. Transgenic approaches also identify α 2 δ-1 as key in mediating the analgesic effects of gabapentinoids, however the precise molecular mechanisms remain unclear. Here we review the current understanding of the pathophysiological role of the α 2 δ-1 subunit, the mechanisms of analgesic action of gabapentinoid drugs and implications for efficacy in the clinic. Despite widespread use, the number needed to treat for gabapentin and pregabalin averages from 3 to 8 across neuropathies. The failure to treat large numbers of patients adequately necessitates a novel approach to treatment selection. Stratifying patients by sensory profiles may imply common underlying mechanisms, and a greater understanding of these mechanisms could lead to more direct targeting of gabapentinoids.

NSs protein of rift valley fever virus promotes posttranslational downregulation of the TFIIH subunit p62.

Science.gov (United States)

Kalveram, Birte; Lihoradova, Olga; Ikegami, Tetsuro

2011-07-01

Rift Valley fever virus (RVFV; family Bunyaviridae, genus Phlebovirus) is an important emerging pathogen of humans and ruminants. Its NSs protein has previously been identified as a major virulence factor that suppresses host defense through three distinct mechanisms: it directly inhibits beta interferon (IFN-β) promoter activity, it promotes the degradation of double-stranded RNA-dependent protein kinase (PKR), and it suppresses host transcription by disrupting the assembly of the basal transcription factor TFIIH through sequestration of its p44 subunit. Here, we report that in addition to PKR, NSs also promotes the degradation of the TFIIH subunit p62. Infection of cells with the RVFV MP-12 vaccine strain reduced p62 protein levels to below the detection limit early in the course of infection. This NSs-mediated downregulation of p62 was posttranslational, as it was unaffected by pharmacological inhibition of transcription or translation and MP-12 infection had no effect on p62 mRNA levels. Treatment of cells with proteasome inhibitors but not inhibition of lysosomal acidification or nuclear export resulted in a stabilization of p62 in the presence of NSs. Furthermore, p62 could be coprecipitated with NSs from lysates of infected cells. These data suggest that the RVFV NSs protein is able to interact with the TFIIH subunit p62 inside infected cells and promotes its degradation, which can occur directly in the nucleus.

Crystallization and preliminary X-ray diffraction analyses of several forms of the CfaB major subunit of enterotoxigenic Escherichia coli CFA/I fimbriae

International Nuclear Information System (INIS)

Li, Yong-Fu; Poole, Steven; Rasulova, Fatima; McVeigh, Annette L.; Savarino, Stephen J.; Xia, Di

2009-01-01

Three fusion proteins were generated in order to resolve the atomic structure of the CFA/I fimbriae of enterotoxigenic E. coli. CfaEB is a fusion of the minor and major CFA/I subunits, while CfaBB and CfaBBB are tandem fusions of two and three repeats, respectively, of the major subunit. Each protein was crystallized and the crystal structures of each of these fusions were determined successively by the molecular-replacement method using the CfaE crystal structure as an initial phasing model. Enterotoxigenic Escherichia coli (ETEC), a major global cause of diarrhea, initiates the pathogenic process via fimbriae-mediated attachment to the small intestinal epithelium. A common prototypic ETEC fimbria, colonization factor antigen I (CFA/I), consists of a tip-localized minor adhesive subunit CfaE and the stalk-forming major subunit CfaB, both of which are necessary for fimbrial assembly. To elucidate the structure of CFA/I at atomic resolution, three recombinant proteins were generated consisting of fusions of the minor and major subunits (CfaEB) and of two (CfaBB) and three (CfaBBB) repeats of the major subunit. Crystals of CfaEB diffracted X-rays to 2.1 Å resolution and displayed the symmetry of space group P2 1 . CfaBB exhibited a crystal diffraction limit of 2.3 Å resolution and had the symmetry of space group P2 1 2 1 2. CfaBBB crystallized in the monoclinic space group C2 and diffracted X-rays to 2.3 Å resolution. These structures were determined using the molecular-replacement method

Development of a Subunit Vaccine for Contagious Bovine ...

International Development Research Centre (IDRC) Digital Library (Canada)

Their work has set the stage for commercial development of a sub-unit vaccine. ... The sub-unit vaccine will be cost-effective, easy to produce, and safe. How it will make a ... IDRC invites applications for the IDRC Doctoral Research Awards.

Core Mediator structure at 3.4 Å extends model of transcription initiation complex.

Science.gov (United States)

Nozawa, Kayo; Schneider, Thomas R; Cramer, Patrick

2017-05-11

Mediator is a multiprotein co-activator that binds the transcription pre-initiation complex (PIC) and regulates RNA polymerase (Pol) II. The Mediator head and middle modules form the essential core Mediator (cMed), whereas the tail and kinase modules play regulatory roles. The architecture of Mediator and its position on the PIC are known, but atomic details are limited to Mediator subcomplexes. Here we report the crystal structure of the 15-subunit cMed from Schizosaccharomyces pombe at 3.4 Å resolution. The structure shows an unaltered head module, and reveals the intricate middle module, which we show is globally required for transcription. Sites of known Mediator mutations cluster at the interface between the head and middle modules, and in terminal regions of the head subunits Med6 (ref. 16) and Med17 (ref. 17) that tether the middle module. The structure led to a model for Saccharomyces cerevisiae cMed that could be combined with the 3.6 Å cryo-electron microscopy structure of the core PIC (cPIC). The resulting atomic model of the cPIC-cMed complex informs on interactions of the submodules forming the middle module, called beam, knob, plank, connector, and hook. The hook is flexibly linked to Mediator by a conserved hinge and contacts the transcription initiation factor IIH (TFIIH) kinase that phosphorylates the carboxy (C)-terminal domain (CTD) of Pol II and was recently positioned on the PIC. The hook also contains residues that crosslink to the CTD and reside in a previously described cradle. These results provide a framework for understanding Mediator function, including its role in stimulating CTD phosphorylation by TFIIH.

The morphological and chemical characteristics of striatal neurons immunoreactive for the alpha1-subunit of the GABA(A) receptor in the rat.

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Waldvogel, H J; Kubota, Y; Trevallyan, S C; Kawaguchi, Y; Fritschy, J M; Mohler, H; Faull, R L

1997-10-01

The distribution, morphology and chemical characteristics of neurons immunoreactive for the alpha1-subunit of the GABA(A) receptor in the striatum of the basal ganglia in the rat brain were investigated at the light, confocal and electron microscope levels using single, double and triple immunohistochemical labelling techniques. The results showed that alpha1-subunit immunoreactive neurons were sparsely distributed throughout the rat striatum. Double and triple labelling results showed that all the alpha1-subunit-immunoreactive neurons were positive for glutamate decarboxylase and immunoreactive for the beta2,3 and gamma2 subunits of the GABA(A) receptor. Three types of alpha1-subunit-immunoreactive neurons were identified in the striatum on the basis of cellular morphology and chemical characteristics. The most numerous alpha1-subunit-immunoreactive neurons were medium-sized, aspiny neurons with a widely branching dendritic tree. They were parvalbumin-negative and were located mainly in the dorsolateral regions of the striatum. Electron microscopy showed that these neurons had an indented nuclear membrane, typical of striatal interneurons, and were surrounded by small numbers of axon terminals which established alpha1-subunit-immunoreactive synaptic contacts with the soma and dendrites. These cells were classified as type 1 alpha1-subunit-immunoreactive neurons and comprised 75% of the total population of alpha1-subunit-immunoreactive neurons in the striatum. The remaining alpha1-subunit-immunoreactive neurons comprised of a heterogeneous population of large-sized neurons localized in the ventral and medial regions of the striatum. The most numerous large-sized cells were parvalbumin-negative, had two to three relatively short branching dendrites and were designated type 2 alpha1-subunit-immunoreactive neurons. Electron microscopy showed that the type 2 neurons were characterized by a highly convoluted nuclear membrane and were sparsely covered with small axon

Human placental Na/sup +/, K/sup +/-ATPase. cap alpha. subunit: cDNA cloning, tissue expression, DNA polymorphism, and chromosomal localization

Energy Technology Data Exchange (ETDEWEB)

Chehab, F.F.; Kan, Y.W.; Law, M.L.; Hartz, J.; Kao, F.T.; Blostein, R.

1987-11-01

A 2.2-kilobase clone comprising a major portion of the coding sequence of the Na/sup +/, K/sup +/-ATPase ..cap alpha.. subunit was cloned from human placenta and its sequence was identical to that encoding the ..cap alpha.. subunit of human kidney and HeLa cells. Transfer blot analysis of the mRNA products of the Na/sup +/, K/sup +/-ATPase gene from various human tissues and cell lines revealed only one band (approx. = 4.7 kilobases) under low and high stringency washing conditions. The levels of expression in the tissues were intestine > placenta > liver > pancreas, and in the cell lines the levels were human erythroleukemia > butyrate-induced colon > colon > brain > HeLa cells. mRNA was undetectable in reticulocytes, consistent with the authors failure to detect positive clones in a size-selected ( > 2 kilobases) lambdagt11 reticulocyte cDNA library. DNA analysis revealed by a polymorphic EcoRI band and chromosome localization by flow sorting and in situ hybridization showed that the ..cap alpha.. subunit is on the short is on the short arm (band p11-p13) of chromosome 1.

Potent and Selective BACE-1 Peptide Inhibitors Lower Brain Aβ Levels Mediated by Brain Shuttle Transport

Directory of Open Access Journals (Sweden)

Nadine Ruderisch

2017-10-01

Full Text Available Therapeutic approaches to fight Alzheimer's disease include anti-Amyloidβ (Aβ antibodies and secretase inhibitors. However, the blood-brain barrier (BBB limits the brain exposure of biologics and the chemical space for small molecules to be BBB permeable. The Brain Shuttle (BS technology is capable of shuttling large molecules into the brain. This allows for new types of therapeutic modalities engineered for optimal efficacy on the molecular target in the brain independent of brain penetrating properties. To this end, we designed BACE1 peptide inhibitors with varying lipid modifications with single-digit picomolar cellular potency. Secondly, we generated active-exosite peptides with structurally confirmed dual binding mode and improved potency. When fused to the BS via sortase coupling, these BACE1 inhibitors significantly reduced brain Aβ levels in mice after intravenous administration. In plasma, both BS and non-BS BACE1 inhibitor peptides induced a significant time- and dose-dependent decrease of Aβ. Our results demonstrate that the BS is essential for BACE1 peptide inhibitors to be efficacious in the brain and active-exosite design of BACE1 peptide inhibitors together with lipid modification may be of therapeutic relevance.

Silica nanoparticles mediated neuronal cell death in corpus striatum of rat brain: implication of mitochondrial, endoplasmic reticulum and oxidative stress

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Parveen, Arshiya; Rizvi, Syed Husain Mustafa; Mahdi, Farzana; Tripathi, Sandeep; Ahmad, Iqbal; Shukla, Rajendra K.; Khanna, Vinay K.; Singh, Ranjana; Patel, Devendra K.; Mahdi, Abbas Ali

2014-11-01

Extensive uses of silica nanoparticles (SiNPs) in biomedical and industrial fields have increased the risk of exposure, resulting concerns about their safety. We focussed on some of the safety aspects by studying neurobehavioural impairment, oxidative stress (OS), neurochemical and ultrastructural changes in corpus striatum (CS) of male Wistar rats exposed to 80-nm SiNPs. Moreover, its role in inducing mitochondrial and endoplasmic reticulum (ER) stress-mediated neuronal apoptosis was also investigated. The results demonstrated impairment in neurobehavioural indices, and a significant increase in lipid peroxide levels (LPO), hydrogen peroxide (H2O2), superoxide (O2 -) and protein carbonyl content, whereas there was a significant decrease in the activities of the enzymes, manganese superoxide dismutase (Mn SOD), glutathione peroxidase (GPx), catalase (CAT) and reduced glutathione (GSH) content, suggesting impaired antioxidant defence system. Protein (cytochrome c, Bcl-2, Bax, p53, caspase-3, caspase 12 and CHOP/Gadd153) and mRNA (Bcl-2, Bax, p53 and CHOP/Gadd153, cytochrome c) expression studies of mitochondrial and ER stress-related apoptotic factors suggested that both the cell organelles were involved in OS-mediated apoptosis in treated rat brain CS. Moreover, electron microscopic studies clearly showed mitochondrial and ER dysfunction. In conclusion, the result of the study suggested that subchronic SiNPs' exposure has the potential to alter the behavioural activity and also to bring about changes in biochemical, neurochemical and ultrastructural profiles in CS region of rat brain. Furthermore, we also report SiNPs-induced apoptosis in CS, through mitochondrial and ER stress-mediated signalling.

Leptin Mediate High Fat Diet Sensitization of Angiotensin II-elicited Hypertension by Upregulating the Brain Renin-Angiotensin System and Inflammation

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Xue, Baojian; Yu, Yang; Zhang, Zhongming; Guo, Fang; Beltz, Terry G.; Thunhorst, Robert L.; Felder, Robert B.; Johnson, Alan Kim

2016-01-01

Obesity is characterized by increased circulating levels of the adipocyte-derived hormone leptin, which can increase sympathetic nerve activity and raise blood pressure. A previous study revealed that rats fed a high fat diet (HFD) have an enhanced hypertensive response to subsequent angiotensin (Ang) II administration that is mediated at least in part by increased activity of brain renin-angiotensin system (RAS) and proinflammatory cytokines (PICs). The present study tested whether leptin mediates this HFD-induced sensitization of Ang II-elicited hypertension by interacting with brain RAS and PICs mechanisms. Rats fed a HFD for 3 weeks had significant increases in white adipose tissue mass, plasma leptin levels and mRNA expression of leptin and its receptors in the lamina terminalis (LT) and hypothalamic paraventricular nucleus (PVN). Central infusion of a leptin receptor antagonist during HFD feeding abolished HFD sensitization of Ang II-elicited hypertension. Furthermore, central infusion of leptin mimicked the sensitizing action of HFD. Concomitant central infusions of the AT1-R antagonist irbesartan, the TNF-α synthesis inhibitor pentoxifylline, or the inhibitor of microglial activation minocycline prevented the sensitization produced by central infusion of leptin. RT-PCR analysis indicated that either HFD or leptin administration upregulated mRNA expression of several components of the RAS and PICs in the LT and PVN. The leptin antagonist and the inhibitors of AT1-R, TNF-α synthesis and microglial activation all reversed the expression of these genes. The results suggest that HFD-induced sensitization of Ang II-elicited hypertension is mediated by leptin through upregulation of central RAS and PICs. PMID:27021010

Liposome-Based Adjuvants for Subunit Vaccines: Formulation Strategies for Subunit Antigens and Immunostimulators

DEFF Research Database (Denmark)

Schmidt, Signe Tandrup; Foged, Camilla; Korsholm, Karen Smith

2016-01-01

be classified into delivery systems or immunostimulators. Liposomes are versatile delivery systems for antigens, and they can carefully be customized towards desired immune profiles by combining them with immunostimulators and optimizing their composition, physicochemical properties and antigen-loading mode......The development of subunit vaccines has become very attractive in recent years due to their superior safety profiles as compared to traditional vaccines based on live attenuated or whole inactivated pathogens, and there is an unmet medical need for improved vaccines and vaccines against pathogens...... of immunostimulators and antigens, respectively, into liposomes, and the choice of immunostimulator should ideally be based on knowledge regarding the specific PRR expression profile of the target APCs. Here, we review state-of-the-art formulation approaches employed for the inclusion of immunostimulators and subunit...

Optimism and the brain: trait optimism mediates the protective role of the orbitofrontal cortex gray matter volume against anxiety.

Science.gov (United States)

Dolcos, Sanda; Hu, Yifan; Iordan, Alexandru D; Moore, Matthew; Dolcos, Florin

2016-02-01

Converging evidence identifies trait optimism and the orbitofrontal cortex (OFC) as personality and brain factors influencing anxiety, but the nature of their relationships remains unclear. Here, the mechanisms underlying the protective role of trait optimism and of increased OFC volume against symptoms of anxiety were investigated in 61 healthy subjects, who completed measures of trait optimism and anxiety, and underwent structural scanning using magnetic resonance imaging. First, the OFC gray matter volume (GMV) was associated with increased optimism, which in turn was associated with reduced anxiety. Second, trait optimism mediated the relation between the left OFC volume and anxiety, thus demonstrating that increased GMV in this brain region protects against symptoms of anxiety through increased optimism. These results provide novel evidence about the brain-personality mechanisms protecting against anxiety symptoms in healthy functioning, and identify potential targets for preventive and therapeutic interventions aimed at reducing susceptibility and increasing resilience against emotional disturbances. © The Author (2015). Published by Oxford University Press. For Permissions, please email: journals.permissions@oup.com.

Auxin-dependent compositional change in Mediator in ARF7- and ARF19-mediated transcription.

Science.gov (United States)

Ito, Jun; Fukaki, Hidehiro; Onoda, Makoto; Li, Lin; Li, Chuanyou; Tasaka, Masao; Furutani, Masahiko

2016-06-07

Mediator is a multiprotein complex that integrates the signals from transcription factors binding to the promoter and transmits them to achieve gene transcription. The subunits of Mediator complex reside in four modules: the head, middle, tail, and dissociable CDK8 kinase module (CKM). The head, middle, and tail modules form the core Mediator complex, and the association of CKM can modify the function of Mediator in transcription. Here, we show genetic and biochemical evidence that CKM-associated Mediator transmits auxin-dependent transcriptional repression in lateral root (LR) formation. The AUXIN/INDOLE 3-ACETIC ACID 14 (Aux/IAA14) transcriptional repressor inhibits the transcriptional activity of its binding partners AUXIN RESPONSE FACTOR 7 (ARF7) and ARF19 by making a complex with the CKM-associated Mediator. In addition, TOPLESS (TPL), a transcriptional corepressor, forms a bridge between IAA14 and the CKM component MED13 through the physical interaction. ChIP assays show that auxin induces the dissociation of MED13 but not the tail module component MED25 from the ARF7 binding region upstream of its target gene. These findings indicate that auxin-induced degradation of IAA14 changes the module composition of Mediator interacting with ARF7 and ARF19 in the upstream region of their target genes involved in LR formation. We suggest that this regulation leads to a quick switch of signal transmission from ARFs to target gene expression in response to auxin.

Catalytic Subunit 1 of Protein Phosphatase 2A Is a Subunit of the STRIPAK Complex and Governs Fungal Sexual Development.

Science.gov (United States)

Beier, Anna; Teichert, Ines; Krisp, Christoph; Wolters, Dirk A; Kück, Ulrich

2016-06-21

The generation of complex three-dimensional structures is a key developmental step for most eukaryotic organisms. The details of the molecular machinery controlling this step remain to be determined. An excellent model system to study this general process is the generation of three-dimensional fruiting bodies in filamentous fungi like Sordaria macrospora Fruiting body development is controlled by subunits of the highly conserved striatin-interacting phosphatase and kinase (STRIPAK) complex, which has been described in organisms ranging from yeasts to humans. The highly conserved heterotrimeric protein phosphatase PP2A is a subunit of STRIPAK. Here, catalytic subunit 1 of PP2A was functionally characterized. The Δpp2Ac1 strain is sterile, unable to undergo hyphal fusion, and devoid of ascogonial septation. Further, PP2Ac1, together with STRIPAK subunit PRO22, governs vegetative and stress-related growth. We revealed in vitro catalytic activity of wild-type PP2Ac1, and our in vivo analysis showed that inactive PP2Ac1 blocks the complementation of the sterile deletion strain. Tandem affinity purification, followed by mass spectrometry and yeast two-hybrid analysis, verified that PP2Ac1 is a subunit of STRIPAK. Further, these data indicate links between the STRIPAK complex and other developmental signaling pathways, implying the presence of a large interconnected signaling network that controls eukaryotic developmental processes. The insights gained in our study can be transferred to higher eukaryotes and will be important for understanding eukaryotic cellular development in general. The striatin-interacting phosphatase and kinase (STRIPAK) complex is highly conserved from yeasts to humans and is an important regulator of numerous eukaryotic developmental processes, such as cellular signaling and cell development. Although functional insights into the STRIPAK complex are accumulating, the detailed molecular mechanisms of single subunits are only partially understood

Dynamic functional brain connectivity for face perception

NARCIS (Netherlands)

Yang, Yuan; Qiu, Yihong; Schouten, Alfred C.

2015-01-01

Face perception is mediated by a distributed brain network comprised of the core system at occipito-temporal areas and the extended system at other relevant brain areas involving bilateral hemispheres. In this study we explored how the brain connectivity changes over the time for face-sensitive

Bidirectional apical-basal traffic of the cation-independent mannose-6-phosphate receptor in brain endothelial cells

DEFF Research Database (Denmark)

Siupka, Piotr; Hersom, Maria N. S.; Lykke-Hartmann, Karin

2017-01-01

Brain capillary endothelium mediates the exchange of nutrients between blood and brain parenchyma. This barrier function of the brain capillaries also limits passage of pharmaceuticals from blood to brain, which hinders treatment of several neurological disorders. Receptor-mediated transport has ...

INTRINSIC REGULATION OF HEMOGLOBIN EXPRESSION BY VARIABLE SUBUNIT INTERFACE STRENGTHS

Science.gov (United States)

Manning, James M.; Popowicz, Anthony M.; Padovan, Julio C.; Chait, Brian T.; Manning, Lois R.

2012-01-01

SUMMARY The expression of the six types of human hemoglobin subunits over time is currently considered to be regulated mainly by transcription factors that bind to upstream control regions of the gene (the “extrinsic” component of regulation). Here we describe how subunit pairing and further assembly to tetramers in the liganded state is influenced by the affinity of subunits for one another (the “intrinsic” component of regulation). The adult hemoglobin dimers have the strongest subunit interfaces and the embryonic hemoglobins are the weakest with fetal hemoglobins of intermediate strength, corresponding to the temporal order of their expression. These variable subunit binding strengths and the attenuating effects of acetylation contribute to the differences with which these hemoglobin types form functional O2-binding tetramers consistent with gene switching. PMID:22129306

Immunochemical aspects of crotoxim and its subunits

International Nuclear Information System (INIS)

Nakazone, A.K.

1979-01-01

Crotamine and crotoxin with the subunits - phospholipase A and crotapotin - were obtained by purification from Crotalus durissus terrificus venom. Interaction studies of the subunits using crotalic antiserum, indicated that: crotoxin is formed of crotapotin and phospholipase A with the molar ratio of 1 to 1; using crotapotin 125 I the presence of a soluble complex was shown with the same antiserum. Immunological precipitation reactions demonstrated that crotapotin is antigenic: crotapotin and phospholipase A presented similar antigenic determinants; crotoxin antiserum reacted with each one of the submits; when the subunits are mixed to form synthetic crotoxin some antigenic determinants are masked in the process of interaction. Crotamine, interacted with crotapotin 1:1, without hidden antigenic determinants crotapotin antigenic site seems to be formed by, at least, one lysine. Enzimatical activity of phospholipase A apreared to be dependent on some reaction conditions when its arginine residues are blocked. Tyrosines of phospholipase A are more susceptible to labelling with 131 I than crotapotin. Gama irradiation of aqueous solutions of the subunits produced modifications in the ultraviolet spectra. A decrease of the enzymatic activity occured as a function of radiation dosis. Immunological activities of crotapotin and phospholipase A were not altered [pt

The Mediator complex: a master coordinator of transcription and cell lineage development.

Science.gov (United States)

Yin, Jing-wen; Wang, Gang

2014-03-01

Mediator is a multiprotein complex that is required for gene transcription by RNA polymerase II. Multiple subunits of the complex show specificity in relaying information from signals and transcription factors to the RNA polymerase II machinery, thus enabling control of the expression of specific genes. Recent studies have also provided novel mechanistic insights into the roles of Mediator in epigenetic regulation, transcriptional elongation, termination, mRNA processing, noncoding RNA activation and super enhancer formation. Based on these specific roles in gene regulation, Mediator has emerged as a master coordinator of development and cell lineage determination. Here, we describe the most recent advances in understanding the mechanisms of Mediator function, with an emphasis on its role during development and disease.

Gz mediates the long-lasting desensitization of brain CB1 receptors and is essential for cross-tolerance with morphine

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Rodríguez-Muñoz María

2009-03-01

Full Text Available Abstract Background Although the systemic administration of cannabinoids produces antinociception, their chronic use leads to analgesic tolerance as well as cross-tolerance to morphine. These effects are mediated by cannabinoids binding to peripheral, spinal and supraspinal CB1 and CB2 receptors, making it difficult to determine the relevance of each receptor type to these phenomena. However, in the brain, the CB1 receptors (CB1Rs are expressed at high levels in neurons, whereas the expression of CB2Rs is marginal. Thus, CB1Rs mediate the effects of smoked cannabis and are also implicated in emotional behaviors. We have analyzed the production of supraspinal analgesia and the development of tolerance at CB1Rs by the direct injection of a series of cannabinoids into the brain. The influence of the activation of CB1Rs on supraspinal analgesia evoked by morphine was also evaluated. Results Intracerebroventricular (icv administration of cannabinoid receptor agonists, WIN55,212-2, ACEA or methanandamide, generated a dose-dependent analgesia. Notably, a single administration of these compounds brought about profound analgesic tolerance that lasted for more than 14 days. This decrease in the effect of cannabinoid receptor agonists was not mediated by depletion of CB1Rs or the loss of regulated G proteins, but, nevertheless, it was accompanied by reduced morphine analgesia. On the other hand, acute morphine administration produced tolerance that lasted only 3 days and did not affect the CB1R. We found that both neural mu-opioid receptors (MORs and CB1Rs interact with the HINT1-RGSZ module, thereby regulating pertussis toxin-insensitive Gz proteins. In mice with reduced levels of these Gz proteins, the CB1R agonists produced no such desensitization or morphine cross-tolerance. On the other hand, experimental enhancement of Gz signaling enabled an acute icv administration of morphine to produce a long-lasting tolerance at MORs that persisted for more than

Brain activation to negative stimuli mediates a relationship between adolescent marijuana use and later emotional functioning.

Science.gov (United States)

Heitzeg, Mary M; Cope, Lora M; Martz, Meghan E; Hardee, Jillian E; Zucker, Robert A

2015-12-01

This work investigated the impact of heavy marijuana use during adolescence on emotional functioning, as well as the brain functional mediators of this effect. Participants (n=40) were recruited from the Michigan Longitudinal Study (MLS). Data on marijuana use were collected prospectively beginning in childhood as part of the MLS. Participants were classified as heavy marijuana users (n=20) or controls with minimal marijuana use. Two facets of emotional functioning-negative emotionality and resiliency (a self-regulatory mechanism)-were assessed as part of the MLS at three time points: mean age 13.4, mean age 19.6, and mean age 23.1. Functional neuroimaging data during an emotion-arousal word task were collected at mean age 20.2. Negative emotionality decreased and resiliency increased across the three time points in controls but not heavy marijuana users. Compared with controls, heavy marijuana users had less activation to negative words in temporal, prefrontal, and occipital cortices, insula, and amygdala. Activation of dorsolateral prefrontal cortex to negative words mediated an association between marijuana group and later negative emotionality. Activation of the cuneus/lingual gyrus mediated an association between marijuana group and later resiliency. Results support growing evidence that heavy marijuana use during adolescence affects later emotional outcomes. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Enhanced Long-Term and Impaired Short-Term Spatial Memory in GluA1 AMPA Receptor Subunit Knockout Mice: Evidence for a Dual-Process Memory Model

Science.gov (United States)

Sanderson, David J.; Good, Mark A.; Skelton, Kathryn; Sprengel, Rolf; Seeburg, Peter H.; Rawlins, J. Nicholas P.; Bannerman, David M.

2009-01-01

The GluA1 AMPA receptor subunit is a key mediator of hippocampal synaptic plasticity and is especially important for a rapidly-induced, short-lasting form of potentiation. GluA1 gene deletion impairs hippocampus-dependent, spatial working memory, but spares hippocampus-dependent spatial reference memory. These findings may reflect the necessity of…

A conserved phenylalanine as a relay between the α5 helix and the GDP binding region of heterotrimeric Gi protein α subunit.

Science.gov (United States)

Kaya, Ali I; Lokits, Alyssa D; Gilbert, James A; Iverson, Tina M; Meiler, Jens; Hamm, Heidi E

2014-08-29

G protein activation by G protein-coupled receptors is one of the critical steps for many cellular signal transduction pathways. Previously, we and other groups reported that the α5 helix in the G protein α subunit plays a major role during this activation process. However, the precise signaling pathway between the α5 helix and the guanosine diphosphate (GDP) binding pocket remains elusive. Here, using structural, biochemical, and computational techniques, we probed different residues around the α5 helix for their role in signaling. Our data showed that perturbing the Phe-336 residue disturbs hydrophobic interactions with the β2-β3 strands and α1 helix, leading to high basal nucleotide exchange. However, mutations in β strands β5 and β6 do not perturb G protein activation. We have highlighted critical residues that leverage Phe-336 as a relay. Conformational changes are transmitted starting from Phe-336 via β2-β3/α1 to Switch I and the phosphate binding loop, decreasing the stability of the GDP binding pocket and triggering nucleotide release. When the α1 and α5 helices were cross-linked, inhibiting the receptor-mediated displacement of the C-terminal α5 helix, mutation of Phe-336 still leads to high basal exchange rates. This suggests that unlike receptor-mediated activation, helix 5 rotation and translocation are not necessary for GDP release from the α subunit. Rather, destabilization of the backdoor region of the Gα subunit is sufficient for triggering the activation process. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Inter-subunit interactions across the upper voltage sensing-pore domain interface contribute to the concerted pore opening transition of Kv channels.

Directory of Open Access Journals (Sweden)

Tzilhav Shem-Ad

Full Text Available The tight electro-mechanical coupling between the voltage-sensing and pore domains of Kv channels lies at the heart of their fundamental roles in electrical signaling. Structural data have identified two voltage sensor pore inter-domain interaction surfaces, thus providing a framework to explain the molecular basis for the tight coupling of these domains. While the contribution of the intra-subunit lower domain interface to the electro-mechanical coupling that underlies channel opening is relatively well understood, the contribution of the inter-subunit upper interface to channel gating is not yet clear. Relying on energy perturbation and thermodynamic coupling analyses of tandem-dimeric Shaker Kv channels, we show that mutation of upper interface residues from both sides of the voltage sensor-pore domain interface stabilizes the closed channel state. These mutations, however, do not affect slow inactivation gating. We, moreover, find that upper interface residues form a network of state-dependent interactions that stabilize the open channel state. Finally, we note that the observed residue interaction network does not change during slow inactivation gating. The upper voltage sensing-pore interaction surface thus only undergoes conformational rearrangements during channel activation gating. We suggest that inter-subunit interactions across the upper domain interface mediate allosteric communication between channel subunits that contributes to the concerted nature of the late pore opening transition of Kv channels.

Inter-subunit interactions across the upper voltage sensing-pore domain interface contribute to the concerted pore opening transition of Kv channels.

Science.gov (United States)

Shem-Ad, Tzilhav; Irit, Orr; Yifrach, Ofer

2013-01-01

The tight electro-mechanical coupling between the voltage-sensing and pore domains of Kv channels lies at the heart of their fundamental roles in electrical signaling. Structural data have identified two voltage sensor pore inter-domain interaction surfaces, thus providing a framework to explain the molecular basis for the tight coupling of these domains. While the contribution of the intra-subunit lower domain interface to the electro-mechanical coupling that underlies channel opening is relatively well understood, the contribution of the inter-subunit upper interface to channel gating is not yet clear. Relying on energy perturbation and thermodynamic coupling analyses of tandem-dimeric Shaker Kv channels, we show that mutation of upper interface residues from both sides of the voltage sensor-pore domain interface stabilizes the closed channel state. These mutations, however, do not affect slow inactivation gating. We, moreover, find that upper interface residues form a network of state-dependent interactions that stabilize the open channel state. Finally, we note that the observed residue interaction network does not change during slow inactivation gating. The upper voltage sensing-pore interaction surface thus only undergoes conformational rearrangements during channel activation gating. We suggest that inter-subunit interactions across the upper domain interface mediate allosteric communication between channel subunits that contributes to the concerted nature of the late pore opening transition of Kv channels.

Structural characterization of recombinant crustacyanin subunits from the lobster Homarus americanus

International Nuclear Information System (INIS)

Ferrari, Michele; Folli, Claudia; Pincolini, Elisa; McClintock, Timothy S.; Rössle, Manfred; Berni, Rodolfo; Cianci, Michele

2012-01-01

The two recombinant apo subunits H1 and H2 from H. americanus have been structurally characterized. Reconstitution studies with astaxanthin reproduced the bathochromic shift of 85–95 nm typical of the natural crustacyanin subunits. Crustacean crustacyanin proteins are linked to the production and modification of carapace colour, with direct implications for fitness and survival. Here, the structural and functional properties of the two recombinant crustacyanin subunits H 1 and H 2 from the American lobster Homarus americanus are reported. The two subunits are structurally highly similar to the corresponding natural apo crustacyanin CRTC and CRTA subunits from the European lobster H. gammarus. Reconstitution studies of the recombinant crustacyanin proteins H 1 and H 2 with astaxanthin reproduced the bathochromic shift of 85–95 nm typical of the natural crustacyanin subunits from H. gammarus in complex with astaxanthin. Moreover, correlations between the presence of crustacyanin genes in crustacean species and the resulting carapace colours with the spectral properties of the subunits in complex with astaxanthin confirmed this genotype–phenotype linkage

Identification of the subunit of cAMP receptor protein (CRP) that functionally interacts with CytR in CRP-CytR-mediated transcriptional repression

DEFF Research Database (Denmark)

Meibom, K L; Kallipolitis, B H; Ebright, R H

2000-01-01

At promoters of the Escherichia coli CytR regulon, the cAMP receptor protein (CRP) interacts with the repressor CytR to form transcriptionally inactive CRP-CytR-promoter or (CRP)(2)-CytR-promoter complexes. Here, using "oriented heterodimer" analysis, we show that only one subunit of the CRP dimer......, the subunit proximal to CytR, functionally interacts with CytR in CRP-CytR-promoter and (CRP)(2)-CytR-promoter complexes. Our results provide information about the architecture of CRP-CytR-promoter and (CRP)(2)-CytR-promoter complexes and rule out the proposal that masking of activating region 2 of CRP...

Probing the functional subunits of the tonoplast H+-ATPase

International Nuclear Information System (INIS)

Randall, S.K.; Lai, S.; Sze, H.

1986-01-01

The tonoplast ATPase of oat roots is composed of at least three polypeptides of 72, 60, and 16 kDa. The 16 kDA polypeptide covalently binds N,N'-dicyclohexylcarbodiimide and is postulated to be a component of the proton channel. Initial studies to identify other subunits indicate that both the 72 and 60 kDa subunits covalently bind 14 C]-7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and [ 14 C]N-ethylamleimide, inhibitors of the tonoplast ATPase. ATP prevents binding of these inhibitors suggesting that both the 72 and 60 kDa subunits are involved in substrate binding. Polyclonal antibody has been made to the 72 kDa subunit. Western blot analysis of tonoplast vesicles reveals single reactive polypeptide (72 kDa). The antibody shows no cross-reactivity towards either the mitochondrial F 1 -ATPase or the plasma membrane ATPase. This antibody specifically inhibits ATP hydrolysis and ATP-dependent H + pumping in native tonoplast vesicles. The authors conclude that the 72 kDa subunit is intimately associated with the catalytic (or ATP-binding) site

Adeno-associated virus vector-mediated transduction in the cat brain.

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Vite, Charles H; Passini, Marco A; Haskins, Mark E; Wolfe, John H

2003-10-01

Adeno-associated virus (AAV) vectors are capable of delivering a therapeutic gene to the mouse brain that can result in long-term and widespread protein production. However, the human infant brain is more than 1000 times larger than the mouse brain, which will make the treatment of global neurometabolic disorders in children more difficult. In this study, we evaluated the ability of three AAV serotypes (1,2, and 5) to transduce cells in the cat brain as a model of a large mammalian brain. The human lysosomal enzyme beta-glucuronidase (GUSB) was used as a reporter gene, because it can be distinguished from feline GUSB by heat stability. The vectors were injected into the cerebral cortex, caudate nucleus, thalamus, corona radiata, internal capsule, and centrum semiovale of 8-week-old cats. The brains were evaluated for gene expression using in situ hybridization and enzyme histochemistry 10 weeks after surgery. The AAV2 vector was capable of transducing cells in the gray matter, while the AAV1 vector resulted in greater transduction of the gray matter than AAV2 as well as transduction of the white matter. AAV5 did not result in detectable transduction in the cat brain.

Constitutive Activation of the G-Protein Subunit G[alpha]s within Forebrain Neurons Causes PKA-Dependent Alterations in Fear Conditioning and Cortical "Arc" mRNA Expression

Science.gov (United States)

Kelly, Michele P.; Cheung, York-Fong; Favilla, Christopher; Siegel, Steven J.; Kanes, Stephen J.; Houslay, Miles D.; Abel, Ted

2008-01-01

Memory formation requires cAMP signaling; thus, this cascade has been of great interest in the search for cognitive enhancers. Given that medications are administered long-term, we determined the effects of chronically increasing cAMP synthesis in the brain by expressing a constitutively active isoform of the G-protein subunit G[alpha]s…

Smuggling Drugs into the Brain: An Overview of Ligands Targeting Transcytosis for Drug Delivery across the Blood–Brain Barrier

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Julia V. Georgieva

2014-11-01

Full Text Available The blood–brain barrier acts as a physical barrier that prevents free entry of blood-derived substances, including those intended for therapeutic applications. The development of molecular Trojan horses is a promising drug targeting technology that allows for non-invasive delivery of therapeutics into the brain. This concept relies on the application of natural or genetically engineered proteins or small peptides, capable of specifically ferrying a drug-payload that is either directly coupled or encapsulated in an appropriate nanocarrier, across the blood–brain barrier via receptor-mediated transcytosis. Specifically, in this process the nanocarrier–drug system (“Trojan horse complex” is transported transcellularly across the brain endothelium, from the blood to the brain interface, essentially trailed by a native receptor. Naturally, only certain properties would favor a receptor to serve as a transporter for nanocarriers, coated with appropriate ligands. Here we briefly discuss brain microvascular endothelial receptors that have been explored until now, highlighting molecular features that govern the efficiency of nanocarrier-mediated drug delivery into the brain.

(−)1-(Benzofuran-2-yl)-2-propylaminopentane, [(−)BPAP], a selective enhancer of the impulse propagation mediated release of catecholamines and serotonin in the brain

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Knoll, Joseph; Yoneda, Fumio; Knoll, Berta; Ohde, Hironori; Miklya, Ildikó

1999-01-01

The brain constituents β-phenylethylamine (PEA) and tryptamine enhance the impulse propagation mediated transmitter release (exocytosis) from the catecholaminergic and serotoninergic neurons in the brain (‘catecholaminergic/serotoninergic activity enhancer, CAE/SAE, effect'). (−)Deprenyl (Selegiline) and (−)1-phenyl-2-propylaminopentane [(−)PPAP] are amphetamine derived CAE substances devoid of the catecholamine releasing property.By changing the aromatic ring in PPAP we developed highly potent and selective CAE/SAE substances, structurally unrelated to the amphetamines. Out of 65 newly synthetized compounds, a tryptamine derived structure, (−)1-(benzofuran-2-yl)-2-propylaminopentane [(−)BPAP] was selected as a potential follower of (−)deprenyl in the clinic and as a reference compound for further analysis of the CAE/SAE mechanism in the mammalian brain.(−)BPAP significantly enhanced in 0.18 μmol 1−1 concentration the impulse propagation mediated release of [3H]-noradrenaline and [3H]-dopamine and in 36 nmol 1−1 concentration the release of [3H]-serotonin from the isolated brain stem of rats. The amount of catecholamines and serotonin released from isolated discrete rat brain regions (dopamine from the striatum, substantia nigra and tuberculum olfactorium, noradrenaline from the locus coeruleus and serotonin from the raphe) enhanced significantly in the presence of 10−12–10−14 M (−)BPAP. BPAP protected cultured hippocampal neurons from the neurotoxic effect of β-amyloid in 10−14 M concentration. In rats (−)BPAP significantly enhanced the activity of the catecholaminergic and serotoninergic neurons in the brain 30 min after acute injection of 0.1 μg kg−1 s.c. In the shuttle box, (−)BPAP in rats was about 130 times more potent than (−)deprenyl in antagonizing tetrabenazine induced inhibition of performance. PMID:10588928

Characterization of Mediator Complex and its Associated Proteins from Rice.

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Samanta, Subhasis; Thakur, Jitendra Kumar

2017-01-01

The Mediator complex is a multi-protein complex that acts as a molecular bridge conveying transcriptional messages from the cis element-bound transcription factor to the RNA Polymerase II machinery. It is found in all eukaryotes including members of the plant kingdom. Increasing number of reports from plants regarding different Mediator subunits involved in a multitude of processes spanning from plant development to environmental interactions have firmly established it as a central hub of plant regulatory networks. Routine isolation of Mediator complex in a particular species is a necessity because of many reasons. First, composition of the Mediator complex varies from species to species. Second, the composition of the Mediator complex in a particular species is not static under all developmental and environmental conditions. Besides this, at times, Mediator complex is used in in vitro transcription systems. Rice, a staple food crop of the world, is used as a model monocot crop. Realizing the need of a reliable protocol for the isolation of Mediator complex from plants, we describe here the isolation of Mediator complex from rice.

Crystal structure of the P pilus rod subunit PapA.

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Denis Verger

2007-05-01

Full Text Available P pili are important adhesive fibres involved in kidney infection by uropathogenic Escherichia coli strains. P pili are assembled by the conserved chaperone-usher pathway, which involves the PapD chaperone and the PapC usher. During pilus assembly, subunits are incorporated into the growing fiber via the donor-strand exchange (DSE mechanism, whereby the chaperone's G1 beta-strand that complements the incomplete immunoglobulin-fold of each subunit is displaced by the N-terminal extension (Nte of an incoming subunit. P pili comprise a helical rod, a tip fibrillum, and an adhesin at the distal end. PapA is the rod subunit and is assembled into a superhelical right-handed structure. Here, we have solved the structure of a ternary complex of PapD bound to PapA through donor-strand complementation, itself bound to another PapA subunit through DSE. This structure provides insight into the structural basis of the DSE reaction involving this important pilus subunit. Using gel filtration chromatography and electron microscopy on a number of PapA Nte mutants, we establish that PapA differs in its mode of assembly compared with other Pap subunits, involving a much larger Nte that encompasses not only the DSE region of the Nte but also the region N-terminal to it.

The Role of Brain-Reactive Autoantibodies in Brain Pathology and Cognitive Impairment

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Simone Mader

2017-09-01

Full Text Available Antibodies to different brain proteins have been recently found to be associated with an increasing number of different autoimmune diseases. They need to penetrate the blood–brain barrier (BBB in order to bind antigens within the central nervous system (CNS. They can target either neuronal or non-neuronal antigen and result in damage either by themselves or in synergy with other inflammatory mediators. Antibodies can lead to acute brain pathology, which may be reversible; alternatively, they may trigger irreversible damage that persists even though the antibodies are no longer present. In this review, we will describe two different autoimmune conditions and the role of their antibodies in causing brain pathology. In systemic lupus erythematosus (SLE, patients can have double stranded DNA antibodies that cross react with the neuronal N-methyl-d-aspartate receptor (NMDAR, which have been recently linked to neurocognitive dysfunction. In neuromyelitis optica (NMO, antibodies to astrocytic aquaporin-4 (AQP4 are diagnostic of disease. There is emerging evidence that pathogenic T cells also play an important role for the disease pathogenesis in NMO since they infiltrate in the CNS. In order to enable appropriate and less invasive treatment for antibody-mediated diseases, we need to understand the mechanisms of antibody-mediated pathology, the acute and chronic effects of antibody exposure, if the antibodies are produced intrathecally or systemically, their target antigen, and what triggers their production. Emerging data also show that in utero exposure to some brain-reactive antibodies, such as those found in SLE, can cause neurodevelopmental impairment since they can penetrate the embryonic BBB. If the antibody exposure occurs at a critical time of development, this can result in irreversible damage of the offspring that persists throughout adulthood.

Casein kinase 2 down-regulation and activation by polybasic peptides are mediated by acidic residues in the 55-64 region of the beta-subunit. A study with calmodulin as phosphorylatable substrate

DEFF Research Database (Denmark)

Meggio, F; Boldyreff, B; Issinger, O G

1994-01-01

to substitute for wild-type beta-subunit as a suppressor of activity toward calmodulin. The only mutations that reduced the ability of the beta-subunit to suppress calmodulin phosphorylation activity, though being compatible with normal reconstitution of CK2 holoenzyme, were those affecting Asp55, Glu57...... are conversely ineffective. The latent "calmodulin kinase" activity of CK2 can also be specifically unmasked by a peptide (alpha[66-86]) reproducing a basic insert of the catalytic subunit. This effect is reversed by equimolar addition of a peptide (beta[55-71]) including the 55-64 acidic stretch of the beta......-subunit. Comparable polylysine stimulation was observed with the holoenzymes reconstituted with either beta wt or the beta mutants capable of assembling with the alpha-subunit, with the notable exception of those bearing Ala substitutions for acidic residues at positions 55, 57, and 59-61. These were nearly...

The GABAB1a isoform mediates heterosynaptic depression at hippocampal mossy fiber synapses

DEFF Research Database (Denmark)

Guetg, Nicole; Seddik, Riad; Vigot, Réjan

2009-01-01

)) receptors mediate heterosynaptic depression of MF transmission, a physiological phenomenon involving transsynaptic inhibition of glutamate release via presynaptic GABA(B) receptors. Our data demonstrate that the difference in GABA(B1a) and GABA(B1b) protein levels at MF terminals is sufficient to produce...... a strictly GABA(B1a)-specific effect under physiological conditions. This consolidates that the differential subcellular localization of the GABA(B1a) and GABA(B1b) proteins is of regulatory relevance.......GABA(B) receptor subtypes are based on the subunit isoforms GABA(B1a) and GABA(B1b), which associate with GABA(B2) subunits to form pharmacologically indistinguishable GABA(B(1a,2)) and GABA(B(1b,2)) receptors. Studies with mice selectively expressing GABA(B1a) or GABA(B1b) subunits revealed...

Muscular subunits transplantation for facial reanimation

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Hazan André Salo Buslik

2006-01-01

Full Text Available PURPOSE: To present an alternative technique for reconstruction of musculocutaneous damages in the face transferring innervated subsegments(subunits of the latissimus dorsi flap for replacement of various facial mimetic muscles. METHODS: One clinical case of trauma with skin and mimetic muscles damage is described as an example of the technique. The treatment was performed with microsurgical transfer of latissimus dorsi muscle subunits. Each subunit present shape and dimensions of the respective mimetic muscles replaced. The origin, insertions and force vectors for the mimicmuscle lost were considered. Each subsegment has its own arterial and venous supply with a motor nerve component for the muscular unit. RESULTS: Pre and one year postoperative photos registration of static and dynamic mimic aspects, as well as digital electromyography digital data of the patients were compared. The transplanted muscular units presented myoeletric activity, fulfilling both the functional and cosmetic aspect. CONCLUSION: This technique seems to be a promising way to deal with the complex musculocutaneous losses of the face as well as facial palsy.

Association of C-Type Lectin Mincle with FcεRIβγ Subunits Leads to Functional Activation of RBL-2H3 Cells through Syk.

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Honjoh, Chisato; Chihara, Kazuyasu; Yoshiki, Hatsumi; Yamauchi, Shota; Takeuchi, Kenji; Kato, Yuji; Hida, Yukio; Ishizuka, Tamotsu; Sada, Kiyonao

2017-04-10

Macrophage-inducible C-type lectin (Mincle) interacts with the γ-subunit of high-affinity IgE receptor (FcεRIγ) and activates Syk by recognizing its specific ligand, trehalose-6,6'-dimycolate, a glycolipid produced by Mycobacterium tuberculosis. It has been suggested that mast cells participate in the immune defense against pathogenic microbes including M. tuberculosis, although the functions are still uncertain. In this study, we examined the Mincle-mediated signaling pathway and cellular responses using RBL-2H3 cells. Mincle formed a protein complex with not only FcεRIγ but also FcεRIβ in a stable cell line expressing myc-tagged Mincle. In addition, engagement of Mincle increased the levels of protein tyrosine phosphorylation and ERK phosphorylation. A pull-down assay demonstrated that cross-linking of Mincle induced binding of FcεRIβγ subunits to the Src homology 2 domain of Syk. Pharmacological and genetic studies indicated that activation of Syk was critical for Mincle-mediated activation of phospholipase Cγ2, leading to the activation of ERK and nuclear factor of activated T cells. Moreover, engagement of Mincle efficiently induced up-regulation of characteristic mast cell genes in addition to degranulation. Taken together, our present results suggest that mast cells contribute to Mincle-mediated immunity through Syk activation triggered by association with the FcεRIβγ complex.

[Liposome-mediated glial growth factor 2 gene therapy in brain injury: an experimental study with rats].

Science.gov (United States)

Xue, Ya-jun; Dong, Yan; Han, Xi; Wei, Mei-yang; Ge, Jun-hui; Cai, Ru-jue; Hu, Guo-han; Luo, Chun; Zhu, Cheng; Lu, Yi-cheng

2006-09-05

hippocampus of the treatment group was 102 +/- 11, significantly more than those of the vector control group and liposome group (67 +/- 8 and 58 +/- 9 respectively, both P < 0.01). Higher level of immunoreactivity with MBP was also detected in the cortex in the rats of the treatment group. Cationic liposome-mediated GGF2 gene therapy effectively promotes the recovery of brain injury.

The metazoan Mediator co-activator complex as an integrative hub for transcriptional regulation.

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Malik, Sohail; Roeder, Robert G

2010-11-01

The Mediator is an evolutionarily conserved, multiprotein complex that is a key regulator of protein-coding genes. In metazoan cells, multiple pathways that are responsible for homeostasis, cell growth and differentiation converge on the Mediator through transcriptional activators and repressors that target one or more of the almost 30 subunits of this complex. Besides interacting directly with RNA polymerase II, Mediator has multiple functions and can interact with and coordinate the action of numerous other co-activators and co-repressors, including those acting at the level of chromatin. These interactions ultimately allow the Mediator to deliver outputs that range from maximal activation of genes to modulation of basal transcription to long-term epigenetic silencing.

Pituitary glycoprotein hormone a-subunit secretion by cirrhotic patients

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Oliveira M.C.

1999-01-01

Full Text Available Secretion of the a-subunit of pituitary glycoprotein hormones usually follows the secretion of intact gonadotropins and is increased in gonadal failure and decreased in isolated gonadotropin deficiency. The aim of the present study was to determine the levels of the a-subunit in the serum of patients with cirrhosis of the liver and to compare the results obtained for eugonadal cirrhotic patients with those obtained for cirrhotic patients with hypogonadotropic hypogonadism. Forty-seven of 63 patients with cirrhosis (74.6% presented hypogonadism (which was central in 45 cases and primary in 2, 7 were eugonadal, and 9 women were in normal menopause. The serum a-subunit was measured by the fluorimetric method using monoclonal antibodies. Cross-reactivity with LH, TSH, FSH and hCG was 6.5, 1.2, 4.3 and 1.1%, respectively, with an intra-assay coefficient of variation (CV of less than 5% and an interassay CV of 5%, and sensitivity limit of 4 ng/l. The serum a-subunit concentration ranged from 36 to 6253 ng/l, with a median of 273 ng/l. The median was 251 ng/l for patients with central hypogonadism and 198 ng/l for eugonadal patients. The correlation between the a-subunit and basal LH levels was significant both in the total sample (r = 0.48, P<0.01 and in the cirrhotic patients with central hypogonadism (r = 0.33, P = 0.02. Among men with central hypogonadism there was a negative correlation between a-subunit levels and total testosterone levels (r = 0.54, P<0.01 as well as free testosterone levels (r = -0.53, P<0.01. In conclusion, although the a-subunit levels are correlated with LH levels, at present they cannot be used as markers for hypogonadism in patients with cirrhosis of the liver.

Visual artistic creativity and the brain.

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Heilman, Kenneth M; Acosta, Lealani Mae

2013-01-01

Creativity is the development of a new or novel understanding--insight that leads to the expression of orderly relationships (e.g., finding and revealing the thread that unites). Visual artistic creativity plays an important role in the quality of human lives, and the goal of this chapter is to describe some of the brain mechanisms that may be important in visual artistic creativity. The initial major means of learning how the brain mediates any activity is to understand the anatomy and physiology that may support these processes. A further understanding of specific cognitive activities and behaviors may be gained by studying patients who have diseases of the brain and how these diseases influence these functions. Physiological recording such as electroencephalography and brain imaging techniques such as PET and fMRI have also allowed us to gain a better understanding of the brain mechanisms important in visual creativity. In this chapter, we discuss anatomic and physiological studies, as well as neuropsychological studies of healthy artists and patients with neurological disease that have helped us gain some insight into the brain mechanisms that mediate artistic creativity. © 2013 Elsevier B.V. All rights reserved.

Immunochemical analysis of Micrococcus lysodeikticus (luteus) F1-ATPase and its subunits.

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Urban, C; Salton, M R

1983-08-31

The F1-ATPase from Micrococcus lysodeikticus has been purified to 95% protein homogeneity in this laboratory and as all other bacterial F1S, possesses five distinct subunits with molecular weights ranging from 60 000 to 10 000 (Huberman, M. and Salton, M.R.J. (1979) Biochim. Biophys. Acta 547, 230-240). In this communication, we demonstrate the immunochemical reactivities of antibodies to native and SDS-dissociated subunits with the native and dissociated F1-ATPase and show that: (1) the antibodies generated to the native or SDS-dissociated subunits react with the native molecule; (2) all of the subunits comprising the F1 are antigenically unique as determined by crossed immunoelectrophoresis and the Ouchterlony double-diffusion techniques; (3) antibodies to the SDS-denatured individual delta- and epsilon-subunits can be used to destabilize the interaction of these specific subunits with the rest of the native F1; and (4) all subunit antibodies as well as anti-native F1 were found to inhibit ATPase activity to varying degrees, the strongest inhibition being seen with antibodies to the total F1 and anti-alpha- and anti-beta-subunit antibodies. The interaction of specific subunit antibodies may provide a new and novel way to study further and characterize the catalytic portions of F1-ATPases and in general may offer an additional method for the examination of multimeric proteins.

Endothelin-1 Mediates Brain Microvascular Dysfunction Leading to Long-Term Cognitive Impairment in a Model of Experimental Cerebral Malaria.

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Brandi D Freeman

2016-03-01

Full Text Available Plasmodium falciparum infection causes a wide spectrum of diseases, including cerebral malaria, a potentially life-threatening encephalopathy. Vasculopathy is thought to contribute to cerebral malaria pathogenesis. The vasoactive compound endothelin-1, a key participant in many inflammatory processes, likely mediates vascular and cognitive dysfunctions in cerebral malaria. We previously demonstrated that C57BL6 mice infected with P. berghei ANKA, our fatal experimental cerebral malaria model, sustained memory loss. Herein, we demonstrate that an endothelin type A receptor (ETA antagonist prevented experimental cerebral malaria-induced neurocognitive impairments and improved survival. ETA antagonism prevented blood-brain barrier disruption and cerebral vasoconstriction during experimental cerebral malaria, and reduced brain endothelial activation, diminishing brain microvascular congestion. Furthermore, exogenous endothelin-1 administration to P. berghei NK65-infected mice, a model generally regarded as a non-cerebral malaria negative control for P. berghei ANKA infection, led to experimental cerebral malaria-like memory deficits. Our data indicate that endothelin-1 is critical in the development of cerebrovascular and cognitive impairments with experimental cerebral malaria. This vasoactive peptide may thus serve as a potential target for adjunctive therapy in the management of cerebral malaria.

Large-scale structural alteration of brain in epileptic children with SCN1A mutation

OpenAIRE

Lee, Yun-Jeong; Yum, Mi-Sun; Kim, Min-Jee; Shim, Woo-Hyun; Yoon, Hee Mang; Yoo, Il Han; Lee, Jiwon; Lim, Byung Chan; Kim, Ki Joong; Ko, Tae-Sung

2017-01-01

Objective: Mutations in SCN1A gene encoding the alpha 1 subunit of the voltage gated sodium channel are associated with several epilepsy syndromes including genetic epilepsy with febrile seizures plus (GEFS+) and severe myoclonic epilepsy of infancy (SMEI). However, in most patients with SCN1A mutation, brain imaging has reported normal or non-specific findings including cerebral or cerebellar atrophy. The aim of this study was to investigate differences in brain morphometry in epileptic chil...

[Beta]-Adrenergic Receptor Activation Rescues Theta Frequency Stimulation-Induced LTP Deficits in Mice Expressing C-Terminally Truncated NMDA Receptor GluN2A Subunits

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Moody, Teena D.; Watabe, Ayako M.; Indersmitten, Tim; Komiyama, Noboru H.; Grant, Seth G. N.; O'Dell, Thomas J.

2011-01-01

Through protein interactions mediated by their cytoplasmic C termini the GluN2A and GluN2B subunits of NMDA receptors (NMDARs) have a key role in the formation of NMDAR signaling complexes at excitatory synapses. Although these signaling complexes are thought to have a crucial role in NMDAR-dependent forms of synaptic plasticity such as long-term…

Differential regulation of thyrotropin subunit apoprotein and carbohydrate biosynthesis by thyroid hormone

International Nuclear Information System (INIS)

Taylor, T.; Weintraub, B.D.

1985-01-01

The regulation of TSH apoprotein and carbohydrate biosynthesis by thyroid hormone was studied by incubating pituitaries from normal and hypothyroid (3 weeks post-thyroidectomy) rats in medium containing [ 14 C]alanine and [ 3 H] glucosamine. After 6 h, samples were sequentially treated with anti-TSH beta to precipitate TSH and free TSH beta, anti-LH beta to clear the sample of LH and free LH beta, then anti-LH alpha to precipitate free alpha-subunit. Total proteins were acid precipitated. All precipitates were subjected to electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, which were then sliced and assayed by scintillation spectrometry. In hypothyroid pituitaries plus medium, [ 14 C]alanine incorporation in combined and free beta-subunits was 26 times normal and considerably greater than the 3.4-fold increase seen in total protein; combined and free alpha-subunits showed no specific increase in apoprotein synthesis. [ 3 H]Glucosamine incorporation in combined alpha- and beta-subunits in hypothyroid samples was 13 and 21 times normal, respectively, and was greater than the 1.9-fold increase in total protein; free alpha-subunit showed no specific increase in carbohydrate synthesis. The glucosamine to alanine ratio, reflecting relative glycosylation of newly synthesized molecules, was increased in hypothyroidism for combined alpha-subunits, but not for combined beta-subunits, free alpha-subunits, or total proteins. In summary, short term hypothyroidism selectively stimulated TSH beta apoprotein synthesis and carbohydrate synthesis of combined alpha- and beta-subunits. Hypothyroidism also increased the relative glycosylation of combined alpha-subunit. Thus, thyroid hormone deficiency appears to alter the rate-limiting step in TSH assembly (i.e. beta-subunit synthesis) as well as the carbohydrate structure of TSH, which may play important roles in its biological function

Vasoactive intestinal peptide is a local mediator in a gut-brain neural axis activating intestinal gluconeogenesis.

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De Vadder, F; Plessier, F; Gautier-Stein, A; Mithieux, G

2015-03-01

Intestinal gluconeogenesis (IGN) promotes metabolic benefits through activation of a gut-brain neural axis. However, the local mediator activating gluconeogenic genes in the enterocytes remains unknown. We show that (i) vasoactive intestinal peptide (VIP) signaling through VPAC1 receptor activates the intestinal glucose-6-phosphatase gene in vivo, (ii) the activation of IGN by propionate is counteracted by VPAC1 antagonism, and (iii) VIP-positive intrinsic neurons in the submucosal plexus are increased under the action of propionate. These data support the role of VIP as a local neuromodulator released by intrinsic enteric neurons and responsible for the induction of IGN through a VPAC1 receptor-dependent mechanism in enterocytes. © 2015 John Wiley & Sons Ltd.

Recombinant Adeno-Associated Virus-Mediated microRNA Delivery into the Postnatal Mouse Brain Reveals a Role for miR-134 in Dendritogenesis in Vivo

DEFF Research Database (Denmark)

Christensen, Mette; Larsen, Lars A; Kauppinen, Sakari

2010-01-01

delivery of microRNAs in vivo by use of recombinant adeno-associated virus (rAAV). rAAV-mediated overexpression of miR-134 in neurons of the postnatal mouse brain provided evidence for a negative role of miR-134 in dendritic arborization of cortical layer V pyramidal neurons in vivo, thereby confirming...

A truncated Kv1.1 protein in the brain of the megencephaly mouse: expression and interaction

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Århem Peter

2005-11-01

Full Text Available Abstract Background The megencephaly mouse, mceph/mceph, is epileptic and displays a dramatically increased brain volume and neuronal count. The responsible mutation was recently revealed to be an eleven base pair deletion, leading to a frame shift, in the gene encoding the potassium channel Kv1.1. The predicted MCEPH protein is truncated at amino acid 230 out of 495. Truncated proteins are usually not expressed since nonsense mRNAs are most often degraded. However, high Kv1.1 mRNA levels in mceph/mceph brain indicated that it escaped this control mechanism. Therefore, we hypothesized that the truncated Kv1.1 would be expressed and dysregulate other Kv1 subunits in the mceph/mceph mice. Results We found that the MCEPH protein is expressed in the brain of mceph/mceph mice. MCEPH was found to lack mature (Golgi glycosylation, but to be core glycosylated and trapped in the endoplasmic reticulum (ER. Interactions between MCEPH and other Kv1 subunits were studied in cell culture, Xenopus oocytes and the brain. MCEPH can form tetramers with Kv1.1 in cell culture and has a dominant negative effect on Kv1.2 and Kv1.3 currents in oocytes. However, it does not retain Kv1.2 in the ER of neurons. Conclusion The megencephaly mice express a truncated Kv1.1 in the brain, and constitute a unique tool to study Kv1.1 trafficking relevant for understanding epilepsy, ataxia and pathologic brain overgrowth.

Catalytic Subunit 1 of Protein Phosphatase 2A Is a Subunit of the STRIPAK Complex and Governs Fungal Sexual Development

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Anna Beier

2016-06-01

Full Text Available The generation of complex three-dimensional structures is a key developmental step for most eukaryotic organisms. The details of the molecular machinery controlling this step remain to be determined. An excellent model system to study this general process is the generation of three-dimensional fruiting bodies in filamentous fungi like Sordaria macrospora. Fruiting body development is controlled by subunits of the highly conserved striatin-interacting phosphatase and kinase (STRIPAK complex, which has been described in organisms ranging from yeasts to humans. The highly conserved heterotrimeric protein phosphatase PP2A is a subunit of STRIPAK. Here, catalytic subunit 1 of PP2A was functionally characterized. The Δpp2Ac1 strain is sterile, unable to undergo hyphal fusion, and devoid of ascogonial septation. Further, PP2Ac1, together with STRIPAK subunit PRO22, governs vegetative and stress-related growth. We revealed in vitro catalytic activity of wild-type PP2Ac1, and our in vivo analysis showed that inactive PP2Ac1 blocks the complementation of the sterile deletion strain. Tandem affinity purification, followed by mass spectrometry and yeast two-hybrid analysis, verified that PP2Ac1 is a subunit of STRIPAK. Further, these data indicate links between the STRIPAK complex and other developmental signaling pathways, implying the presence of a large interconnected signaling network that controls eukaryotic developmental processes. The insights gained in our study can be transferred to higher eukaryotes and will be important for understanding eukaryotic cellular development in general.