58 documentos corresponden a la consulta.
Palabras contadas: messenger: 95, rna: 350
Caceres, J.F. - Krainer, A.R. - Kornblihtt, A.R.
EMBO Rep. 2007;8(8):730-734
2007
Temas: arginine - messenger RNA - protein - serine - transcription factor - alternative RNA splicing - cancer - conference paper - gene expression - gene mutation
Descripción: Fil:Caceres, J.F. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.
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Pearson, J.L. - Robinson, T.J. - Muñoz, M.J. - Kornblihtt, A.R. - Garcia-Blanco, M.A.
J. Biol. Chem. 2008;283(12):7949-7961
2008
Temas: Binding energy - Binding sites - Biochemistry - Gene expression - Nucleic acids - Polymers - Proteins - RNA - Targets - Transcription factors
Descripción: The transcription factor TCERG1 (also known as CA150) associates with RNA polymerase II holoenzyme and alters the elongation efficiency of reporter transcripts. TCERG1 is also found as a component of highly purified spliceosomes and has been implicated in splicing. To elucidate the function of TCERG1, we used short interfering RNA-mediated knockdown followed by en masse gene expression analysis to identify its cellular targets. Analysis of data from HEK293 and HeLa cells identified high confidence targets of TCERG1. We found that targets of TCERG1 were enriched in microRNA-binding sites, suggesting the possibility of post-transcriptional regulation. Consistently, reverse transcription-PCR analysis revealed that many of the changes observed upon TCERG1 knockdown were because of differences in alternative mRNA processing of the 3′-untranslated regions. Furthermore, a novel computational approach, which can identify alternatively processed events from conventional microarray data, showed that TCERG1 led to widespread alterations in mRNA processing. These findings provide the strongest support to date for a role of TCERG1 in mRNA processing and are consistent with proposals that TCERG1 couples transcription and processing. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.
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Cramer, P. - Srebrow, A. - Kadener, S. - Werbajh, S. - De La Mata, M. - Melen, G. - Nogués, G. - Kornblihtt, A.R.
FEBS Lett. 2001;498(2-3):179-182
2001
Temas: Carboxy-terminal domain - Coupling - mRNA processing - RNA polymerase II - messenger RNA - RNA polymerase II - capping phenomenon - carboxy terminal sequence - cell nucleus - nonhuman
Descripción: A large body of work has proved that transcription by RNA polymerase II and pre-mRNA processing are coordinated events within the cell nucleus. Capping, splicing and polyadenylation occur while transcription proceeds, suggesting that RNA polymerase II plays a role in the regulation of these events. The presence and degree of phosphorylation of the carboxy-terminal domain of RNA polymerase II large subunit is important for functioning of the capping enzymes, the assembly of spliceosomes and the binding of the cleavage/polyadenylation complex. Nuclear architecture and gene promoter structure have also been shown to play key roles in coupling between transcription and splicing. © 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.
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Baez, M.V. - Boccaccio, G.L.
J. Biol. Chem. 2005;280(52):43131-43140
2005
Temas: Degradation - Genes - Proteins - RNA - Deadenylation - Gene expression - RNA-binding proteins - Cytology - messenger RNA - protein mSmaug 1
Descripción: Cytoplasmic events depending on RNA-binding proteins contribute to the fine-tuning of gene expression. Sterile α motif-containing RNA-binding proteins constitute a novel family of post-transcriptional regulators that recognize a specific RNA sequence motif known as Smaug recognition element (SRE). The Drosophila member of this family, dSmaug, triggers the translational repression and deadenylation of maternal mRNAs by independent mechanisms, and the yeast homologue Vts1 stimulates degradation of SRE-containing messengers. Two homologous genes are present in the mammalian genome. Here we showed that hSmaug 1, encoded in human chromosome 14, represses the translation of reporter transcripts carrying SRE motifs. When expressed in fibroblasts, hSmaug 1 forms cytoplasmic granules that contain polyadenylated mRNA and the RNA-binding proteins Staufen, TIAR, TIA-1, and HuR. Smaug 1 foci are distinct from degradation foci. The murine protein mSmaug 1 is expressed in the central nervous system and is abundant in post-synaptic densities, a subcellular region where translation is tightly regulated by synaptic stimulation. Biochemical analysis indicated that mSmaug 1 is present in synaptoneurosomal 20 S particles. These results suggest a role for mammalian Smaug 1 in RNA granule formation and translation regulation in neurons. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.
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Schwede, A. - Manful, T. - Jha, B.A. - Helbig, C. - Bercovich, N. - Stewart, M. - Clayton, C.
Nucleic Acids Res. 2009;37(16):5511-5528
2009
Temas: chromatin assembly factor 1 - gene product - messenger RNA - protein EP - ribosome protein - unclassified drug - EP procyclin protein, Trypanosoma brucei - membrane protein - mRNA deadenylase - phosphoglycerate kinase
Descripción: Removal of the poly(A) tail is the first step in the degradation of many eukaryotic mRNAs. In metazoans and yeast, the Ccr4/Caf1/Not complex has the predominant deadenylase activity, while the Pan2/Pan3 complex may trim poly(A) tails to the correct size, or initiate deadenylation. In trypanosomes, turnover of several constitutively-expressed or long-lived mRNAs is not affected by depletion of the 5'-3' exoribonuclease XRNA, but is almost completely inhibited by depletion of the deadenylase CAF1. In contrast, two highly unstable mRNAs, encoding EP procyclin and a phosphoglycerate kinase, PGKB, accumulate when XRNA levels are reduced. We here show that degradation of EP mRNA was partially inhibited after CAF1 depletion. RNAi-targeting trypanosome PAN2 had a mild effect on global deadenylation, and on degradation of a few mRNAs including EP. By amplifying and sequencing degradation intermediates, we demonstrated that a reduction in XRNA had no effect on degradation of a stable mRNA encoding a ribosomal protein, but caused accumulation of EP mRNA fragments that had lost substantial portions of the 5' and 3' ends. The results support a model in which trypanosome mRNAs can be degraded by at least two different, partially independent, cytoplasmic degradation pathways attacking both ends of the mRNA. © 2009 The Author(s).
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Nogués, G. - Kadener, S. - Cramer, P. - De la Mata, M. - Fededa, J.P. - Blaustein, M. - Srebrow, A. - Kornblihtt, A.R.
IUBMB Life 2003;55(4-5):235-241
2003
Temas: Alternative splicing - Elongation - mRNA processing - RNA polymerase II - Transcription - calcitonin gene related peptide - fibronectin - Hermes antigen - messenger RNA - protein
Descripción: The realization that the mammalian proteomic complexity is achieved with a limited number of genes demands a better understanding of alternative splicing regulation. Promoter control of alternative splicing was originally described by our group in studies performed on the fibronectin gene. Recently, other labs extended our findings to the cystic fibrosis, CD44 and CGRP genes strongly supporting a coupling between transcription and pre-mRNA splicing. A possible mechanism that would fit in these results is that the promoter itself is responsible for recruiting splicing factors, such as SR proteins, to the site of transcription, possibly through transcription factors that bind the promoter or the transcriptional enhancers. An alternative model, discussed more extensively in this review, involves modulation of RNA pol II (pol II) elongation rate. The model is supported by findings that cis- and trans-acting factors that modulate pol II elongation on a particular template also provoke changes in the alternative splicing balance of the encoded mRNAs.
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Sala, C.D. - Soler-Bistué, A.J.C. - Korprapun, L. - Zorreguieta, A. - Tolmasky, M.E.
PLoS ONE 2012;7(10)
2012
Temas: messenger RNA - ribonuclease P - transfer RNA - article - bacterial cell - binding affinity - binding site - complex formation - controlled study - Escherichia coli
Descripción: EGS (external guide sequence) technology is a promising approach to designing new antibiotics. EGSs are short antisense oligoribonucleotides that induce RNase P-mediated cleavage of a target RNA by forming a precursor tRNA-like complex. The ftsZ mRNA secondary structure was modeled and EGSs complementary to two regions with high probability of being suitable targets were designed. In vitro reactions showed that EGSs targeting these regions bound ftsZ mRNA and elicited RNase P-mediated cleavage of ftsZ mRNA. A recombinant plasmid, pEGSb1, coding for an EGS that targets region "b" under the control of the T7 promoter was generated. Upon introduction of this plasmid into Escherichia coli BL21(DE3)(pLysS) the transformant strain formed filaments when expression of the EGS was induced. Concomitantly, E. coli harboring pEGSb1 showed a modest but significant inhibition of growth when synthesis of the EGSb1 was induced. Our results indicate that EGS technology could be a viable strategy to generate new antimicrobials targeting ftsZ. © 2012 Sala et al.
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Landau, A.M. - Lokstein, H. - Scheller, H.V. - Lainez, V. - Maldonado, S. - Prina, A.R.
Plant Physiol. 2009;151(4):1802-1811
2009
Temas: messenger RNA - pigment - plant extract - vegetable protein - article - barley - chloroplast - cytoplasm - gene expression regulation - genetics
Descripción: A cytoplasmically inherited chlorophyll-deficient mutant of barley (Hordeum vulgare) termed cytoplasmic line 3 (CL3), displaying a viridis (homogeneously light-green colored) phenotype, has been previously shown to be affected by elevated temperatures. In this article, biochemical, biophysical, and molecular approaches were used to study the CL3 mutant under different temperature and light conditions. The results lead to the conclusion that an impaired assembly of photosystem I (PSI) under higher temperatures and certain light conditions is the primary cause of the CL3 phenotype. Compromised splicing of ycf3 transcripts, particularly at elevated temperature, resulting from a mutation in a noncoding region (intron 1) in the mutant ycf3 gene results in a defective synthesis of Ycf3, which is a chaperone involved in PSI assembly. The defective PSI assembly causes severe photoinhibition and degradation of PSII. © 2009 American Society of Plant Biologists.
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Rocha-Viegas, L. - Vicent, G.P. - Barañao, J.L. - Beato, M. - Pecci, A.
J. Biol. Chem. 2006;281(45):33959-33970
2006
Temas: Derivatives - Enzyme inhibition - Genes - Hormones - RNA - Tissue - Cell death - Hormonal treatment - Lymphoid cells - Thymocytes
Descripción: The bcl-X gene plays a critical role in apoptosis. Six different isoforms generated by tissue-specific promoter usage and alternative splicing were described. Some of them exert opposite effects on cell death. In mammary epithelial cells glucocorticoids induce bcl-X expression and increase the ratio bcl-XL (antiapoptotic)/bcl-XS (apoptotic) by activating P4 promoter, which contains two hormone response elements. Here we show that, on mouse thymocytes and T lymphocyte derivative S49 cells, glucocorticoids inhibited transcription from P4 and decreased the ratio bcl-X L/bcl-XS favoring apoptosis. Upon hormonal treatment, glucocorticoid receptor (GR), steroid receptor coactivator-1, and RNA polymerase II were transiently recruited to P4 promoter, whereas STAT5B was also recruited but remained bound. Concomitant with the release of GR, silencing mediator for retinoic acid receptor and thyroid hormone receptor and histone deacetylase 3 were recruited, histone H3 was deacetylated, and RNA polymerase II left the promoter. Inhibition of STAT5 activity reverted glucocorticoid repression to activation of transcription and was accompanied by stable recruitment of GR and RNA polymerase II to P4. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.
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Schor, I.E. - Llères, D. - Risso, G.J. - Pawellek, A. - Ule, J. - Lamond, A.I. - Kornblihtt, A.R.
PLoS ONE 2012;7(11)
2012
Temas: enhanced green fluorescent protein - heterochromatin protein 1 - heterochromatin protein 1alpha - histone H3 - histone H4 - messenger RNA - nuclear protein - protein SRSF1 - protein SRSF2 - trichostatin A
Descripción: Chromatin structure is an important factor in the functional coupling between transcription and mRNA processing, not only by regulating alternative splicing events, but also by contributing to exon recognition during constitutive splicing. We observed that depolarization of neuroblastoma cell membrane potential, which triggers general histone acetylation and regulates alternative splicing, causes a concentration of SR proteins in nuclear speckles. This prompted us to analyze the effect of chromatin structure on splicing factor distribution and dynamics. Here, we show that induction of histone hyper-acetylation results in the accumulation in speckles of multiple splicing factors in different cell types. In addition, a similar effect is observed after depletion of the heterochromatic protein HP1α, associated with repressive chromatin. We used advanced imaging approaches to analyze in detail both the structural organization of the speckle compartment and nuclear distribution of splicing factors, as well as studying direct interactions between splicing factors and their association with chromatin in vivo. The results support a model where perturbation of normal chromatin structure decreases the recruitment efficiency of splicing factors to nascent RNAs, thus causing their accumulation in speckles, which buffer the amount of free molecules in the nucleoplasm. To test this, we analyzed the recruitment of the general splicing factor U2AF65 to nascent RNAs by iCLIP technique, as a way to monitor early spliceosome assembly. We demonstrate that indeed histone hyper-acetylation decreases recruitment of U2AF65 to bulk 3′ splice sites, coincident with the change in its localization. In addition, prior to the maximum accumulation in speckles, ~20% of genes already show a tendency to decreased binding, while U2AF65 seems to increase its binding to the speckle-located ncRNA MALAT1. All together, the combined imaging and biochemical approaches support a model where chromatin structure is essential for efficient co-transcriptional recruitment of general and regulatory splicing factors to pre-mRNA. © 2012 Schor et al.
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Alló, M. - Schor, I.E. - Muñoz, M.J. - De La Mata, M. - Agirre, E. - Valcárcel, J. - Eyras, E. - Kornblihtt, A.R.
Cold Spring Harbor Symp. Quant. Biol. 2010;75:103-111
2010
Temas: double stranded RNA - fibronectin - messenger RNA - mitogen activated protein kinase - nerve cell adhesion molecule - polypyrimidine tract binding protein - RNA polymerase II - small interfering RNA - trichostatin A - histone
Descripción: Alternative splicing affects more than 90% of human genes. Coupling between transcription and splicing has become crucial in the complex network underlying alternative splicing regulation. Because chromatin is the real template for nuclear transcription, changes in its structure, but also in the "reading" and "writing" of the histone code, could modulate splicing choices. Here, we discuss the evidence supporting these ideas, from the first proposal of chromatin affecting alternative splicing, performed 20 years ago, to the latest findings including genome-wide evidence that nucleosomes are preferentially positioned in exons. We focus on two recent reports from our laboratories that add new evidence to this field. The first report shows that a physiological stimulus such as neuron depolarization promotes intragenic histone acetylation (H3K9ac) and chromatin relaxation, causing the skipping of exon 18 of the neural cell adhesion molecule gene. In the second report, we show how specific histone modifications can be created at targeted gene regions as a way to affect alternative splicing: Using small interfering RNAs (siRNAs), we increased the levels of H3K9me2 and H3K27me3 in the proximity of alternative exon 33 of the human fibronectin gene, favoring its inclusion into mature messenger RNA (mRNA) through a mechanism that recalls RNAmediated transcriptional gene silencing. © 2010 Cold Spring Harbor Laboratory Press.
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Colman-Lerner, A. - Fischman, M.L. - Lanuza, G.M. - Bissell, D.M. - Kornblihtt, A.R. - Barañao, J.L.
Endocrinology 1999;140(6):2541-2548
1999
Temas: cyclic AMP - estradiol - fibronectin - messenger RNA - recombinant protein - RNA precursor - transforming growth factor beta - alternative RNA splicing - animal cell - animal tissue
Descripción: This study was aimed at testing the hypothesis that different forms of fibronectin (FN), produced as a consequence of the alternative splicing of the precursor messenger RNA, play specific roles during development of the ovarian follicle. In particular, we were interested in determining the effect of the ED-I (also termed ED-A) type III repeat, which is absent in the plasma form. Analysis of FN levels in follicular fluids corresponding to different stages of development of bovine follicles revealed marked changes in the concentrations of ED-I + FN, whereas total FN levels remained relatively constant. ED-I + FN levels were higher in small follicles, corresponding to the phase of granulosa cell proliferation. The hypothesis of a physiological role for ED-I + FN was further supported by the finding of a regulation of the alternative splicing of FN in primary cultures of bovine granulosa cells by factors known to control ovarian follicular development. cAMP produced a 10-fold decrease in the relative proportion of the ED-I region. In contrast, transforming growth factor-β elicited a 2-fold stimulation of overall FN synthesis and a 4-fold increase in the synthesis of ED-I containing FN. This effect was evident at the protein (Western blots) and messenger RNA (Northern blots) levels. Although a negative correlation (P < 0.001) was detected between ED-I + FN and estradiol levels in follicular fluid, this steroid was unable to modulate in vitro the alternative splicing of FN. A possible mitogenic effect of ED-I + FN was suggested by the observation that a recombinant peptide corresponding to the ED-I domain stimulated DNA synthesis in a bovine granulosa cell line (BGC-1), whereas a peptide corresponding to the flanking type III sequences had no effect. The hypothesis of ED-I + FN as a growth regulatory factor was further strengthened by the fact that depletion of FN from BGC-1-conditioned medium, which contained ED-I + FN, abrogated its mitogenic activity, whereas plasma FN was without effect. We propose that changes in the primary structure of FN may mediate some of the effects of gonadotropin and intraovarian factors during follicular development.
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Luquet, C.M. - Weihrauch, D. - Senek, M. - Towle, D.W.
J. Exp. Biol. 2005;208(19):3627-3636
2005
Temas: Arginine kinase - Crab - Gene expression - Gill - Na+/K+-ATPase - Na+/K+/2Cl - cotransporter - Quantitative PCR - V-type H+-ATPase - messenger RNA - primer DNA
Descripción: Using quantitative real-time PCR, the expression of mRNAs encoding three transport-related proteins and one putative housekeeping protein was analyzed in anterior and posterior gills of the euryhaline crab Chasmagnathus granulatus following transfer from isosmotic conditions (30‰ salinity) to either dilute (2‰) or concentrated (45‰) seawater. Modest changes were observed in the abundance of mRNAs encoding the housekeeping protein arginine kinase and the vacuolar-type H+-ATPase B-subunit, both of which were highly expressed under all conditions. By contrast, the expression of Na +/K+-ATPase α-subunit mRNA and Na+/K +/2Cl- cotransporter mRNA was strongly responsive to external salinity. During acclimation to dilute seawater, cotransporter mRNA increased 10-20-fold in posterior gills within the first 24 h while Na +/K+-ATPase α-subunit mRNA increased 35-55-fold. During acclimation to concentrated seawater, cotransporter mRNA increased 60-fold by 96 h and Na+/K+-ATPase α-subunit increased approximately 25-fold in posterior gills. Our results indicate a complex pattern of transcriptional regulation dependent upon the direction of salinity change and the developmental background of the gills.
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Dominguez, P.G. - Frankel, N. - Mazuch, J. - Balbo, I. - Iusem, N. - Fernie, A.R. - Carrari, F.
Plant Physiol. 2013;161(3):1486-1500
2013
Temas: abscisic acid - carbon - carrier protein - gibberellic acid - gibberellin - glucose - messenger RNA - phytohormone - vegetable protein - article
Descripción: Asr (for ABA, stress, ripening) genes are exclusively found in the genomes of higher plants, and the encoded proteins have been found localized both to the nucleus and cytoplasm. However, before the mechanisms underlying the activity of ASR proteins can be determined, the role of these proteins in planta should be deciphered. Results from this study suggest that ASR is positioned within the signaling cascade of interactions among glucose, abscisic acid, and gibberellins. Tobacco (Nicotiana tabacum) transgenic lines with reduced levels of ASR protein showed impaired glucose metabolism and altered abscisic acid and gibberellin levels. These changes were associated with dwarfism, reduced carbon dioxide assimilation, and accelerated leaf senescence as a consequence of a fine regulation exerted by ASR to the glucose metabolism. This regulation resulted in an impact on glucose signaling mediated by Hexokinase1 and Snf1-related kinase, which would subsequently have been responsible for photosynthesis, leaf senescence, and hormone level alterations. It thus can be postulated that ASR is not only involved in the control of hexose uptake in heterotrophic organs, as we have previously reported, but also in the control of carbon fixation by the leaves mediated by a similar mechanism. © 2013 American Society of Plant Biologists. All Rights Reserved.
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Gomez-Sanchez, C.E. - Zhou, M.Y. - Cozza, E.N. - Morita, H. - Foecking, M.F. - Gomez-Sanchez, E.P.
ENDOCRINOLOGY 1997;138(8):3369-3373
1997
Temas: 18 hydroxydeoxycorticosterone - aldosterone - corticosterone - messenger RNA - synthetase - amygdaloid nucleus - animal tissue - article - brain - cerebellum
Descripción: Messenger RNA (mRNA) for enzymes involved in adrenal steroid biosynthesis are expressed in the brain, and the coded enzymes have been shown to be active. The expression of mRNA for the cytochrome P-450 enzyme aldosterone synthase, crucial for the final step in the synthesis of aldosterone and the synthesis of aldosterone was studied in several anatomic areas of the rat brain. Expression of the mRNA for the aldosterone synthase was demonstrated by RT-PCR/Southern blot in adrenal, aorta, hypothalamus, hippocampus, amygdala, cerebrum, and cerebellum. Incubation of brain minces from intact and adrenalectomized rats demonstrated the synthesis of corticosterone and aldosterone from endogenous precursors. Incubations of brain mince, with [1,23H]-deoxycorticosterone, followed by extraction and three different successive TLCs, demonstrated the presence of labeled aldosterone, corticosterone, and 18-hydroxy-deoxycorticosterone. Incubation, in the presence of 10 μM cortisol or metyrapone, inhibited the synthesis of aldosterone or both aldosterone and corticosterone, respectively. These studies indicate that the rat brain has the enzymatic machinery for the synthesis of adrenal corticosteroids and is capable of synthesizing aldosterone. Aldosterone synthesized in the brain might play a paracrine role in the regulation of blood pressure.
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Taranda, J. - Ballestero, J.A. - Hiel, H. - De Souza, F.S.J. - Wedemeyer, C. - Gómez-Casati, M.E. - Lipovsek, M. - Vetter, D.E. - Fuchs, P.A. - Katz, E. - Elgoyhen, A.B.
JARO J. Assoc. Res. Otolaryngol. 2009;10(3):397-406
2009
Temas: Acetylcholine - Efferent medial olivocochlear - Nicotinic cholinergic receptors - SK2 channel - Transgenic mice - acetylcholine - complementary DNA - messenger RNA - nicotinic acetylcholine receptor alpha10 - nicotinic receptor
Descripción: Efferent inhibition of cochlear hair cells is mediated by α9α10 nicotinic cholinergic receptors (nAChRs) functionally coupled to calcium-activated, small conductance (SK2) potassium channels. Before the onset of hearing, efferent fibers transiently make functional cholinergic synapses with inner hair cells (IHCs). The retraction of these fibers after the onset of hearing correlates with the cessation of transcription of the Chrna10 (but not the Chrna9) gene in IHCs. To further analyze this developmental change, we generated a transgenic mice whose IHCs constitutively express α10 into adulthood by expressing the α10 cDNA under the control of the Pou4f3 gene promoter. In situ hybridization showed that the α10 mRNA is expressed in IHCs of 8-week-old transgenic mice, but not in wild-type mice. Moreover, this mRNA is translated into a functional protein, since IHCs from P8-P10 α10 transgenic mice backcrossed to a Chrna10 -/- background (whose IHCs have no cholinergic function) displayed normal synaptic and acetylcholine (ACh)-evoked currents in patch-clamp recordings. Thus, the α10 transgene restored nAChR function. However, in the α10 transgenic mice, no synaptic or ACh-evoked currents were observed in P16-18 IHCs, indicating developmental down-regulation of functional nAChRs after the onset of hearing, as normally observed in wild-type mice. The lack of functional ACh currents correlated with the lack of SK2 currents. These results indicate that multiple features of the efferent postsynaptic complex to IHCs, in addition to the nAChR subunits, are down-regulated in synchrony after the onset of hearing, leading to lack of responses to ACh. © 2009 Association for Research in Otolaryngology.
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Soler Bistué, A.J.C. - Martín, F.A. - Vozza, N. - Ha, H. - Joaquín, J.C. - Zorreguieta, A. - Tolmasky, M.E.
Proc. Natl. Acad. Sci. U. S. A. 2009;106(32):13230-13235
2009
Temas: Aminoglycoside - Antibiotic resistance - Antisense - Nucleic acids analogs - RNase P - aac(6') Ib enzyme - amikacin - antisense oligonucleotide - bacterial DNA - bacterial enzyme
Descripción: Inhibition of bacterial gene expression by RNase P-directed cleavage is a promising strategy for the development of antibiotics and pharmacological agents that prevent expression of antibiotic resistance. The rise in multiresistant bacteria harboring AAC(6′)-Ib has seriously limited the effectiveness of amikacin and other aminoglycosides. We have recently shown that recombinant plasmids coding for external guide sequences (EGS), short antisense oligoribonucleotides (ORN) that elicit RNase P-mediated cleavage of a target mRNA, induce inhibition of expression of aac(6′)-Ib and concomitantly induce a significant decrease in the levels of resistance to amikacin. However, since ORN are rapidly degraded by nucleases, development of a viable RNase P-based antisense technology requires the design of nuclease resistant RNA analog EGSs. We have assayed a variety of ORN analogs of which selected LNA/DNA co-oligomers elicited RNase P-mediated cleavage of mRNA in vitro. Although we found an ideal configuration of LNA/DNA residues, there seems not to be a correlation between number of LNA substitutions and level of activity. Exogenous administration of as low as 50 nM of an LNA/DNA co-oligomer to the hyperpermeable E. coli AS19 harboring the aac(6′)-Ib inhibited growth in the presence of amikacin. Our experiments strongly suggest an RNase P-mediated mechanism in the observed antisense effect.
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Quaglino, A. - Salierno, M. - Pellegrotti, J. - Rubinstein, N. - Kordon, E.C.
BMC Cell Biol. 2009;10
2009
Temas: leukemia inhibitory factor - messenger RNA - mitogen activated protein kinase 1 - protein c fos - protein kinase B - silicone - STAT3 protein - leukemia inhibitory factor - Lif protein, mouse - messenger RNA
Descripción: Background: Shortly after weaning, a complex multi-step process that leads to massive epithelial apoptosis is triggered by tissue local factors in the mouse mammary gland. Several reports have demonstrated the relevance of mechanical stress to induce adaptive responses in different cell types. Interestingly, these signaling pathways also participate in mammary gland involution. Then, it has been suggested that cell stretching caused by milk accumulation after weaning might be the first stimulus that initiates the complete remodeling of the mammary gland. However, no previous report has demonstrated the impact of mechanical stress on mammary cell physiology. To address this issue, we have designed a new practical device that allowed us to evaluate the effects of radial stretching on mammary epithelial cells in culture. Results: We have designed and built a new device to analyze the biological consequences of applying mechanical stress to cells cultured on flexible silicone membranes. Subsequently, a geometrical model that predicted the percentage of radial strain applied to the elastic substrate was developed. By microscopic image analysis, the adjustment of these calculations to the actual strain exerted on the attached cells was verified. The studies described herein were all performed in the HC11 non-tumorigenic mammary epithelial cell line, which was originated from a pregnant BALB/c mouse. In these cells, as previously observed in other tissue types, mechanical stress induced ERK1/2 phosphorylation and c-Fos mRNA and protein expression. In addition, we found that mammary cell stretching triggered involution associated cellular events as Leukemia Inhibitory Factor (LIF) expression induction, STAT3 activation and AKT phosphorylation inhibition. Conclusion: Here, we show for the first time, that mechanical strain is able to induce weaning-associated events in cultured mammary epithelial cells. These results were obtained using a new practical and affordable device specifically designed for such a purpose. We believe that our results indicate the relevance of mechanical stress among the early post-lactation events that lead to mammary gland involution. © 2009 Quaglino et al., licensee BioMed Central Ltd.
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Fustiñana, M.S. - Ariel, P. - Federman, N. - Freudenthal, R. - Romano, A.
BMC Neurosci. 2010;11
2010
Temas: amyloid precursor protein - beta amyloid precursor protein like protein - messenger RNA - unclassified drug - amyloid precursor protein - complementary DNA - messenger RNA - amino acid sequence - amino terminal sequence - animal experiment
Descripción: Background: Human β-amyloid, the main component in the neuritic plaques found in patients with Alzheimer's disease, is generated by cleavage of the β-amyloid precursor protein. Beyond the role in pathology, members of this protein family are synaptic proteins and have been associated with synaptogenesis, neuronal plasticity and memory, both in vertebrates and in invertebrates. Consolidation is necessary to convert a short-term labile memory to a long-term and stable form. During consolidation, gene expression and de novo protein synthesis are regulated in order to produce key proteins for the maintenance of plastic changes produced during the acquisition of new information.Results: Here we partially cloned and sequenced the beta-amyloid precursor protein like gene homologue in the crab Chasmagnathus (cappl), showing a 37% of identity with the fruit fly Drosophila melanogaster homologue and 23% with Homo sapiens but with much higher degree of sequence similarity in certain regions. We observed a wide distribution of cappl mRNA in the nervous system as well as in muscle and gills. The protein localized in all tissues analyzed with the exception of muscle. Immunofluorescence revealed localization of cAPPL in associative and sensory brain areas. We studied gene and protein expression during long-term memory consolidation using a well characterized memory model: the context-signal associative memory in this crab species. mRNA levels varied at different time points during long-term memory consolidation and correlated with cAPPL protein levels. Conclusions: cAPPL mRNA and protein is widely distributed in the central nervous system of the crab and the time course of expression suggests a role of cAPPL during long-term memory formation. © 2010 Fustiñana et al; licensee BioMed Central Ltd.
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Perez-Pepe, M. - Slomiansky, V. - Loschi, M. - Luchelli, L. - Neme, M. - Thomas, M.G. - Boccaccio, G.L.
PLoS ONE 2012;7(12)
2012
Temas: focal adhesion kinase - focal adhesion kinase 56D - initiation factor 2alpha - messenger RNA - phosphoprotein phosphatase 1 - transcription factor - translation initiation factor 2alpha - unclassified drug - analytic method - article
Descripción: The spontaneous and reversible formation of foci and filaments that contain proteins involved in different metabolic processes is common in both the nucleus and the cytoplasm. Stress granules (SGs) and processing bodies (PBs) belong to a novel family of cellular structures collectively known as mRNA silencing foci that harbour repressed mRNAs and their associated proteins. SGs and PBs are highly dynamic and they form upon stress and dissolve thus releasing the repressed mRNAs according to changes in cell physiology. In addition, aggregates containing abnormal proteins are frequent in neurodegenerative disorders. In spite of the growing relevance of these supramolecular aggregates to diverse cellular functions a reliable automated tool for their systematic analysis is lacking. Here we report a MATLAB Script termed BUHO for the high-throughput image analysis of cellular foci. We used BUHO to assess the number, size and distribution of distinct objects with minimal deviation from manually obtained parameters. BUHO successfully addressed the induction of both SGs and PBs in mammalian and insect cells exposed to different stress stimuli. We also used BUHO to assess the dynamics of specific mRNA-silencing foci termed Smaug 1 foci (S-foci) in primary neurons upon synaptic stimulation. Finally, we used BUHO to analyze the role of candidate genes on SG formation in an RNAi-based experiment. We found that FAK56D, GCN2 and PP1 govern SG formation. The role of PP1 is conserved in mammalian cells as judged by the effect of the PP1 inhibitor salubrinal, and involves dephosphorylation of the translation factor eIF2α. All these experiments were analyzed manually and by BUHO and the results differed in less than 5% of the average value. The automated analysis by this user-friendly method will allow high-throughput image processing in short times by providing a robust, flexible and reliable alternative to the laborious and sometimes unfeasible visual scrutiny. © 2012 Perez-Pepe et al.
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