Mammalian RNA-dependent deaminases and edited mRNAs.

The past year has witnessed major progress in the field of mammalian nuclear RNA editing. Two new sequence-related RNA-dependent adenosine deaminases, distantly related to the previously characterized double-stranded RNA adenosine deaminase DRADA/dsRAD, have been molecularly characterized. One of these deaminases edits in vitro with precision for the molecular determinant that controls the Ca2+ permeability of fast synaptic glutamate-gated cation channels. This deaminase, like DRADA, is expressed in many tissues and the search is now on for more substrates of these RNA-editing enzymes. Moreover, the physiological role of the apolipoprotein B RNA editing enzyme APOBEC-1 has been investigated in genetically manipulated mice.

Control of kinetic properties of AMPA receptor channels by nuclear RNA editing.

AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor channels mediate the fast component of excitatory postsynaptic currents in the central nervous system. Site-selective nuclear RNA editing controls the calcium permeability of these channels, and RNA editing at a second site is shown here to affect the kinetic aspects of these channels in rat brain. In three of the four AMPA receptor subunits (GluR-B, -C, and -D), intronic elements determine a codon switch (AGA, arginine, to GGA, glycine) in the primary transcripts in a position termed the R/G site, which immediately precedes the alternatively spliced modules "flip" and "flop." The extent of editing at this site progresses with brain development in a manner specific for subunit and splice form, and edited channels possess faster recovery rates from desensitization.

Relative abundance of subunit mRNAs determines gating and Ca2+ permeability of AMPA receptors in principal neurons and interneurons in rat CNS.

Recording of glutamate-activated currents in membrane patches was combined with RT-PCR-mediated AMPA receptor (AMPAR) subunit mRNA analysis in single identified cells of rat brain slices. Analysis of AMPARs in principal neurons and interneurons of hippocampus and neocortex and in auditory relay neurons and Bergmann glial cells indicates that the GluR-B subunit in its flip version determines formation of receptors with relatively slow gating, whereas the GluR-D subunit promotes assembly of more rapidly gated receptors. The relation between Ca2+ permeability of AMPAR channels and the relative GluR-B mRNA abundance is consistent with the dominance of this subunit in determining the Ca2+ permeability of native receptors. The results suggest that differential expression of GluR-B and GluR-D subunit genes, as well as splicing and editing of their mRNAs, account for the differences in gating and Ca2+ permeability of native AMPAR channels.

GABAB receptor antagonist SGS742 improves spatial memory and reduces protein binding to the cAMP response element (CRE) in the hippocampus.

Memory storage in the brain requires protein synthesis initiated through signaling pathways that control transcription. Such mechanisms are under active investigation for therapies in disorders involving cognitive dysfunction. Long-term memory can be improved by inhibiting activation or reducing expression of transcription factors such as ATF4/CREB2 and some C/EBP family members which appear to serve as memory suppressors. Here, we provide evidence that GABAB receptor antagonists may enhance cognition, at least in part, by this mechanism. We tested a GABAB receptor antagonist, SGS742 (CGP36742), on hippocampal-dependent memory and hippocampal nuclear CRE-binding activity in rats. As a result, acute in vivo administration of SGS742 both improved memory and reduced total hippocampal CRE-binding activity of which a large proportion in the basal state could be immunoneutralized with CREB2 antibodies. Consistent with its activity on information storage mechanisms, acute SGS742 effectively improved long-term memory in retrograde protocols, in which drug was given at times when memory formation can be interrupted by blocking new protein production. In conclusion, GABAB antagonists may provide a pharmacological therapy for cognitive impairment, sharing mechanistic features with genetic approaches to reduce CREB2 activity and to augment long-term memory.

Candidate editases for GluR channels in single neurons of rat hippocampus and cerebellum.

RNA editing by site selective adenosine deamination changes codons in several nuclear transcripts in the mammalian brain and affects critical properties of the encoded proteins, as exemplified by the calcium permeability of AMPA receptor channels. The recently cloned RNA dependent adenosine deaminases ADAR1, ADAR2 and ADAR3 form a small family of sequence-related candidate editases which are expressed in brain and other tissues at distinct levels and patterns. We have employed single-cell polymerase chain reaction of hippocampal CA1 and CA3 pyramidal neurons and cerebellar Purkinje and Bergmann glial cells in an attempt to evaluate the expression of these enzymes at a cellular level. We found ADAR2 expressed in all cells analyzed; approximately 50% of the cells co-expressed ADAR1 or ADAR3. The differential ADAR expression revealed by our study might underlie the distinct editing efficiencies and selectivities in different GluR subunit transcripts.

Tryptophan mutants of human C5a anaphylatoxin: a fluorescence anisotropy decay and energy transfer study.

Three mutants of the anaphylatoxin C5a were prepared with positions 2, 64 and 70, respectively, substituted by tryptophan. The last mutant was additionally labelled at Cys27 for fluorescence energy transfer (FET) measurements. The structural integrity and biological activity of the molecules were not affected. Fluorescence anisotropy decay (FAD) measurements showed that the rotational correlation time for tryptophan decreases in the order: [Trp2]rhC5a > [Trp64]rhC5a > [Trp70]rhC5a, indicating an increasing mobility of the side chain. Measurements of the fluorescence energy transfer from Trp70 to the 1,5-AEDANS group at Cys27 yielded a distance distribution of 2.4 +/- 0.8 nm. This value is compatible with the C-terminal chain being arranged as a slightly stretched helix pointing away from the body of the molecule.

Neural correlates of ideomotor effect anticipations.

How does our mind produce physical, goal-directed action of our body? For about 200years, philosophers and psychologists hypothesized the transformation from mind to body to rely on the anticipation of an action's sensory consequences. Whereas this hypothesis received tremendous support from behavioral experiments, the neural underpinnings of action control via such ideomotor effect anticipations are virtually unknown. Using functional magnetic resonance imaging, the present study identified the inferior parietal cortex and the parahippocampal gyrus as key regions for this type of action control - setting the stage for a neuroscientific framework for explaining action control by ideomotor effect anticipations and thus enabling a synthesis of psychological and neuroscientific approaches to human action.

The neural substrate of the ideomotor principle revisited: evidence for asymmetries in action-effect learning.

Ideomotor theory holds that the perception or anticipatory imagination of action effects activates motor tendencies toward the action that is known to produce these effects, herein referred to as ideomotor response activation (IRA). IRA presupposes that the agent has previously learned which action produces which effects, and that this learning process has created bidirectional associations between the sensory effect codes and the motor codes producing the sensory effects. Here, we refer to this process as ideomotor learning. In the presented fMRI study, we adopted a standard two-phase ideomotor learning paradigm; a mixed between/within-subjects design allowed us to assess the neural substrate of both, IRA and ideomotor learning. We replicated earlier findings of a hand asymmetry in ideomotor processing with significantly stronger IRA by left-hand than right-hand action effects. Crucially, we traced this effect back to more pronounced associative learning for action-contingent effects of the left hand compared with effects of the right hand. In this context, our findings point to the caudate nucleus and the angular gyrus as central structures of the neural network underlying ideomotor learning.

Alimentação e prática de atividades físicas de crianças: hábitoscotidianos e culturais / Food and activities of children’s physical practice: daily habits and culture

Objetivo: conhecer os hábitos cotidianos de alimentação e prática de atividades físicas de crianças entre sete e dez anos de idade, de uma unidade básica de educação de um município do Sul de Santa Catarina. Método: pesquisa qualitativa descritiva e exploratória, desenvolvida com 24 crianças em uma unidade de ensino, vinculada a Estratégia de Saúde da família. Os dados foram coletados no período de outubro de 2012, por meio de entrevista semiestruturada e analisados a luz do referencial teórico de Madeleine Leininger. Resultados: o cotidiano das crianças e de suas famílias nem sempre auxiliam nos hábitos saudáveis, padrões culturais podem influenciar neste processo, consequentemente refletindo no ambiente escolar. Conclusão: parcerias entre a Estratégia de Saúde da Família (ESF), escola e família são importantes para traçar estratégias que fomentam as práticas de educação em saúde desta comunidade.

Objective: to learn about the everyday habits of food and physical activity of children from sevento ten years of age, from a basic unit of education at a city in the south of Santa Catarina. Methods:qualitative descriptive and exploratory study, developed with 24 children in a unit of education,linked to the Family Health Strategy. The data was collected in October 2012, through semistructuredinterviews, and analyzed through the theoretical framework of Madeleine Leininger.Results: the daily life of children and their families do not always assist in healthy habits; culturalpatterns may influence this process, consequently reflecting in the school environment. Conclusion:partnerships among the Family Health Strategy (FHS), school, and family are important to devisestrategies that foster the practices of health education in this community.

Dysgerminoma in Turner's syndrome.

The importance of the Y-chromosome for the germ cell tumour development in gonadal dysgenesis has been emphasized many times. In contrast, only two cases of dysgerminoma or gonadoblastoma had been published so far in the XO-Turner's syndrome. With this report, another case of Turner's syndrome developing a dysgerminoma in a gonadal streak is presented. No Y-chromosome containing stemline could be detected in the patient nor in the tumour. A primary genetic etiology or a mechanism related to early secondary regression or dysgenesis of the gonad are discussed as causative factors in germ cell tumour development within gonadal streaks.

Tad1p, a yeast tRNA-specific adenosine deaminase, is related to the mammalian pre-mRNA editing enzymes ADAR1 and ADAR2.

We have identified an RNA-specific adenosine deaminase (termed Tad1p/scADAT1) from Saccharomyces cerevisiae that selectively converts adenosine at position 37 of eukaryotic tRNAAla to inosine. The activity of purified recombinant Tad1p depends on the conformation of its tRNA substrate and the enzyme was found to be inactive on all other types of RNA tested. Mutant strains in which the TAD1 gene is disrupted are viable but lack Tad1p enzyme activity and their tRNAAla is not modified at position A37. Transformation of the mutant cells with the TAD1 gene restored enzyme activity. Tad1p has significant sequence similarity with the mammalian editing enzymes which act on specific precursor-mRNAs and on long double-stranded RNA. These findings suggest an evolutionary link between pre-mRNA editing and tRNA modification.

RED2, a brain-specific member of the RNA-specific adenosine deaminase family.

The mammalian RNA-specific adenosine deaminases DRADA/dsRAD (alias ADAR) and RED1 (alias ADARB1) have been implicated in the site-selective editing of brain-expressed pre-mRNAs for glutamate receptor subunits and of antigenomic RNA of hepatitis delta virus. These enzymes are expressed in many if not all tissues, predicting an as yet unappreciated significance for adenosine deamination-mediated recoding of gene transcripts in the mammalian organism. We now report the molecular cloning of cDNA for RED2 (alias ADARB2), a third member of the RNA-specific adenosine deaminase family in the rodent. RED2 is closely sequence-related to RED1 but appears to be expressed only in the brain, where expression is widespread reaching highest levels in olfactory bulb and thalamus. RED2 further differs from RED1 in having a 54-residue amino-terminal extension which includes an arginine-rich motif. Different from DRADA and RED1, recombinantly expressed RED2 did not deaminate adenosines in extended synthetic dsRNA or in GluR-B pre-mRNA. However, a chimera of RED1 and RED2 edited the GluR-B Q/R and R/G sites with moderate efficiency. Our data suggest that RED2 may edit brain-specific transcripts with distinct structural features.

Editing of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor GluR-B pre-mRNA in vitro reveals site-selective adenosine to inosine conversion.

In neurons of the mammalian brain primary transcripts of genes encoding subunits of glutamate receptor channels can undergo RNA editing, leading to altered properties of the transmitter-activated channel. Editing of these transcripts is a nuclear process that targets specific adenosines and requires a double-stranded RNA structure configured from complementary exonic and intronic sequences. We show here that the two independent editing sites in alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor GluR-B pre-mRNA are edited with positional accuracy by nuclear extract from HeLa cells. Nucleotide analysis by thin layer chromatography of the edited RNA sequences revealed selective adenosine to inosine conversion, most likely reflecting the participation of double-stranded RNA adenosine deaminase. Our results predict the presence of inosine-containing codons in other mammalian mRNAs.

A mammalian RNA editing enzyme.

Editing of RNA by site-selective adenosine deamination alters codons in brain-expressed pre-messenger RNAs for glutamate receptor (GluR) subunits including a codon for a channel determinant (Q/R site) in GluR-B, which controls the Ca2+ permeability of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors. Editing of GluR pre-mRNAs requires a double-stranded RNA (dsRNA) structure formed by exonic and intronic sequences and is catalysed by an unknown dsRNA adenosine deaminase. Here we report the cloning of complementary DNA for RED1, a dsRNA adenosine deaminase expressed in brain and peripheral tissues that efficiently edits the Q/R site in GluR-B pre-mRNA in vitro. This site is poorly edited by DRADA, which is distantly sequence-related to RED1. Both deaminases edit the R/G site in GluR-B pre-mRNA, indicating that members of an emerging gene family catalyse adenosine deamination in nuclear transcripts with distinct but overlapping substrate specificities.

Structural requirements for RNA editing in glutamate receptor pre-mRNAs by recombinant double-stranded RNA adenosine deaminase.

Pre-mRNAs for brain-expressed ionotropic glutamate receptor subunits undergo RNA editing by site-specific adenosine deamination, which alters codons for molecular determinants of channel function. This nuclear process requires double-stranded RNA structures formed by exonic and intronic sequences in the pre-mRNA and is likely to be catalyzed by an adenosine deaminase that recognizes these structures as a substrate. DRADA, a double-stranded RNA adenosine deaminase, is a candidate enzyme for L-glutamate-activated receptor channel (GluR) pre-mRNA editing. We show here that DRADA indeed edits GluR pre-mRNAs, but that it displays selectivity for certain editing sites. Recombinantly expressed DRADA, both in its full-length form and in an N-terminally truncated version, edited the Q/R site in GluR6 pre-mRNA and the R/G site but not the Q/R site of GluR-B pre-mRNA. This substrate selectivity correlated with the base pairing status and sequence environment of the editing-targeted adenosines. The Q/R site of GluR-B pre-mRNA was edited by an activity partially purified from HeLa cells and thus differently structured editing sites in GluR pre-mRNAs appear to be substrates for different enzymatic activities.

Orthodontics as a risk factor for temporomandibular disorders (TMD). II.

Debate about orthodontic treatment as a risk factor for temporomandibular disorders (TMD) led to this study. This report, the second in a series, concerns findings from a longitudinal study in which 30 new orthodontic patients have been enrolled annually since 1983. The method of Helkimo was used to collect TMD data before initiation of orthodontic treatment, and at annual intervals after debanding. Treatment was by fixed edgewise appliances. Data from a pretreatment and at least one posttreatment Helkimo examination were available for 109 patients. Follow-up data were available for 92 patients in the first year after debanding, with the corresponding sample sizes declining to 56, 33, 19, 11, and 7 for the second through the sixth posttreatment years, respectively. Primary analyses involved comparison of mean scores from the Helkimo 25-point dysfunction index scale. There were no significant differences between mean pretreatment and posttreatment Helkimo scores for any of the various groupings except for small, clinically unimportant improvements seen in the 12 to 24 month subgroup of 55 patients and in the 48 to 60 month subgroup of 11 patients. With average follow-up time of about 2 years for the 109 patients, 90% had Helkimo scores that stayed the same or improved, and 10% had scores that increased or worsened from 2 to 5 Helkimo points. We conclude that the orthodontic treatment experienced by our sample was not an important etiologic factor for TMD.