Transcriptome-Wide Binding Sites for Components of the Saccharomyces cerevisiae Non-Poly(A) Termination Pathway: Nrd1, Nab3, and Sen1

RNA polymerase II synthesizes a diverse set of transcripts including both protein-coding and non-coding RNAs. One major difference between these two classes of transcripts is the mechanism of termination. Messenger RNA transcripts terminate downstream of the coding region in a process that is coupled to cleavage and polyadenylation reactions. Non-coding transcripts like Saccharomyces cerevisiae snoRNAs terminate in a process that requires the RNA–binding proteins Nrd1, Nab3, and Sen1. We report here the transcriptome-wide distribution of these termination factors. These data sets derived from in vivo protein–RNA cross-linking provide high-resolution definition of non-poly(A) terminators, identify novel genes regulated by attenuation of nascent transcripts close to the promoter, and demonstrate the widespread occurrence of Nrd1-bound 3′ antisense transcripts on genes that are poorly expressed. In addition, we show that Sen1 does not cross-link efficiently to many expected non-coding RNAs but does cross-link to the 3′ end of most pre–mRNA transcripts, suggesting an extensive role in mRNA 3′ end formation and/or termination

Mitochondria of the Yeasts Saccharomyces cerevisiae and Kluyveromyces lactis Contain Nuclear rDNA-Encoded Proteins

In eukaryotes, the nuclear ribosomal DNA (rDNA) is the source of the structural 18S, 5.8S and 25S rRNAs. In hemiascomycetous yeasts, the 25S rDNA sequence was described to lodge an antisense open reading frame (ORF) named TAR1 for Transcript Antisense to Ribosomal RNA. Here, we present the first immuno-detection and sub-cellular localization of the authentic product of this atypical yeast gene. Using specific antibodies against the predicted amino-acid sequence of the Saccharomyces cerevisiae TAR1 product, we detected the endogenous Tar1p polypeptides in S. cerevisiae (Sc) and Kluyveromyces lactis (Kl) species and found that both proteins localize to mitochondria. Protease and carbonate treatments of purified mitochondria further revealed that endogenous Sc Tar1p protein sub-localizes in the inner membrane in a Nin-Cout topology. Plasmid-versions of 5′ end or 3′ end truncated TAR1 ORF were used to demonstrate that neither the N-terminus nor the C-terminus of Sc Tar1p were required for its localization. Also, Tar1p is a presequence-less protein. Endogenous Sc Tar1p was found to be a low abundant protein, which is expressed in fermentable and non-fermentable growth conditions. Endogenous Sc TAR1 transcripts were also found low abundant and consistently 5′ flanking regions of TAR1 ORF exhibit modest promoter activity when assayed in a luciferase-reporter system. Using rapid amplification of cDNA ends (RACE) PCR, we also determined that endogenous Sc TAR1 transcripts possess heterogeneous 5′ and 3′ ends probably reflecting the complex expression of a gene embedded in actively transcribed rDNA sequence. Altogether, our results definitively ascertain that the antisense yeast gene TAR1 constitutes a functional transcription unit within the nuclear rDNA repeats

A review of elliptical and disc galaxy structure, and modern scaling laws

A century ago, in 1911 and 1913, Plummer and then Reynolds introduced their models to describe the radial distribution of stars in `nebulae'. This article reviews the progress since then, providing both an historical perspective and a contemporary review of the stellar structure of bulges, discs and elliptical galaxies. The quantification of galaxy nuclei, such as central mass deficits and excess nuclear light, plus the structure of dark matter halos and cD galaxy envelopes, are discussed. Issues pertaining to spiral galaxies including dust, bulge-to-disc ratios, bulgeless galaxies, bars and the identification of pseudobulges are also reviewed. An array of modern scaling relations involving sizes, luminosities, surface brightnesses and stellar concentrations are presented, many of which are shown to be curved. These 'redshift zero' relations not only quantify the behavior and nature of galaxies in the Universe today, but are the modern benchmark for evolutionary studies of galaxies, whether based on observations, N-body-simulations or semi-analytical modelling. For example, it is shown that some of the recently discovered compact elliptical galaxies at 1.5 < z < 2.5 may be the bulges of modern disc galaxies.Comment: Condensed version (due to Contract) of an invited review article to appear in "Planets, Stars and Stellar Systems"(www.springer.com/astronomy/book/978-90-481-8818-5). 500+ references incl. many somewhat forgotten, pioneer papers. Original submission to Springer: 07-June-201

Leftward Lateralization of Auditory Cortex Underlies Holistic Sound Perception in Williams Syndrome

BACKGROUND: Individuals with the rare genetic disorder Williams-Beuren syndrome (WS) are known for their characteristic auditory phenotype including strong affinity to music and sounds. In this work we attempted to pinpoint a neural substrate for the characteristic musicality in WS individuals by studying the structure-function relationship of their auditory cortex. Since WS subjects had only minor musical training due to psychomotor constraints we hypothesized that any changes compared to the control group would reflect the contribution of genetic factors to auditory processing and musicality. METHODOLOGY/PRINCIPAL FINDINGS: Using psychoacoustics, magnetoencephalography and magnetic resonance imaging, we show that WS individuals exhibit extreme and almost exclusive holistic sound perception, which stands in marked contrast to the even distribution of this trait in the general population. Functionally, this was reflected by increased amplitudes of left auditory evoked fields. On the structural level, volume of the left auditory cortex was 2.2-fold increased in WS subjects as compared to control subjects. Equivalent volumes of the auditory cortex have been previously reported for professional musicians. CONCLUSIONS/SIGNIFICANCE: There has been an ongoing debate in the neuroscience community as to whether increased gray matter of the auditory cortex in musicians is attributable to the amount of training or innate disposition. In this study musical education of WS subjects was negligible and control subjects were carefully matched for this parameter. Therefore our results not only unravel the neural substrate for this particular auditory phenotype, but in addition propose WS as a unique genetic model for training-independent auditory system properties

Why do General Practitioners Decline Training to Improve Management of Medically Unexplained Symptoms?

BACKGROUND: General practitioners’ (GPs) communication with patients presenting medically unexplained symptoms (MUS) has the potential to somatize patients’ problems and intensify dependence on medical care. Several reports indicate that GPs have negative attitudes about patients with MUS. If these attitudes deter participation in training or other methods to improve communication, practitioners who most need help will not receive it. OBJECTIVE: To identify how GPs’ attitudes to patients with MUS might inhibit their participation with training to improve management. DESIGN: Qualitative study. PARTICIPANTS: GPs (N = 33) who had declined or accepted training in reattribution techniques in the context of a research trial. APPROACH: GPs were interviewed and their accounts analysed qualitatively. RESULTS: Although attitudes that devalued patients with MUS were common in practitioners who had declined training, these coexisted, in the same practitioners, with evidence of intuitive and elaborate psychological work with these patients. However, these practitioners devalued their psychological skills. GPs who had accepted training also described working psychologically with MUS but devalued neither patients with MUS nor their own psychological skills. CONCLUSIONS: GPs’ attitudes that suggested disengagement from patients with MUS belied their pursuit of psychological objectives. We therefore suggest that, whereas negative attitudes to patients have previously been regarded as the main barrier to involvement in measures to improve patient management, GPs devaluing of their own psychological skills with these patients may be more important

Autoinhibition of TBCB regulates EB1-mediated microtubule dynamics

Tubulin cofactors (TBCs) participate in the folding, dimerization, and dissociation pathways of the tubulin dimer. Among them, TBCB and TBCE are two CAP-Gly domain-containing proteins that interact and dissociate the tubulin dimer. Here we show how TBCB localizes at spindle and midzone microtubules during mitosis. Furthermore, the motif DEI/M-COO– present in TBCB, which is similar to the EEY/F-COO– element characteristic of EB proteins, CLIP-170, and α-tubulin, is required for TBCE–TBCB heterodimer formation and thus for tubulin dimer dissociation. This motif is responsible for TBCB autoinhibition, and our analysis suggests that TBCB is a monomer in solution. Mutants of TBCB lacking this motif are derepressed and induce microtubule depolymerization through an interaction with EB1 associated to microtubule tips. TBCB is also able to bind to the chaperonin complex CCT containing α-tubulin, suggesting that it could escort tubulin to facilitate its folding and dimerization, recycling or degradation

Hippocampal state-dependent behavioral reflex to an identical sensory input in rats.

We examined the local field potential of the hippocampus to monitor brain states during a conditional discrimination task, in order to elucidate the relationship between ongoing brain states and a conditioned motor reflex. Five 10-week-old Wistar/ST male rats underwent a serial feature positive conditional discrimination task in eyeblink conditioning using a preceding light stimulus as a conditional cue for reinforced trials. In this task, a 2-s light stimulus signaled that the following 350-ms tone (conditioned stimulus) was reinforced with a co-terminating 100-ms periorbital electrical shock. The interval between the end of conditional cue and the onset of the conditioned stimulus was 4±1 s. The conditioned stimulus was not reinforced when the light was not presented. Animals successfully utilized the light stimulus as a conditional cue to drive differential responses to the identical conditioned stimulus. We found that presentation of the conditional cue elicited hippocampal theta oscillations, which persisted during the interval of conditional cue and the conditioned stimulus. Moreover, expression of the conditioned response to the tone (conditioned stimulus) was correlated with the appearance of theta oscillations immediately before the conditioned stimulus. These data support hippocampal involvement in the network underlying a conditional discrimination task in eyeblink conditioning. They also suggest that the preceding hippocampal activity can determine information processing of the tone stimulus in the cerebellum and its associated circuits

ALDH1A2 (RALDH2) genetic variation in human congenital heart disease

Abstract\ud \ud \ud \ud Background\ud \ud Signaling by the vitamin A-derived morphogen retinoic acid (RA) is required at multiple steps of cardiac development. Since conversion of retinaldehyde to RA by retinaldehyde dehydrogenase type II (ALDH1A2, a.k.a RALDH2) is critical for cardiac development, we screened patients with congenital heart disease (CHDs) for genetic variation at the ALDH1A2 locus.\ud \ud \ud \ud Methods\ud \ud One-hundred and thirty-three CHD patients were screened for genetic variation at the ALDH1A2 locus through bi-directional sequencing. In addition, six SNPs (rs2704188, rs1441815, rs3784259, rs1530293, rs1899430) at the same locus were studied using a TDT-based association approach in 101 CHD trios. Observed mutations were modeled through molecular mechanics (MM) simulations using the AMBER 9 package, Sander and Pmemd programs. Sequence conservation of observed mutations was evaluated through phylogenetic tree construction from ungapped alignments containing ALDH8 s, ALDH1Ls, ALDH1 s and ALDH2 s. Trees were generated by the Neighbor Joining method. Variations potentially affecting splicing mechanisms were cloned and functional assays were designed to test splicing alterations using the pSPL3 splicing assay.\ud \ud \ud \ud Results\ud \ud We describe in Tetralogy of Fallot (TOF) the mutations Ala151Ser and Ile157Thr that change non-polar to polar residues at exon 4. Exon 4 encodes part of the highly-conserved tetramerization domain, a structural motif required for ALDH oligomerization. Molecular mechanics simulation studies of the two mutations indicate that they hinder tetramerization. We determined that the SNP rs16939660, previously associated with spina bifida and observed in patients with TOF, does not affect splicing. Moreover, association studies performed with classical models and with the transmission disequilibrium test (TDT) design using single marker genotype, or haplotype information do not show differences between cases and controls.\ud \ud \ud \ud Conclusion\ud \ud In summary, our screen indicates that ALDH1A2 genetic variation is present in TOF patients, suggesting a possible causal role for this gene in rare cases of human CHD, but does not support the hypothesis that variation at the ALDH1A2 locus is a significant modifier of the risk for CHD in humans.Work supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) 01/000090; 00/030722; 01/142381; 02/113402; 03/099982; 04/116068; 04/157044 and Conselho Nacional de Desenvolvimento Científico e Tecnológico 481872/20078. We would like to thank the careful work and thoughtful suggestions of the two reviewers responsible for the reviewing editorial process.Work supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) 01/00009-0; 00/03072-2; 01/14238-1; 02/11340-2; 03/09998-2; 04/11606-8; 04/15704-4 and Conselho Nacional de Desenvolvimento Científico e Tecnológico 481872/2007-8. We would like to thank the careful work and thoughtful suggestions of the two reviewers responsible for the reviewing editorial process

Opposing effects of monomeric and pentameric C-reactive protein on endothelial progenitor cells

C-reactive protein (CRP) has been linked to the pathogenesis of atherosclerosis. The dissociation of native, pentameric (p)CRP to monomeric (m)CRP on the cell membrane of activated platelets has recently been demonstrated. The dissociation of pCRP to mCRP may explain local pro-inflammatory reactions at the site of developing atherosclerotic plaques. As a biomarker, pCRP predicts cardiovascular adverse events and so do reduced levels and function of circulating endothelial progenitor cells (EPCs). We hypothesised that mCRP and pCRP exert a differential effect on EPC function and differentiation. EPCs were treated with mCRP or pCRP for 72 h, respectively. Phenotypical characterisation was done by flow cytometry and immunofluorescence microscopy, while the effect of mCRP and pCRP on gene expression was examined by whole-genome gene expression analysis. The functional capacity of EPCs was determined by colony forming unit (CFU) assay and endothelial tube formation assay. Double staining for acetylated LDL and ulex lectin significantly decreased in cells treated with pCRP. The length of tubuli in a matrigel assay with HUVECs decreased significantly in response to pCRP, but not to mCRP. The number of CFUs increased after pCRP treatment. RNA expression profiling demonstrated that mCRP and pCRP cause highly contradictory gene regulation. Interferon-responsive genes (IFI44L, IFI44, IFI27, IFI 6, MX1, OAS2) were among the highly up-regulated genes after mCRP, but not after pCRP treatment. In conclusion, EPC phenotype, genotype and function were differentially affected by mCRP and pCRP, strongly arguing for differential roles of these two CRP conformations. The up-regulation of interferon-inducible genes in response to mCRP may constitute a mechanism for the local regulation of EPC function

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

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