A functional alternative splicing mutation in human tryptophan hydroxylase-2.

The brain serotonergic system has an essential role in the physiological functions of the central nervous system and dysregulation of serotonin (5-HT) homeostasis has been implicated in many neuropsychiatric disorders. The tryptophan hydroxylase-2 (TPH2) gene is the rate-limiting enzyme in brain 5-HT synthesis, and thus is an ideal candidate gene for understanding the role of dysregulation of brain serotonergic homeostasis. Here, we characterized a common, but functional single-nucleotide polymorphism (SNP rs1386493) in the TPH2 gene, which decreases efficiency of normal RNA splicing, resulting in a truncated TPH2 protein (TPH2-TR) by alternative splicing. TPH2-TR, which lacks TPH2 enzyme activity, dominant-negatively affects full-length TPH2 function, causing reduced 5-HT production. The predicted mRNA for TPH2-TR is present in postmortem brain of rs1386493 carriers. The rs13864923 variant does not appear to be overrepresented in either global or multiplex depression cohorts. However, in combination with other gene variants linked to 5-HT homeostasis, this variant may exhibit important epistatic influences.

Restoring orbital thinking from real space descriptions: bonding in classical and non-classical transition metal carbonyls.

A combined strategy that unifies our interacting quantum atoms approach (IQA), a chemically intuitive energetic perspective within the quantum theory of atoms in molecules (QTAIM), the domain natural orbitals obtained by the diagonalization of the charge-weighted domain-averaged Fermi hole (DAFH), and the statistical analyses of chemical bonding provided by the electron number distribution functions (EDF) is presented. As shown, it allows for recovering traditional orbital images from the orbital invariant descriptions of QTAIM. It does also provide bonding indices (like bond orders) and bond energetics, all in a per orbital basis, still invariant manner, using a single unified framework. The procedure is applied to show how the Dewar, Chatt, and Ducanson model of bonding in simple transition metal carbonyls may be recovered in the real space. The balance between the number of σ-donated and π-backdonated electrons is negative in classical compounds and positive in non-classical ones. The energetic strength of backdonation is, however, smaller than that of donation. Our technique surpasses conventional orbital models by providing physically sound, quantitative energetics of chemical bonds (or interactions) together with effective one-electron pictures, all for arbitrary wavefunctions.

Convergence of the multipole expansion for 1,2 Coulomb interactions: The modified multipole shifting algorithm.

A force field that accounts for the quantum chemical reality of interacting atoms must include Coulomb interactions between bonded atoms. The short-range nature of such 1,2 interactions necessitates atomic multipole moments in addition to point charges. However, the close proximity of bonded atoms would normally lead to a divergent multipolar expansion. A special algorithm presented here, within the scope of the previously presented multipole shifting method [M. Rafat and P. L. A. Popelier, J. Chem. Phys. 124, 144102 (2006)], shows that convergence can nevertheless be achieved by a suitable selection of multipole displacements. The algorithm is applied to improve the convergence of the multipolar expansion within the quantum theory of atoms in molecules approach.

Expectations of nursing degree students: A longitudinal analysis.

BACKGROUND: The expectations of students regarding their studies have a strong influence on their academic performance and personal training, and they are closely related with their academic and professional future. OBJECTIVES: To analyze initial expectations and how they are fulfilled, in a cohort of nursing students during the four years of their degree program. DESIGN: Creation and validation of a questionnaire and longitudinal study. SETTING: University nursing school in Catalonia (Spain). PARTICIPANTS: 339 students of the nursing degree. METHODS: Two questionnaires were constructed with 10 items each, of a mixed nature: some items were previously validated in prior studies while other new items were made and included, following a review of the literature. The questionnaires were designed to assess the expectations of the students at the beginning of the academic year (CUDEX questionnaire) and the fulfillment of these at the end of the year (CUDEX-C questionnaire). RESULTS: Internal consistency (Cronbach's alpha) for the CUDEX questionnaire and the CUDEX-C was 0.70. Factorial analysis of the expectations questionnaires suggested a three-factor model, with an acceptable internal consistency for each of the factors. Statistically significant differences were found between the initial expectations and their fulfillment at the end of the academic year for all the factors and in all four years of the degree program. CONCLUSIONS: The three-factor model of the expectations (Academic adaptation, personal development, and academic performance) shows a good fit for the several samples and time points. The nursing students feel that not all of their expectations were fulfilled during their studies, and there were significant differences between their initial expectations and those fulfilled, in terms of all three factors.

Steric repulsions, rotation barriers, and stereoelectronic effects: a real space perspective.

Widely used chemical concepts like Pauli repulsion or hyperconjugation, and their role in determining rotation barriers or stereoelectronic effects, are analyzed from the real space perspective of the interacting quantum atoms approach (IQA). IQA emerges from the quantum theory of atoms in molecules (QTAIM), but is free from the equilibrium geometry constraint of the former. A framework with both electronically unrelaxed and relaxed wavefunctions is presented that leads to an approximate correspondence between the IQA concepts and those used in the EDA (energy decomposition analysis) or NBO (natural bond orbital) procedures. We show that no net force acts upon the electrons in an electronically relaxed system, so that any reasonable definition of Pauli repulsion must involve unrelaxed state functions. Using antisymmetrized fragments clarifies that Pauli repulsions are energetically connected to the IQA deformation energies, leaving footprints in the finally relaxed states. Similarly, EDA or NBO hyperconjugative stabilizations are found to be naturally related to the IQA electron delocalization patterns. Applications to the rotation barrier of ethane and other simple systems are presented, and the very often forgotten role of electrostatic contributions in determining preferred conformations is highlighted.

Nuclear spreads: I. Visualization of bipartite ribosomal RNA domains.

Visualization of nuclear architecture is key to the understanding of the association between RNA synthesis and processing. This architecture is obscured by the high density of components in most nuclei. We have developed a method of spreading nuclei and nucleoli that reduces overlap of weakly associated components. Strong interactions among nuclear components are not disrupted by this method. Spread nucleoli remained structurally distinct and functionally competent in ribosomal RNA synthesis. Nascent ribosomal RNA colocalized with RNA polymerase I and fibrillarin, a protein required for processing of ribosomal RNA. Colocalization of nascent transcripts and fibrillarin was seen in nucleoli spread over several microns, suggesting a strong interaction. These data suggest that nucleoli are superassemblies of bipartite domains, each composed of a ribosomal RNA synthesis center tightly associated with areas likely to be involved in ribosomal RNA processing.

Molecular basis of latency in pathogenic human viruses.

Several human viruses are able to latently infect specific target cell populations in vivo. Analysis of the replication cycles of herpes simplex virus, Epstein-Barr virus, and human immunodeficiency virus suggests that the latent infections established by these human pathogens primarily result from a lack of host factors critical for the expression of viral early gene products. The subsequent activation of specific cellular transcription factors in response to extracellular stimuli can induce the expression of these viral regulatory proteins and lead to a burst of lytic viral replication. Latency in these eukaryotic viruses therefore contrasts with latency in bacteriophage, which is maintained primarily by the expression of virally encoded repressors of lytic replication.

Using pseudopotentials within the interacting quantum atoms approach.

A general strategy to extend the interacting quantum atoms (IQA) approach to pseudopotential or effective core potential electronic structure calculations is presented. With the protocol proposed here, the scope of IQA thinking opens to chemical bonding problems in heavy-atom systems, as well as to larger molecules than those presently allowed by computational limitations. We show that, provided that interatomic surfaces are computed from core-reconstructed densities, reasonable results are obtained by integrating reduced density matrices built from the pseudowave functions. Comparison with all-electron results in a few test systems shows that exchange-correlation energies are better reproduced than Coulombic contributions, an effect which is traced to inadequate atomic populations and leakage of the core population into the surrounding quantum atoms.

A connection between domain-averaged Fermi hole orbitals and electron number distribution functions in real space.

We show in this article how for single-determinant wave functions the one-electron functions derived from the diagonalization of the Fermi hole, averaged over an arbitrary domain Omega of real space, and expressed in terms of the occupied canonical orbitals, describe coarse-grained statistically independent electrons. With these domain-averaged Fermi hole (DAFH) orbitals, the full electron number distribution function (EDF) is given by a simple product of one-electron events. This useful property follows from the simultaneous orthogonality of the DAFH orbitals in Omega, Omega(')=R(3)-Omega, and R(3). We also show how the interfragment (shared electron) delocalization index, delta(Omega,Omega(')), transforms into a sum of one-electron DAFH contributions. Description of chemical bonding in terms of DAFH orbitals provides a vivid picture relating bonding and delocalization in real space. DAFH and EDF analyses are performed on several test systems to illustrate the close relationship between both concepts. Finally, these analyses clearly prove how DAFH orbitals well localized in Omega or Omega(') can be simply ignored in computing the EDFs and/or delta(Omega,Omega(')), and thus do not contribute to the chemical bonding between the two fragments.

Rabies control in Mexico.

Rabies in dogs was unknown in the Americas before the arrival of the Spanish "Conquistadores". Until the mid-1980s rabies in animals and, in turn in humans, changed little from year to year, with the number of dog vaccinations reported annually rarely reaching one million. In Mexico, the national rabies control programme using mass parenteral vaccination of dogs started in 1990 with about seven million dogs vaccinated the same year. The number of vaccinated dogs exceeded 10 and 15 million in 1995 and 2005, respectively. Modern cell culture-based inactivated rabies virus vaccines were used. A key factor for the success of the dog rabies control program was the supply of potent canine rabies vaccines. Between 1990 and 2005, more than 150 million vaccine doses from 300 lots were administered. Each lot was tested for potency prior to use in the field. The required minimum content of rabies virus antigen for vaccines was 2 IU, in accord with WHO standards. Testing revealed antigen contents ranging from 3.28 to 5.59 IU. As a result of the mass dog vaccination campaigns, human rabies cases due to dog-mediated rabies decreased from 60 in 1990 to 0 in 2000. The number of rabies cases in dogs decreased from 3,049 in 1990 to 70 cases last year.

Endocarditis due to vancomycin-resistant Enterococcus raffinosus successfully treated with linezolid: case report and review of literature.

Enterococcus raffinosus is scarcely found in clinical samples and even less frequently as etiologic agent of endocarditis. We are herein presenting one case of mitral prosthetic-valve endocarditis in a 77-y-o male due to a vancomycin-resistant Enterococcus raffinosus isolate, successfully treated with 6 weeks of linezolid, and a two-year follow up.

Transcriptional cofactor CA150 regulates RNA polymerase II elongation in a TATA-box-dependent manner.

Tat protein strongly activates transcription from the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) by enhancing the elongation efficiency of RNA polymerase II complexes. Tat-mediated transcriptional activation requires cellular cofactors and specific cis-acting elements within the HIV-1 promoter, among them a functional TATA box. Here, we have investigated the mechanism by which one of these cofactors, termed CA150, regulates HIV-1 transcription in vivo. We present a series of functional assays that demonstrate that the regulation of the HIV-1 LTR by CA150 has the same functional requirements as the activation by Tat. We found that CA150 affects elongation of transcription complexes assembled on the HIV-1 promoter in a TATA-box-dependent manner. We discuss the data in terms of the involvement of CA150 in the regulation of Tat-activated HIV-1 gene expression. In addition, we also provide evidence suggesting a role for CA150 in the regulation of cellular transcriptional processes.

The spliceosome assembly pathway in mammalian extracts.

A mammalian splicing commitment complex was functionally defined by using a template commitment assay. This complex was partially purified and shown to be a required intermediate for complex A formation. The productive formation of this commitment complex required both splice sites and the polypyrimidine tract. U1 small nuclear ribonucleoprotein (snRNP) was the only spliceosomal U snRNP required for this formation. A protein factor, very likely U2AF, is probably involved in the formation of the splicing commitment complex. From the kinetics of appearance of complex A and complex B, it was previously postulated that complex A represents a functional intermediate in spliceosome assembly. Complex A was partially purified and shown to be a required intermediate for complex B (spliceosome) formation. Thus, a spliceosome pathway is for the first time supported by direct biochemical evidence: RNA+U1 snRNP+?U2 auxiliary factor+?Y----CC+U2 snRNP+Z----A+U4/6,5 snRNPs+ beta----B.

An intronic splicing silencer causes skipping of the IIIb exon of fibroblast growth factor receptor 2 through involvement of polypyrimidine tract binding protein.

Alternative splicing of fibroblast growth factor receptor 2 (FGF-R2) transcripts involves the mutually exclusive usage of exons IIIb and IIIc to produce two different receptor isoforms. Appropriate splicing of exon IIIb in rat prostate cancer DT3 cells requires a previously described cis element (ISAR, for "intronic splicing activator and repressor") which represses the splicing of exon IIIc and activates the splicing of exon IIIb. This element is nonfunctional in rat prostate AT3 cells, which repress exon IIIb inclusion and splice to exon IIIc. We have now identified an intronic element upstream of exon IIIb that causes repression of exon IIIb splicing. Deletion of this element abrogates the requirement for ISAR in order for exon IIIb to be spliced in DT3 cells and causes inappropriate inclusion of exon IIIb in AT3 cells. This element consists of two intronic splicing silencer (ISS) sequences, ISS1 and ISS2. The ISS1 sequence is pyrimidine rich, and in vitro cross-linking studies demonstrate binding of polypyrimidine tract binding protein (PTB) to this element. Competition studies demonstrate that mutations within ISS1 that abolish PTB binding in vitro alleviate splicing repression in vivo. Cotransfection of a PTB-1 expression vector with a minigene containing exon IIIb and the intronic splicing silencer element demonstrate PTB-mediated repression of exon IIIb splicing. Furthermore, all described PTB isoforms were equally capable of mediating this effect. Our results support a model of splicing regulation in which exon IIIc splicing does not represent a default splicing pathway but rather one in which active repression of exon IIIb splicing occurs in both cells and in which DT3 cells are able to overcome this repression in order to splice exon IIIb.

An intronic sequence element mediates both activation and repression of rat fibroblast growth factor receptor 2 pre-mRNA splicing.

Alternative splicing of fibroblast growth factor receptor 2 (FGF-R2) is an example of highly regulated alternative splicing in which exons IIIb and IIIc are utilized in a mutually exclusive manner in different cell types. The importance of this splicing choice is highlighted by studies which indicate that deregulation of the FGF-R2 splicing is associated with progression of prostate cancer. Loss of expression of a IIIb exon-containing isoform of FGF-R2 [FGF-R2 (IIIb)] accompanies the transition of a well-differentiated, androgen-dependent rat prostate cancer cell line, DT3, to the more aggressive, androgen-independent AT3 cell line. We have used transfection of rat FGF-R2 minigenes into DT3 and AT3 cancer cell lines to study the mechanisms that control alternative splicing of rat FGF-R2. Our results support a model in which an important cis-acting element located in the intron between these alternative exons mediates activation of splicing using the upstream IIIb exon and repression of the downstream IIIc exon in DT3 cells. This element consists of 57 nucleotides (nt) beginning 917 nt downstream of the IIIb exon. Analysis of mutants further demonstrates that an 18-nt "core sequence" within this element is most crucial for its function. Based on our observations, we have termed this sequence element ISAR (for intronic splicing activator and repressor), and we suggest that factors which bind this sequence are required for maintenance of expression of the FGF-R2 (IIIb) isoform.

CA150, a nuclear protein associated with the RNA polymerase II holoenzyme, is involved in Tat-activated human immunodeficiency virus type 1 transcription.

Maximal human immunodeficiency virus type 1 (HIV-1) gene expression requires specific cellular factors in addition to the virus-encoded trans-activator protein Tat and the RNA element TAR. We developed a functional assay, based on transcriptional activation in vitro, to identify these cellular factors. Here, we describe the purification and molecular cloning of CA150, a nuclear protein that is associated with the human RNA polymerase II holoenzyme and is involved in Tat-dependent HIV-1 transcriptional activation. The sequence of CA150 contains an extensive glutamine- and alanine-rich repeat that is found in transcriptional modulators such as GAL11 and SSN6 in Saccharomyces cerevisiae and Zeste in Drosophila melanogaster. Immunodepletion of CA150 abolished Tat trans activation in vitro. Moreover, overexpression of a mutant CA150 protein specifically and dramatically decreased Tat-mediated activation of the HIV-1 promoter in vivo, strongly suggesting a role for CA150 in HIV-1 gene regulation. Immunoprecipitation experiments demonstrated that both CA150 and Tat associate with the RNA polymerase II holoenzyme. Furthermore, we found that functional Tat associates with the holoenzyme whereas activation-deficient Tat mutants do not. Thus, we propose that Tat action is transduced via an RNA polymerase II holoenzyme that contains CA150.

RNA binding by Sxl proteins in vitro and in vivo.

Sxl has been proposed to regulate splicing of specific target genes by directly interacting with their pre-mRNAs. We have therefore examined the RNA-binding properties of Sxl protein in vitro and in vivo. Gel shift and UV cross-linking assays with a purified recombinant MBP-Sxl fusion protein demonstrated preferential binding to RNAs containing poly(U) tracts, and the protein footprinted over the poly(U) region. The protein did not appear to recognize either branch point or AG dinucleotide sequences, but an adenosine residue at the 5' end of the poly(U) tract enhanced binding severalfold. MBP-Sxl formed two shifted complexes on a tra regulated acceptor site RNA; the doubly shifted form may have been stabilized by protein-protein interactions. Consistent with its proposed role in pre-mRNA processing, in nuclear extracts Sxl was found in large ribonucleoprotein (RNP) complexes which sedimented significantly faster than bulk heterogeneous nuclear RNP and small nuclear RNPs. Anti-Sxl staining of polytene chromosomes showed Sxl protein at a number of chromosomal locations, among which was the Sxl locus itself. Sxl protein could also be targeted to a new chromosomal site carrying a transgene containing splicing regulatory sequences from the Sxl gene, following transcriptional induction. After prolonged heat shock, all Sxl protein was restricted to the heat-induced puff at the hs93D locus. In contrast, a presumptive small nuclear RNP protein was observed at several heat puffs following shock.

The transcription elongation factor CA150 interacts with RNA polymerase II and the pre-mRNA splicing factor SF1.

CA150 represses RNA polymerase II (RNAPII) transcription by inhibiting the elongation of transcripts. The FF repeat domains of CA150 bind directly to the phosphorylated carboxyl-terminal domain of the largest subunit of RNAPII. We determined that this interaction is required for efficient CA150-mediated repression of transcription from the alpha(4)-integrin promoter. Additional functional determinants, namely, the WW1 and WW2 domains of CA150, were also required for efficient repression. A protein that interacted directly with CA150 WW1 and WW2 was identified as the splicing-transcription factor SF1. Previous studies have demonstrated a role for SF1 in transcription repression, and we found that binding of the CA150 WW1 and WW2 domains to SF1 correlated exactly with the functional contribution of these domains for repression. The binding specificity of the CA150 WW domains was found to be unique in comparison to known classes of WW domains. Furthermore, the CA150 binding site, within the carboxyl-terminal half of SF1, contains a novel type of proline-rich motif that may be recognized by the CA150 WW1 and WW2 domains. These results support a model for the recruitment of CA150 to repress transcription elongation. In this model, CA150 binds to the phosphorylated CTD of elongating RNAPII and SF1 targets the nascent transcript.

Regulation of 2',5'-oligoadenylate synthetase gene expression by interferons and platelet-derived growth factor.

In murine BALB/c 3T3 cell cultures, either beta interferon or platelet-derived growth factor (PDGF) enhanced expression of the 2',5'-oligoadenylate synthetase mRNA and protein. The time course of induction in response to beta interferon was similar to that in response to PDGF. Of several growth factors known to be present in clotted blood serum (i.e., epidermal growth factor, transforming growth factor beta, and PDGF), only PDGF enhanced expression of 2',5'-oligoadenylate synthetase. The linkage of an interferon response element-containing segment from the 5'-flanking region of a human or murine 2',-5'-oligoadenylate synthetase gene made a heterologous gene responsive to interferon. The expression of such a gene construct in transfected cells was also induced by PDGF. Induction by PDGF was inhibited by mono- or polyclonal antibodies to murine interferon, which suggested that induction by PDGF requires interferon. Both PDGF and interferon induced nuclear factors that bound to this interferon response element-containing segment in vitro.

Spliceosome-mediated RNA trans-splicing as a tool for gene therapy.

We have developed RNA molecules capable of effecting spliceosome-mediated RNA trans-splicing reactions with a target messenger RNA precursor (pre-mRNA). Targeted trans-splicing was demonstrated in a HeLa nuclear extract, cultured human cells, and H1299 human lung cancer tumors in athymic mice. Trans-splicing between a cancer-associated pre-mRNA encoding the beta-subunit of human chorionic gonadotropin gene 6 and pre-trans-splicing molecule (PTM) RNA was accurate both in vitro and in vivo. Comparison of targeted versus nontargeted trans-splicing revealed a moderate level of specificity, which was improved by the addition of an internal inverted repeat encompassing the PTM splice site. Competition between cis- and trans-splicing demonstrated that cis-splicing can be inhibited by trans-splicing. RNA repair in a splicing model of a nonfunctional lacZ transcript was effected in cells by a PTM, which restored significant beta-galactosidase activity. These observations suggest that spliceosome-mediated RNA trans-splicing may represent a general approach for reprogramming the sequence of targeted transcripts, providing a novel approach to gene therapy.