Autoregulation of TRA-2 pre -mRNA splicing

One of the most elegant and tightly regulated mechanisms for control of gene expression is alternative pre-mRNA splicing. Despite the importance of regulated splicing in a variety of biological processes relatively little is understood about the mechanisms by which specific alternative splice choices are made and regulated. The transformer-2 (tra-2) gene encodes a splicing regulator that controls the use of alternative splicing pathways in the sex determination cascade of D. melanogaster and is particularly interesting because it directs the splicing of several distinct pre-mRNAs in different manners. The tra-2 protein positively regulates the splicing of both doublesex (dsx) and fruitless (fru) pre-mRNAs. Additionally tra-2 controls exuperantia (exu) by directing the choices between splicing and cleavage/polyadenylation and autoregulates the tra-2 pre-mRNA processing by repressing the removal of a specific intron (called M1). The goal of this study is to identify the molecular mechanisms by which TRA-2 protein affects the alternative splicing of pre-mRNA deriving from the tra-2 gene itself.^ The autoregulation of M1 splicing plays a key role in regulation of the relative levels of two functionally distinct TRA-2 protein isoforms expressed in the male germline. We have examined whether the structure, function, and regulation of tra-2 are conserved in Drosophila virilis, a species diverged from D. melanogaster by over 60 million years. We find that the D. virilis homolog of tra-2 produces alternatively spliced RNAs encoding a set of protein isoforms analogous to those found in D. melanogaster. When introduced into the genome of D. melanogaster, this homolog can functionally replace the endogenous tra-2 gene for both normal female sexual differentiation and spermatogenesis. Examination of alternative pre-mRNAs produced in D. virilis testes suggests that the germline-specific autoregulation of tra-2 function is accomplished by a strategy similar to that used in D. melanogaster.^ To identify elements necessary for regulation of tra-2 M1 splicing, we mutagenized evolutionarily conserved sequences within the tra-2 M1 intron and flanking exons. Constructs containing these mutations were used to generate transgenic fly lines that have been tested for their ability to carry out autoregulation. These transgenic fly experiments elucidated several elements that are necessary for setting up a context under which tissue-specific regulation of M1 splicing can occur. These elements include a suboptimal 3$\sp\prime$ splice site, an element that has been conserved between D. virilis and D. melanogaster, and an element that resembles the 3$\sp\prime$ portion of a dsx repeat and other splicing enhancers.^ Although important contextual features of the tra-2 M1 intron have been delineated in the transgenic fly experiments, the specific RNA sequences that interact directly with the TRA-2 protein were not identified. Using Drosophila nuclear extracts from Schneider cells, we have shown that recombinant TRA-2 protein represses M1 splicing in vitro. UV crosslinking analysis suggests that the TRA-2 protein binds to several different sites within and near the M1 intron. ^

Stability and potential application of spliced nuclear pre -mRNA introns

A major portion of this thesis work was dedicated to study the nature and significance of spliced introns. The initial work was focused on studying the IVS1$\sb{\rm C\beta 1}$ intron from a T-cell receptor (TCR)-$\beta$ gene. Compared to an intron lariat control from adenovirus pre-mRNA that was spliced in vitro, IVS1$\sb{\rm C\beta 1}$ was debranched less efficiently by HeLa S100 extracts, although IVS1$\sb{\rm C\beta 1}$ also used the consensus branchpoint in vivo. Subcellular-fractionation analysis showed that most IVS1$\sb{\rm C\beta 1}$ lariats cofractionated with pre-mRNA in the nucleus, consistent with the possibility that intron degradation releases splicing factors which will be available for further rounds of splicing. The half-life of IVS1$\sb{\rm C\beta 1}$ from the endogenous TCR-$\beta$ gene was measured using the general transcription inhibitor actinomycin D to be about $\sim$15 min, which was similar to that of unstable mRNAs such as c-myc mRNA.^ The general transcription inhibitor DRB was also used for intron stability analysis. Unexpectedly, DRB decreased intron and pre-mRNA levels only initially, it later increased the levels of intron-containing RNAs. Inhibition of transcription initiation appeared to be the major early effect (the reduction phase); whereas enhanced premature transcription termination was dominant later (the induction phase).^ Having established the procedures for studying in vivo spliced introns, this approach was applied to study the mechanism of nonsense-mediated downregulation (NMD), a phenomena in which premature termination codons (PTCs) decrease the levels of mRNAs. In this study, the novel intron-oriented approach was applied to study the mechanism of NMD. The levels of spliced introns immediately upstream and downstream of a PTC-bearing exon in a TCR-$\beta$ gene were identified and analyzed along with their pre-mRNA. Although PTC reduced the mRNA levels by 4 to 9 fold, the steady-state levels of spliced introns and the pre-mRNA-to-intron ratios were not significantly altered, indicating that the PTC did not significantly inhibit TCR-$\beta$ RNA splicing. Consistent with this conclusion, the half-lives of the PTC$\sp+$ and PTC$\sp-$ pre-mRNA were similar. The protein synthesis inhibitor cyclohexmide (CHX) upregulated the levels of the PTC$\sp+$ mRNA over 10 fold without affecting the levels of the spliced introns, suggesting that the reversal effect of CHX was through stabilization, not production. These results indicated that inhibition of splicing could not be the major mechanism for the NMD pathway of the TCR-$\beta$ gene, instead, suggesting that mRNA destabilization may be more important. (Abstract shortened by UMI.) ^

Regulations of SOX9 activity during chondrogenesis

Sox9 is a transcription factor required for chondrocyte differentiation and cartilage formation. In an effort to identify SOX9 interacting protein(s), we screened a chondrocyte cDNA library with a modified yeast two-hybrid method, Son of Sevenless (SOS) recruitment system (SRS). The catalytic subunit of cyclic AMP-dependent protein kinase A (PKA-Cα) and a new long form of c-Maf transcription factor (Lc-Maf) were found to interact specifically with SOX9. We showed here that two PKA phosphorylation consensus sites of SOX9 could be phosphorylated by PKA in vitro as well as in vivo. PKA phosphorylation of SOX9 increases its DNA binding and transcriptional activities on a Col2a1 chondrocyte-specific enhancer. Mutations of these two PKA phosphorylation sites markedly decreased the activation of SOX9 by PKA. ^ To test whether parathyroid hormone-related peptide (PTHrP) signaling results in SOX9 phosphorylation, we generated a phosphospecific antibody that specifically recognizes SOX9 that is phosphorylated at serine 181 (S 181) one of the two consensus PKA phosphorylation sites. Addition of PTHrP to COS7 cells cotransfected with SOX9 and PTH/PTHrP receptor strongly increased phosphorylation of SOX9 at S181; this phosphorylation was blocked by a PKA-specific inhibitor. In similar experiments we showed that PTHrP increased the activity of a SOX9-dependent Col2a1 enhancer. This increase in activity was abolished when a SOX9 mutant was used containing serine-to-alanine substitution in the two consensus PKA phosphorylation sites of SOX9. Using our phosphospecific SOX9 antibody we showed by immunohistochemistry of mouse embryos that Sox9 phosphorylated at S181 was localized almost exclusively in the pre-hypertrophic zone of the growth plate, an area corresponding to the major site of expression of PTH/PTHrP receptor. In contrast, no phosphorylation of Sox9 at S181 was detected in growth plates of PTH/PTHrP receptor null mutant mice. Sox9, regardless of phosphorylation state, was present in all chondrocytes of both genotypes except in hypertrophic chondrocytes. Thus, Sox9 is a target of PTHrP signaling and the PTHrP-dependent phosphorylation of SOX9 enhances its transcriptional activity. ^ In order to investigate the in vivo function of Sox9 phosphorylation by PKA, we are generating a mouse model of mutant Sox9 harboring point mutations in two PKA phosphorylation sites. Preliminary results indicated that heterozygous mice containing half amount of mutant Sox9 that can not be phosphorylated by PKA have normal skeletal phenotype and homozygous mice are being generated. ^ Lc-Maf encodes an extra ten amino acids at the carboxyl terminus of c-Maf and contains a completely different 3′ untranslated region. The interaction between SOX9 and Lc-Maf was further confirmed by co-immunoprecipitation and GST-pull down assays, which mapped the interacting domains of SOX9 to HMG DNA binding domain and that of Lc-Maf to basic leusine zipper motif. In situ hybridizations showed that RNA of Lc-Maf coexpressed with those of Sox9 and Col2a1 in areas of mesenchymal condensation during the early stages of mouse embryo development. A DNA binding site of Lc-Maf was identified at the 5′ part of a 48-bp Col2a1 enhancer element near the HMG binding site of SOX9. Lc-Maf and SOX9 synergistically activated a luciferase reporter plasmid containing a Col2al enhancer and increased the transcription of endogenous Col2a1 gene. In summary, Lc-Maf is the first identified SOX9-interating protein during chondrogenesis and may be an important activator of Col2a1 gene. ^

PEECE II mesocosm experiment: Dynamics of extracellular enzyme activities in seawater under changed atmospheric pCO2, 2011

As part of the PeECE II mesocosm project, we investigated the effects of pCO2 levels on the initial step of heterotrophic carbon cycling in the surface ocean. The activities of microbial extracellular enzymes hydrolyzing 4 polysaccharides were measured during the development of a natural phytoplankton bloom under pCO2 conditions representing glacial (190 µatm) and future (750 µatm) atmospheric pCO2. We observed that (1) chondroitin hydrolysis was variable throughout the pre-, early- and late-bloom phases, (2) fucoidanase activity was measurable only in the glacial mesocosm as the bloom developed, (3) laminarinase activity was low and constant, and (4) xylanase activity declined as the bloom progressed. Concurrent measurements of microbial community composition, using denaturing-gradient gel electrophoresis (DGGE), showed that the 2 mesocosms diverged temporally, and from one another, especially in the late-bloom phase. Enzyme activities correlated with bloom phase and pCO2, suggesting functional as well as compositional changes in microbial communities in the different pCO2 environments. These changes, however, may be a response to temporal changes in the development of phytoplankton communities that differed with the pCO2 environment. We hypothesize that the phytoplankton communities produced dissolved organic carbon (DOC) differing in composition, a hypothesis supported by changing amino acid composition of the DOC, and that enzyme activities responded to changes in substrates. Enzyme activities observed under different pCO2 conditions likely reflect both genetic and population-level responses to changes occurring among multiple components of the microbial loop.

Low current performance of the Bern medical cyclotron down to the pA range

A medical cyclotron accelerating H- ions to 18 MeV is in operation at the Bern University Hospital (Inselspital). It is the commercial IBA 18/18 cyclotron equipped with a specifically conceived 6 m long external beam line ending in a separate bunker. This feature is unique for a hospital-based facility and makes it possible to conduct routine radioisotope production for PET diagnostics in parallel with multidisciplinary research activities, among which are novel particle detectors, radiation biophysics, radioprotection, radiochemistry and radiopharmacy developments. Several of these activities, such as radiobiology experiments for example, require low current beams down to the pA range, while medical cyclotrons are designed for high current operation above 10 μA. In this paper, we present the first results on the low current performance of a PET medical cyclotron obtained by ion source, radio-frequency and main coil tuning. With this method, stable beam currents down to (1.5+/- 0.5 ) pA were obtained and measured with a high-sensitivity Faraday cup located at the end of the beam transport line.

The Production Technology of Philippine Domestic Banks in the Pre-Asian Crisis Periods: Estimation of Cost Function in the 1990-96 Period

This is to analyzes the operational behavior and technical progress among Philippine domestic banks, using micro-level data on individual banks. First, we summarize their major business activities and gain insight on how the structure is changing. Then, we formally estimate the cost function of Philippine domestic banks using panel data covering a seven-year period (1990-96). The presence of economies of scale and economies of scope is investigated and technical progress in the banking industry is measured. In addition, the results of analysis for the Philippines are compared with those of similar studies on Thailand conducted by the author previously.

Redes de información y conocimiento climatológico en el mundo hispánico del siglo XVIII

Durante la Ilustración, el imperio español alcanzó su máxima amplitud y las instituciones oficiales incrementaron su apoyo a las ciencias. Para defender sus fronteras y ejercer con eficacia el poder político, económico y religioso, la Corona y la Iglesia necesitaban obtener información precisa --incluida la climatológica-- de las posesiones españolas y de los pobladores de éstas. Fueron varios los procedimientos empleados para ello: sistema de cuestionarios y relaciones geográficas, estudios medico-topográficos, visitas e inspecciones oficiales, expediciones político-científicas, correspondencia epistolar, artículos periodísticos, etc. Dichos procedimientos fueron aplicados por redes de informadores cuyas actuaciones se basaban en la división del trabajo, el reparto de colaboradores en diferentes lugares, el uso de códigos de comunicación comprensibles, el envío de los resultados a los superiores jerárquicos y la toma de decisiones por las autoridades competentes. Las redes de información estaban sometidas a dictámenes que normalizaban su creación y continuidad temporal, daban forma a su estructura interna, especificaban sus cometidos y obligaban a cumplir protocolos y plazos. En su seno se idearon planes de investigación integrados en el estudio general de la Tierra, el ser humano y la cultura. El beneficio de las actuaciones de sus miembros se plasmó en cubrir grandes ámbitos geográficos con el consiguiente ahorro de tiempo, esfuerzos y medios. En sus correspondientes contextos, los miembros de las redes efectuaron estudios climatológicos conforme a intereses, imposiciones y circunstancias específicas. Así, los médicos se interesaron por las condiciones climáticas que influían en la salud humana; los funcionarios reales y los ingenieros militares describieron los climas locales y regionales aptos para el fomento y el control político, jurídico y educativo de los habitantes de los territorios hispánicos; los expedicionarios estudiaron las interacciones entre los fenómenos naturales y las influencias de los accidentes geográficos en los climas; los clérigos se interesaron por los aspectos estéticos, apologéticos y contemplativos de los climas; finalmente, en la prensa de la época se publicaron registros meteorológicos periódicos y trabajos climatológicos varios. En definitiva, el saber climatológico en el mundo hispánico ilustrado aportó algunos rasgos esenciales a la climatología en una etapa pre-fundacional de esta disciplina. Dichos rasgos se desarrollaron generalmente en una escala local o regional y se refirieron a los siguientes asuntos: el calor como principal agente de las modificaciones atmosféricas, de la formación de vapor acuoso y de las precipitaciones; la influencia del suelo en el aumento de humedad y calor en el aire; el poder de los vientos para trasladar de un lugar a otro el frío o el calor, el vapor de agua, los fenómenos atmosféricos y los agentes responsables de las enfermedades contagiosas; las propiedades del aire atmosférico y su capacidad para interaccionar con el medio ambiente; la condición estática y repetitiva de los climas, si bien se admitió que dichos fenómenos podían sufrir modificaciones; la corroboración experimental de las diferencias climáticas entre las zonas tropicales y medias del planeta; la refutación de que la naturaleza americana y sus habitantes eran inferiores a los europeos; y la demostración de que los principios rectores de los fenómenos físicos del Viejo y el Nuevo Mundo eran idénticos. Desde el último tercio del siglo XVIII, los documentos producidos por los componentes de las redes de información incluyeron datos meteorológicos. Pero no siempre se emplearon los mismos instrumentos de medida ni se siguieron los mismos protocolos de indagación en idénticas condiciones. Además, y salvo excepciones, los períodos durante los cuales se recabaron datos atmosféricos fueron relativamente cortos, y los expertos no efectuaron las mismas operaciones aritméticas con los parámetros. Por esta razón, y por la orientación utilitaria de los ilustrados hispánicos, el saber climatológico no obtuvo en el período y en el ámbito geográfico considerados resultados teóricos apreciables; en cambio, dio lugar a una gran cantidad de actividades prácticas con aplicaciones a la medicina, la agricultura, la náutica, el fomento, la prevención de riesgos naturales, etc. La principal utilidad de este trabajo consiste en servir de complemento a los procedimientos actualmente en uso en historia de la hidrología y en climatología histórica. ABSTRACT During the Enlightenment, the Spanish Empire achieved its highest length and State institutions increased their support to sciences. In order to defend their frontiers and to exercise political, economical and religious power, the Crown and the Church needed exact information --including the climatologic one-- about its possessions and its habitants. Some of the procedures employed to get that objective were: system of questionnaires and geographic relations, medical-topographic studies, official visits and inspections, political-scientific expeditions, direct mail, journalistic articles, etc. Those procedures were applied by informers´ networks which obtained, manned and transmitted every kind of data about the natural and moral reality of the Hispanic territories; their actions were based on the division of tasks, the distribution of collaborators at several places, the use of understandable communication codes and the sending of results to the hierarchical superiors; after, the competent authorities took decisions. The information networks were subjected to rules witch regulated its creation, temporary continuity, interior structure, objectives, protocols and periods. Their memberships invented plans about the general research of the Earth, the human beings and the culture; and they contributed to get benefits because of covering large geographic frames and economizing time, effort and means. According to their specifics contexts, concerns, impositions and circumstances, the informers performed climatologic tasks. Thus, the physicians were interested in the climatic conditions which affected to human health; the royal officers and military engineers described the most propitious climates to patronage and political, lawful and educative control of inhabitants of Hispanic territories; the participants in politic-scientific expeditions studied the interactions among natural phenomena and the influence of geographic aspects on the climate; the clergymen underlined the esthetic, apologetic and contemplative face of climates; finally, in the newspapers were published a lot of meteorological data and climatologic works. So, the climatologic knowledge in the Hispanic enlightened world added these essential aspects --referred in a local and regional area-- during the pre-foundational epoch of the climatology: the heat as first agent of atmospheric modifications, aqueous vapor and precipitations; the influx of the land in the increment of humidity and heat of the air; the power of the winds to convey the cold, the heat, the aqueous vapor, the atmospheric phenomena and the agents which caused contagious maladies; the properties of the air and its faculty to mediate with the environs; the static and repetitive condition of the climate and its possibility to experience change; the experimental confirmation of climatic varieties between tropical and central areas of the planet; the negation of the inferiority of the American nature and inhabitants; the demonstration about the equality of the rules which conducted physical phenomena in the Old and the New world. Since the last third part of the eighteenth century, the documents produced by the members of the networks included meteorological data. But the informers were not used to employ the same measure instruments and homogeneous protocols completion in the same conditions. Exceptions besides, the times of taking atmospheric data, usually were very short; and the experts did not carry out the same arithmetical operations with parameters. Because of this reason and the utilitarian guidance of the informers, during the Hispanic Enlightenment, it was not possible to obtain theoretic conclusions about climatologic knowledge; but there were a lot of practical activities applied to Medicine, Agriculture, Navigation, patronage, prevention of natural risks, etc. The main utility of this work consist in favoring the present procedures of the History of Hydrology and Historic Climatology.

Inhibition of interferon γ induced interleukin 12 production: A potential mechanism for the anti-inflammatory activities of tumor necrosis factor

Inflammation is associated with production of cytokines and chemokines that recruit and activate inflammatory cells. Interleukin (IL) 12 produced by macrophages in response to various stimuli is a potent inducer of interferon (IFN) γ production. IFN-γ, in turn, markedly enhances IL-12 production. Although the immune response is typically self-limiting, the mechanisms involved are unclear. We demonstrate that IFN-γ inhibits production of chemokines (macrophage inflammatory proteins MIP-1α and MIP-1β). Furthermore, pre-exposure to tumor necrosis factor (TNF) inhibited IFN-γ priming for production of high levels of IL-12 by macrophages in vitro. Inhibition of IL-12 by TNF can be mediated by both IL-10-dependent and IL-10-independent mechanisms. To determine whether TNF inhibition of IFN-γ-induced IL-12 production contributed to the resolution of an inflammatory response in vivo, the response of TNF+/+ and TNF−/− mice injected with Corynebacterium parvum were compared. TNF−/− mice developed a delayed, but vigorous, inflammatory response leading to death, whereas TNF+/+ mice exhibited a prompt response that resolved. Serum IL-12 levels were elevated 3-fold in C. parvum-treated TNF−/− mice compared with TNF+/+ mice. Treatment with a neutralizing anti-IL-12 antibody led to resolution of the response to C. parvum in TNF−/− mice. We conclude that the role of TNF in limiting the extent and duration of inflammatory responses in vivo involves its capacity to regulate macrophage IL-12 production. IFN-γ inhibition of chemokine production and inhibition of IFN-γ-induced IL-12 production by TNF provide potential mechanisms by which these cytokines can exert anti-inflammatory/repair function(s).

Phosphoinositide Signaling Pathways in Nuclei Are Associated with Nuclear Speckles Containing Pre-mRNA Processing Factors

Phosphoinositide signal transduction pathways in nuclei use enzymes that are indistinguishable from their cytosolic analogues. We demonstrate that distinct phosphatidylinositol phosphate kinases (PIPKs), the type I and type II isoforms, are concentrated in nuclei of mammalian cells. The cytosolic and nuclear PIPKs display comparable activities toward the substrates phosphatidylinositol 4-phosphate and phosphatidylinositol 3-phosphate. Indirect immunofluorescence revealed that these kinases were associated with distinct subnuclear domains, identified as “nuclear speckles,” which also contained pre-mRNA processing factors. A pool of nuclear phosphatidylinositol bisphosphate (PIP2), the product of these kinases, was also detected at these same sites by monoclonal antibody staining. The localization of PIPKs and PIP2 to speckles is dynamic in that both PIPKs and PIP2 reorganize along with other speckle components upon inhibition of mRNA transcription. Because PIPKs have roles in the production of most phosphatidylinositol second messengers, these findings demonstrate that phosphatidylinositol signaling pathways are localized at nuclear speckles. Surprisingly, the PIPKs and PIP2 are not associated with invaginations of the nuclear envelope or any nuclear membrane structure. The putative absence of membranes at these sites suggests novel mechanisms for the generation of phosphoinositides within these structures.

Molecular cloning and characterization of a murine pre-B-cell growth-stimulating factor/stromal cell-derived factor 1 receptor, a murine homolog of the human immunodeficiency virus 1 entry coreceptor fusin

Pre-B-cell growth-stimulating factor/stromal cell-derived factor 1 (PBSF/SDF-1) is a member of the CXC group of chemokines that is initially identified as a bone marrow stromal cell-derived factor and as a pre-B-cell stimulatory factor. Although most chemokines are thought to be inducible inflammatory mediators, PBSF/SDF-1 is essential for perinatal viability, B lymphopoiesis, bone marrow myelopoiesis, and cardiac ventricular septal formation, and it has chemotactic activities on resting lymphocytes and monocytes. In this paper, we have isolated a cDNA that encodes a seven transmembrane-spanning-domain receptor, designated pre-B-cell-derived chemokine receptor (PB-CKR) from a murine pre-B-cell clone, DW34. The deduced amino acid sequence has 90% identity with that of a HUMSTSR/fusin, a human immunodeficiency virus 1 (HIV-1) entry coreceptor. However, the second extracellular region has lower identity (67%) compared with HUMSTSR/fusin. PB-CKR is expressed during embryo genesis and in many organs and T cells of adult mice. Murine PBSF/SDF-1 induced an increase in intracellular free Ca2+ in DW34 cells and PB-CKR-transfected Chinese hamster ovary (CHO) cells, suggesting that PB-CKR is a functional receptor for murine PBSF/SDF-1. Murine PBSF/SDF-1 also induced Ca2+ influx in fusin-transfected CHO cells. On the other hand, considering previous results that HIV-1 does not enter murine T cells that expressed human CD4, PB-CKR may not support HIV-1 infection. Thus, PB-CKR will be an important tool for functional mapping of HIV-1 entry coreceptor fusin and for understanding the function of PBSF/SDF-1 further.

Fetal and maternal contributions to risk of pre-eclampsia: population based study

Objective: To use familial patterns of recurrence of pre-eclampsia to investigate whether paternal genes expressed in the fetus contribute to the mother’s risk of pre-eclampsia and whether mother’s susceptibility to pre-eclampsia is related to maternal inheritance by mitochondrial DNA.