The three α1-adrenoceptor subtypes show different spatio-temporal mechanisms of internalization and ERK1/2 phosphorylation.

We analyzed the kinetic and spatial patterns characterizing activation of the MAP kinases ERK 1 and 2 (ERK1/2) by the three α1-adrenoceptor (α1-AR) subtypes in HEK293 cells and the contribution of two different pathways to ERK1/2 phosphorylation: protein kinase C (PKC)-dependent ERK1/2 activation and internalization-dependent ERK1/2 activation. The different pathways of phenylephrine induced ERK phosphorylation were determined by western blot, using the PKC inhibitor Ro 31-8425, the receptor internalization inhibitor concanavalin A and the siRNA targeting ß-arrestin 2. Receptor internalization properties were studied using CypHer5 technology and VSV-G epitope-tagged receptors. Activation of α1A- and α1B-ARs by phenylephrine elicited rapid ERK1/2 phosphorylation that was directed to the nucleus and inhibited by Ro 31-8425. Concomitant with phenylephrine induced receptor internalization α1A-AR, but not α1B-AR, produced a maintained and PKC-independent ERK phosphorylation, which was restricted to the cytosol and inhibited by ß-arrestin 2 knockdown or concanavalin A treatment. α1D-AR displayed constitutive ERK phosphorylation, which was reduced by incubation with prazosin or the selective α1D antagonist BMY7378. Following activation by phenylephrine, α1D-AR elicited rapid, transient ERK1/2 phosphorylation that was restricted to the cytosol and not inhibited by Ro 31-8425. Internalization of the α1D-AR subtype was not observed via CypHer5 technology. The three α1-AR subtypes present different spatio-temporal patterns of receptor internalization, and only α1A-AR stimulation translates to a late, sustained ERK1/2 phosphorylation that is restricted to the cytosol and dependent on ß-arrestin 2 mediated internalization.

The mouse neurotrophin receptor trkB gene is transcribed from two different promoters.

We have analysed a 7-kb region upstream of the mouse trkB coding sequence. The region showed promoter activity in transient transfection experiments and conferred tissue-specific expression to a reporter gene. Deletion analysis of this region demonstrated the presence of two alternative promoters named P1 and P2 that have been mapped by RNase protection. P1 has been located to 1.8 kb and P2 to 0.5 kb upstream of the trkB translation start site. From the P1 promoter, alternative splicing generates various transcripts. Interestingly, P2 is located in an intron of the transcripts produced from the P1 promoter. This peculiar arrangement results in different mRNA species that encode the same protein(s) but differ in their 5'-untranslated regions. In addition, transcription of the trkB locus results in two different trkB isoforms (kinase and truncated receptors) originated by alternative splicing of the mRNA, that possess differential spatial and temporal expression patterns. Using RT-PCR, we demonstrated that there was no linkage between promoter usage and alternative splicing, since transcripts initiated from each promoter encoded both kinase and truncated receptor proteins.

Structural features of a regulatory nucleosome.

DNA sequences from the long terminal repeat of the mouse mammary tumor virus (MMTV-LTR) position nucleosomes both in vivo and in vitro. Here, were present chromatin reconstitution experiments showing that MMTV-LTR sequences from -236 to +204 accommodate two histone octamers in positions compatible with the in vivo data. This positioning is not influenced by the length of the DNA fragment and occurs in linear as well as in closed circular DNA molecules. MMTV-LTR DNA sequences show an intrinsic bendability that closely resembles its wrapping around the histone octamer. We propose that bendability is responsible for the observed rotational nucleosome positioning. Translational nucleosome positioning seems also to be determined by the DNA sequence. These data, along with the results from reconstitution experiments with insertion mutants, support a modular model of nucleosome phasing on MMTV-LTR, where the actual positioning of the histone octamer results from the additive effect of multiple features of the DNA sequence.

Stimulation of the myelin basic protein gene expression by 9-cis-retinoic acid and thyroid hormone: activation in the context of its native promoter.

Thyroid hormone plays an important role in brain development, in part by regulating myelination. Previous studies have shown that the myelin basic protein (MBP) promoter is activated by thyroid hormone (T3) via a T3-response element (T3RE) at position -186. Surprisingly, although MBP levels are initially decreased in hypothyroid neonates, they approach later control levels, in most brain regions, despite persistent hypothyroidism. We have studied the T3-independent transcriptional regulation of this gene, using transient transfection assays. We found that, in the absence of T3, the RXR ligand, 9-cis-retinoic acid (9cRA) was able to stimulate transcription of the MBP promoter in a dose-dependent manner. This activation was unaffected by the mutation or deletion of the T3RE and required DNA sequences located between positions -162/+60. Accordingly, this MBP promoter fragment bound RXR in vitro. The 9cRA-dependent activation of the MBP promoter required the presence of both, the DNA binding and the ligand-dependent transactivation domain (AF-2) in RXR. Furthermore, as T3, 9cRA was able to stimulate MBP expression in the CG-4 cell line after differentiation to oligodendrocytes and increased the number of cells expressing the MBP protein in primary rat optic nerve glial cell cultures.

Transcriptional control by steroid hormones.

Gene regulation by steroid hormones leads to induction or repression of particular sets of genes. These effects are mediated by intracellular hormone receptors that, in the unliganded state, are maintained in an inactive form by unknown mechanisms possibly involving association with other cellular proteins. Induction of the mouse mammary tumor virus (MMTV) requires binding of the hormone receptor to a complex hormone-responsive element (HRE) located between 75 and 190 bp upstream from the start of transcription. The interaction of several receptor molecules with the four receptor binding sites in the HRE is highly cooperative on circular DNA molecules and each individual site is needed for optimal induction. In chromatin the HRE is precisely organized in phased nucleosomes. Following hormone treatment and receptor binding, changes in chromatin structure are detected that correlate with binding of transcription factors, including nuclear factor I, to the MMTV promoter. However, though nuclear factor I acts as a basal transcription factor on the MMTV promoter it does not cooperate with the hormone receptors in terms of binding to free DNA, and mutation of the nuclear factor I binding site does not eliminate hormonal stimulation. This residual induction is mediated by octamer motifs, upstream of the TATA box, that bind the ubiquitous transcription factor OTF-1. Mutation of these octamer motifs does not influence basal transcription in vitro, but completely abolishes the stimulatory effect of progesterone receptor.

Astrocytes in culture express the full-length Trk-B receptor and respond to brain derived neurotrophic factor by changing intracellular calcium levels: effect of ethanol exposure in rats.

Although cultured astroglial cells were reported to express exclusively the truncated non-catalytic Trk B receptor for brain-derived neurotrophic factor (BDNF), we detect here, using a sensitive ribonuclease protection assay, mRNAs for both truncated (TrkB-T) and the full length catalytic (TrkB-fl) form of BDNF receptor in developing cortical astrocytes and neurons in culture. Cortical neurons and immature astroglia, such as radial glia and proliferating astrocytes, express both the protein and mRNAs for TrkB-fl and TrkB-T, whereas the differentiation of astrocytes leads to a decrease in the trkB-fl mRNA, being the truncated TrkB the predominant receptor in differentiating and confluent astrocytes. The levels of TrkB-fl expression in proliferating and differentiating astrocytes and neurons correlates with the cell response to BDNF, monitored by the rise in intracellular [Ca(2+)](i). Foetal exposure to ethanol alters astroglial development and delays the reduction in trkB-fl mRNA levels observed with differentiation of astrocytes. These results demonstrate that immature astrocytes are able to express the catalytic Trk B receptors and to respond to BDNF with the activation of conventional signal transduction pathways. The results suggest that this signalling pathway is more activated in ethanol-exposed cells.

The extent of the Aznalcóllar pyritic sludge spill and its effects on soils.

This paper presents some initial results from the Instituto Tecnológico Geominero de España's (ITGE) study of the Aznalcóllar mine spill. The spatial distribution of the pyritic sludge released was surveyed by using remote sensing data, aerial photography, and more than 700 field measurements on the sludge thickness. Initial estimation of the extent of the sludge was provided by radar data. Maps at 1:10,000 scale, drawn on the basis of field data and interpretation of aerial photos, show the distribution of the sludge, divided into 168 subsections on the basis of average thickness. GIS analysis provided estimates of the area and volume of the sludge. Three approaches were followed in order to survey the effects of the spill on the Guadiamar river alluvial soils: (1) Mineralogical and chemical characterization of the sludge and its evolution until its removal. Alteration products of the pyritic sludge were also analyzed. (2) Determination of geochemical background of soils in the Guadiamar river basin, in order to establish the content of heavy metals and other elements in the soil before the spill. (3) Assessment of the sludge effect on soils caused by the acid water and the deposited sludge, by comparison of the heavy metal content of soil under the sludge layer with that of background soil. Finally, an airborne multispectral survey was carried out over the Aznalcóllar-Doñana area to evaluate its efficiency for monitoring soil condition during and after sludge removal.

[Role of antithrombin iii in cardiac surgery]. / Papel de la antitrombina iii en cirugía cardiaca.

Coagulation of blood is of multidisciplinary interest. Cardiac surgery produces major changes in the delicate balance between pro-and anti-coagulant serum factors. The role of antithrombin iii has been analysed after finding evidence that associated decreased levels of protein activity to postoperative morbidity and mortality. Supplementing exogenous antithrombin is considered with the aim of optimising outcomes. Its intrinsic anticoagulant and anti-inflammatory properties have stimulated a growing interest, and suggests new lines of research.

Role of GHF-1 in the regulation of the rat growth hormone gene promoter by thyroid hormone and retinoic acid receptors.

In non-pituitary HeLa cells the unliganded thyroid hormone or retinoic acid receptors cause a strong activation of the rat growth hormone promoter that is repressed by their ligands. In contrast, after expression of the pituitary-specific transcription factor GHF-1, thyroid hormone and retinoic acid produce a stimulation similar to that found in pituitary cells. Therefore, GHF-1 changes a ligand-dependent inhibition into a ligand-dependent activation. The essential role of GHF-1 on the rat growth hormone promoter was also demonstrated with AF-2-defective T3 receptor mutants that show a normal activation of this promoter in the presence of GHF-1. Furthermore, a truncated T3 receptor, which lacks the N-terminus and the DNA binding domain, was able to stimulate this promoter in the presence of GHF-1 and exogenous RXR receptors, suggesting the importance of protein to protein interactions in this regulation. This study shows that the final transcriptional effect depends not only on the type of regulatory promoter response elements but also on the presence of other transcriptional activators, in the case of the growth hormone promoter, the tissue-specific transcription factor GHF-1, which plays a coactivator-like role in this promoter.

Induction of the heat-shock response by carbon dioxide in Chironomus thummi.

The effects of a set of stress treatments on gene expression of Chironomus thummi salivary gland cells have been analyzed. Among the treatments assayed, only during recovery from carbon dioxide have we observed a response similar to that previously described after heat-shock treatment: induction of the heat-shock puffs and synthesis of the heat-shock polypeptides. In these conditions, puffing and transcription of telomeric regions were observed, which led to the appearance of the temperature-inducible telomeric Balbiani ring T-BR-III. Other treatments failed to induce the heat-shock response, despite promoting real stress conditions to C. thummi larvae or salivary gland cells.

Characterization of the ligand-dependent transactivation domain of thyroid hormone receptor.

Transcriptional activation by nuclear receptors is achieved through autonomous activation functions (AFs), a constitutive N-terminal AF-1 and a C-terminal, ligand-dependent AF-2 that comprises a motif conserved between nuclear receptors. We have performed an extensive mutational analysis of the putative AF-2 domain of chicken thyroid hormone receptor alpha (cT3R alpha). We show that the AF-2 region mediates transactivation as well as transcriptional interference (squelching), not only between the thyroid hormone and vitamin (type II) receptors, but also between type II and steroid hormone (type I) receptors. Transcriptional activation and interference require equivalent doses of the cognate ligand, and mutations in the conserved motif that reduce ligand-induced transactivation also impair transcriptional interference. When fused to the Gal4 DNA binding domain, a 35 amino acid long fragment containing the conserved motif is able to transactivate and squelch, albeit in a ligand-independent manner. Our results define the AF-2 of cT3R alpha as an autonomous transactivation domain that, in its natural context, is governed by ligand. We propose that AF-2 is probably part of a surface for interaction with either a general transcription factor or a putative bridging factor, that might be utilized by type I and II receptors.

Identification of the spI products of Balbiani ring genes in Chironomus thummi.

The spI fraction of high molecular weight secretory proteins was analysed in Chironomus thummi. These proteins are encoded by giant Balbiani ring (BR) genes which develop specifically in salivary gland cells. Each component of the spI fraction was studied electrophoretically from early and middle 4th instar larvae and prepupae, as well from galactose-treated larvae where changes in the relative puffing pattern of BR1 and BR2 are known to occur. The spI fraction consists of at least two bands with electrophoretic mobilities slower than those of the spI components of Camptochironomus. The slow migrating component remains throughout the 4th larval instar, while the amount of the faster component changes, being abundant in early 4th instar and prepupae, but not present (or very weak) in middle 4th instar. The correlated shifts in BR puffing pattern during these developmental stages suggest that the slow and fast components are encoded by BR2 and BR1. The spI fraction is modified by galactose treatment, the fast component being induced in parallel with a decrease in the slow component. These changes are correlated with changes in the steady-state levels of RNA: an increase in BR1 RNA and a decrease in BR2 RNA, and of proteins. These proteins could correspond to the spIb and spIa fractions allocated to BR2 and BR1, respectively, in Camptochironomus. After galactose treatment a new faster band sometimes appears, that could correspond to the spIc fraction of Camptochironomus. A possible spId equivalent was also identified. In conclusion the main features of the spI family in C. thummi are similar to those of spI in Camptochironomus.

Telomeric DNA sequences differentially activated by heat shock in two Chironomus subspecies.

The patterns of puffing, transcription and protein synthesis under heat shock were analysed in polytene nuclei of Chironomus thummi piger, in comparison with those obtained in the closely related subspecies C. th. thummi. Most chromosomal heat shock puffs, as well as heat shock induced polypeptides, in C. th. piger paralleled those previously reported for C. th. thummi. Nevertheless, we found a striking difference in behaviour in the induction of telomeric Balbiani rings by heat shock in the two subspecies. Although homologous sequences were present at all the telomeres in both subspecies, they were not always transcriptionally activated by heat shock. The most frequently puffed telomeres were that of chromosome III R in C. th. thummi and that of chromosome IV R in piger. Transcription of the same sequences from both telomeric Balbiani rings (T-BR-III and T-BR-IV) occurred under heat shock. The enigmatic behaviour of telomeres and the functional significance of T-BRs are discussed in relation to possible equivalents in other Diptera.

Correlation between the activity of a 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole-insensitive puff and the synthesis of major heat-shock polypeptide, hsp70, in Chironomus thummi.

The induction of puff III-A3b, a major heat-shock puff in Chironomus thummi salivary cells, was insensitive to the transcription inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), whereas no transcriptional activity could be detected at the other heat-shock puffs in the presence of this drug. In these conditions, a polypeptide with the same Mr and isoform pattern as those of the major heat-shock polypeptide, hsp70, was synthesized. These results suggest that hsp70 is encoded by locus III-A3b. In addition to DRB insensitivity, incorporation of [3H]UTP on puff III-A3b took place in an in vitro transcription assay under low-salt conditions (100 mM NaCl); no labelling could be detected at the other heat-shock puffs under these conditions. Although DRB has been reported as a specific inhibitor of RNA polymerase II-directed transcription, and although the low-salt conditions were not propitious for the activity of this enzyme, RNA polymerase II was detected on puff III-A3b and on the other heat-shock puffs by immunofluorescence with anti-RNA polymerase II antibodies.

Heat-shock induction and cytoplasmic localization of transcripts from telomeric-associated sequences in Chironomus thummi.

Transcription of telomeric-associated sequences has been detected in the salivary gland cells of the larvae Chironomus thummi. In this species, a heat shock induces puffing at some telomeres, especially at one of the telomeres of chromosome III. We found that this process was concomitant with an increase in the overall telomeric transcript levels. Transcription was also observed in all the telomeres under control conditions, by in situ hybridization, even when these telomeres appeared to be in a nonpuffed state. The telomeric transcripts were found in both, the nuclei and, at higher levels, in the cytoplasmic extracts of salivary gland cells. The heat-shock activation, however, appeared to be restricted to the nuclear level. Telomeric transcription and the peculiar behavior of C. thummi telomeres after a heat shock are discussed.

Retinoid-dependent in vitro transcription mediated by the RXR/RAR heterodimer.

The effects of retinoids on gene regulation are mediated by retinoic acid receptors (RARs) and retinoid X receptors (RXRs). Here, we provide the first biochemical evidence that, in vitro, ligand governs the transcriptional activity of RXR alpha/RAR alpha by inducing conformational changes in the ligand-binding domains. Using limited proteolytic digestion we show that binding of the cognate ligand causes a conformational change in the carboxy-terminal part of the receptor. We also show that recombinant RXR alpha/RAR alpha is partially active in the absence of exogenously added ligand. Trans-activation depends critically on the ligand-dependent transcriptional activation function AF-2 of RAR alpha. Full activation by recombinant RXR alpha/RAR alpha, however, requires the addition of either all-trans RA, 9-cis RA, or other RAR-specific agonists, whereas an RAR alpha-specific antagonist abolishes trans-activation. Intriguingly, the ligand-dependent AF-2 of RXR does not contribute to the level of transcription from the RAR beta 2 promoter in vitro even when the cognate ligand (9-cis RA) is bound. Thus, the major role of RXR in trans-activation of the RAR beta 2 promoter is to serve as an auxiliary factor required for the binding of RAR which, in turn, is directly responsible for transcriptional activity.

Transcriptional repression of neurotrophin receptor trkB by thyroid hormone in the developing rat brain.

Expression of the neurotrophin receptor trkB is regulated by thyroid hormone (T3) during development of the rat brain. trkB mRNA levels, coding for the full-length and the truncated isoforms, are increased in the cerebral cortex of neonatal experimental hypothyroid animals. Run-on transcription assays with nuclei from postnatal day 15, hypothyroid, and control cerebral cortices demonstrated that an increase in the transcription rate of the trkB gene accounts for the observed effect. Transient transfection experiments using a reporter plasmid containing a 7-kilobase pair DNA fragment upstream of the mouse trkB gene showed that unliganded thyroid hormone receptor (T3R) increases promoter activity, whereas addition of T3 reverses that activity below basal levels. Deletion analysis shows that the T3-dependent repression requires binding of the T3R to a specific region located downstream of the transcription start site. This region, at nucleotide position -465/-432, contains an array of thyroid hormone response half-sites that bind preferentially T3R as heterodimers with retinoid X receptor and whose deletion causes loss of the T3-dependent repression. These half-sites are able to confer negative regulation by T3 to a heterologous promoter, thus indicating the functionality of these sequences. These results demonstrate that, in the developing rat brain, T3 down-regulates the expression of the trkB gene through the active repression of a novel negative response element located downstream of its transcription initiation site.

Alcohol exposure alters the expression pattern of neural cell adhesion molecules during brain development.

Neural cell adhesion molecules (NCAMs) play critical roles during development of the nervous system. The aim of this study is to investigate the possible effect of ethanol exposure on the pattern of expression and sialylation of NCAM isoforms during postnatal rat brain development because alterations in NCAM content and distribution have been associated with defects in cell migration, synapse formation, and memory consolidation, and deficits in these processes have been observed after in utero alcohol exposure. The expression of NCAM isoforms in the developing cerebral cortex of pups from control and alcohol-fed mothers was assessed by western blotting, ribonuclease protection assay, and immunocytochemistry. The highly sialylated form of NCAM [polysialic acid (PSA)-NCAM] is mainly expressed during the neonatal period and then is down-regulated in parallel with the appearance of NCAM 180 and NCAM 140. Ethanol exposure increases PSA-NCAM levels during the neonatal period, delays the loss of PSA-NCAM, decreases the amount of NCAM 180 and NCAM 140 isoforms, and reduces sialyltransferase activity during postnatal brain development. Neuraminidase treatment of ethanol-exposed neonatal brains leads to more intense band degradation products, suggesting a higher content of NCAM polypeptides carrying PSA in these samples. However, NCAM mRNA levels are not changed by ethanol. Immunocytochemical analysis demonstrates that ethanol triggers an increase in PSA-NCAM immunolabeling in the cytoplasm of astroglial cells, accompanied by a decrease in immunogold particles over the plasma membrane. These findings indicate that ethanol exposure during brain development alters the pattern of NCAM expression and suggest that modification of NCAM could affect neuronal-glial interactions that might contribute to the brain defects observed after in utero alcohol exposure.

Cooperativity in transactivation between retinoic acid receptor and TFIID requires an activity analogous to E1A.

In embryonal carcinoma (EC) cells retinoic acid (RA) strongly induces transcription from the RA receptor beta 2 (RAR beta 2) promoter through an RA response element (RARE) located in close proximity to the TATA box. Here we demonstrate that recombinant human TATA box-binding protein, hTFIID, and RAR functionally cooperate in transactivation of the RAR beta 2 promoter in EC cells in a strictly RA-dependent manner. We demonstrate that the core domain of hTFIID is sufficient to mediate RAR-dependent transcription and that Drosophila, but not yeast, TFIID can substitute for hTFIID. In COS cells ectopic expression of the E1A protein is a prerequisite for hTFIID and RAR to cooperate in transactivation. We propose a model for transcriptional regulation of the RAR beta 2 promoter in EC cells in which RAR, following activation by RA, functionally interacts with hTFIID via an E1A-like activity present in EC cells.