Biomedical subjects

G Cibelli

Publications and source records attributed to G Cibelli.

14 recordsLinked to original sources

PubMed2001

Regulation and composition of activator protein 1 (AP-1) transcription factors controlling collagenase and c-Jun promoter activities.

The activator protein 1 (AP-1) transcription factor is composed of heterodimers of the Fos/activating transcription factor (ATF) and Jun subfamilies of basic-region leucine-zipper (B-ZIP) proteins. In order to determine the identities of individual B-ZIP proteins in various AP-1 complexes we tested the effect of dominant-negative mutants to the B-ZIP proteins c-Fos, ATF2, ATF4 and CCAAT-enhancer-binding protein (C/EBP) on the activities of the collagenase and c-Jun promoters. These dominant-negative mutants inhibit DNA binding of wild-type B-ZIP proteins in a leucine-zipper-dependent fashion. Transcription of a collagenase promoter/reporter gene was induced in HepG2 hepatoma cells by expression of c-Fos and c-Jun, administration of PMA ("TPA") or by expression of a truncated form of MEK (mitogen-activated/extracellular-signal-regulated kinase kinase) kinase-1, MEKK1Delta. In all cases, the dominant-negative mutants A-Fos and A-ATF2 decreased collagenase promoter activity. However, A-ATF4 and A-C/EBP had no effect. A-Fos and A-ATF2 also reduced MEKK1Delta-induced stimulation of the c-Jun promoter. In contrast, constitutive c-Jun promoter activity was blocked solely by A-ATF2, strongly suggesting that ATF2 and/or an ATF2-dimerizing protein are of major importance for c-Jun transcription in unstimulated cells. These results demonstrate that AP-1 transcription factors of different compositions control c-jun gene transcription in resting or stimulated cells.

Amino Acid Sequence↗

PubMed2001

Modular structure of cAMP response element binding protein 2 (CREB2).

The transcription factor cAMP-response element binding protein 2 (CREB2), a member of the family of basic region leucine zipper proteins, has been suggested to function in the brain as a repressor of long-term memory. Using recombinant proteins we show that CREB2 binds in vitro to the palindromic cAMP response element derived from the secretogranin II gene. Recent studies of the chromogranin B, secretogranin II and enkephalin genes showed that CREB2 functioned as a repressor of cAMP-induced transcription. We analyzed the ability of CREB2 to repress transcription using model promoters. A molecular dissection of the CREB2 molecule revealed that CREB2 lacks a transferable repressor domain suggesting that CREB2 may function solely as a "passive" transcriptional repressor. In contrast, "active" repressor domains derived from the thyroid hormone receptor alpha or the NK10 zinc finger protein containing a "Krüppel associated box" could be transfered to a heterologous DNA-binding domain and functioned as fusion proteins in repressing transcription of a reporter gene. In addition, a strong activation domain located at the N-terminus was identified in the CREB2 protein suggesting that CREB2 may act as an activator of transcription by binding to different genetic regulatory elements.

Activating Transcription Factor 4↗

PubMed2001

Corticotropin-releasing factor triggers neurite outgrowth of a catecholaminergic immortalized neuron via cAMP and MAP kinase signalling pathways.

Corticotropin-releasing factor (CRF), a neuropeptide of 41 amino acids, acts as the major physiological regulator of the basal and stress-induced release of corticotropin (ACTH), beta-endorphin and other proopiomelanocortin-derived peptides from the anterior pituitary gland. In addition to its endocrine activity, CRF displays extrahypophysiotropic effects, mainly as a regulator of stress responses. We show here that CRF may additionally function as a differentiating factor in immortalized noradrenergic neuronal CATH.a cells that express CRF receptor type I and resemble locus coeruleus-derived neurons. CRF triggers morphological changes in CATH.a cells including the appearance of extended long, slender neurites with prominent growth cones. CRF-treated CATH.a cells exhibit a morphology similar to locus coeruleus neurons in primary culture. CRF-induced neurite outgrowth of CATH.a cells was blocked by addition of inhibitors for cAMP-dependent protein kinase or extracellular signal-regulated protein kinase (ERK), a subtype of the mitogen-activated protein kinases. The participation of ERK within the CRF signalling cascade was further confirmed by Western blot experiments, with antibodies directed against the phosphorylated form of ERK, and also with transcription-based assays. We conclude that CRF functions as a differentiating factor of CATH.a cells via the cAMP and the MAP kinase signalling pathways.

Animals↗

PubMed2001

Biological activity of mammalian transcriptional repressors.

Research on the regulation of transcription in mammals has focused in recent years mainly on the mechanism of transcriptional activation. However, transcriptional repression mediated by repressor proteins is a common regulatory mechanism in mammals and might play an important role in many biological processes. To understand the molecular mechanism of transcriptional repression, the activity of eight mammalian repressors or repressor domains was investigated using a set of model promoters in combination with two different transcriptional detection methods. The repressors studied were: REST, the thyroid hormone receptors alpha and beta, the zinc finger protein NK10 containing a 'krüppel-associated box' (KRAB), repressor domains derived from the proteins Egr-1, Oct2A and Dr1 and the repressor/activator protein YY1. Here we show that the repressor domains of REST, Egr-1, the thyroid hormone receptors alpha< and beta and NK10 were transferable to a heterologous DNA-binding domain and repressed transcription from proximal and distal positions. Moreover, these repressor domains also blocked the activity of a strong viral enhancer in a 'remote position'. Thus, these domains are 'general' transcriptional repressor domains. The 'krüppel-associated box' was the most powerful repressor domain tested. In contrast, the repressor domains derived from Oct2A and Dr1 were inactive when fused to a heterologous DNA-binding domain. The repressor domain of YY1 exhibited transcriptional repression activity only in one of the transcriptional assay systems. The recruitment of histone deacetylases to the proximity of the basal transcriptional apparatus was recently discussed as a mechanism for some mammalian transcriptional repressor proteins. Here we show here that histone deacetylase 2, targeted to the reporter gene via DNA-protein interaction, functions as a transcriptional repressor protein regardless of the location of its binding site within the transcription unit.

Animals↗

PubMed1999

Nuclear targeting of cAMP response element binding protein 2 (CREB2).

The transcription factor cAMP response element binding protein 2 (CREB2) belongs to a family of proteins containing a basic region as DNA-binding domain and a leucine zipper as a dimerization domain in its C-terminus. Using indirect immunofluorescence labeling of cells we show that CREB2 is a nuclear protein. To identify the signal(s) required for nuclear targeting of CREB2, various domains of the protein were expressed in COS cells as fusion proteins with glutathione S-transferase and their cellular location assayed by indirect immunofluorescence using antibodies directed against the glutathione S-transferase moiety of the fusion proteins. The results show that the nuclear targeting signal is located in the C-terminal part of the molecule. Deletion mutagenesis revealed that the basic region of CREB2, encompassing amino acids 280 to 300, is sufficient for sorting CREB2 to the nucleus. Single point mutations of basic amino acids within the basic region of CREB2 identified the sequence KKLKK (amino acids 280 to 284) as important for nuclear targeting. Thus, the basic region of CREB2 is necessary not only for tethering CREB2 to DNA but also for sorting CREB2 to the nucleus. However, sequences outside the basic region are additionally required for efficient nuclear sorting of CREB2.

Activating Transcription Factor 4↗

PubMed1999

Corticotropin-releasing factor and vasoactive intestinal polypeptide activate gene transcription through the cAMP signaling pathway in a catecholaminergic immortalized neuron.

Corticotropin-releasing factor (CRF) and vasoactive intestinal polypeptide (VIP) are neuropeptides displaying a variety of short-term effects in the nervous system. It is shown here in transfection experiments of an immortalized noradrenergic locus coeruleus-like cell line that both CRF and VIP also trigger a signaling cascade capable of activating gene transcription. To elucidate the signaling pathway leading to transcriptional induction, cells were transfected with an inhibitor for cAMP-dependent protein kinase, targeted to the nucleus via a nuclear-localization signal. Transcriptional induction of a reporter gene by CRF and VIP was blocked in these cells, indicating that the cAMP-dependent protein kinase is required for transducing CRF and VIP generated signals into the nucleus. Additionally, transfection experiments with a reporter gene containing cAMP response elements in its regulatory region demonstrate that CRF and VIP receptor activation induce transcription through this genetic regulatory element. We conclude that long-term effects of CRF and VIP in neurons are likely to be mediated by the transcriptional regulation of CRF and VIP-responsive genes via the cAMP signaling pathway.

Animals↗

PubMed1996

A (G+C)-rich motif in the aldolase C promoter functions as a constitutive transcriptional enhancer element.

The enzyme fructose-1,6-bisphosphate aldolase consists of three isozymes that are expressed in a tissue-specific manner. Using antibodies against aldolase B and C, it is shown that aldolase C is expressed in virtually all neuronal cell lines derived from the central and peripheral nervous system. Recently, experiments with transgenic mice indicated that a (G+C)-rich region of the aldolase C promoter might function as a neuron-specific control element of the rat aldolase C gene [Thomas, M., Makeh, I., Briand, P., Kahn, A. & Skala, H. (1993) Eur. J. Biochem. 218, 143-151). To functionally analyse this element, a plasmid consisting of four copies of this (G+C)-rich sequence, a TATA box, and the rabbit beta-globin gene as reporter was constructed. This plasmid was transfected into neuronal and nonneuronal cell lines and transcription was monitored by RNase protection mapping of the beta-globin mRNA. It is shown that the (G+C)-rich element of the aldolase C promoter directs transcription in neuronal as well as in nonneuronal cells. In contrast, the synapsin I promoter, used as a control for neuron-specific gene expression, directed transcription only in neuronal cells. In gel-retardation assays, two major DNA-protein complexes were detected with the (G+C)-rich element of the aldolase C promoter used as a DNA probe and nuclear extracts from brain and liver as a source for DNA-binding proteins. These DNA-proteins interactions could be impaired by a DNA probe that contained an Sp1-binding site, indicating that Sp1 or an Sp1-related factor binds to the aldolase C promoter (G+C)-rich element. This was confirmed by supershift analysis with antibodies specific for Sp1. The zinc finger transcription factor zif268/egr-1, also known to recognize a (G+C)-rich consensus site, did not, however, bind to the (G+C)-rich motif of the aldolase C promoter, nor could it stimulate transcription in transactivation assays from this control region. From these data, we conclude that the (G+C)-rich element of the aldolase C promoter functions as a constitutive transcriptional response element mediated by Sp1 and Sp1-related transcription factors.

Animals↗

PubMed1996

Identification of a functional cAMP response element in the secretogranin II gene.

Secretogranin II is an acidic secretory protein with a widespread distribution in secretory granules of neuronal and endocrine cells. The secretogranin II gene contains, like other members of the granin family, a cAMP response element (CRE) in its upstream region. To investigate the functional significance of this motif, intracellular cAMP levels were increased in a neuronal cell line derived from the septal region of the brain and the level of secretogranin II gene expression was analysed. It was found that increased cAMP levels did, in fact, induce secretogranin II gene expression. To analyse the cis-acting sequence responsible for this induction, a hybrid gene containing the upstream region of the mouse secretogranin II gene fused to beta-globin as a reporter was constructed. Transfection analysis revealed that cAMP-induced transcription of the secretogranin II promoter/beta-globin gene in septal and insulinoma cells. DNA-protein binding assays showed that recombinant CRE-binding protein (CREB), produced in bacteria or human cells, bound in a sequence-specific manner to the secretogranin II promoter CRE. Moreover, deletion mutagenesis revealed that the CRE motif is a bifunctional genetic regulatory element in that it mediates basal as well as cAMP-stimulated transcription. Interestingly, cAMP had no effect upon secretogranin II gene transcription in PC12 and neuroblastoma cells. An increase in the intracellular cAMP concentration activated a GAL4-CREB fusion protein upon transcription in neuroblastoma cells indicating the integrity of the cAMP signaling pathway to the nucleus. Basal as well as cAMP-stimulated transcription, directed from the secretogranin II promoter was, however, impaired in insulinoma cells by overexpression of CREB-2, a negative-acting CRE-binding protein. These results indicate that competitive effects are likely to occur between CRE-bound transcriptional activators and repressors. We conclude that cAMP-stimulated induction of secretogranin II gene transcription is mediated by the CRE motif in a cell-type-specific manner, and is likely to depend on the balance between positive and negative CRE-binding proteins in a particular cell type.

1-Methyl-3-isobutylxanthine↗

PubMed1996

Neuron-specific gene expression of synapsin I. Major role of a negative regulatory mechanism.

The synapsins are a family of neuron-specific phosphoproteins that selectively bind to small synaptic vesicles in the presynaptic nerve terminal. The human synapsin I gene was functionally analyzed to identify control elements directing the neuron-specific expression of synapsin I. By directly measuring the mRNA transcripts of a reporter gene, we demonstrate that the proximal region of the synapsin I promoter is sufficient for directing neuron-specific gene expression. This proximal region is highly conserved between mouse and human. Deletion of a putative binding site for the zinc finger protein, neuron-restrictive silencer factor/RE-1 silencing transcription factor (NRSF/REST), abolished neuron-specific expression of the reporter gene almost entirely, allowing constitutively acting elements of the promoter to direct expression in a non-tissue-specific manner. These constitutive transcriptional elements are present as a bipartite enhancer, consisting of the region upstream (nucleotides -422 to -235) and downstream (nucleotides -199 to -143) of the putative NRSF/REST-binding site. The latter contains a motif identical to the cAMP response element. Both regions are not active or are only weakly active in promoting transcription on their own and show no tissue-specific preference. From these data we conclude that neuron-specific expression of synapsin I is accomplished by a negative regulatory mechanism via the NRSF/REST binding motif.

Animals↗

PubMed1996

Synapsin-like molecules in Aplysia punctata and Helix pomatia: identification and distribution in the nervous system and during the formation of synaptic contacts in vitro.

The distribution and biochemical features of the synapsin-like peptides recognized in Aplysia and Helix by various antibodies directed against mammalian synapsins were studied. The peptides can be extracted at low pH and are digested by collagenase; further, they can be phosphorylated by both protein kinase A and Ca2+/calmodulin-dependent protein kinase II. In the ganglia of both snails, they are associated with the soma of most neurons and with the neuropil; punctate immunostaining is present along the neurites. Using cocultures of a Helix serotoninergic neuron and of its target cell, we analysed the redistribution of the synapsin-like peptides during the formation of active synaptic contacts. When the presynaptic neuron is plated in isolation, both synapsin and serotonin immunoreactivities are restricted to the distal axonal segments and to the growth cones; in the presence of the target, the formation of a chemical connection is accompanied by redistribution of the synapsin and serotonin immunoreactivities that concentrate in highly fluorescent round spots scattered along the newly grown neurites located close to the target cell. Almost every spot that is stained for serotonin is also positive for synapsin. In the presynaptic cell plated alone, the number of these varicosity-like structures is substantially stable throughout the whole period; by contrast, when the presynaptic cell synapses the target, their number increases progressively parallel to the increase in the mean amplitude of cumulative excitatory postsynaptic potentials recorded at the same times. The data indicate that mollusc synapsin-like peptides to some extent resemble their mammalian homologues, although they are not exclusively localized in nerve terminals and their expression strongly correlates with the formation of active synaptic contacts.

Animals↗

PubMed1994

Differential regulation of chromogranin B and synapsin I gene promoter activity by cAMP and cAMP-dependent protein kinase.

cAMP has neutrotrophic effects in the nervous system. We have investigated whether there is a correlation between cAMP-induced neurite outgrowth and induction of chromogranin B and synapsin I gene expression. These genes encode marker proteins of distinct populations of vesicles in neurons, neuroendocrine and endocrine cells, and in addition, they contain a cAMP response element (CRE) in their upstream regions, making it likely that cAMP-induced neuronal differentiation might be accompanied by increased transcription of these genes. We increased intracellular cAMP levels in neuronal and neuroendocrine cells and analyzed the levels of chromogranin B and synapsin I mRNA. Our data revealed that, while chromogranin B mRNA was in fact induced following cAMP stimulation, synapsin I mRNA was not affected. To analyze the cis-acting sequences, we constructed hybrid genes containing the upstream region of the mouse chromogranin B gene fused to a reporter gene. Similar plasmids containing the synapsin I or the glucagon promoter were constructed. Transfections of neuronal and endocrine cells, together with deletion mutagenesis, revealed that the CRE of the chromogranin B gene mediated the effect of cAMP upon transcription. This effect was mimicked by overexpression of the catalytic subunit of the cAMP-dependent protein kinase. In addition, overexpression of the negative-acting CRE-binding protein CREB-2 revealed that the chromogranin B CRE functions as a bifunctional genetic regulatory element in that it mediates basal as well as cAMP-stimulated transcription. Synapsin I gene expression, however, was not induced by either elevated intracellular cAMP concentration or by overexpression of protein kinase A, although a similar pattern of proteins, including CREB, bound to the synapsin I and chromogranin B CRE in vitro. Thus while the CRE element in the chromogranin B gene promoter is responsive to cAMP, the same element, when present in the synapsin I promoter, does not confer cAMP inducibility.

1-Methyl-3-isobutylxanthine↗

PubMed1989

The mouse neuronal cell surface protein F3: a phosphatidylinositol-anchored member of the immunoglobulin superfamily related to chicken contactin.

Several members of the Ig superfamily are expressed on neural cells where they participate in surface interactions between cell bodies and processes. Their Ig domains are more closely related to each other than to Ig variable and constant domains and have been grouped into the C2 set. Here, we report the cloning and characterization of another member of this group, the mouse neuronal cell surface antigen F3. The F3 cDNA sequence contains an open reading frame that could encode a 1,020-amino acid protein consisting of a signal sequence, six Ig-like domains of the C2 type, a long premembrane region containing two segments that exhibit sequence similarity to fibronectin type III repeats and a moderately hydrophobic COOH-terminal sequence. The protein does not contain a typical transmembrane segment but appears to be attached to the membrane by a phosphatidylinositol anchor. Antibodies against the F3 protein recognize a prominent 135-kD protein in mouse brain. In fetal brain cultures, they stain the neuronal cell surface and, in cultures maintained in chemically defined medium, most prominently neurites and neurite bundles. The mouse f3 gene maps to band F of chromosome 15. The gene transcripts detected in the brain by F3 cDNA probes are developmentally regulated, the highest amounts being expressed between 1 and 2 wk after birth. The F3 nucleotide and deduced amino acid sequence show striking similarity to the recently published sequence of the chicken neuronal cell surface protein contactin. However, there are important differences between the two molecules. In contrast to F3, contactin has a transmembrane and a cytoplasmic domain. Whereas contactin is insoluble in nonionic detergent and is tightly associated with the cytoskeleton, about equal amounts of F3 distribute between buffer-soluble, nonionic detergent-soluble, and detergent-insoluble fractions. Among other neural cell surface proteins, F3 most resembles the neuronal cell adhesion protein L1, with 25% amino acid identity between their extracellular domains. Based on its structural similarity with known cell adhesion proteins of nervous tissue and with L1 in particular, we propose that F3 mediates cell surface interactions during nervous system development.

Amino Acid Sequence↗

PubMed1984

Hypokalemic myopathy associated with liquorice ingestion.

A case of hypokalemic myopathy with systolic hypertension due to chronic liquorice ingestion is described. The clinical features quickly receded on potassium replacement therapy and discontinuation of liquorice ingestion. The relationship between the clinical features and the blood values and instrumental data is shown. The aldosterone-like action of the active principle of liquorice is underlined.

Foodborne Diseases↗

PubMed1984

Heterogeneous models for blood-cerebrospinal fluid barrier permeability to serum proteins in normal and abnormal cerebrospinal fluid/serum protein concentration gradients.

Cerebrospinal fluid (CSF)/serum concentration gradients (Q) of individual proteins (albumin, IgG, alpha 2-macroglobulin) have been studied in controls and in patients in whom the lumbar CSF flow is altered (medullary compression) or the blood-CSF barrier (BCB) function impaired (acute idiopathic polyneuropathy and acute meningoencephalitis). The analysis of relationships among protein Q has been performed by total and multiple regressions and the actual BCB permeability to individual proteins has been interpreted according to the accepted theoretical porous or vesicular BCB models. The exponential Q-IgG vs. Q-albumin total regression, and the poor Q-alpha 2-macroglobulin vs. Q-albumin regression found in controls, together with the different multiple regressions among proteins and the high Q-IgG vs. Q-albumin partial regression coefficients found in medullary compression, acute idiopathic polyneuropathy and acute meningoencephalitis, indicated that different permeability mechanisms can be postulated. Heterogeneous, fairly independent permeability BCB mechanisms maintain the normal CSF/serum protein concentration gradient. Pinocytotic vesicles or pores of radius exceeding 1000-1500 A, probably located at the capillary endothelium, account for the main serum-derived CSF protein fraction(s) with large hydrodynamic radius (R). A more selective endothelial vesicular transport with a radius of 250 A transfers a negligible amount of protein from serum into CSF. Proteins with small R also enter the CSF through a set of selective pores of radius 120 A, probably at the level of the choroidal epithelium. Pinocytotic vesicles with a radius of 250 A and increased rate of formation induce the accumulation of proteins below an obstruction of lumbar CSF flow. An increased formation rate of vesicles with a radius of 450 A can explain the increased capillary permeability in nerve roots in acute idiopathic polyneuropathy. Loss of selectivity was the main feature of BCB in acute meningoencephalitis, and it seemed to be due to pores or vesicles with a radius larger than 1000-1500 A. The heterogeneity of BCB mechanisms must be taken into account when the intrathecal synthesis of a protein, also derived from serum (for example IgG), has to be measured.

Blood Proteins↗