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R Bravo

Publications and source records attributed to R Bravo.

At least 163 records · Page 9Linked to original sources
PubMed1993

Expression of immediate early gene proteins following axotomy and inhibition of axonal transport in the rat central nervous system.

The expression of the immediate early gene-encoded proteins c-Jun, Jun B, Jun D, c-Fos, Fos B and Krox-24 in central neurons following transection of, or inhibition of, axonal transport in their axons was investigated in the rat using immunocytochemistry. Transection of the medial forebrain bundle, which produces an essentially complete axotomy of neurons in the ipsilateral mammillary nucleus, substantia nigra pars compacta, ventral tegmental area and parafascicularis, induced the expression of c-Jun, Jun D and, to a lesser extent, Krox-24, in these nuclei. Microinjection of colchicine into the medial forebrain bundle to chemically inhibit axonal transport similarly induced the expression of these proteins in these areas. The expression of the proteins was first evident 24 h after transection, reached a maximum at 48 h and was still present after 10 days. However, after 30 days the proteins were absent from the substantia nigra, ventral tegmentum and parafascicularis, and were still present only in the mammillary nuclei. The other immediate early genes, Jun B, c-Fos and Fos B, were never expressed above the basal levels seen in untreated rats. Transection of the corpus callosum and the hippocampal commissure, which produces only a partial axotomy of neurons in the cerebral cortex and hippocampus, respectively, did not induce the expression of any of the genes in these neurons. Microinjection of colchicine or vinblastine to produce a localized inhibition of axonal transport in the cerebral cortex, hippocampus, thalamus and cerebellum also induced the expression of c-Jun, Jun D and, again to a lesser extent, Krox-24, in neurons surrounding the injection site. In contrast to this selective expression, administration of the neuronal excitant metrazole induced the expression of all six immediate early gene proteins in central nervous system neurons. These results demonstrate that transection of, or inhibition of, transport in the axons of central neurons induces a particular pattern of expression of transcriptionally operating immediate early genes that may be related to the regenerative competency of the neurons.

Animals↗
PubMed1993

Induction and suppression of immediate-early genes in the rat brain by a selective alpha-2-adrenoceptor agonist and antagonist following noxious peripheral stimulation.

The effect of medetomidine, a highly selective alpha-2-adrenoceptor agonist, on noxious stimulation-induced expression of immediate-early genes was studied in the central nervous system of the rat. The expressions of c-JUN, JUN B, c-FOS FOS B and KROX-24 proteins were investigated by immunocytochemistry following the application of formalin (5%, 50 microliters) into the plantar skin of one hindpaw. Medetomidine (100 or 300 micrograms/kg i.p.) was administered 12 min or 5 min before the application of formalin. Atipamezole (1.5 mg/kg i.p.), and alpha-2-adrenoceptor antagonist, administered simultaneously with medetomidine (300 micrograms/kg), was used to reverse the alpha-2-adrenergic effects. The rats were killed and perfused 90 min after formalin injection. Formalin induced expression of all studied proteins in the ipsilateral spinal dorsal horn and the contralateral parabrachial nucleus, and in the medial thalamus bilaterally. Both medetomidine doses administered 12 min before formalin strongly suppressed the expression of c-FOS in the spinal dorsal horn; the suppression was stronger in the deep (III-VI) than in the superficial (I and II) laminae of the dorsal horn (76% and 86% for 100 micrograms/kg dose vs 97% and 99% for 300 micrograms/kg dose, respectively). However, application of medetomidine 5 min before formalin did not reduce the expression of immediate-early genes. In the parabrachial nucleus, both medetomidine doses also produced a significant suppression of c-FOS expression (68%). In contrast, medetomidine at the dose of 100 micrograms/kg was ineffective in the medical thalamus. Only the higher dose of medetomidine (300 micrograms/kg) produced a suppression by 29% and 46% in centromedian and paraventricular nuclei, respectively. Atipamezole produced a significant attenuation in spinal cord and a complete reversal in parabrachial nucleus of the medetomidine-induced suppression. However, in the medial thalamus, atipamezole produced a dramatic increase of formalin-induced c-FOS expression when compared with formalin injection alone. The expression of c-JUN, JUN B, FOS B and KROX-24 proteins paralleled that of c-FOS. It is concluded that the expression of immediate-early gene encoded proteins is more strongly suppressed by alpha-2-adrenoceptor agonists in spinal and parabrachial than in medial thalamic neurons. The increased expression of immediate-early genes in medical thalamus following atipamezole treatment may be explained by increased release of noradrenaline and the consequent activation of alpha-1- and beta-adrenoceptors. Compared with the previously reported effects of behaviorally equipotent doses of morphine, the suppression of c-FOS expression in the spinal cord was stronger following medetomidine than that following morphine.(ABSTRACT TRUNCATED AT 400 WORDS)

Adrenergic alpha-Agonists↗
PubMed1993

Induction and suppression of immediate early genes in specific rat brain regions by the non-competitive N-methyl-D-aspartate receptor antagonist MK-801.

The expression pattern of six different immediate early gene-encoded proteins was examined in the rat forebrain after intraperitoneal administration of MK-801, a non-competitive N-methyl-D-aspartate receptor antagonist, at doses of 3 mg/kg and 0.3 mg/kg, respectively. Following MK-801 treatment, the presence of c-FOS, FOS B, KROX-24, c-JUN, JUN B, and JUN D were investigated by immunocytochemistry with specific antisera at different time intervals up to 48 h. Selective induction of all six immediate early genes was found in layer III neurons of the posterior cingulate and retrosplenial cortex. More complex effects were observed in the neocortex: MK-801 did not influence constitutive expression of different FOS and JUN proteins, but caused marked induction of c-FOS, FOS B, JUN B and JUN D, mainly in layer IV, but also in layers V and VI. In contrast, strong neocortical constitutive expression of KROX-24 was almost abolished by MK-801 administration, and replaced by an expression pattern similar to that of FOS and JUN proteins. Subcortical areas such as the hypothalamus and thalamus demonstrated an induction of a subset of immediate early genes (c-fos, fos B, Krox-24, jun B). Injection of MK-801 caused the same distributional pattern of immediate early gene expression irrespective of the dose given, but the extent of changes was stronger after 3 mg/kg, and altered levels of immunoreactivity persisted longer. In many experimental paradigms, immediate early genes are induced by N-methyl-D-aspartate receptor-mediated mechanisms. This induction can readily be blocked by N-methyl-D-aspartate receptor antagonists like MK-801. Our data, however, indicate that MK-801 itself causes immediate early gene expression in specific neuronal populations. In the present study MK-801-elicited expression of immediate early gene-encoded proteins seems to identify reversibly injured neurons, mainly in layer III of the posterior cingulate and retrosplenial cortex. These neurons have previously been shown to be the principal target of N-methyl-D-aspartate receptor antagonist toxicity. Since immediate early gene induction precedes heat-shock protein expression as well as pathomorphological changes, and is induced in additional cortical cell populations, it seems to be a more rapid and more sensitive indicator of non-lethal neuronal injury.

Animals↗
PubMed1993

Differential transcription and translation of immediate early genes in the gerbil hippocampus after transient global ischemia.

Excitotoxic activation of glutamate receptors is thought to be a key event for the molecular pathogenesis of postischemic delayed neuronal death of CA-1 neurons in the gerbil hippocampus. Glutamate receptor stimulation also causes induction of transcription factors that belong to the class of immediate early genes. We examined the expression of six different immediate early genes in the gerbil hippocampus after transient global ischemia. Comparative analysis of c-fos and Krox-24 expression was carried out in the same animals at the transcriptional and translational level by in situ hybridization and immunocytochemistry. Postischemic synthesis of four additional immediate early gene (IEG)-encoded proteins (FOS-B, c-JUN, JUN-B, and JUN-D) was investigated by immunocytochemistry at recirculation intervals between 1 and 48 h. After 5 min of ischemia, transcription of c-fos and Krox-24 mRNA was induced in all hippocampal subpopulations with peak expression at 1 h after recirculation. In vulnerable CA-1 neurons, increased transcription of c-fos and Krox-24 was not followed by translation into protein. Induction of immediate early gene-encoded proteins was restricted to neuronal populations less vulnerable to brief ischemia and identified neurons that are targets of glutamate receptor-mediated neurotoxicity but that are destined to survive. Our data indicate an asynchronous synthesis and persistence of individual IEG-encoded proteins in these neurons. The staggered induction implies that combinatorial changes of transcription factors allow a differential postischemic regulation of target gene expression both spatially and over time.

Animals↗
PubMed1993

Expression of PILOT, a putative transcription factor, requires two signals and is cyclosporin A sensitive in T cells.

Few known genes (IL-2, members of the IL-8 family, interferon-gamma) are induced in T cells only through the combined effect of phorbol myristic acetate (PMA) and a Ca(2+)-ionophore, and expression of only these genes can be fully suppressed by Cyclosporin A (CyA). We have identified a putative transcription factor, designated PILOT, with an identical dual signal requirement for expression. Induction of the PILOT gene is detectable in human T cells 20 min following activation in the presence of cycloheximide and is fully suppressed by CyA. The PILOT protein has a calculated M(r) of 42.6 kDa and contains three zinc fingers of the C2H2-type at the carboxyl-terminus which are highly homologous to the zinc finger regions of the transcription factors EGR1, EGR2, and pAT 133. In contrast to T cells, in fibroblasts PILOT gene expression requires only one signal (PMA) and is not affected by CyA. This observation directly demonstrates the existence of a Ca2+ signal-dependent regulatory element obligatory for expression of some genes in T cells but not in fibroblasts. This differential expression model will be valuable in the dissection of the dual signal pathway in T cells and the effects of CyA upon it.

Amino Acid Sequence↗
PubMed1993

Both N- and C-terminal domains of RelB are required for full transactivation: role of the N-terminal leucine zipper-like motif.

RelB, a member of the Rel family of transcription factors, can stimulate promoter activity in the presence of p50-NF-kappa B or p50B/p49-NF-kappa B in mammalian cells. Transcriptional activation analysis reveals that the N and C termini of RelB are required for full transactivation in the presence of p50-NF-kappa B. RelB/p50-NF-kappa B hybrid molecules containing the Rel homology domain of p50-NF-kappa B and the N and C termini of RelB have high transcriptional activity compared with wild-type p50-NF-kappa B. The N and C termini of RelB cooperate in transactivation in cis or trans configuration. Alterations in the structure of the leucine zipper-like motif present in the N terminus of RelB significantly decrease the transcriptional capacity of RelB and of different RelB/p50-NF-kappa B hybrid molecules.

Amino Acid Sequence↗
PubMed1993

The NFAT-1 DNA binding complex in activated T cells contains Fra-1 and JunB.

Activation of T cells induces transcription of the interleukin-2 (IL-2) gene. IL-2 expression is regulated through the binding of transcription factors to multiple sites within the IL-2 enhancer. One such cis-acting element within the IL-2 enhancer is the NFAT-1 (nuclear factor of activated T cells) binding site. NFAT-1 binding activity is absent in resting cells but is induced upon T-cell activation. The induction of NFAT-1 binding activity can be inhibited by cyclosporin A, potentially accounting for the ability of cyclosporin A to inhibit IL-2 production by T cells. We have previously reported that the NFAT-1 binding complex is composed of at least two proteins and that the 5' portion of the NFAT-1 sequence acts as a binding site for one or more proteins from the Ets family of transcription factors. We now report that the 3' portion of the NFAT-1 sequence contains a variant AP-1 binding site. NFAT-1 binding can be specifically inhibited by oligonucleotides containing a consensus AP-1 site. Moreover, mutation of the AP-1 site at the 3' end of the NFAT-1 sequence inhibits both NFAT-1 binding and the ability of the NFAT-1 binding site to activate expression from a reporter plasmid upon T-cell activation. Since AP-1 sites bind dimeric protein complexes composed of individual members of the Fos and Jun families of transcription factors, we used antibodies specific for individual Fos and Jun family members to determine whether they are present in the NFAT-1 binding complex. These experiments demonstrated that the NFAT-1 binding complex contains JunB and Fra-1 proteins. Northern (RNA) blot analyses demonstrate that both fra-1 and junB mRNAs are induced upon T-cell activation, although fra-1 mRNA is present even in quiescent T cells. Of interest, junB is not expressed in quiescent T cells, and it is induced with kinetics that are similar to those for the induction of IL-2 mRNA expression. Taken together, these results suggested that the JunB-Fra-1 heterodimer is the inducible nuclear component of the NFAT-1 binding activity and that JunB expression regulates the formation of the heterodimer. In addition, these data indicated that specific heterodimers of Fos and Jun family members may have selective roles in the induction of transcription during cellular activation.

Enhancer Elements, Genetic↗
PubMed1993

The product of a novel growth factor-activated gene, fic, is a biologically active "C-C"-type cytokine.

We have characterized a new member of the superfamily of proinflammatory peptides encoded by a growth factor-inducible gene, fic, previously isolated by differential screening of a cDNA library of mRNA from serum-stimulated NIH 3T3 cells. Immunoprecipitation analyses showed that the protein was rapidly induced following serum stimulation and secreted unglycosylated into the medium. The fic protein, FIC, shows highest sequence homology (57%) to human and rabbit monocyte chemoattractant protein 1 (MCP-1), an established monocyte activator. To determine the biological activity of FIC and to compare it with that of mouse MCP-1 (muMCP-1), both proteins were expressed in the baculovirus system. FIC and muMCP-1 were purified to near homogeneity by a two-step chromatography protocol. Both proteins elicited changes in intracellular calcium concentration in human monocytes. The effect was dependent on external Ca2+ and was inhibited by pretreatment of cells with pertussis toxin. FIC did not desensitize human monocytes to the three related cytokines muMCP-1, human MCP-1 (huMCP-1), and huMCP-2. However, pretreatment with muMCP-1 or huMCP-1, but not with huMCP-2, desensitized human monocytes to FIC. Specific binding of [125I]FIC was found in human monocytes, mouse monocytic cultured cells, and human endothelial cells but not in lymphocytes, neutrophils, or primary mouse fibroblasts. Scatchard analysis of the binding of [125I]FIC to human monocytes showed the presence of two classes of receptors, with apparent KdS of 1.2 and 7.7 nM and receptor numbers per cell of 2,400 and 6,300, respectively. FIC, muMCP-1, and huMCP-1 competed to the same extent for the binding of [125I]FIC to human monocytes, contrary to huMCP-2, which competed very ineffectively, if at all.

Amino Acid Sequence↗
PubMed1993

Structure, mapping, and expression of erp, a growth factor-inducible gene encoding a nontransmembrane protein tyrosine phosphatase, and effect of ERP on cell growth.

We have characterized a growth factor-inducible gene, erp, and demonstrated that it encodes a 367-amino-acid nontransmembrane tyrosine phosphatase protein with significant similarity to the vaccinia virus H1 protein. Immunoprecipitation analyses show that the erp protein, ERP, is rapidly induced following serum stimulation of quiescent fibroblasts. ERP has been expressed as a fusion protein with glutathione S-transferase and shown to have tyrosine as well as serine protein phosphatase activity. The enzymatic activity of ERP depends on the presence of reducing agents such as dithiothreitol, and its tyrosine phosphatase activity is inhibited by sodium vanadate, a potent inhibitor of protein tyrosine phosphatases. The number of stable NIH 3T3 clones obtained after transfection with a vector expressing the complete ERP protein is reduced more than 90% compared with that after transfection with a vector expressing a mutated inactive ERP protein. The remaining ERP-expressing clones present a significant increase in the proportion of bi- and multinucleated cells and a decrease in proliferation rate. Studies on the genomic structure reveal that the erp transcription unit is 2.8 kbp long and split into four exons. The erp gene maps to the 17A2-17C region of the murine genome. Our results demonstrate that the protein product of the immediate-early gene erp has a negative effect on cell proliferation.

3T3 Cells↗
PubMed1993

Expression of relB transcripts during lymphoid organ development: specific expression in dendritic antigen-presenting cells.

We have studied the expression of the relB gene during mouse development using in situ hybridization and immunocytochemical analysis. The results show that the expression of the relB gene is highly restricted to a subpopulation of cells that colonize the lymphoid tissues and that appear very late during the process of hematopoietic diversification. RNA transcripts of relB are very low or undetectable in early and late embryos. Low relB expression is observed in the thymus at late stages of embryogenesis but rapidly increases after birth. In adult lymphoid tissues, relB is detected in the medullary region of the thymus, the periarterial lymphatic sheaths of the spleen, and the deep cortex of the lymph nodes, which correspond to the regions where T cells of mature phenotype and interdigitating dendritic cells are present. Using double immunofluorescent labeling of thymic cell suspensions, we have identified the interdigitating dendritic cells as the target of RelB expression. These cells are part of a system of antigen-presenting cells that function in the induction of several immune responses, such as, tolerance, sensitization of MHC-restricted T cells, rejection of organ transplants and formation of T-dependent antibodies. Our observations indicate that RelB may play a particular role in the signal transduction pathway that regulate dendritic cell differentiation and its cellular responses.

Animals↗
PubMed1993

Long-lasting expression of JUN and KROX transcription factors and nitric oxide synthase in intrinsic neurons of the rat brain following axotomy.

In adult rats, the medial forebrain bundle (MFB) and mammillothalamic tract (MT) were unilaterally transected, resulting in axotomy of neurons in numerous areas such as the substantia nigra (SN), ventral tegmental area (VTA), nucleus (ncl.) mammillaris (MnM), and ncl. parafascicularis of the thalamus (PF). In these areas, expression of the transcription factor proteins c-JUN, JUN B, JUN D, c-FOS, FOS B, KROX-20, KROX-24, and CREB was investigated by immunocytochemistry up to 150 d. In parallel, the expression of nitric oxide synthase (NOS) was investigated both immunocytochemically and by the NADPH-diaphorase reaction (NDP), and the antibody against NOS was further characterized. The colocalization of c-JUN with NDP or NOS was also studied in the axotomized neurons. c-JUN and JUN D became visible in nuclei of many neurons of the ipsilateral MnM, PF, VTA, and SN (predominantly in the pars compacta and those double labeled by tyrosine hydroxylase, TH) after 36 hr, not after 24 hr, following transection of MFB and MT. In MnM, c-JUN and JUN D persisted at a nearly maximal level for up to 150 d. In PF, these proteins returned to control levels after 75 d. Expression of c-JUN and JUN D declined in the VTA after 30 d, but in the SN, it already declined after only 10 d. KROX-24 had a later onset of expression, being visible after 3 d in all investigated areas, and its pattern was similar to that of JUN proteins, although labeling was visible in fewer nuclei and declined earlier. JUN B, c-FOS, FOS B, and KROX-20 were not expressed in these areas, and substantial alterations of CREB immunoreactivity (CREB-IR) could not be detected. A subset of SN neurons (predominantly in the pars reticularis and negative for TH) presented an early and transient expression of all studied JUN, FOS, and KROX-24 proteins within 3 hr of transection that declined between 24 hr and 48 hr to basal levels. This expression pattern is typical of that caused by transynaptic stimulation (probably due to excitation of descending striatal neurons running within the MFB) and was clearly distinct from that evoked by c-JUN, JUN D, and KROX-24 IRs after 36 hr (predominantly in the pars compacta). An ipsilateral increase in NOS and NDP became visible in many neurons of the MnM after 10 d, but not after 5 d, and this persisted up to 150 d. The temporospatial pattern of NDP was similar to the pattern of NOS-IR.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Oxidoreductases↗
PubMed1993

Expression of the nontransmembrane tyrosine phosphatase gene erp during mouse organogenesis.

We have studied the expression of the nontransmembrane tyrosine phosphatase gene erp during mouse development using in situ hybridization analysis. The results show that during the early postimplantational stages of development, erp expression is observed only in maternally derived decidual cells surrounding the developing embryo. At day 10.5, erp is weakly expressed in the embryo in the neural tube, hind gut, and other embryonic structures. However, in 12.5-day embryos, erp is present in most organs, with the highest expression restricted to the developing neural system. During later development, at day 17.5, the levels of erp decline in some neural structures but remain high in others, like the dorsal root ganglia. High levels of erp expression are maintained in several parts of adult brain, such as cortical layers, thalamus, hypothalamus, and hippocampus. High levels of erp transcripts are also observed in the cerebellar cortex, in the Purkinje cell layer, and in the granular cell layer. In all tissues analyzed, the expression of erp corresponds to regions undergoing terminal cell differentiation and/or regions where cell proliferation has declined.

Animals↗
PubMed1992

The transcription factor CREB, but not immediate-early gene encoded proteins, is expressed in activated microglia of lumbar spinal cord following sciatic nerve transection in the rat.

Expression of CREB, JUN, FOS and KROX-24 proteins was investigated in glial cells of the lumbar spinal cord. In untreated rats, CREB, c-JUN and JUN D were present in glial cells of the ventral and dorsal horn. Following sciatic nerve transection, the number of CREB immunoreactive glial cells increased in both the ipsilateral ventral and dorsal horns between 24 h and 48 h, reached a maximum after 5 days and returned to control levels after 20 days. Counterstaining with Cresyl violet, a general stain of cells, revealed that the increase of CREB positive glial cells was congruent with the increase of the number of glial cells. Staining with GFAP, a marker for astrocytes, showed an increase in intensity of labelling but no change in number of GFAP labelled cells. This indicates a constitutive expression of CREB in activated microglia. The number of glial cells labelled by c-JUN and JUN D did not change, and glial cells were not labelled by FOS and KROX-24 proteins following sciatic nerve transection. These findings demonstrate that proliferation and differentiation of glial cells in vivo can occur in absence of JUN, FOS and KROX proteins.

Animals↗
PubMed1992

Structure and mapping of the fosB gene. FosB downregulates the activity of the fosB promoter.

We have determined the genomic structure of the fosB gene and shown that it consists of 4 exons and 3 introns at positions also found in the c-fos gene. By deletion analysis we have characterized a region upstream of the TATA box which is the promoter region of the gene. Several consensus sequences have been identified, including an SRE and AP-1 binding site whose relative positions are identical to those in the 5' upstream region of the c-fos gene. We have also shown that FosB and c-Fos can downregulate the activity of the fosB promoter to a similar extent. The fosB gene is located in the [A1-B1] region of mouse chromosome 7.

3T3 Cells↗