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Biomedical subjects

R Bravo

Publications and source records attributed to R Bravo.

At least 109 records · Page 6Linked to original sources
PubMed1995

Block of c-Fos and JunB expression by antisense oligonucleotides inhibits light-induced phase shifts of the mammalian circadian clock.

Light-induced phase shifts of circadian rhythmic locomotor activity are associated with the expression of c-Jun, JunB, c-Fos and FosB transcription factors in the rat suprachiasmatic nucleus, as shown in the present study. In order to explore the importance of c-Fos and JunB, the predominantly expressed AP-1 proteins for the phase-shifting effects of light, we blocked the expression of c-Fos and JunB in the suprachiasmatic nucleus of male rats, housed under constant darkness, by intracerebroventricular application of 2 microliters of 1 mM antisense phosphorothioate oligodeoxynucleotides (ASO) specifically directed against c-fos and junB mRNA. A light pulse (300 lux for 1 h) at circadian time 15 induced a significant phase shift (by 125 +/- 15 min) of the circadian locomotor activity rhythm, whereas application of ASO 6 h before the light pulse completely prevented this phase shift. Application of control nonsense oligodeoxynucleotides had no effect. ASO strongly reduced the light-induced expression of c-Fos and JunB proteins. In contrast, light pulses with or without the control nonsense oligodeoxynucleotides evoked strong nuclear c-Fos and JunB immunoreactivity in the rat suprachiasmatic nucleus. These results demonstrate for the first time that inducible transcription factors such as c-Fos and JunB are an essential part of fundamental biological processes in the adult mammalian nervous system, e.g. of light-induced phase shifts of the circadian pacemaker.

Animals↗
PubMed1995

Multiorgan inflammation and hematopoietic abnormalities in mice with a targeted disruption of RelB, a member of the NF-kappa B/Rel family.

RelB, a member of the NF-kappa B/Rel family of transcription factors, has been implicated in the constitutive expression of kappa B-regulated genes in lymphoid tissues. We have generated mice carrying a germline mutation of the relB gene, resulting in the absence of RelB protein and a dramatic reduction of constitutive kappa B-binding activity in thymus and spleen. Mice homozygous for the disrupted relB locus had phenotypic abnormalities including multifocal, mixed inflammatory cell infiltration in several organs, myeloid hyperplasia, splenomegaly due to extramedullary hematopoiesis, and a reduced population of thymic dendritic cells. RelB-deficient animals also had an impaired cellular immunity, as observed in contact sensitivity experiments. Thus, RelB plays a decisive role in the hematopoietic system, and its absence cannot be functionally compensated by any other member of the NF-kappa B/Rel family.

Aging↗
PubMed1995

Transection of rat fimbria-fornix induces lasting expression of c-Jun protein in axotomized septal neurons immunonegative for choline acetyltransferase and nitric oxide synthase.

The fimbria-fornix (FF) fiber tract was unilaterally transected in adult rats by a stereotaxic knife cut. In the axotomized neurons of the medial septal nucleus (MS) and ventral diagonal band of Broca (VDB), the expression of Jun, Fos, Krox, CREB transcription factors, choline acetyltransferase (ChAT) and nitric oxide synthase (NOS) were studied by immunocytochemistry. In addition, NADPH-diaphorase (NDP) and acetylcholine esterase (AChE) were visualized by activity assays. For retrograde tracing of axotomized neurons, either HRP-coupled gold was injected in the entorhinal cortex prior to axotomy, or Fast Blue was injected into the transection site subsequently to FF transection. Following FF transection c-Jun and in a less extend JunD were expressed in axotomized MS and VDB neurons. Expression levels rose at 24 h, but not at 18 h, postaxotomy, reached their maximal levels between 5 and 7 days, and then gradually declined. Up to 100 days, c-Jun was still present in a substantial number of septal neurons. JunB, Krox-20, Krox-24, c-Fos, and pan-Fos immunoreactivities (IR) were not detectable in axotomized septal neurons and CREB-IR did not change compared to the intact contralateral side. ChAT-IR dramatically declined over 36 h, and furthermore AChE reactivity had substantially fallen after 5 days. The number and intensity of cytoplasmic neuronal NOS-IR and NDP which generated congruent temporospatial patterns gradually fell between 3 and 5 days postaxotomy. The surviving neurons labeled by NOS and NDP showed a high coexpression of c-Jun, whereas c-Jun was almost completely absent in neurons stained for ChAT and AChE. Finally, ChAT-IR and NDP reaction labeled different subpopulations. Our findings demonstrate a lasting expression of the c-Jun transcription factor in axotomized MS and VDB neurons that might indicate the regenerative propensity of damaged neurons. The decrease of NOS and NDP in MS and VDB neurons demonstrates that neuronal populations respond to axotomy with an individual regulation of NOS expression.

Acetylcholinesterase↗
PubMed1995

Krox20 may play a key role in the stabilization of long-term potentiation.

Long-term potentiation-inducing stimulation of the perforant path was followed in dentate gyrus granule cells by a dramatic increase of mRNA and protein for Krox20, a zinc-finger-containing transcription factor. Induction of Krox20 required stimulation sufficient to induce LTP and was prevented by NMDA antagonists CPP and MK-801, which block LTP induction. Krox20 protein increased within 20 min of tetanization, was maximal between 1 and 8 h, and was still significantly elevated at 24 h after LTP induction. This prolonged appearance is in striking contrast with the more transient induction of the related molecule, Krox24. The elevation in the mRNA for Krox20 and Krox24 was of similar duration, suggesting that the Krox20 protein has a greater stability and may play a key role in the stabilization of long-term potentiation.

Animals↗
PubMed1995

Angiotensin II induces a complex activation of transcription factors in the rat brain: expression of Fos, Jun and Krox proteins.

We investigated the effects of intracerebroventricular injection of angiotensin II on neuronal immediate early gene-encoded protein synthesis in the brain of conscious rats. The expression of seven immediate early gene-encoded transcription factors (c-Fos, FosB, c-Jun, JunB, JunD, Krox-20 (Egr-2) and Krox-24 (NGFI-A, Egr-1, Zif/268) was assessed simultaneously. Angiotensin II (1, 10, 100 ng) induced a dose-dependent expression of c-Fos and Krox-24 in the subfornical organ, the median preoptic area and in the paraventricular nucleus and supraoptic nucleus of the hypothalamus, regions known to be involved in the central osmoregulatory and neuroendocrine actions of angiotensin II. FosB expression was induced four hours after icv injection of the highest dose of angiotensin II in the median preoptic area and paraventricular nucleus, c-Jun expression was restricted to the median preoptic area, subfornical organ and paraventricular nucleus, and JunB was only induced in the median preoptic area and subfornical organ. In these above mentioned regions, JunD exhibited a high basal staining, which was not visibly altered by angiotensin II. Krox-20 was not induced by angiotensin II. Intracerebroventricular injections of isotonic saline did not induce immediate early gene expression in any of the above brain areas. The angiotensin II-AT1 receptor antagonist, losartan, applied intracerebroventricular five minutes prior to angiotensin II, prevented the angiotensin II-induced immediate early gene protein expression. Losartan alone had no effects on immediate early gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin II↗
PubMed1995

Efficacy and safety of alpha-interferon treatment for chronic hepatitis C in HIV-infected patients. HIV-Hepatitis Spanish Study Group.

The efficacy and safety of recombinant alpha-interferon (IFN) therapy for chronic hepatitis C (CHC) was assessed in 57 HIV-infected individuals with CD4+ T cells above 200/mm3 and compared to the response obtained in 21 HIV-negative patients with CHC. IFN 5 megaU was given three times a week subcutaneously for 3 months. In responding patients, IFN 3 megaU three times a week was additionally administered for 9 months. After 8 months follow-up in HIV-infected patients, 38% (22/57) achieved normal (complete response, CR) alanine aminotransferase (ALT) values. Partial response (PR) was seen in 21% (12/57), and 40% (23/57) did not respond. Patients with CD4+ cells above 500/mm3 achieved CR in 58% (14/24) of cases compared to 24% (8/33) among those having a lower CD4+ count (P < 0.01). Females attained CR in 60% (9/15) of cases, and men in only 30.9% (13/42) (P < 0.01). No serious side effects or opportunistic infections were observed during the study period. However, three (5.2%) patients showed a dramatic fall in total CD4+ T cell count after beginning IFN therapy. Among 21 HIV-negative patients, after 8 months follow-up, CR was achieved in 10 (47%), PR in four (19%), and seven (33%) did not respond. We concluded that IFN therapy seems to be well tolerated and useful in HIV-infected patients suffering CHC. The rate of CR was not significantly different compared to that observed in HIV-negative patients (38% vs. 47%), relative risk (RR) = 0.67 (0.19-2.37).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1995

Characterization of elements determining the dimerization properties of RelB and p50.

Members of the Rel/NF-kappa B family of transcription factors share a region of approximately 300 amino acids which mediates dimerization and sequence-specific binding to DNA. Here we report a detailed characterization of the dimerization domain of RelB. The structural core sufficient to form stable Rel/NF-kappa B dimeric complexes consists of about 110 residues. The dimerization and DNA binding properties of more than 50 RelB mutants were analyzed by using p50 and p52 as partners. We present evidence that amino acids of a conserved element in the dimerization domain play a role in the recognition of a kappa B DNA target sequence. The analysis of hybrid molecules with dimerization domains containing different parts of p50 and RelB allowed us to identify some important structural elements determining homo- and heterodimerization properties. Furthermore, we were able to rescue the dimerization-defective mutant RelB-N287D by the introduction of a counteracting mutation intramolecularly (cis), and also intermolecularly (trans) by a mutation in the NF-kappa B dimerization partner p50. Correspondingly, a dimerization defective p50 mutant was effectively rescued by RelB-N287D.

Amino Acid Sequence↗
PubMed1995

Overexpression of RelA in transgenic mouse thymocytes: specific increase in levels of the inhibitor protein I kappa B alpha.

RelA (p65) is one of the strongest activators of the Rel/NF-kappa B family. As a first step to elucidate the mechanisms that regulate its activity in vivo, we have generated transgenic mice overexpressing RelA in the thymus. Although the levels of RelA were significantly increased in thymocytes of transgenic mice, the overall NF-kappa B-binding activity in unstimulated cells was not augmented compared with that in control thymocytes. This could be explained by the dramatic increase of endogenous I kappa B alpha levels observed in RelA-overexpressing cells in both cytoplasmic and nuclear compartments. The ikba mRNA levels were not augmented by overexpressed RelA, but I kappa B alpha inhibitor was found to be stabilized through association with RelA. Although a fraction of RelA was associated with cytoplasmic p105, no changes in the precursor levels were observed. Upon stimulation of RelA-overexpressing thymocytes with phorbol 12-myristate 13-acetate and lectin (phytohemaglutinin), different kappa B-binding complexes, including RelA homodimers, were partially released from I kappa B alpha. Association of RelA with I kappa B alpha prevented complete degradation of the inhibitor. No effect of phorbol 12-myristate 13-acetate-lectin treatment was detected on RelA associated with p105. Our data indicate that cytoplasmic retention of overexpressed RelA by I kappa B alpha is the major in vivo mechanism controlling the potential excess of NF-kappa B activity in long-term RelA-overexpressing thymocytes.

Animals↗
PubMed1995

Synovial fibroblast-like cells strongly express jun-B and C-fos proto-oncogenes in rheumatoid- and osteoarthritis.

To identify recently activated cells in the synovial membrane (SM) of patients with rheumatoid arthritis (RA), the in situ expression of the proto-oncogenes jun-B, c-jun, jun-D, and c-fos was assessed by means of immunohistochemistry and in situ hybridization techniques. SM from patients with osteoarthritis (OA) or joint trauma (JT), as well as from normals (No) were used as controls. Numerous cells expressing high levels of jun-B and c-fos were found within lining layer and diffuse infiltrates in the vicinity of inflammatory cells, but only a few in lymphoid follicles and endothelia. The positive cells were spindle-shaped, CD14- and CD3-negative and, in addition, expressed mRNA for collagen alpha 2 (I) and alpha 1 (III), indicating that they were fibroblasts. In control OA, JT, and even No SM, individual fibroblast-like cells stained as strongly as in RA; however, the density of positive cells was substantially lower. In RA SM, fibroblasts, but not lymphocytes or macrophages, appear to undergo in situ activation. Quantitative differences among RA, OA, and JT may be related to different degrees of inflammatory infiltration.

Arthritis, Rheumatoid↗
PubMed1995

Neuronal regulation of c-fos, c-jun, and junB immediate-early genes in rat adrenal medulla.

We have applied in situ hybridization histochemistry, Northern analysis, and immunocytochemistry to study the regulation of the immediate-early gene (IEG) c-fos, c-jun, and junB mRNAs and the respective proteins in the rat adrenal medulla. Electrophoresis mobility shift assay was used to examine changes in AP-1 DNA binding activity. In nontreated rats the mRNA and protein levels for these three IEGs were low. Reflex stimulation of adrenal medulla elicited by a single capsaicin injection induced a rapid and marked elevation in the mRNA levels for these IEGs. Stimulation with nicotine also caused a drastic increase in the mRNA levels, whereas muscarine only induced moderate elevations. c-fos and c-jun were induced strongly in adrenaline cells and only weakly in noradrenaline cells. junB was upregulated mainly in adrenaline cells. The AP-1 DNA binding activity was low in control adrenals, whereas a marked increase was observed after nicotine treatment. Treatment of the animals with a nicotinic (chlorisondamine) or a muscarinic (atropine) receptor antagonist did not change the expression of IEGs studied. The combination of the two drugs, however elevated the mRNA levels for all three IEGs, especially for junB. Pretreatment of the rats with chlorisondamine alone or in combination with atropine diminished the capsaicin-induced increase in c-fos, whereas atropine alone was less efficient. Increase in c-jun mRNA was not affected by these drugs. The capsaicin-induced elevation of junB mRNA levels was not influenced by chlorisondamine or atropine alone, whereas both combined potentiated the effect of capsaicin. The present results demonstrate that neurotransmitters released from splanchnic nerve terminals induce expression of c-fos, c-jun, and junB in adrenal chromaffin cells which results in increased AP-1 DNA binding activity. Although stimulation of both nicotinic and muscarinic cholinergic receptors may mediate the induction of these IEGs, it is possible that also another neurotransmitter(s), in addition to ACh, released from splanchnic nerve terminals is involved in the regulation of their expression, especially of c-jun and junB.

Adrenal Medulla↗
PubMed1995

Expression and characterization of mouse cFos protein using the baculovirus expression system: ability to form functional AP-1 complex with coexpressed cJun protein.

The products of the proto-oncogenes c-fos and c-jun play important roles in cell growth control, differentiation, and malignant transformation. Purified oncogenic proteins are essential tools in cell growth control studies. Here we describe the production of mouse cFos and cJun oncoproteins in Sf9 insect cells using the baculovirus expression system. The mouse c-fos cDNA was subcloned into two different baculovirus expression vectors, namely pVL1392 and pVLMH6, to generate, respectively, nonfusion and His-fusion cFos oncoproteins in insect cells. The products were characterized by immunofluorescence, immunoblotting, immunoprecipitation, and ability to bind to the in vitro translated JunB protein to form the AP-1 complex. A His-cJun fusion protein was also produced in insect cells using the pVLMH6 baculovirus vector. Coexpression of cFos and cJun in insect cells yielded functional AP-1 complexes as judged from gel retardation assays. The results point to the possibility of using insect cells for structural and functional studies of different Fos/Jun AP-1 complexes.

Animals↗
PubMed1994

Expression of the chemokine N51/KC in the thymus and epidermis of transgenic mice results in marked infiltration of a single class of inflammatory cells.

Transgenic mice expressing the chemokine N51/KC in thymus, skin, and tongue showed a marked infiltration of a single class of inflammatory cells (neutrophils) in the sites of transgene expression. In the thymus, neutrophils were most numerous in the cortex and juxta-medullary regions, often forming aggregates or clusters. A similar, but less intense, neutrophilic infiltrate occurred in close proximity to the epidermal basal layer of the tongue and skin. No morphologic evidence of injury was observed in the thymus, skin, or tongue of these transgenic mice, indicating that N51/KC expression induces recruitment but not inflammatory activation of neutrophils. The lack of activation in the thymus resulted in a large senescent neutrophilic population that was phagocytosed by thymic macrophages and epithelial-reticular cells. These results indicate that N51/KC is a neutrophil chemoattractant in vivo and establish these transgenic mice as effective models to study the phenomena of recruitment and clearance of neutrophils, events that are critical for the initiation and resolution of the inflammatory response.

Aging↗
PubMed1994

Differential interactions of Rel-NF-kappa B complexes with I kappa B alpha determine pools of constitutive and inducible NF-kappa B activity.

The Rel-NF-kappa B family of transcription factors plays a crucial role in the regulation of genes involved in inflammatory and immune responses. We demonstrate that in vivo, in contrast to the other members of the family, RelB associates efficiently only with NF-kappa B1 (p105-p50) and NF-kappa B2 (p100-p52), but not with cRel or p65. The RelB-p52 heterodimers display a much lower affinity for I kappa B alpha than RelB-p50 heterodimers or p65 complexes. However, similarly to the other Rel-NF-kappa B complexes, RelB-p52 can upregulate the synthesis of I kappa B alpha leading to the cytoplasmic trapping of dimers which have a higher affinity for the inhibitor. We suggest that a hierarchy of interactions between I kappa B alpha and the different Rel-NF-kappa B complexes governs their cellular distribution. This results in the presence of two distinct pools of NF-kappa B activity which differ in their composition: one a constitutive nuclear and the other an inducible cytoplasmic activity.

B-Lymphocytes↗
PubMed1994

Monocyte chemotactic protein-2, monocyte chemotactic protein-3, and fibroblast-induced cytokine. Three new chemokines induce chemotaxis and activation of basophils.

Cytokine-dependent mediator release from basophils and mast cells may play an important role in the pathogenesis of allergic and inflammatory conditions. Many C-C chemokines have been found to activate basophils and mast cells. We investigated the effect of three newly identified C-C chemokines, monocyte chemotactic protein-2 and -3 (MCP-2, MCP-3) and fibroblast-induced cytokine (FIC) on basophils and mast cells. We found that all three cytokines induced histamine secretion from basophils in a dose-dependent manner. The secretion of histamine was a Ca(2+)-dependent process. MCP-3 was the most potent activator of basophils. MCP-3 and FIC activated basophils from all study subjects, whereas the histamine release by MCP-2 was donor-dependent. The histamine-releasing activity of MCP-2, MCP-3, and FIC was compared with that of MCP-1, RANTES, and macrophage inflammatory protein-1 alpha using basophils from 10 donors. MCP-1 was the most potent among all the C-C chemokines. However, MCP-3 was nearly as potent. MCP-2, MCP-3, and FIC induced significant chemotaxis of basophils. None of the cytokines activated mouse peritoneal mast cells. The synthesis of mRNA for MCP-3 was investigated by reverse-transcription PCR using allergen-stimulated PBMC and bronchoalveolar lavage cells. Both MNC and bronchoalveolar lavage cells expressed mRNA for MCP-3. The results of this study indicate that MCP-2, MCP-3, and FIC are novel histamine-releasing factors.

Allergens↗
PubMed1994

A C-terminal domain in FosB, absent in FosB/SF and Fra-1, which is able to interact with the TATA binding protein, is required for altered cell growth.

Transcriptional regulation in eukaryotes is thought to occur through interactions between specific transcription factors and the general transcription machinery. We show that the regulatory protein FosB, but not FosB/SF or Fra-1, specifically and stably associates with the TATA box binding protein (TBP) and the multiprotein complex TFIID. The binding to TBP is specified by the last 55 C-terminal amino acids of FosB, requiring a small amino acid sequence, termed the 'TBP binding motif' (TBM). Deletion of the TBM affects transcriptional activity slightly, but it is adjacent to a proline-rich sequence which constitutes the major transactivation domain. However, both regions are required for the transformation of Rat-1A cells by FosB. Transfection experiments demonstrate that inhibition of transactivation due to excess levels of Gal4-FosB (squelching) can be partially relieved by the co-expression of TBP, which establishes that TFIID is a functional target of FosB. Since TBP binding is not exhibited by FosB/SF or Fra-1, we suggest that the activity mediated by the TBP interaction is one differentiating characteristic that distinguishes the FosB functions from those of FosB/SF and Fra-1.

Amino Acid Sequence↗