Novelty, but Not Operant Aversive Learning, Enhances Fos and Egr-1 Expression in the Medial Prefrontal Cortex and Hippocampal Areas of Rats

Immediate early genes (IEG) are presumed to be activated in response to stress, novelty, and learning. Evidence supports the involvement of prefrontal and hippocampal areas in stress and learning, but also in the detection of novel events. This study examined whether a previous experience with shocks changes the pattern of Fos and Egr-1 expression in the medial prefrontal cortex (mPFC), the hippocampal cornus ammonis 1 (CA1), and dentate gyrus (DG) of adult male Wistar rats that learned to escape in an operant aversive test. Subjects previously exposed to inescapable footshocks that learned to escape from Shocks were assigned to the treated group (EXP). Subjects from Group Novelty (NOV) rested undisturbed during treatment and also learned to escape in the test. The nonshock group (NSH) rested undisturbed in both sessions. Standard immunohistochemistry procedures were used to detect the proteins in brain sections. The results show that a previous experience with shocks changed the pattern of IEG expression, then demonstrating c-fos and egr-1 induction as experience-dependent events. Compared with NSH and EXP an enhanced Fos expression was detected in the mPFC and CA1 subfield of Group NOV, which also exhibited increased Egr-1 expression in the mPFC and DG in comparison to NSH. No differences were found in the DG for Fos, or in the CA1 for Egr-1. Novelty, and not the operant aversive escape learning, seems to have generated IEG induction. The results suggest novel stimuli as a possible confounding factor in studies on Fos and/or Egr-1 expression in aversive conditions.

Neuronal NOS Inhibitor and Conventional Antidepressant Drugs Attenuate Stress-induced Fos Expression in Overlapping Brain Regions

Recent evidence indicates that the administration of inhibitors of neuronal nitric oxide synthase (nNOS) induces antidepressant-like effects in animal models such as the forced swimming test (FST). However, the neural circuits involved in these effects are not yet known. Therefore, this study investigated the expression of Fos protein, a marker of neuronal activity, in the brain of rats submitted to FST and treated with the preferential nNOS inhibitor, 7-nitroindazole (7-NI), or with classical antidepressant drugs (Venlafaxine and Fluoxetine). Male Wistar rats were submitted to a forced swimming pretest (PT) and, immediately after, started receiving a sequence of three ip injections (0, 5, and 23 h after PT) of Fluoxetine (10 mg/kg), Venlafaxine (10 mg/kg), 7-NI (30 mg/kg) or respective vehicles. One hour after the last drug injection the animals were submitted to the test session, when immobility time was recorded. After the FST they were sacrificed and had their brains removed and processed for Fos immunohistochemistry. Independent group of non-stressed animals received the same drug treatments, or no treatment (naive). 7-NI, Venlafaxine or Fluoxetine reduced immobility time in the FST, an antidepressant-like effect. None of the treatments induce significant changes in Fos expression per se. However, swimming stress induced significant increases in Fos expression in the following brain regions: medial prefrontal cortex, nucleus accumbens, locus coeruleus, raphe nuclei, striatum, hypothalamic nucleus, periaqueductal grey, amygdala, habenula, paraventricular nucleus of hypothalamus, and bed nucleus of stria terminalis. This effect was attenuated by 7-NI, Venlafaxine or Fluoxetine. These results show that 7-NI produces similar behavioral and neuronal activation effects to those of typical antidepressants, suggesting that these drugs share common neurobiological substrates.

Molekulare Mechanismen Proteinkinase C-induzierter Nox4-Hochregulation in humanen Endothelzellen

Eine verstärkte Transkription von NADPH-Oxidasen (Nox) wird mit der Entstehung von atherosklerotischer Veränderungen in Verbindung gebracht. Die Arbeit unserer Gruppe zeigte, dass die Aktivität der Proteinkinase C (PKC) zu einer Nox4-Hochregulation führt, der dominanten NOX Isoform in endothelialen Zellen. Die vorliegende Arbeit zielte auf die Aufdeckung der dowm-stream gelegenen Mechanismen. Die Behandlung von humanen EA.hy 926-Zellen mit dem PKC Aktivator Phorbol-12-Myristat-13-Acetat (PMA) für 48 h führte in eine signifikante Nox4-mRNA-Hochregulation, welche mittels PKC-Inhibitoren oder PKC alpha siRNA abgewendet werden konnte. PMA führte zu einer andauernden Aktivierung der MAP-Kinase Erk1/2. Die PMA vermittelte Nox4-Expression konnte durch Erk1/2-Inhibitoren oder durch Erk1/2-Knock-down geblockt werden. Down-stream konnte die Involvierung der Erk1/2-Substarte Elk-1 und c-Fos mittels siRNA-Experimente gezeigt werden. Darüber hinaus blockte die Inhibierung der Histondeacetylasen (HDACs) mit Scriptaid oder durch HDAC3-Knock-down mittels siRNA die PMA-induzierte Nox4-Expression in EA.hy 926-Zellen, weswegen eine Rolle für HADC3 in der Regulation der Nox4-Expression angezeigt wurde. Abschließend reduzierte ein Knock-down von p53 (siRNA) deutlich die basale Expression von Nox4, hatte aber nur einen kleinen Effekt auf die PMA-induzierte Nox4-Expression. Zusammenfassend zeigen die Daten der vorliegenden Arbeit, dass in einer PKC alpha induzierten Nox4-mRNA-Hochregulation Erk1/2, Elk-1, cFos und HDAC3 involviert sind.

The transcriptional regulator megakaryoblastic leukemia-1 mediates serum response factor-independent activation of tenascin-C transcription by mechanical stress

The extracellular matrix protein tenascin-C (TNC) is up-regulated in processes influenced by mechanical stress, such as inflammation, tissue remodeling, wound healing, and tumorigenesis. Cyclic strain-induced TNC expression depends on RhoA-actin signaling, the pathway that regulates transcriptional activity of serum response factor (SRF) by its coactivator megakaryoblastic leukemia-1 (MKL1). Therefore, we tested whether MKL1 controls TNC transcription. We demonstrate that overexpression of MKL1 strongly induces TNC expression in mouse NIH3T3 fibroblasts and normal HC11 and transformed 4T1 mammary epithelial cells. Part of the induction was dependant on SRF and a newly identified atypical CArG box in the TNC promoter. Another part was independent of SRF but required the SAP domain of MKL1. An MKL1 mutant incapable of binding to SRF still strongly induced TNC, while induction of the SRF target c-fos was abolished. Cyclic strain failed to induce TNC in MKL1-deficient but not in SRF-deficient fibroblasts, and strain-induced TNC expression strongly depended on the SAP domain of MKL1. Promoter-reporter and chromatin immunoprecipitation experiments unraveled a SAP-dependent, SRF-independent interaction of MKL1 with the proximal promoter region of TNC, attributing for the first time a functional role to the SAP domain of MKL1 in regulating gene expression.

Selective elevation of c-{\it myc} RNA transcripts during clonal evolution of N-methyl-N-nitrosourea induced thymic lymphomas in AKR/J mice

Untreated AKR mice develop spontaneous thymic lymphomas by 6-12 months of age. Lymphoma development is accelerated when young mice are injected with the carcinogen N-methyl-N-nitrosourea (MNU). Selected molecular and cellular events were compared during the latent period preceding "spontaneous" (retrovirally-induced) and MNU-induced thymic lymphoma development in AKR mice. These studies were undertaken to test the hypothesis that thymic lymphomas induced in the same inbred mouse strain by endogenous retroviruses and by a chemical carcinogen develop by different mechanisms.^ Immunofluorescence analysis of differentiation antigens showed that most MNU-induced lymphomas express an immature CD4-8+ profile. In contrast, spontaneous lymphomas represent each of the major lymphocyte subsets. These data suggest involvement of different target populations in MNU-induced and spontaneous lymphomas. Analyses at intervals after MNU treatment revealed selective expansion of the CD4-8+ J11d+ thymocyte subset at 8-10 weeks post-MNU in 68% of the animals examined, suggesting that these cells are targets for MNU-induced lymphomagenesis. Untreated age-matched animals showed no selective expansion of thymocyte subsets.^ Previous data have shown that both spontaneous and MNU-induced lymphomas are monoclonal or oligoclonal. Distinct rearrangement patterns of the J$\sb2$ region of the T-cell receptor $\beta$-chain showed emergence of clonal thymocyte populations beginning at 6-7 weeks after MNU treatment. However, lymphocytes from untreated animals showed no evidence of clonal expansion at the time intervals investigated.^ Activation of c-myc frequently occurs during development of B- and T- cell lymphomas. Both spontaneous and MNU-induced lymphomas showed increased c-myc transcript levels. Increased c-myc transcription was first detected at 6 weeks post-MNU, and persisted throughout the latent period. However, untreated animals showed no increases in c-myc transcripts at the time intervals examined. Another nuclear oncogene, c-fos, did not display a similar change in RNA transcription during the latent period.^ These results supports the hypothesis that MNU-induced and spontaneous tumors develop by multi-step pathways which are distinct with respect to the target cell population affected. Clonal emergence and c-myc deregulation are important steps in the development of both MNU-induced and spontaneous tumors, but the onset of these events is later in spontaneous tumor development. ^

Targeted overexpression of protein kinase C β2 isoform in myocardium causes cardiomyopathy

Increased cardiovascular mortality occurs in diabetic patients with or without coronary artery disease and is attributed to the presence of diabetic cardiomyopathy. One potential mechanism is hyperglycemia that has been reported to activate protein kinase C (PKC), preferentially the β isoform, which has been associated with the development of micro- and macrovascular pathologies in diabetes mellitus. To establish that the activation of the PKCβ isoform can cause cardiac dysfunctions, we have established lines of transgenic mice with the specific overexpression of PKCβ2 isoform in the myocardium. These mice overexpressed the PKCβ2 isoform transgene by 2- to 10-fold as measured by mRNA, and proteins exhibited left ventricular hypertrophy, cardiac myocyte necrosis, multifocal fibrosis, and decreased left ventricular performance without vascular lesions. The severity of the phenotypes exhibited gene dose-dependence. Up-regulation of mRNAs for fetal type myosin heavy chain, atrial natriuretic factor, c-fos, transforming growth factor, and collagens was also observed. Moreover, treatment with a PKCβ-specific inhibitor resulted in functional and histological improvement. These findings have firmly established that the activation of the PKCβ2 isoform can cause specific cardiac cellular and functional changes leading to cardiomyopathy of diabetic or nondiabetic etiology.

Distinct regions of c-Mpl cytoplasmic domain are coupled to the JAK-STAT signal transduction pathway and Shc phosphorylation.

c-Mpl, a member of the hematopoietic cytokine receptor family, is the receptor for thrombopoietin. To investigate signal transduction by c-Mpl, a chimeric receptor, composed of the extracellular domain of human growth hormone receptor and the intracellular domain of c-Mpl, was introduced into the interleukin 3-dependent cell line Ba/F3. In response to growth hormone, this chimeric receptor induced growth in the absence of interleukin 3. Deletion analysis of the 123-amino acid intracellular domain indicated that the elements responsible for this effect are present within the 63 amino acids proximal to the transmembrane domain. Mutation of the recently described box 1 motif abrogated the proliferative response. Tyrosine phosphorylation of the tyrosine kinase JAK-2 and activation of STAT proteins were dependent on box 1 and sequences within 63 amino acids of the plasma membrane. STAT proteins activated by thrombopoietin in a megakaryocytic cell line were purified and shown to be STAT1 and STAT3. A separate region located at the C terminus of the c-Mpl intracellular domain was found to be required for induction of Shc phosphorylation and c-fos mRNA accumulation, suggesting involvement of the Ras signal transduction pathway. Thus, at least two distinct regions are involved in signal transduction by the c-Mpl.

Virus de la hepatitis C: variabiliad en las regiones del CORE y NS5A y su impacto en la infección por el genotipo 1b

La infección por el virus de la hepatitis C (VHC) tiene un gran impacto a nivel mundial, considerándose una de las principales causas de hepatitis crónica, enfermedad hepática terminal y hepatocarcinoma. Hasta la incorporación de los nuevos agentes antivirales, el genotipo 1b era el que tenía tasas más bajas de respuesta viral sostenida (RVS); y a su vez, el de mayor prevalencia en nuestro medio. Lo que ha llevado a la búsqueda de marcadores predictores de respuesta, con el fin de identificar a aquellos pacientes que pudieran beneficiarse del tratamiento. Estudios previos han mostrado que la variabilidad genómica en la región codificante de la proteína C del Core y en la región comprendida entre los aminoácidos 2209 y 2248 (región determinante de la sensibilidad al IFN: ISDR) de la región que codifica la proteína no-­‐estructural 5A (NS5A) están relacionados con la respuesta al tratamiento con biterapia. Es por ello, que el objetivo de nuestro estudio es caracterizar a nivel genómico ambas regiones y analizar su impacto en pacientes con infección crónica por el genotipo 1b. Se analizan, además, otros factores basales implicados en la respuesta: sexo, edad y carga viral basal...

Could leprosy reaction episodes be exacerbated by oral infections?

INTRODUCTION: This study evaluated whether leprosy reactions could be associated with oral infection. METHODS: Leprosy patients (n = 38) with (Group I) and without (Group II) oral infections were selected. Reactions were identified from the clinical and histopathological features associated with serum C-reactive protein (CRP) and10kDa interferon-gamma-induced protein (IP-10) levels, determined before and after elimination of the foci of infection. RESULTS: Group I presented more reactions than group II did, and improvement of the reactions after dental treatment. Serum CRP and IP-10 did not differ before and after the dental treatment, but differed between the groups. CONCLUSIONS: Oral infection could be an exacerbating factor in leprosy reactions.

Effects of an intervention in eating habits and physical activity in Japanese-Brazilian women with a high prevalence of metabolic syndrome in Bauru, São Paulo State, Brazil

We evaluated the impact of a lifestyle intervention on the cardiometabolic risk profile of women participating in the Study on Diabetes and Associated Diseases in the Japanese-Brazilian Population in Bauru. This was a non-controlled experimental study including clinical and laboratory values at baseline and after a 1-year intervention period. 401 Japanese-Brazilian women were examined (age 60.8±11.7 years), and 365 classified for metabolic syndrome (prevalence = 50.6%). Subjects with metabolic syndrome were older than those without (63.0±10.0 vs. 56.7±11.6 years, p < 0.01). After intervention, improvements in variables were found, except for C-reactive protein. Body mass index and waist circumference decreased, but adiposity reduction was more pronounced in the abdominal region (87.0±9.7 to 84.5±11.2cm, p < 0.001). Intervention-induced differences in total cholesterol, LDL, and post-challenge glucose were significant; women who lost more than 5% body weight showed a better profile than those who did not. The lifestyle intervention in Japanese-Brazilian women at high cardiometabolic risk improved anthropometric and laboratory parameters, but it is not known whether such benefits will persist and result in long-term reduction in cardiovascular events.

Effect of cyhalothrin on Ehrlich tumor growth and macrophage activity in mice

Cyhalothrin, a pyrethroid insecticide, induces stress-like symptoms, increases c-fos immunoreactivity in the paraventricular nucleus of the hypothalamus, and decreases innate immune responses in laboratory animals. Macrophages are key elements in cellular immune responses and operate at the tumor-host interface. This study investigated the relationship among cyhalothrin effects on Ehrlich tumor growth, serum corticosterone levels and peritoneal macrophage activity in mice. Three experiments were done with 10 experimental (single gavage administration of 3.0 mg/kg cyhalothrin daily for 7 days) and 10 control (single gavage administration of 1.0 mL/kg vehicle of cyhalothrin preparation daily for 7 days) isogenic BALB/c mice in each experiment. Cyhalothrin i) increased Ehrlich ascitic tumor growth after ip administration of 5.0 x 106 tumor cells, i.e., ascitic fluid volume (control = 1.97 ± 0.39 mL and experimental = 2.71 ± 0.92 mL; P < 0.05), concentration of tumor cells/mL in the ascitic fluid (control = 111.95 ± 16.73 x 106 and experimental = 144.60 ± 33.18 x 106; P < 0.05), and total number of tumor cells in the ascitic fluid (control = 226.91 ± 43.22 x 106 and experimental = 349.40 ± 106.38 x 106; P < 0.05); ii) increased serum corticosterone levels (control = 200.0 ± 48.3 ng/mL and experimental = 420.0 ± 75.5 ng/mL; P < 0.05), and iii) decreased the intensity of macrophage phagocytosis (control = 132.3 ± 19.7 and experimental = 116.2 ± 4.6; P < 0.05) and oxidative burst (control = 173.7 ± 40.8 and experimental= 99.58 ± 41.7; P < 0.05) in vitro in the presence of Staphylococcus aureus. These data provide evidence that cyhalothrin simultaneously alters host resistance to Ehrlich tumor growth, hypothalamic-pituitary-adrenocortical (HPA) axis function, and peritoneal macrophage activity. The results are discussed in terms of data suggesting a link between stress, HPA axis activation and resistance to tumor growth.

Behavioral and Autonomic Responses to Acute Restraint Stress Are Segregated within the Lateral Septal Area of Rats

Background: The Lateral Septal Area (LSA) is involved with autonomic and behavior responses associated to stress. In rats, acute restraint (RS) is an unavoidable stress situation that causes autonomic (body temperature, mean arterial pressure (MAP) and heart rate (HR) increases) and behavioral (increased anxiety-like behavior) changes in rats. The LSA is one of several brain regions that have been involved in stress responses. The aim of the present study was to investigate if the neurotransmission blockade in the LSA would interfere in the autonomic and behavioral changes induced by RS. Methodology/Principal Findings: Male Wistar rats with bilateral cannulae aimed at the LSA, an intra-abdominal datalogger (for recording internal body temperature), and an implanted catheter into the femoral artery (for recording and cardiovascular parameters) were used. They received bilateral microinjections of the non-selective synapse blocker cobalt chloride (CoCl(2), 1 mM/ 100 nL) or vehicle 10 min before RS session. The tail temperature was measured by an infrared thermal imager during the session. Twenty-four h after the RS session the rats were tested in the elevated plus maze (EPM). Conclusions/Significance: Inhibition of LSA neurotransmission reduced the MAP and HR increases observed during RS. However, no changes were observed in the decrease in skin temperature and increase in internal body temperature observed during this period. Also, LSA inhibition did not change the anxiogenic effect induced by RS observed 24 h later in the EPM. The present results suggest that LSA neurotransmission is involved in the cardiovascular but not the temperature and behavioral changes induced by restraint stress.

Tooth Movement Activates the Central Amygdala and the Lateral Hypothalamus by the Magnitude of the Force Applied

Objective: To determine if the magnitude of the force used to induce incisor tooth movement promotes distinct activation in cells in the central amygdala (CEA) and lateral hypothalamus (LH) of rats. Also, the effect of morphine on Fos immunoreactivity (Fos-IR) was investigated in these nuclei. Materials and Methods: Adult male rats were anesthetized and divided into six groups: only anesthetized (control), without orthodontic appliance (OA), OA but without force, OA activated with 30g or 70g, OA with 70g in animals pretreated with morphine (2 mg/kg, intraperitoneal). Three hours after the onset of the experiment the rats were reanesthetized and perfused with 4% paraformaldehyde. The brains were removed and fixed, and sections containing CEA and LH were processed for Fos protein immunohistochemistry. Results: The results show that in the control group, the intramuscular injection of a ketamine/xylazine mixture did not induce Fos-IR cells in the CEA or in the LH. Again, the without force group showed a little Fos-IR. However, in the 70g group the Fos-IR was the biggest observed (P < .05, Tukey) in the CEA and LH compared with the other groups. In the 30g group, the Fos-IR did not differ from the control group, the without OA group, and the without force group. Furthermore, pretreatment with morphine in the 70g group reduced Fos-IR in these regions. Conclusions: Tooth movement promotes Fos-IR in the CEA and LH according to the magnitude of the force applied. (Angle Orthod. 2010;80:111-115.)

Chronic ethanol has region-selective effects on Egr-1 and Egr-3 DNA-binding activity and protein expression in the rat brain

This study focused on the DNA-binding activity and protein expression of the transcription factors Egr-1 and Egr-3 in the rat brain cortex and hippocampus after chronic or acute ethanol exposure. DNA-binding activity was reduced in both regions after chronic ethanol exposure and was restored to the level of the pair-fed group at 16 h of withdrawal. Cortical Egr-1 protein levels were not altered by chronic ethanol exposure but increased 16 h after withdrawal, thus mirroring DNA-binding activity. In contrast, Egr-3 protein levels did not undergo any change. There was no change in the level of either protein in the hippocampus. Immunohistochemistry revealed a region-selective change in immunopositive cells in the cortex and hippocampus. Finally, an acute bolus dose of ethanol did not affect Egr DNA-binding activity and ethanol treatment did not alter the DNA-binding activity or protein levels of the transcription factor Spl. These observations suggest that chronic exposure to ethanol has region-selective effects on the DNA-binding activity and protein expression of Egr-1 and Egr-3 transcription factors in the rat brain. These changes occur after prolonged ethanol exposure and may thus reflect neuroadaptive changes associated with physical dependency and withdrawal. These effects are also transcription factor-selective. Clearly, protein expression is not the sole mediator of the changes in DNA-binding activity and chronic ethanol exposure must have effects on modulatory agents of Egr DNA-binding activity. (C) 2000 Elsevier Science Ltd, All rights reserved.