Transcription factor expression, RNA synthesis and NADPH-diaphorase across the rat brain and exposure to spatial novelty.

The molecular hypothesis of learning and memory processes is based on changes in synaptic weights in neural networks. Aim of this study was to map neural traces of exposure to a spatial novelty were mapped by (i) the transcription factors (TFs) c-fos, c-jun and jun-B using Northern blot and immunocytochemistry (ICC), (ii) RNA synthesis by (3)H-uridine autoradiography and RNA level, (iii) NADPH-diaphorase (NADPH-d) expression by histochemistry. Thus, adult male albino rats were exposed to a Làt-maze and sacrificed at different times. Non-exposed rats served as controls. The latter showed a low constitutive expression of TF, RNA synthesis and NADPH-d across the brain. Northern blots showed a three-fold increase in TFs in exposed versus non-exposed rats in the cerebral cortex. ICC showed in exposed rats several TFs positive cells in the granular and pyramidal layers of the hippocampus and later in all layers of the somatosensory cortex, in the granular layer of the cerebellar cortex. The TF-positivity was stronger in rats exposed for the first time, and was time and NMDA-dependent. Autoradiography for RNA synthesis showed positive cells in the ependyma, hippocampus and cerebellum 6h after testing, and in the somatosensory cortex 24h later. In addition, exposure to novelty induced NADPH-d in the dorsal hippocampus, the caudate-putamen, all the layers of the somatosensory cortex. and the cerebellum. The positivity was absent immediately after exposure, appeared within 2h and disappeared 24h later. A strong neuronal discharge by the convulsant pentylenetetrazol, strongly induced TFs but not din not affect NADPH-d 2h later. Thus, data suggest that the processing of spatial and emotional components of experience activates neural networks across different organization levels of the CNS.

The stimulation of inducible nitric-oxide synthase by the prion protein fragment 106--126 in human microglia is tumor necrosis factor-alpha-dependent and involves p38 mitogen-activated protein kinase.

A synthetic peptide consisting of amino acid residues 106-126 of the human prion protein (PrP-(106--126)) has been previously demonstrated to be neurotoxic and to induce microglial activation. The present study investigated the expression of the inducible form of the nitric-oxide synthase (NOS-II) in human microglial cells treated with PrP-(106--126). Using reverse transcriptase-polymerase chain reaction, we found that PrP-(106--126) induces NOS-II gene expression after 24 h of treatment and that this effect is accompanied by a peak of nuclear factor kappa B (NF-kappa B) binding at 30 min as evaluated by electrophoretic mobility shift assay. Since our previous data demonstrated tumor necrosis factor-alpha (TNF-alpha) to be a potent inducer of NOS-II in these cells, we analyzed the expression of this cytokine in PrP-(106--126)-treated microglia. PrP-(106--126) caused the release of TNF-alpha as detected by enzyme-linked immunosorbent assay, and a blocking antibody, anti-TNF-alpha, abolished NOS-II induction elicited by this peptide. Moreover, PrP-(106-126) activates p38 mitogen-activated protein kinase, and the inhibition of this pathway determines the ablation of NF-kappa B binding induced by this fragment peptide.

Cyclin D1 is an early target in hepatocyte proliferation induced by thyroid hormone (T3).

The thyroid hormone (T3) affects cell growth, differentiation, and regulates metabolic functions via its interaction with the thyroid hormone nuclear receptors (TRs). The mechanism by which TRs mediate cell growth is unknown. To investigate the mechanisms responsible for the mitogenic effect of T3, we have determined changes in activation of transcription factors, mRNA levels of immediate early genes, and levels of proteins involved in the progression from G1 to S phase of the cell cycle. We show that hepatocyte proliferation induced by a single administration of T3 to Wistar rats occurred in the absence of activation of AP-1, NF-kappa B, and STAT3 or changes in the mRNA levels of the immediate early genes c-fos, c-jun, and c-myc. These genes are considered to be essential for liver regeneration after partial hepatectomy (PH). On the other hand, T3 treatment caused an increase in cyclin D1 mRNA and protein levels that occurred much more rapidly compared to liver regeneration after 2/3 PH. The early increase in cyclin D1 expression was associated with accelerated onset of DNA synthesis, as demonstrated by a 20-fold increase of bromodeoxyuridine-positive hepatocytes at 12 h after T3 treatment and by a 20-fold increase in mitotic activity at 18 h. An early increase of cyclin D1 expression was also observed after treatment with nafenopin, a ligand of a nuclear receptor (peroxisome proliferator-activated receptor alpha) of the same superfamily of steroid/thyroid receptors. T3 treatment also resulted in increased expression of cyclin E, E2F, and p107 and enhanced phosphorylation of pRb, the ultimate substrate in the pathway leading to transition from G1 to S phase. The results demonstrate that cyclin D1 induction is one of the earlier events in hepatocyte proliferation induced by T3 and suggest that this cyclin might be a common target responsible for the mitogenic activity of ligands of nuclear receptors.

Molecular features of primary mediastinal B-cell lymphoma: involvement of p16INK4A, p53 and c-myc.

Primary mediastinal B-cell lymphoma (PMBL) shows chromosome 9p anomalies in 50% of cases. Based on reports that p16INK4A gene, located on this chromosomal arm, is frequently altered in aggressive lymphomas, we analysed for alterations of this gene in 27 cases of PMBL, which were part of a series of 32 PMBL cases that have been characterized for alterations in c-myc, p53, N-ras, bcl-1, bcl-2, bcl-6 and for Epstein-Barr virus (EBV) infection. Four cases showed p16INK4A gene anomalies, including three with promoter methylation and one homozygous deletion. Eight PMBLs showed c-myc rearrangements. Three additional cases showed sequence variations in the c-myc P2 promoter, two of which consisted of the same germline variation involving a novel polymorphic XhoI site. Four tumours contained p53 gene mutations and three had clonal EBV infection. One case had a bcl-6 rearrangement. In conclusion, our study shows that p16INK4, c-myc and p53 alterations occur in 15%, 25% and 13% of PMBLs, respectively. EBV monoclonality was found in 9% of cases, whereas no abnormality was detected in bcl-1, bcl-2 and N-ras. Thus, none of the common genetic aberrations seen in other types of non-Hodgkin's lymphomas appears to be stringently involved in the pathogenesis of this unique lymphoma type.

Correlation of poly(ADP-ribose)polymerase and p53 expression levels in high-grade lymphomas.

The steady-state levels of mRNA for the poly(ADP-ribose)polymerase (PARP), c-myc, p53, and histone H3 genes were investigated in 31 high-grade B-cell lymphomas by northern blot analysis. The panel included 15 nodal large B-cell lymphomas, nine mediastinal large B-cell lymphomas, and seven sporadic Burkitt's lymphomas. The PARP mRNA level was significantly higher in lymphomas than in control tissues and corresponded with the amount of PARP protein, as assessed by immunoblot analysis in six samples. The level of PARP mRNA was positively correlated with that of p53 mRNA. No correlation was found between the mRNA expression levels of PARP and histone H3, suggesting that PARP expression levels are independent of the proliferation rate of neoplastic cells. In this setting, the strong correlation between PARP and p53 suggests that the high expression of PARP may be associated with ongoing DNA damage in high-grade lymphomas.

Rapid inactivation of NOS-I by lipopolysaccharide plus interferon-gamma-induced tyrosine phosphorylation.

Human astrocytoma T67 cells constitutively express a neuronal NO synthase (NOS-I) and, following administration of lipopolysaccharide (LPS) plus interferon-gamma (IFNgamma), an inducible NOS isoform (NOS-II). Previous results indicated that a treatment of T67 cells with the combination of LPS plus IFNgamma, by affecting NOS-I activity, also inhibited NO production in a very short time. Here, we report that under basal conditions, a NOS-I protein of about 150 kDa was weakly and partially tyrosine-phosphorylated, as verified by immunoprecipitation and Western blotting. Furthermore, LPS plus IFNgamma increased the tyrosine phosphorylation of NOS-I, with a concomitant inhibition of its enzyme activity. The same effect was observed in the presence of vanadate, an inhibitor of phosphotyrosine-specific phosphatases. On the contrary, genistein, an inhibitor of protein-tyrosine kinases, reduced tyrosine phosphorylation of NOS-I, enhancing its enzyme activity. Finally, using reverse transcriptase-polymerase chain reaction, we have observed that a suboptimal induction of NOS-II mRNA expression in T67 cells was enhanced by vanadate (or L-NAME) and inhibited by genistein. Because exogenous NO has been found to suppress NOS-II expression, the decrease of NO production that we have obtained from the inactivation of NOS-I by LPS/IFNgamma-induced tyrosine phosphorylation provides the best conditions for NOS-II expression in human astrocytoma T67 cells.

In vivo hepatocyte proliferation is inducible through a TNF and IL-6-independent pathway.

Recent studies in mice harboring a targeted disruption of genes encoding TNF receptor 1 (TNFR-1) or Interleukin 6 (IL-6) suggested a critical role for TNF and IL-6 in initiation of liver regeneration after 2/3 partial hepatectomy. However, hepatocyte proliferation can also occur following treatment with agents that do not induce tissue loss (primary mitogens). To determine whether the above cytokines could also be involved in mitogen-induced liver cell proliferation, we studied the hepatocyte proliferative response after treatment with primary mitogens in mice knock-out for TNFR-1 or IL-6. Our results showed no difference in the proliferative response of the liver between the wild type and the knock-out mice following treatment with the mitogens 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), or the peroxisome proliferator, ciprofibrate, suggesting that TNF or IL-6 may not play a major role in this type of proliferation. Gel shift assay indicated that TCPOBOP-induced hepatocyte proliferation is not associated with activation of STAT3 transcription factor, a major target of IL-6 and other growth factors/cytokines. Our results thus indicate that hepatocyte proliferation can be induced by at least two different pathways; compensatory regeneration being TNF and IL-6-dependent, and mitogen-induced direct hyperplasia which does not require TNF or IL-6.

Liver cell proliferation induced by nafenopin and cyproterone acetate is not associated with increases in activation of transcription factors NF-kappaB and AP-1 or with expression of tumor necrosis factor alpha.

Our previous studies have shown a different pattern of immediate early gene and growth factor gene expression between compensatory liver regeneration occurring after cell loss/death and direct hyperplasia induced by primary mitogens. In the present study, modifications in the activation of two transcription factors, NF-kappaB and AP-1; steady-state levels of tumor necrosis factor alpha (TNF-alpha) messenger RNA (mRNA); and induction of the inducible nitric oxide synthase (iNOS) were examined in rat liver during different types of cell proliferation. Compensatory regeneration was induced in male Wistar rats by partial hepatectomy of two thirds (PH) or a necrogenic dose of CCl4 (2 mL/kg), whereas direct hyperplasia was induced by a single administration of the primary mitogens lead nitrate (LN, 100 micromol/kg), cyproterone acetate (CPA, 60 mg/kg), or nafenopin (NAF, 200 mg/kg). Liver regeneration after treatment with CCl4 was associated with an increase in steady-state levels of TNF-alpha mRNA, activation of NF-kappaB and AP-1, and induction of iNOS. A strong and prolonged activation of NF-kappaB but not of AP-1 was observed in LN-induced hyperplasia. LN also induced an increase in hepatic levels of TNF-alpha and iNOS mRNA. On the other hand, direct hyperplasia induced by two other primary mitogens, NAF and CPA, occurred in the complete absence of modifications in the hepatic levels of TNF-alpha mRNA, activation of NF-kappaB and AP-1, or induction of iNOS, although the number of hepatocytes entering S phase 18 to 24 hours after NAF was similar to that seen after PH. These results add further support to the hypothesis that cell proliferation occurring in the absence of cell loss/death may be triggered by unknown signaling pathways different from those responsible for the transition of hepatocytes from G0 to G1 after PH or cell necrosis.

On the presence of DNA polymerase alpha in human lymphocyte nuclei and chromosomes.

Experiments were carried out to correlate the cytological localization of DNA polymerase alpha with the presence of its specific mRNA in human lymphocytes studied at different times after phytohaemagglutinin stimulation. Our data indicated that in resting cells it is not possible to detect DNA polymerase alpha protein or mRNA by Northern hybridization. By contrast, in stimulated cells the detection of mRNA specific for DNA polymerase alpha synthesis is possible after 16 h phytohaemagglutin stimulation, whereas immunolocalization is possible after only 4 h stimulation. Observation of cytological preparations from cells stimulated for times long enough to obtain mitoses surprisingly showed an intense immunoreaction in mitotic chromosomes treated with monoclonal antibodies to DNA polymerase alpha.

TPA and cycloheximide modulate the activation of NF-kappa B and the induction and stability of nitric oxide synthase transcript in primary neonatal rat hepatocytes.

12-O-Tetradecanoylphorbol 13-acetate (TPA) elicited a transient increase in the transcription of the inducible nitric oxide synthase (iNOS) gene coupled with a shortening of the half-life of its mRNA in primary neonatal rat hepatocytes. These effects of TPA were preceded by a surge in nuclear translocation of the transcription factor NF-kappa B, and followed by a mounting accumulation of NO-2 in the growth medium. Even cycloheximide (CHX) added by itself elicited an early, sustained activation of NF-kappa B followed by an intense induction of iNOS gene expression, irrespective of what degree of protein synthesis inhibition was brought about by the several concentrations tested. When given together, TPA and CHX exerted additive effects on hepatocellular iNOS mRNA levels. These results suggest the likelihood of an ordered sequence of events by which an activated NF-kappa B mediates the induction of iNOS gene expression in TPA- and/or CHX-treated primary hepatocytes.

Induction of nitric oxide synthase mRNA expression. Suppression by exogenous nitric oxide.

The reactive nitrogen species, nitric oxide (NO), plays an important role in the pathogenesis of neurodegenerative diseases. The suppression of NO production may be fundamental for survival of neurons. Here, we report that pretreatment of human ramified microglial cells with nearly physiological levels of exogenous NO prevents lipopolysaccharide (LPS)/tumor necrosis factor alpha (TNF alpha)-inducible NO synthesis, because by affecting NF-kappa B activation it inhibits inducible Ca(2+)-independent NO synthase isoform (iNOS) mRNA expression. Using reverse transcriptase polymerase chain reaction, we have found that both NO donor sodium nitroprusside (SNP) and authentic NO solution are able to inhibit LPS/TNF alpha-inducible iNOS gene expression; this effect was reversed by reduced hemoglobin, a trapping agent for NO. The early presence of SNP during LPS/TNF alpha induction is essential for inhibition of iNOS mRNA expression. Furthermore, SNP is capable of inhibiting LPS/TNF alpha-inducible nitrite release, as determined by Griess reaction. Finally, using electrophoretic mobility shift assay, we have shown that SNP inhibits LPS/TNF alpha-elicited NF-kappa B activation. This suggests that inhibition of iNOS gene expression by exogenous NO may be ascribed to a decreased NF-kappa B availability.

Protective effect of NO on gastric lesions and inhibition of expression of gastric inducible NOS by flurbiprofen and its nitro-derivative, nitroflurbiprofen.

Nitroflurbiprofen (NFP) causes significantly less gastric lesions than flurbiprofen (FP), probably because of its capacity to release nitric oxide (NO) in the stomach. Lipopolysaccharide (LPS), which induces the expression of an inducible type of NO synthase (iNOS) in rat stomach, also reduces gastric mucosal damage elicited by FP. Furthermore, both FP and NFP decrease significantly the amount of mRNA encoding iNOS induced by LPS in the stomach. The inhibitory effect of NFP seems to be due at least in part to its ability to release NO.

Nitric oxide in the liver: physiopathological roles.

Many of the known roles of arginine (e.g. in immune function, wound healing, and protection against ammonia intoxication) are mediated by a metabolic pathway synthesising nitric oxide (NO) in the liver. Contrary to some of the current views, liver-produced NO may be basically beneficial, as it exerts both protective actions against tissue injury and cytotoxic effects on invading microorganisms, parasites, or tumor cells. An ongoing equilibrium between NO and other NO-reactive compounds (e.g. O2 and non-heme iron-sulphur-containing moieties) appears to be important in this respect, even under critical conditions. Thus, NO may prevent liver tissue harm from oxidant stress. Only when this putative counterbalance is upset by an uncontrolled, prolonged and/or massive production of NO, liver tissue damage may occur leading to hepatic inflammation or even tumor development. Moreover, the currently available data support the working hypothesis that hepatocytes partake not only to immunoregulatory processes, but even to immune defence mechanisms. Thus, the liver constitutes an excellent model for investigations into the crosstalks regulating the production of NO which take place among not only the various networks operating inside a single hepatic cell, but even the individual types of liver cells.

Differential expression pattern of jun B and c-jun in the rat brain during the 24-h cycle.

Data from a previous report [3] demonstrated that the proto-oncogene c-fos mRNA expression undergoes basally a circadian fluctuation in the rat brain. The present study was designed to verify by means of Northern blot hybridization the eventual occurrence of a spontaneous oscillation in the expression of other two proto-oncogenes, jun B and c-jun, during 24 h. Rats were either entrained to a light-dark photoperiod or maintained under constant darkness or light. During the dark period, as well as the subjective night, the jun B mRNA levels in the cerebral cortex and striatum were 4-6 times higher than in the light hours or subjective day. No consistent oscillation was found in the c-jun mRNA expression during 24 h in any of the examined brain regions. These results suggest the possibility of different interactions of the c-fos, jun B and c-jun gene products throughout a 24-h period in discrete brain regions.

Poly(ADP-ribose) polymerase: early involvement in glutamate-induced neurotoxicity in cultured cerebellar granule cells.

Glutamate neurotoxicity is correlated with an increase of cytosolic free Ca2+. In some cell systems, activation of Ca2+ dependent endonucleases or formation of free radicals can damage DNA and activate the chromatin bound enzyme poly(ADP-ribose) polymerase (pADPRP). We have investigated whether pADPRP may be involved in glutamate neurotoxicity in vitro. Cerebellar granule cells at 12 days in culture when treated with a toxic dose of glutamate (100 microM) showed a rapid and transient increase of polyADP-ribose immunoreactivity. Cellular immunostaining was heterogeneous and returned to control levels after washout of glutamate. In the same cell preparations glutamate elicited a marked increase in enzyme protein immunoreactivity which persisted at later times. Non-toxic doses of glutamate did not affect immunostaining. In another set of experiments, pADPRP mRNA was increased 30 min after glutamate. In order to investigate the role of pADPRP in glutamate-mediated neurotoxicity, structurally different inhibitors of pADPRP (3-aminobenzamide, benzamide,3-aminophthalhydrazide) and their inactive analogues (benzoic acid and phthalimide) were tested in this model. Addition of the inhibitors to cultures 60 min before and during the 30 min of glutamate treatment prevented neuronal death by 60-100%, assessed 24 hr later. Glutamate-induced Ca2+ influx was not affected. Inactive analogues failed to afford neuroprotection. These data indicate that not only is pADPRP activated by the early, possibly Ca(2+)-mediated mechanisms initiated by glutamate, but that it might also actively contribute to the subsequent neuronal death.

Different programs of gene expression are associated with different phases of the 24h and sleep-wake cycles.

The Fos and Jun proteins are encoded by proto-oncogenes acting as immediate early genes in that they are rapidly induced by different kinds of stimuli in the nervous system. These two proteins bind to DNA regulating gene transcription, and thus determining the specificity of the neuronal response to the applied stimulation. We investigated whether the expression of these genes undergoes a variation during 24h in the absence of exogenous stimulation. Male Wistar adult (200 gr. body weight) rats, kept under a 12h/12h light-dark cycle, were sacrificed every 4h starting at 0700. The expression of c-fos, c-jun and jun B mRNAs was studied in six different brain areas by means of Northern blot hybridization, c-fos expression was also studied with in situ hybridization and immunohistochemistry. In basal conditions c-fos expression displayed a highly significant spontaneous oscillation, with the highest level during the darkness hours and the lowest during the light hours. Parallel levels of jun B expression were found in the cortex and striatum, whereas c-jun mRNA remained constantly high throughout 24 h. The periodicity of c-fos and jun B oscillation persisted also when the animals were exposed for 6 days to constant (24h/24h) light or darkness. Such oscillation could instead be inverted by manipulating the rest-activity cycle, i.e. keeping the animals awake during the light hours and allowing them to sleep during the dark hours. We then verified whether the expression of fos and jun could be correlated with states of wakefulness (W) and sleep (S), monitored with EEG recording under behavioral control.(ABSTRACT TRUNCATED AT 250 WORDS)

c-fos mRNA is spontaneously induced in the rat brain during the activity period of the circadian cycle.

The basal expression of the proto-oncogene c-fos was studied by Northern blot analysis in different regions of the rat brain during 24 h. A striking spontaneous oscillation of c-fos mRNA expression was detected in animals kept in basal conditions with a 12 h light/12 h dark cycle. In these animals c-fos mRNA was just detectable during the rest hours (morning through afternoon), and was high during the activity hours (night). The periodicity of this oscillation persisted and became free-running when the animals were exposed for 6 consecutive days to constant light or darkness. It was thus demonstrated that the fluctuation of c-fos expression is circadian and is not created by the light-dark cycle, but the latter exerts a synchronizing effect. The oscillation of c-fos mRNA was modified by manipulations of the rest-activity cycle. In particular, the fluctuation observed in basal conditions was inverted, keeping the animals awake during the rest hours (diurnal) and allowing them to sleep in the activity period (nocturnal). These data indicated a close relationship between the oscillation of c-fos expression and the rest-activity cycle. Finally, electroencephalographic (EEG) monitoring was performed under behavioural control for 3 h before the animals were killed. These experiments confirmed that, irrespective of the time of day, the EEG pattern typical of a state of sleep (including both slow waves and paradoxical sleep) was associated with low or undetectable c-fos levels, whereas the protracted EEG desynchronization corresponding to wakefulness was associated with high c-fos expression.(ABSTRACT TRUNCATED AT 250 WORDS)