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.

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.

Activation of human monocyte-derived macrophages by interferon gamma is accompanied by increase of poly(ADP-ribose) polymerase activity.

We have investigated poly(ADP-ribosyl)ation processes in human monocyte-derived macrophages and the effect of the activating cytokine, interferon gamma (IFN-gamma) on these processes. IFN-gamma was shown to increase the activity of poly(ADP-ribose) polymerase in human macrophages. A 2-3-fold enhancement of poly(ADP-ribose) polymerase activity was observed after 3-4 h of incubation with IFN-gamma, whose effects were dose-dependent and maximal at 20-50 U/ml. Staining with anti-poly(ADP-ribose) antibodies and purification of ADP-ribosylated nuclear proteins by affinity chromatography over boronate agarose showed that enhancement of poly(ADP-ribose) polymerase activity by IFN-gamma was accompanied by accumulation of poly(ADP-ribose) polymers in nuclear proteins. The effects of IFN-gamma on poly(ADP-ribose) polymerase activity were not due to an enhanced accumulation of the message for the enzyme, indicating that the activation of the enzyme activity was due to post-transcriptional modifications. IFN-gamma was shown to induce DNA strand breaks in human macrophages. This phenomenon followed the same time-course and was evident with the same doses of IFN-gamma that increased poly(ADP-ribose) polymerase activity. Since poly(ADP-ribose) polymerase is known to require DNA nicks for its activity, the capability of IFN-gamma to induce DNA strand breaks can explain its effects on poly(ADP-ribosyl)ation processes.

Changes in activity and mRNA levels of poly(ADP-ribose) polymerase during rat liver regeneration.

ADP-ribosylation of nuclear proteins, catalysed by the enzyme poly(ADP-ribose) polymerase, is involved in the regulation of different cellular processes of DNA metabolism. To further clarify the role of the enzyme during proliferating activity of mammalian cells, we have studied the control of gene expression in regenerating rat liver. The changes in activity and mRNA levels were analysed during the early and late phases of the compensatory model. When enzyme activity was measured in isolated liver nuclei obtained at different times after hepatectomy, two different phases were observed: an early wave occurring before the onset of DNA synthesis, and a second one, starting several hours after the onset of DNA synthesis and returning to control values at later times. The evaluation of the enzymatic level in nuclear extracts and by activity gel analysis showed a more gradual increase starting 1 day after hepatectomy, in concomitance with the peak of DNA synthesis. By using a specific murine cDNA probe, a significant enhancement of mRNA levels for poly(ADP-ribose) polymerase was observed during liver regeneration, slightly preceding the onset of DNA synthesis. The results obtained show that changes in poly(ADP-ribose) polymerase activity, during liver regeneration, are associated both to early events preceding the increase in DNA synthesis and to later phases of the cell proliferation process.

Increase of poly(ADP-ribose) polymerase mRNA levels during TPA-induced differentiation of human lymphocytes.

The non-mitogenic stimulation of human peripheral blood mononuclear cells (PBMC) with low concentrations of the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) caused a progressive increase in the percent fraction of the cells that were positive for the early activating antigen CD69. At the same time, it caused a progressive increase in the steady-state levels of poly(ADP-ribose) polymerase (pADPRP) transcripts. A further increase in TPA concentration, while inducing the maximal expression of the levels of CD69 activating surface antigen, both in the presence or in the absence of proliferative activity, did not evoke any additional hightening of pADPRP mRNA levels. Time course of PBMC stimulation with a non-mitogenic dose of TPA showed an early increase in the accumulation of pADPRP mRNA, which changed at 8-16 h, and remained high for several days thereafter. On the basis of these data, we suggest that the increase in pADPRP mRNA may be associated with the commitment of human lymphocytes from a quiescent (G0) to an activated (G1) state.

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.

Involvement of DNA polymerase beta in proliferation of rat liver induced by lead nitrate or partial hepatectomy.

We have studied the expression pattern of DNA polymerase beta in two different models of in vivo cell proliferation. Both mRNA levels and enzyme activity of DNA polymerase beta markedly increased before and/or during DNA synthesis in proliferating hepatocytes in mitogen-treated and partially hepatectomized rats. The time-courses of the expression of the gene coding for DNA polymerase beta were significantly different in the two cell systems. A 5-fold increase in DNA polymerase beta mRNA was observed 8 h after lead nitrate administration, i.e. well before the onset of DNA synthesis. In the regenerative liver cells a 3-fold increase in the amount of mRNA was observed 24-48 h after partial hepatectomy, the event being coincident with extensive DNA synthesis. In both systems, the increase of mRNA levels was always paralleled by an increase in enzyme activity, suggesting that DNA polymerase beta activity may be regulated at a pre-translational level.

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.

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.

Analysis of the methylation pattern of c-Ha-ras oncogene in human prostatic cancer.

To determine the methylation pattern of c-Ha-ras oncogene in prostatic tissue and to identify possible changes of methylation associated with cancer, high molecular weight DNA was extracted from 7 normal and 6 carcinomatous human prostates. Analysis of the samples was performed by cleaving DNA with the restriction endonucleases Msp I, Hpa II and Cfo I, and by Southern hybridizing the DNA digests with the 32P-labelled c-Ha-ras (pT24-C3) probe. Several discrete fragments were obtained with Hpa II and Cfo I digestion while the Msp I pattern showed fewer and smaller bands. Therefore, c-Ha-ras appears to be partially methylated. While a considerable polymorphism of the sequence 5'-CCGG-3' was observed at several Msp I sites in all cases, no significant differences could be evidenced in the methylation patterns of normal and neoplastic prostatic DNA samples extracted and purified from each patient.

[Salivary levels of immunoglobulin and dental caries in children]. / Livelli salivari di immunoglobuline e carie dentale in eta' infantile.

IgG, IgA, IgM and lysozyme levels were determined in whole unstimulated saliva from ten children aged seven-nine years. IgA levels were significantly higher (p less than 0.05) in caries-susceptible subjects when compared with the caries-free ones. High levels of IgG seemed to correlate with a high DMF score. No correlation was found between lysozyme levels and DMF score.

Effects of lead on red blood cell membrane proteins.

The effects of lead on red blood cell (RBC) membrane proteins were studied in two groups of workers with different lead exposure levels: Group I (6 subjects employed in a battery plant) with a mean blood lead of 40.1 (SD = 3.7) micrograms/100 ml; Group II (5 workers employed in different industries) with a mean blood lead of 60.6 (SD = 8.0) micrograms/100 ml, compared with a control group with mean blood lead of 15.6 (SD = 9.3) micrograms/100 ml. The analysis of RBC membrane polypeptides was carried out by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and by using a densitometer for percentage measurement of the bands corresponding to protein fractions. The results show a very significant decrease in Band 3 (anion channel) and 4.1 in more exposed workers (Group II) only. The effects of lead on RBC membrane proteins seem to be evident at blood-lead levels higher (greater than 50 micrograms/100 ml) than those previously reported in literature. These results confirm the effects of lead on membrane proteins, even if the exact mechanism, particularly the influence of proteolysis and the meaning of the interference, still needs to be investigated thoroughly.

Regulation of poly(ADP-ribose) polymerase gene expression in mitogen-stimulated human peripheral blood mononuclear cells.

The level of mRNA for poly(ADP-ribose) polymerase in human PBMC increased 8 h after addition of PHA, reaching its maximum (9-fold over the basal level) 3-4 days after the stimulation and decreasing thereafter. mRNA maximum slightly preceded in time the maximal value of DNA synthesis. The half-life of poly(ADP-ribose) polymerase mRNA, which is 1.2 h in quiescent PBMC, increased up to 3.4 h in stimulated PBMC. This PHA-induced stabilization of the mRNA for poly(ADP-ribose) polymerase could account for the accumulation of the transcript in mitogen-treated PBMC.

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.

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.

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.

The in utero initiation with DMN alters the complement of cytosolic glutathione S-transferases and the phenobarbital-induced expression of c-jun and c-myc oncogenes in primary neonatal rat hepatocytes.

As revealed by a novel in utero-in vitro hepatocarcinogenesis model, a single exposure to dimethylnitrosamine (DMN) elicited in postnatal (and fetal) primary rat hepatocytes (i) immunocytochemically detectable, widespread increases in their complement of the alpha, mu and especially pi classes of cytosolic glutathione S-transferases (GSTs); and (ii) changed patterns (with respect to controls) of the phenobarbital (PB)-evoked increases in steady state levels of c-jun and c-myc mRNA's. These results indicate that DMN causes both transient cytotoxic effects and a broad, permanent initiation in fetal proliferating hepatocytes.