Regulation of cerebral microvessels by glutamatergic mechanisms

We studied changes in glutamate receptors, expression of immediate early genes, and AP-1 DNA binding activity in the brains of phenobarbital (PB)-dependent and -withdrawn rats to investigate the possible involvement of activation of glutamate receptors in PB withdrawal syndrome. PB-dependent rats were prepared by feeding drug-admixed food for 5 weeks. Autoradiographic analysis showed that binding of [3H(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imin e (MK-801), an antagonist of N-methyl-D-aspartic acid (NMDA) receptors, increased significantly in the cerebral cortices of PB-dependent and 24-h-withdrawn rats. However, [3H]MK-801 binding in the hippocampus and [3H]6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and [3H]kainic acid binding in the hippocampus and cerebral cortex were essentially unchanged in both groups. PB withdrawal seizures were followed by increased expression of c-fos mRNA in the hippocampus and cerebral cortex and of c-jun mRNA in the cerebral cortex. The induction of c-fos and c-jun mRNA was suppressed by administration of MK-801. Furthermore, PB withdrawal enhanced AP-1 DNA binding activity in the brain. The present findings suggest functional enhancement of glutamatergic neurotransmission during the development of PB withdrawal syndrome.     

Kainate-evoked modulation of gene expression in rat brain

Kainate is a glutamate analog that produces neuronal excitation resulting in seizures within hours following its intraperitoneal injection into adult rats. Then, at 2-3 days after the treatment, neurodegeneration of apoptotic character can be observed in limbic system. As a consequence, plastic reorganization and glial reactivation phenomena occur. These physiological and pathological responses are reflected by specific changes in gene expression, that can be dissected according to their spatio-temporal patterns. The early phase of gene expression observed in all hippocampal subfields appears to reflect a sudden burst of spiking activity. Changes in mRNA levels restricted to dentate gyrus are suggestive of a link to neuronal plasticity. The late gene expression response implies its correlation either to neuronal cell death or glial reactivation, depending on cellular localization of gene products. Thus analysis of the temporal and spatial gene expression pattern in the hippocampus after kainate treatment may provide clues revealing specific phenomena to which gene expression could be attributed.     

Ethanol exposure increases expression of c-jun and junD in human neuroblastoma cells

The aim of this study was to investigate the effect of ethanol exposure on the expression of fos and jun genes. Exposure of human neuroblastoma SH-SY5Y cells to ethanol for 2-4 days caused a dose-dependent increase in c-jun and junD mRNA levels, whereas mRNAs for c-fos, fosB and junB were not detectable in control or ethanol-treated cells. Four days of ethanol exposure also enhanced the AP-1 binding activity. Experiments with actinomycin D demonstrated that ethanol did not influence the degradation of c-jun and junD mRNAs. These results demonstrate that long-term exposure to ethanol increases c-jun and junD expression. This effect may be one of the mechanisms through which ethanol influences the gene regulatory system in neuronal cells.     

Cortical induction of c-fos by intrastriatal endothelin-1 is mediated via NMDA receptors

Endothelin (ET) is a potent vasoconstrictor which has also been proposed to act as a neuromodulator. We have investigated the action of ET-1 on neurones in vivo, using c-fos as a marker of neuronal activation. Intrastriatal injection of ET-1 caused seizures and barrel rolling which were prevented by pretreatment with the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 and attenuated by the nitric-oxide synthase inhibitor N omega-nitro-L-arginine (L-NNA). In association with these behaviours, a dramatic increase in c-fos mRNA expression was seen in the cerebral cortex. This increase was blocked by both MK-801 and L-NNA. We suggest that ET-1 modulates the activity of cortical afferents to the striatum, and causes seizures via an NMDA receptor-dependent mechanism.     

Induction of c-fos and c-jun proto-oncogene expression by asbestos is ameliorated by N-acetyl-L-cysteine in mesothelial cells

Asbestos fibers cause dose-dependent, persistent increases in mRNA levels of c-jun and c-fos proto-oncogenes in rat pleural mesothelial (RPM) cells, the progenitor cells of asbestos-induced mesothelioma (N. Heintz, Y. M. W. Janssen, and B. T. Mossman. Proc. Natl. Acad. Sci. USA, 90: 3299-3303, 1993). Here we report that addition of N-acetyl-L-cysteine decreases asbestos-mediated induction of c-fos and c-jun mRNA levels in a dose-dependent fashion. Exposure of RPM cells to asbestos causes depletion of total cellular glutathione, a response that can be abolished by pretreatment with N-acetyl-L-cysteine. Pretreatment of cells with buthionine sulfoximine, an agent which diminishes glutathione pools, increases the magnitude of induction of c-fos and c-jun mRNA by asbestos. To determine whether asbestos-induced effects on proto-oncogene expression could be attributed to extracellular generation of active oxygen species (AOS), RPM cells were exposed to H2O2 or xanthine and xanthine oxidase, a generating system of AOS. These oxidant stresses did not decrease cellular glutathione levels nor alter mRNA levels of c-fos or c-jun. However, increased mRNA levels of manganese-containing superoxide dismutase and heme oxygenase were observed, indicating that RPM cells respond to AOS by increased expression of genes encoding antioxidant enzymes. These data indicate that the signaling pathways leading to c-fos/c-jun proto-oncogene induction by asbestos are not triggered directly by formation of extracellular AOS. However, intracellular thiol levels appear to influence the expression of c-fos and c-jun, suggesting a redox-sensitive component in the signaling cascade which modulates gene expression of c-fos and c-jun by asbestos.     

Neuronal stress response and neuronal cell damage after cardiocirculatory arrest in rats

Cardiocirculatory arrest is the most common clinical cause of global cerebral ischemia. We studied neuronal cell damage and neuronal stress response after cardiocirculatory arrest and subsequent cardiopulmonary resuscitation in rats. The temporospatial cellular reactions were assessed by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labeling (TUNEL) staining of DNA fragments, in situ hybridization (heat shock protein hsp70; immediate early genes c-fos and c-jun), and immunocytochemical (HSP70; and myeloperoxidase, specific marker of polymorphonuclear leukocytes [PMNL]) techniques. Cardiac arrest of 10 minutes' duration was induced in mechanically ventilated male Sprague-Dawley rats anesthetized with nitrous oxide and halothane. After cardiopulmonary resuscitation, animals were allowed to reperfuse spontaneously for 6 hours, 24 hours, 3 days, and 7 days (n = 6 per group). Five sham-operated animals were controls. The TUNEL staining revealed an early onset degeneration in the thalamic reticular nucleus (TRN) at 6 hours that peaked at 3 days. In contrast, degeneration was delayed in the hippocampal CA1 sector, showing an onset at 3 days and a further increase in the number of TUNEL-positive cells at 7 days. A minor portion of TUNEL-positive nuclei in the CA1 sector showed condensed chromatin and apoptotic bodies, whereas all nuclei in the TRN revealed more diffuse staining. After 6 hours of reperfusion, levels of mRNA for hsp70 and c-jun were elevated in circumscribed areas of cortex, in all hippocampal areas, and in most nuclei of thalamus, but not in the TRN. After 24 hours, a strong expression of mRNA for hsp70 and c-jun could be observed in the second layer of the cortex and in hippocampal CA1 sector; hsp70 also was observed in hippocampal CA3 sector. Some animals showed expression of hsp70 and c-jun in the dentate gyrus. After 3 days, hsp70 and c-jun were detected mainly in the CA1 sector of hippocampus. At 7 days, mRNA for both returned to control values. Therefore, delayed cell degeneration in the CA1 sector corresponds to a prolonged expression of hsp70 and c-jun in this area. In situ hybridization studies for c-fos revealed a strong signal in CA3 and dentate gyrus and a less prominent signal in TRN at 6 hours. At 24 hours, CA4 and amygdalae were positive, whereas at 3 and 7 days, the signal reached control levels; no prolonged or secondary expression was observed in the CA1 sector. Immunohistochemical study confirmed translation of HSP70 in various areas corresponding to the detection of mRNA, including the CA1 sector. The number of PMNL increased significantly at 6 hours and 7 days after cardiac arrest; PMNL were distributed disseminately and were not regionally associated with neuronal cell damage. The current data support the view that CA1 neurons might undergo an apoptosis-associated death after cardiac arrest, but PMNL are not directly involved in this process. The marked differences in the time course and the characteristics of TUNEL staining and the neuronal stress response in CA1 sector and TRN point to different mechanisms of neuronal injury in the two selectively vulnerable areas.     

Selective expression of the immediate early gene c-jun in axotomized rat medial septal neurons is not related to neuronal degeneration

In the present study, we use the anatomically well defined septohippocampal projection to study the molecular events involved in the reaction of neurons to axotomy. The expression of three immediate early genes (c-fos, c-jun, and jun B) was investigated in rat septohippocampal neurons after axotomy by bilateral fimbria-fornix transection (FFT). Moreover, the extent of retrograde degeneration in the septal complex was assessed by analyzing DNA fragmentation. In a postoperative time course analysis, a strong increase of c-jun immunoreactivity (IR) was observed in the nuclei of neurons in the medial septum/diagonal band complex (MSDB) 2 and 6 d postaxotomy, which was followed by a decline after 12 d and 3 weeks, respectively. Nine weeks after FFT, c-jun IR had disappeared. The c-jun-positive MS neurons were identified as former septohippocampal projection cells by double-labeling with the retrogradely transported tracer Fluoro-Gold injected into the hippocampus before axotomy. In line with the immunocytochemical data, there was a massive induction of c-jun mRNA in the axotomized MS neurons as visualized by in situ hybridization histochemistry. c-fos mRNA and c-fos or jun B IR were not detectable in either unoperated or lesioned medial septal neurons. Experiments using the TdT-mediated deoxyuridine triphosphate nick-end-labeling technique, designed to detect nuclear DNA fragmentation in degenerating neurons, complemented this study. During the postoperative time range studied, MS neurons did not exhibit DNA fragmentation. We conclude that MSDB neurons survive axotomy by FFT and display characteristic changes in gene expression.     

Diarrhea

The antisense oligonucleotide approach has been established as a tool to analyse the functional role of c-fos gene expression in the striatum. Studies on the distribution and cellular localization of microinjected oligonucleotides, as well as their effect on gene expression, demonstrate that the action of the c-fos oligonucleotides can be used to evaluate the role of c-fos gene expression selectively in neuronal function. Antisense oligonucleotides to c-fos inhibit both basal and stimulated c-fos expression in the striatum, which leads to characteristic changes in behavioural and biochemical parameters, as evaluated by analysis of rotational behaviour and dual probe in vivo microdialysis for the neurotransmitter GABA, respectively. These observations could be explained by a postulated D1/NMDA receptor interaction in the striatonigral GABA pathway controlled by the immediate early gene c-fos. We concluded that c-fos might be involved not only in the control of long-term changes in the cellular phenotype but also in control of firing rate.     

Roles of glutamate receptor in c-fos gene expression and epilepsy susceptibility in rats

We use NMDA to induce expression of c-fos mRNA as a marker to observe the activity of NMDA receptor in neurons during development, and compare the activity of NMDA receptor between audiogenic epilepsy -prone (P77PMC) and audiogenic epilepsy resistant rats brain. In primary culture of rats cerebral cortical neurons NMDA induced c-fos mRNA expression exhibits dose and time-dependent changes, which can be prevented by antagonists. During the development of neurons, the NMDA -induced c-fos mRNA expression reaches a maximum at day 24. NMDA-induced c-fos mRNA expression of P77PMC rats is higher than that of controls during 6 to 24 days in vitro with significant difference (P < 0.05) at day 18. To present changes in c-fos mRNA expression induced by NMDA in cultured P77PMC rat cortical neurons may be one of the factors related to susceptibility of epilepsy in P77PMC rats.     

Human papillomavirus infection and esophageal cancer: a nationwide seroepidemiologic case-control study in Sweden

BACKGROUND: Despite being immunogenic, gastric cancers overcome antitumour immune responses by mechanisms that have yet to be fully elucidated. Fas ligand (FasL) is a molecule that induces Fas receptor mediated apoptosis of activated immunocytes, thereby mediating normal immune downregulatory roles including immune response termination, tolerance acquisition, and immune privilege. Colon cancer cell lines have previously been shown to express FasL and kill lymphoid cells by Fas mediated apoptosis in vitro. Many diverse tumours have since been found to express FasL suggesting that a "Fas counterattack" against antitumour immune effector cells may contribute to tumour immune escape. AIM: To ascertain if human gastric tumours express FasL in vivo, as a potential mediator of immune escape in stomach cancer. SPECIMENS: Thirty paraffin wax embedded human gastric adenocarcinomas. METHODS: FasL protein was detected in gastric tumours using immunohistochemistry; FasL mRNA was detected in the tumours using in situ hybridisation. Cell death was detected in situ in tumour infiltrating lymphocytes using terminal deoxynucleotidyl transferase mediated dUTP nick end labelling (TUNEL). RESULTS: Prevalent expression of FasL was detected in all 30 resected gastric adenocarcinomas examined. In the tumours, FasL protein and mRNA were co-localised to neoplastic gastric epithelial cells, confirming expression by the tumour cells. FasL expression was independent of tumour stage, suggesting that it may be expressed throughout gastric cancer progression. TUNEL staining disclosed a high level of cell death among lymphocytes infiltrating FasL positive areas of tumour. CONCLUSIONS: Human gastric adenocarcinomas express the immune downregulatory molecule, FasL. The results suggest that FasL is a prevalent mediator of immune privilege in stomach cancer.