The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) inhibits C2-ceramide-induced cell death through blockade of the mitochondrial apoptotic pathway in rat cerebellar granule neurones. However, the gene induction processes and transcription factors involved in the antiapoptotic effect of PACAP remain unknown. Here, we show that PACAP and C2-ceramide activate activator protein-1 (AP-1) DNA binding in a dose-and time-dependent manner, but generate different AP-1 dimers. Thus, PACAP increased the proportion of c-Fos and Jun D while C2-ceramide increased c-Jun and reduced c-Fos in AP-1 complexes. In addition, PACAP strongly activated c-Fos gene expression while C2-ceramide markedly increased c-Jun phosphorylation. The effect of PACAP on c-Fos expression was blocked by the mitogen-activated protein kinase/extracellular signalregulated kinase (MEK) inhibitor, U0126, while phosphorylation of c-Jun induced by C2-ceramide was abrogated by the protein phosphatase 2A (PP2A) inhibitor, okadaic acid. Transfection of immature granule cells with c-Fos siRNA, which strongly reduced basal and PACAP-stimulated levels of the protein, totally prevented the stimulatory effect of PACAP on Bcl-2 expression. The present study demonstrates that AP-1 complexes containing c-Fos mediate the effect of PACAP on Bcl-2 gene expression in cerebellar granule neurones. Our data also indicate that different AP-1 dimers are associated with the pro-apoptotic effect of C2-ceramide and the antiapoptotic effect of PACAP.

BackgroundA comparative characterization of the oral mucosa in various animals is needed to identify the best animal model(s) for nonclinical evaluation of sublingual immunotherapy products. With this aim, we studied the histological characteristics and immune cell infiltrates of oral mucosae from common animal species.MethodsThree oral regions (i.e. ventral surface of the tongue, mouth floor and cheek) obtained from eight animal species, including rodents (i.e. mice, rats, hamsters, guinea pigs) and non-rodents (i.e. rabbits, dogs, minipigs and monkeys) were characterized by histology and immunohistology in comparison with a human tongue.ResultsRodents exhibit a thin keratinized epithelium with low epithelial extensions, whereas non-rodents, most particularly minipigs and monkeys, display a non-keratinized epithelium with larger rete ridges, similarly to humans. Glycogen-rich cells in the superficial epithelial layers are observed in samples from both minipigs, monkeys and humans. Comparable immune subpopulations detected in the 3 oral regions from rodent and non-rodent species include MHC-II+ antigen presenting cells, mostly CD163+ macrophages, located in the lamina propria (LP) and muscle tissue in the vicinity of resident CD3+CD4+ T cells. Limited numbers of mast cells are also detected in the LP and muscle tissue from all species.ConclusionThe oral mucosae of minipigs and monkeys are closest to that of humans, and the immune networks are quite similar between all rodents and non-rodents. Taking into account the ethical and logistical difficulties of performing research in the latter species, rodents and especially mice, should preferentially be used for pharmacodynamics/efficacy studies. Our data also support the use of minipigs to perform biodistribution and safety studies of sublingual immunotherapy products.

Severe impairment of the beta-oxidation of fatty acids, as a consequence of a single factor or a combination of different causes, leads to microvesicular steatosis of the liver. In an effort to understand the mechanism(s) leading to the development of acute fatty liver of pregnancy in some women, we determined the effects of pregnancy on the mitochondrial oxidation of fatty acids in mice. In vivo, the rate of oxidation of the whole fatty-acid chain length was determined by measuring the rate of exhalation of [14C]CO2 after intragastric administration of a tracer dose of [U-14C]palmitic acid. [14C]CO2 exhalation was not significantly decreased at 14 days of gestation, but it had declined by 40% at 18 days of gestation (i.e., 24 to 48 hr before delivery). The rate of first beta-oxidation cycle was assessed by measuring the rate of [14C]CO2 exhalation after administration of [1-14C]octanoic acid, [1-14C]butyric acid or [1-14C]palmitic acid. [14C]CO2 exhalation had declined by 60%, 46%, and 24% after administration of [1-14C]octanoic acid, [1-14C]butyric acid and [1-14C]palmitic acid, respectively, in 18-day-pregnant mice. Total hepatic lipids and triglycerides, expressed per gram of liver, remained unchanged in 18-day-pregnant mice. In vitro, the rate of mitochondrial beta-oxidation (expressed per milligram of protein) had decreased by 47% at 18 days' gestation with [U-14C]palmitic acid as substrate and by 33% with [1-14C]octanoic acid but remained unchanged with [1-14C]palmitic acid. The activity of the tricarboxylic acid cycle, assessed by the formation of [14C]CO2 from [1-14C]acetic acid, had decreased by 24%. We conclude that the mitochondrial oxidation of fatty acids decreased during late-term pregnancy in mice as a consequence of both decreased mitochondrial beta-oxidation of medium-chain fatty acids, and decreased activity of the tricarboxylic acid cycle. We suggest that this effect, in combination with other factors, may contribute to the development of fatty liver of pregnancy in some pregnant women.

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