Prediction of relaxin-3-induced downstream pathway resulting in anxiolytic-like behaviors in rats based on a microarray and peptidome analysis.

The effect of the intracerebroventricular (i.c.v.) injection of relaxin-3 (RLX3) was evaluated using anxiety-related behavioral tests in rats. RLX3-injected animals showed normal locomotion activity in a habituated environment and declined anxiety cognition in the elevated plus maze test and the shock probe-burying test. The measurement of spontaneous locomotor activity in a novel environment also suggested that RLX3 reduced the stress response. To elucidate the regulatory mechanisms of the downstream signaling pathways underlying RLX3 activity and its relation to anxiolytic and hyperphagic behavior phenotypes, RLX3-i.c.v.-injected rat hypothalamic responses were examined using a microarray analysis. Ingenuity Pathway Analysis software listed the phenotype-relating genes and they showed characteristic expression patterns in the rat hypothalamus. When peptidome data sets for the same listed genes was analyzed using a semi-quantitative approach, the expressions of two neuropeptides were found to have increased. One of these neuropeptides, oxytocin (Oxt), exhibited increased expression in both the microarray and the peptidomic analysis, and a Western blot analysis validated the mass spectrometry results. A cross-omics data analysis is useful for predicting downstream signaling pathways, and the anxiolytic-like behavior of RLX3 may be mediated by an oxytocin signaling pathway in rats. These results suggest that RLX3 acts as an anxiolytic peptide and that the downstream pathways mediated by its receptors may be potential candidates for the treatment of anxieties in the future.

Synthesis and structure-activity relationships of 8-substituted-2-aryl-5-alkylaminoquinolines: Potent, orally active corticotropin-releasing factor-1 receptor antagonists.

We previously reported a series of 8-methyl-2-aryl-5-alkylaminoquinolines as a novel class of corticotropin-releasing factor-1 (CRF(1)) receptor antagonists. A critical issue encountered for this series of compounds was low aqueous solubility at physiological pH (pH 7.4). To address this issue, derivatization at key sites (R(2), R(3), R(5), R(5'), and R(8)) was performed and the relationships between structure and solubility were examined. As a result, it was revealed that introduction of a methoxy substituent at the C(8) position had a positive impact on the solubility of the derivatives. Consequently, through in vivo and in vitro biological studies, compound 21d was identified as a potent, orally active CRF(1) receptor antagonist with improved physicochemical properties.

Structural alterations in outer arms of IgG oligosaccharides in patients with Werner syndrome.

Werner syndrome (WS) is a heredofamilial disorder characterized by clinicopathological premature aging. In healthy individuals, structural alteration of serum IgG oligosaccharides is known to be an aging phenotype. In the present study, we determined and compared oligosaccharide structures of serum IgG among WS patients, healthy age-sex-matched individuals, and healthy elderly individuals from both sexes in order to reveal whether WS patients exhibit an aging phenotype in terms of IgG oligosaccharide structure. Sialylation and galactosylation levels of IgG oligosaccharides from WS patients were similar to those from healthy elderly individuals in which sialylation and galactosylation levels were significantly lower than those from the healthy age-sex-matched individuals. In contrast, the bisecting N-acetylglucosaminylation level of IgG oligosaccharides from WS patients was comparable to that from the healthy age-sex-matched controls and significantly lower than that of the healthy elderly controls. There was no significant sexual difference in these modifications of IgG oligosaccharides. These results suggest that WS patients exhibit an aging phenotype for structural alterations such as sialylation and galactosylation in the outer arms of IgG oligosaccharides.

Orexin-A (hypocretin-1) is possibly involved in generation of anxiety-like behavior.

Orexins (hypocretins) are neuropeptides expressed specifically in neurons in the lateral hypothalamic area and are known to be involved in the regulation of vigilance and feeding behavior. However, the relationship between orexin and emotional behaviors like anxiety is still poorly understood. Therefore, in this report we evaluated the effect of intracerebroventricular injection of orexin-A in two major anxiety tests, the light-dark exploration test (mouse) and the elevated plus-maze test (mouse, rat). Orexin increased time spent in the dark compartment in the light-dark test and time spent on the closed arms in the elevated plus-maze test. These results were not caused by a hypothetical sedative or activity-inducing effect of orexin-A because spontaneous locomotor activity did not alter upon orexin-A application under novel conditions. We therefore suggest an anxiogenic effect of orexin-A. To our knowledge, this is the first report about a relationship between orexin-A and anxiety.

Effect of E6060 [4-{5-[7-fluoro-4-(trifluoromethyl)benzo[b]furan-2-yl]-1H-2-pyrrolyl}benzoic acid], a novel subtype-selective retinoid, on lupus-like nephritis in female (NZBxNZW)F1 mice.

Disease amelioration by retinoids in various nephritic models has been reported from either immunological or pathophysiologic viewpoints. It has also been reported that retinoids exert immunosuppressive effects in a retinoic acid receptor (RAR)-alpha-dependent manner. In particular, synthetic retinoid agonists with selectivity to RAR-alpha have been reported to have a remarkable disease-ameliorating effect in some immune disease models via their potent immunosuppressive activities; however, there has been no report in which the effect of RAR-alpha-selective agonists in the nephritic models was examined. In this report, we investigated the effect of a newly synthesized RAR-alpha-selective retinoid agonist, E6060 [4-{5-[7-fluoro-4-(trifluoromethyl)benzo[b]furan-2-yl]-1H-2-pyrrolyl}benzoic acid], on the disease progression in a murine lupus nephritis model. Female (NZBxNZW)F1 mice were prophylactically treated with E6060 from 5 months of age, and their nephritic (proteinuria, blood urea nitrogen) and immunological parameters (serum anti-DNA autoantibodies and total serum immunoglobulins) were monitored with age up to 10 months old. E6060 at 0.03 and 0.1 mg/kg (once daily, p.o.) significantly improved survival rate and prevented the development of proteinuria in (NZBxNZW)F1 mice. Anti-DNA autoantibodies and total serum IgG were also significantly reduced in the E6060-treated mice. Among IgG isotypes, IgG2a was substantially reduced by E6060 treatment, indicating reduced T helper 1 responses in E6060-treated mice. In accordance with this possibility, elevation of serum interleukin-12 (p40) in old female (NZBxNZW)F1 mice was significantly inhibited by E6060 treatment. Our data suggest that the RAR-alpha-selective retinoid E6060 is a promising candidate of new remedy for lupus nephritis in systemic lupus erythematosus patients.

Prevention of acute and chronic allograft rejection by a novel retinoic acid receptor-alpha-selective agonist.

To investigate the involvement of retinoic acid receptor (RAR)-alpha in allograft rejection, we investigated the effect of a novel selective agonist to the receptor, ER-38925, in a mouse cardiac allograft model. Prophylactic treatment with ER-38925 inhibited the acute rejection of the mouse cardiac allograft (BALB/c --> C3H/HeN) at 0.3 and 3 mg/kg, and its effect was enhanced in combination with tacrolimus. In this model, ER-38925 remarkably inhibited cytotoxic T lymphocyte induction and alloantigen-stimulated production of cytokines, i.e. IL-2, IL-12 and IFN-gamma. In the chronic rejection model, combined treatment with tacrolimus and ER-38925 reduced the grade and incidence of arteriosclerosis in the cardiac allografts significantly more potently than tacrolimus monotherapy. ER-38925 inhibited the proliferation of rat aortic smooth muscle cells stimulated in vitro, presumably through the induction of a cyclin-dependent kinase inhibitor, p27(kip-1). Those results provide a rationale for using RAR-alpha agonists as immunosuppressants in human organ transplantation.