Anosmin-1 is essential for neural crest and cranial placodes formation in Xenopus.

During embryogenesis vertebrates develop a complex craniofacial skeleton associated with sensory organs. These structures are primarily derived from two embryonic cell populations the neural crest and cranial placodes, respectively. Neural crest cells and cranial placodes are specified through the integrated action of several families of signaling molecules, and the subsequent activation of a complex network of transcription factors. Here we describe the expression and function of Anosmin-1 (Anos1), an extracellular matrix protein, during neural crest and cranial placodes development in Xenopus laevis. Anos1 was identified as a target of Pax3 and Zic1, two transcription factors necessary and sufficient to generate neural crest and cranial placodes. Anos1 is expressed in cranial neural crest progenitors at early neurula stage and in cranial placode derivatives later in development. We show that Anos1 function is required for neural crest and sensory organs development in Xenopus, consistent with the defects observed in Kallmann syndrome patients carrying a mutation in ANOS1. These findings indicate that anos1 has a conserved function in the development of craniofacial structures, and indicate that anos1-depleted Xenopus embryos represent a useful model to analyze the pathogenesis of Kallmann syndrome.

Identification of Pax3 and Zic1 targets in the developing neural crest.

The neural crest (NC) is a multipotent population of migratory cells unique to the vertebrate embryo, contributing to the development of multiple organ systems. Transcription factors pax3 and zic1 are among the earliest genes activated in NC progenitors, and they are both necessary and sufficient to promote NC fate. In order to further characterize the function of these transcription factors during NC development we have used hormone inducible fusion proteins in a Xenopus animal cap assay, and DNA microarray to identify downstream targets of Pax3 and Zic1. Here we present the results of this screen and the initial validation of these targets using quantitative RT-PCR, in situ hybridization and morpholinos-mediated knockdown. Among the targets identified we found several well-characterized NC-specific genes, including snail2, foxd3, gbx2, twist, sox8 and sox9, which validate our approach. We also obtained several factors with no known function in Xenopus NC, which represent novel regulators of NC fate. The comprehensive characterization of Pax3 and Zic1 targets function in the NC gene regulatory network, are essential to understanding the mechanisms regulating the emergence of this important cell population.

IL-6, VEGF, KC and RANTES are a major cause of a high irritant dermatitis to phthalic anhydride in C57BL/6 inbred mice.

BACKGROUND: In previous studies, several strains of mice were used as chemical-induced skin irritation models to identify immunological hazards and elucidate the molecular and cellular mechanisms by which irritant dermatitis disease occur. BALB/c and C57BL/6 mice have been used for most of these experiments. Although there are some differences in the immune response to chemical allergens between these strains, few studies have been conducted to determine what regulatory factors contribute to these variations. METHODS: To investigate the cause of high responses to skin irritation in C57BL/6 mice that are widely used to study atopic dermatitis, changes in various immune-related factors such as ear thickness, myeloperoxidase activity, lymph node weight, IgE concentration and cytokine concentration were measured in C57BL/6 and BALB/c mice following phthalic anhydride (PA) treatment. RESULTS: Based on analysis of the skin irritation, C57BL/6 mice showed a greater skin irritation to PA than BALB/c mice, although the IgE concentration and auricular lymph node weight did not contribute to this difference in the response. However, the concentration of several cytokines and chemokines (interleukin [IL]-6 and vascular endothelial growth factor [VEGF], keratinocyte-derived chemokine [KC] and regulated on activation normal T cell expressed and secreted [RANTES]) were significantly higher in C57BL/6 mice than BALB/c mice following treatment with PA. CONCLUSIONS: Our results suggest that several of the cytokines and chemokines secreted from irritant site could contribute to the regulation mechanism responsible for the difference in the skin irritation among various strains of mice following exposure to PA.

Overexpression of human selenoprotein M differentially regulates the concentrations of antioxidants and H2O2, the activity of antioxidant enzymes, and the composition of white blood cells in a transgenic rat.

Selenoprotein is associated with a variety of serious diseases, including infectious diseases, neurodegenerative disorders, cancer and cardiovascular disease. The aim of this study was to produce a new transgenic (Tg) rat expressing human selenoprotein M (SelM) in order to examine the protective function of the antioxidant status in vivo. To achieve this, a new lineage of Tg rats was produced by the microinjection of pCMV/GFP-hSelM constructs into a fertilized rat egg. Several conclusions can be drawn based on the results of the present study. The human SelM gene was successfully expressed at both the transcription and protein levels in the CMV/GFP-hSelM Tg rats. This Tg rat showed a different enzyme activity for the antioxidant protein in the various tissues examined. In response to the 2,2'-azobiz(2-amidinopropane) dihydrochloride (AAPH) injection, the Tg rats showed a lower level of antioxidant and H2O2 concentration as the activity of the antioxidant enzyme was maintained at a higher level in the Tg rats than in the non-Tg rats. Also, the neutrophil-to-lymphocyte ratio was significantly increased in this Tg rat, even though the level of corticosterone remained unchanged in both genotypes. Thus, the results of this study demonstrated that the CMV/GFP-hSelM Tg rat can serve as an animal model for the maintenance of a high level of antioxidant status and can be used to study the biological function of selenoprotein in infectious diseases, cardiovascular disease and cancer.

The effects of long-duration, low-temperature ground transportation on physiological and biochemical indicators of stress in mice.

Transportation can cause stress to laboratory animals and alter physiological characteristics that may confound experimental results. The authors investigated stress-related effects of 3-4 h of transportation by truck in two strains of mice (C57BL/6, which are known to be aggressive, and ICR, which are less aggressive). Transported mice had sufficient space and access to water, though temperature in the truck was lower than what is usually recommended. Transportation affected the following parameters in both strains of mice: (i) serum corticosterone concentrations, (ii) expression of the chaperone proteins Hsp70 and Grp78 in various tissues and (iii) concentrations of serological enzymes that are associated with liver disease. These parameters also differed substantially between the two strains of mice.

α-Isocubebenol alleviates scopolamine-induced cognitive impairment by repressing acetylcholinesterase activity.

α-Isocubebenol (ICO) isolated from Schisandra chinensis fruit was recently shown to exert neuroprotective properties with significant anti-neuroinflammatory effects. Here, we present evidence of the novel effects of ICO on alleviation of cognitive impairment. To confirm these effects, ICR mice were pretreated with two different doses of ICO for 3 weeks and scopolamine (SP) to induce memory impairment for the last 7days of the period. A passive avoidance test showed that ICO pretreatment recovered memory impairment in SP treated mice, although there was no difference between the two doses. Acetylcholinesterase (AChE) activity was significantly decreased in the SP+ICO treated group compared with the SP+Vehicle treated group. Additionally, significant recovery of the number of apoptotic cells and the ratio of apoptosis proteins (Bcl-2/Bax) were detected in the SP+ICO treated group than the SP+Vehicle treated group. Moreover, ICO treatment attenuated the decrease of ERK phosphorylation by SP treatment. These results indicate that ICO from S. chinensis fruit could be applied as an active pharmaceutical ingredient for cognitive improvement in Alzheimer's disease (AD).

Ethyl acetate extract from Asparagus cochinchinensis exerts anti­inflammatory effects in LPS­stimulated RAW264.7 macrophage cells by regulating COX­2/iNOS, inflammatory cytokine expression, MAP kinase pathways, the cell cycle and anti-oxidant activity.

Asparagus cochinchinesis (A. cochinchinesis) is a medicine traditionally used to treat fever, cough, kidney disease, breast cancer, inflammatory disease and brain disease in northeast Asian countries. Although numerous studies of the anti­inflammatory effects of A. cochinchinesis have been conducted, the underlying mechanisms of such effects in macrophages remain to be demonstrated. To investigate the mechanism of suppressive effects on the inflammatory response in macrophages, alterations of the nitric oxide (NO) level, the cell viability, inducible nitric oxide synthase (iNOS) and cyclooxygenase­2 (COX­2) expression levels, inflammatory cytokine expression, the mitogen-activated protein kinase (MAPK) signaling pathway, cell cycle arrest and reactive oxygen species (ROS) levels were measured in lipopolysaccharide (LPS)-activated RAW264.7 cells following treatment with ethyl acetate extract from A. cochinchinesis root (EaEAC). RAW264.7 cells pretreated two different concentrations of EaEAC prior to LPS treatment exhibited no significant toxicity. The concentration of NO was significantly decreased in the EaEAC + LPS treated group compared with the vehicle + LPS treated group. A similar decrease in mRNA transcript level of COX­2, iNOS, pro-inflammatory cytokines [tumor necrosis factor­α and interleukin (IL)­1ß] and anti­inflammatory cytokines (IL­6 and IL­10) was detected in the EaEAC + LPS treated group compared with the vehicle + LPS treated group, although the decrease rate varied. Enhancement of the phosphorylation of MAPK family members following LPS treatment was partially rescued in the EaEAC pretreated group, and the cell cycle was arrested at the G2/M phase. Furthermore, the EaEAC pretreated group exhibited a reduced level of ROS generation compared with the vehicle + LPS treated group. Taken together, these results suggest that EaEAC suppresses inflammatory responses through inhibition of NO production, COX­2 expression and ROS production, as well as differential regulation of inflammatory cytokines and cell cycle in RAW264.7 cells. In addition, these results provide strong evidence to suggest that EaEAC may be considered as an important candidate for the treatment of particular inflammatory diseases.

Saponin-enriched extract of Asparagus cochinchinensis alleviates airway inflammation and remodeling in ovalbumin-induced asthma model.

Asthma is a chronic inflammatory disease characterized by T-lymphocyte and eosinophil infiltration, mucus overproduction and airway hyper-responsiveness. The present study examined the therapeutic effects and action mechanism of a saponin-enriched extract of Asparagus cochinchinensis (SEAC) on airway inflammation and remodeling in an ovalbumin (OVA)-induced asthma model. To accomplish this, alterations of the nitric oxide (NO) level, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression levels, as well as variations in immune cell numbers, immunoglobulin E (IgE) concentration, histopathological structure and inflammatory cytokine levels were measured in lipopolysaccharide (LPS)-activated RAW264.7 cells or an OVA-induced mouse model of asthma treated with SEAC. The concentration of NO and mRNA levels of COX-2 and iNOS were significantly decreased in the SEAC + LPS-treated RAW264.7 cells compared with the vehicle + LPS-treated RAW264.7 cells. Additionally, in the OVA-induced asthma model, the number of immune cells in the bronchoalveolar lavage fluid, the concentration of OVA-specific IgE, the infiltration of inflammatory cells, the bronchial thickness and the levels of the inflammatory mediators interleukin-4 (IL-4), IL-13 and COX-2 were significantly lower in the OVA + SEAC­treated group compared with the OVA + vehicle­treated group. In addition, a significant reduction in goblet cell hyperplasia, peribronchiolar collagen layer thickness and VEGF expression for airway remodeling was detected in the OVA + SEAC­treated group compared with the OVA + vehicle­treated group. These findings indicate that SEAC is a suppressor of airway inflammation and remodeling, and may therefore be useful as an anti-inflammatory drug for the treatment of asthma.

Beneficial effect of diosgenin as a stimulator of NGF on the brain with neuronal damage induced by Aß-42 accumulation and neurotoxicant injection.

To investigate the beneficial effects of diosgenin (DG) on the multiple types of brain damage induced by Aß-42 peptides and neurotoxicants, alterations in the specific aspects of brain functions were measured in trimethyltin (TMT)-injected transgenic 2576 (TG) mice that had been pretreated with DG for 21 days. Multiple types of damage were successfully induced by Aß-42 accumulation and TMT injection into the brains of TG mice. However, DG treatment significantly reduced the number of Aß-stained plaques and dead cells in the granule cells layer of the dentate gyrus. Significant suppression of acetylcholinesterase (AChE) activity and Bax/Bcl-2 expression was also observed in the DG treated TG mice (TG+DG group) when compared with those of the vehicle (VC) treated TG mice (TG+VC group). Additionally, the concentration of nerve growth factor (NGF) was dramatically enhanced in TG+DG group, although it was lower in the TG+VC group than the non-transgenic (nTG) group. Furthermore, the decreased phosphorylation of downstream members in the TrkA high affinity receptor signaling pathway in the TG+VC group was significantly recovered in the TG+DG group. A similar pattern was observed in p75(NTR) expression and JNK phosphorylation in the NGF low affinity receptor signaling pathway. Moreover, superoxide dismutase (SOD) activity was enhanced in the TG+DG group, while the level of malondialdehyde (MDA), a marker of lipid peroxidation, was lower in the TG+DG group than the TG+VC group. These results suggest that DG could exert a wide range of beneficial activities for multiple types of brain damage through stimulation of NGF biosynthesis.

A novel function for Egr4 in posterior hindbrain development.

Segmentation of the vertebrate hindbrain is an evolutionarily conserved process. Here, we identify the transcription factor early growth response 4 (egr4) as a novel regulator of posterior hindbrain development in Xenopus. egr4 is specifically and transiently expressed in rhombomeres 5 and 6 (r5/r6), and Egr4 knockdown causes a loss of mafb/kreisler and krox20/egr2 expression in r5/r6 and r5, respectively. This phenotype can be fully rescued by injection of frog or mouse Egr4 mRNA. Moreover Egr4-depleted embryos exhibit a specific loss of the neural crest stream adjacent to r5, and have inner ear defects. While the homeodomain protein vHnf1/Hnf1b directly activates Mafb and Krox20 expression in the mouse hindbrain to specify r5, we show that in Xenopus this process is indirect through the activation of Egr4. We provide evidence that rearrangements in the regulatory sequences around egr4 and mafb genes may account for this difference.

Detection of allergenic compounds using an IL-4/luciferase/CNS-1 transgenic mice model.

The interleukin-4 (IL-4) signaling cascade has been identified as a potentially important pathway in the development of allergies. The principal objective of this study was to produce novel transgenic (Tg) mice harboring the luciferase gene under the control of the human IL-4 promoter and the enhancer of IL-4 (CNS-1), in an effort to evaluate three types of allergens including a respiratory sensitizer, vaccine additives, and crude extracts of natural allergens in vivo. A new lineage of Tg mice was generated by the microinjection of pIL-4/Luc/CNS-1 constructs into a fertilized mice egg. The luciferase activity was successfully regulated by the IL-4 promoter in splenocytes cultured from IL-4/Luc/CNS-1 Tg mice. From the first five founder lines, one (#57) evidencing a profound response to ovalbumin was selected for use in evaluating the allergens. Additionally, the lungs, thymus, and lymph nodes of IL-4/Luc/CNS-1 Tg mice evidenced high luciferase activity in response to allergens such as phthalic anhydride (PA), trimellitic anhydride, ovalbumin, gelatin, Dermatophagoides pteronyssinus extracts, and Japanese cedar pollen, whereas key allergy-related indicators including ear thickness, Immunoglobulin E concentration, and the infiltration of inflammatory leukocytes in response to PA were unaltered in the Tg mice relative to the non-Tg mice. Furthermore, the expression levels of endogenous type 2 helper T cells cytokines and proinflammatory cytokines were similarly increased in these organs of IL-4/Luc/CNS-1 Tg mice in response to allergens. These results indicate that IL-4/Luc/CNS-1 Tg mice may be used as an animal model for the evaluation and prediction of the human body response to a variety of allergens originating from the environment and from certain industrial products.

Overexpression of Insulin Degrading Enzyme could Greatly Contribute to Insulin Down-regulation Induced by Short-Term Swimming Exercise.

Exercise training is highly correlated with the reduced glucose-stimulated insulin secretion (GSIS), although it enhanced insulin sensitivity, glucose uptake and glucose transporter expression to reduce severity of diabetic symptoms. This study investigated the impact of short-term swimming exercise on insulin regulation in the Goto-Kakizaki (GK) rat as a non-obese model of non-insulin-dependent diabetes mellitus. Wistar (W/S) and GK rats were trained 2 hours daily with the swimming exercise for 4 weeks, and then the changes in the metabolism of insulin and glucose were assessed. Body weight was markedly decreased in the exercised GK rats compare to their non-exercised counterpart, while W/S rats did not show any exercise-related changes. Glucose concentration was not changed by exercise, although impaired glucose tolerance was improved in GK rats 120 min after glucose injection. However, insulin concentration was decreased by swimming exercise as in the decrease of GSIS after running exercise. To identify the other cause for exercise-induced insulin down-regulation, the changes in the levels of key factors involved in insulin production (C-peptide) and clearance (insulin-degrading enzyme; IDE) were measured in W/S and GK rats. The C-peptide level was maintained while IDE expression increased markedly. Therefore, these results showed that insulin down-regulation induced by short-term swimming exercise likely attributes to enhanced insulin clearance via IDE over-expression than by altered insulin production.

Characterization of changes in global gene expression in the brain of neuron-specific enolase/human Tau23 transgenic mice in response to overexpression of Tau protein.

Tau is a neuronal phosphoprotein responsible for the formation of the neurofibrillary tangles in Alzheimer's disease. To characterize the changes in global gene expression in the brain of transgenic mice that overexpress human Tau23 protein in response to the increase of Tau23 phosphorylation, total RNA extracted from the hippocampus of 12-month-old transgenic and wild-type mice was converted to cDNA, labeled with biotin and hybridized to oligonucleotide microarrays. The microarray results were confirmed by real-time RT-PCR and Western blotting method. It was determined that 43 genes were up-regulated and 8 genes were down-regulated by Tau23 in transgenic mice compared to controls, based on the arbitrary difference in the 2-fold change. Among the up-regulated transcripts, those encoding for transporter and oxidoreductase were dramatically over-represented, followed by those related to regulatory molecule, cytoskeletal protein, signaling molecule, and extracellular matrix protein. Genes encoding for transcription factor, regulatory molecule, miscellaneous function, and chaperone were significantly reduced in the down-regulated group. The major genes in the up-regulated categories included Ecrg4, Folr1, Defb11, Aqp1 and Soctdc1. The major genes in the down-regulated categories were Ncor1, Gpm6a, and Hspd1. These results indicate that the microarray analysis identifies several gene functional groups and individual genes that respond to a sustained increase in Tau23 phosphorylation levels in the brain of transgenic mice. In addition, the results suggest the microarray test is a useful tool for increased understanding of the role of Tau23 protein in regulating neurodegenerative disorders.