The purpose of this study was to determine whether Endothelin-1 (ET-1) induced cellular responses in bovine corneal endothelial cells (BCECs) involves MAPK pathway by phosphorylating ERK1/2 protein kinase and to find out the phosphorylation patterns of ERK1/2 in confluent and sub-confluent cells. BCECs were isolated from bovine corneas and cultured in medium supplemented with 10% serum. Confluent (contact inhibited) and sub-confluent (actively growing cells) serum starved cells grown in T-75 flasks were treated with 10nM Endothelin-1. The control cells were left untreated. Total cellular protein was isolated using RIPA buffer and quantified according to the Peterson modification of the Lowry method. The level of expression of phosphorylated ERK1/2 (pp44, pp42) proteins relative to overall ERK1/2 (p44, p42) was determined by western blotting technique. Densitometry analysis of immunoblots revealed differential phosphorylation patterns in confluent and sub-confluent cultures. The pERK1/2 levels were significantly increased at 15 min and 24 hrs after post incubation with ET-1, whereas following the initial rise levels declined to 6hrs of incubation with ET-1 in confluent cultures. In sub-confluent cultures pERK1/2 levels increased gradually to 6hrs of incubation with ET-1, returning to pre-incubation levels at 24hrs. In conclusion, ET-1 treatment was shown to induce phosphorylation of ERK1/2 in BCEC. ET-1 treatment in confluent and sub confluent BCEC exhibited time dependent phosphorylation of ERK1/2. ET-1 treatment affected the phosphorylation pattern distinctively in confluent and sub-confluent BCEC. These observations led to the conclusion that ET-1 induced cellular events in BCEC may involve the MAPK cascade and that these ET-1 induced MAPK cascades may exhibit a negative feedback mechanism, suggested by a distinctive oscillations in pERK 1/2 levels. The contrasting effects of ET-1 in confluent and subconfluent cells may suggest a density dependent phosphatase activity.

Zebrafish, Danio rerio, have become an important model for developmental studies and have several advantages over other model systems. These advantages include (1) the easy accessibility of zebrafish embryos for direct observation of their development and (2) their suitability for systematic mutagenesis studies for the identification of genes regulating the development of various tissues and organs, including the skeletal system. Recently, it has been reported that glutamate receptors are expressed in many types of bone cells and regulate bone physiological functions. In the present study, we have examined the effects of a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist¡Xdextromethorphan¡Xon the development of the axial skeleton in zebrafish embryos by using calcein stain. Our results revealed that dextromethorphan significantly attenuates the formation of the axial skeleton and that it is inhibited on pretreatment with glutamate. Moreover, immunohistochemical analysis revealed protein level expression of the NMDA subunit NR1 in the axial region of zebrafish. Our results also indicate that attenuation of NMDA receptor activity-induced change in the axial skeleton may be related to heat-shock protein and extracellular signal-regulated kinase (ERK) signalings. In conclusion, we suggest that the NMDA receptor plays an important role in the development of the axial skeleton. However, further studies are required on the cellular mechanisms of glutamate regulated bone formation.

Bidens pilosa L. is a traditional Chinese herbal medicine of which was considered as a potential COX2 inhibitor and anti-inflammatory agent. The objective of this study is to discriminate the protective effect of aqueous extract of Bidens pilosa L. leaves (BPLAE) against TAA-induced live fibrosis using an animal model. The herb extracts were administrated via intraperitoneal injection once per week (1.25, 2.5 g/kg), and thioacetamide (200 mg/kg) was injected three times per week and the mice were sacrificed at week 4 and week 8, respectively. Immunohistochemistry staining, Hematoxylin-eosin (HE) staining, Sirius red staining were carried out to evaluate the pathological alterations of mouse livers; in addition, Western blotting was performed to measure the differential expression of £\-smooth muscle actin (£\-SMA) between different treatment groups (vehicle, week 4 and week 8). Hepatic hydroxyproline was also detected in order to compare difference in collagen formation of each group. The results showed that Bidens pilosa L. effectively reduced amount of hepatic hydroxyproline and £\-SMA protein in mice with fibrotic liver induced by TAA. Moreover, in histiopathological exam, the BPLAE treated mice demonstrated a lower collagen and £\-SMA expression, which indicated that BPLAE might reduce degree and severity of liver fibrosis in mice. In conclusion, these results suggested that BPLAE potentially against fibrogenesis in TAA- induced mice liver fibrosis. Additionally, we found that BPLAE might involve in the signaling pathway of MAPK (ERK1/ERK2), which reduced the phosporylation level of p44 but not p42. Further studies using cell base assay to confirm the inhibiting role of BPLAE against cell proliferation or migration is warrant.

Les mâles sexuellement expérimentés présentent des modifications à long terme de l'arborisation dendritique, de modifications épigénétiques ainsi qu'une augmentation des niveaux d'expression de protéines associées à la neurotransmission glutamatergique et à la microglie dans l'aire pré-optique médiane (mPOA). En revanche, les concentrations plasmatiques et hypothalamiques en hormones stéroïdes ainsi que les propriétés du système nitrergique, connues pour être modulés par l'expérience sexuelle chez le rat, ne sont pas modifiées. Dans un second temps, l'implication de la voie de signalisation ERK1/2 dans la réponse comportementale associée à l'expérience sexuelle a été étudiée. Nous avons montré que cette voie de signalisation, activée dans la mPOA lors de l'accouplement, est potentialisée par l'expérience sexuelle. Nous avons ensuite démontré que son activation est possible par une action rapide (30 minutes) des stéroïdes. Enfin, nous avons montré que l'inhibition de la voie ERK1/2 avant un premier accouplement n'altère pas la mise en place de l'expérience sexuelle mais diminue de façon réversible la motivation sexuelle des mâles. Ainsi, l'expérience sexuelle est à l'origine de modifications structurales et biochimiques à long terme de la mPOA. Ces modifications, différentes de celles connues chez le rat, sont associées à une potentialisation de la voie de signalisation ERK1/2 activée de façon transitoires durant l'accouplement. Ces résultats mettent en évidence la nécessité d'élaborer un nouveau modèle, différent de celui établi chez le rat, permettant d'expliquer l'amélioration comportementale associée à l'expérience sexuelle chez la souris mâle. / Sexually experimented males exhibit long term modifications of the dendritic arborization, epigenetic modifications and increased levels of microglia and glutamate associated protein within the hypothalamic medial preoptic area (mPOA). However, hypothalamic and plasmatic concentration of steroid hormones and the nitrergic system are not impacted, contrary to data obtained in rat.The involvement of the ERK1/2 signaling pathway in the induction of sexual experience has also been studied. We showed that ERK1/2 pathway was activated within the mPOA during mating. This activation was increased in sexually experienced males. Furthermore, we showed ex vivo on hypothalamic slices that sex steroids were capable of rapidly (30 min) activate this pathway. Finally, the inhibition of ERK1/2 phosphorylation before the first mating did not disrupt the induction of sexual experience but decreased sexual motivation in a reversible manner.Taken together, these results indicate that long lasting and transitory plasticity mechanisms leading to sexual experience are different between rat and mouse. This indicate the necessity to elaborate a new molecular model associated with the behavioral improvement induced by sexual experience in male mouse.