YAP contributes to DNA methylation remodeling upon mouse embryonic stem cell differentiation

The Yes-associated protein YAP, one of the major effectors of the Hippo pathway together with its related protein TAZ, mediates a range of cellular processes from proliferation and death to morphogenesis. YAP and TAZ regulate a large number of target genes, acting as co-activators of DNA-binding transcription factors or as negative regulators of transcription by interacting with the nucleosome remodeling and histone deacetylase complexes. YAP is expressed in self-renewing embryonic stem cells (ESCs), although it is still debated whether it plays any crucial roles in the control of either stemness or differentiation. Here we show that the transient downregulation of YAP in mouse ESCs perturbs cellular homeostasis, leading to the inability to differentiate properly. Bisulfite genomic sequencing revealed that this transient knockdown caused a genome-wide alteration of the DNA methylation remodeling that takes place during the early steps of differentiation, suggesting that the phenotype we observed might be due to the dysregulation of some of the mechanisms involved in regulation of ESC exit from pluripotency. By gene expression analysis we identified two molecules which could have a role in the altered genome-wide methylation profile: the long non-coding RNA Ephemeron, whose rapid upregulation is crucial for ESCs transition into epiblast, and the methyltransferase-like protein Dnmt3l, which, during the embryo development, cooperates with Dnmt3a and Dnmt3b to contribute to the de novo DNA methylation that governs early steps of ESC differentiation. These data suggest a new role for YAP in the governance of the epigenetic dynamics of exit from pluripotency

Prepuberal stimulation of 5-HT7-R by LP-211 in a rat model of hyper-activity and attention-deficit: permanent effects on attention, brain amino acids and synaptic markers in the fronto-striatal interface

The cross-talk at the prefronto-striatal interface involves excitatory amino acids, different receptors, transducers and modulators. We investigated long-term effects of a prepuberal, subchronic 5-HT7-R agonist (LP-211) on adult behaviour, amino acids and synaptic markers in a model for Attention-Deficit/Hyperactivity Disorder (ADHD). Naples High Excitability rats (NHE) and their Random Bred controls (NRB) were daily treated with LP-211 in the 5th and 6th postnatal week. One month after treatment, these rats were tested for indices of activity, non selective (NSA), selective spatial attention (SSA) and emotionality. The quantity of L-Glutamate (L-Glu), L-Aspartate (L-Asp) and L-Leucine (L-Leu), dopamine transporter (DAT), NMDAR1 subunit and CAMKIIα, were assessed in prefrontal cortex (PFC), dorsal (DS) and ventral striatum (VS), for their role in synaptic transmission, neural plasticity and information processing. Prepuberal LP-211 (at lower dose) reduced horizontal activity and (at higher dose) increased SSA, only for NHE but not in NRB rats. Prepuberal LP-211 increased, in NHE rats, L-Glu in the PFC and L-Asp in the VS (at 0.250 mg/kg dose), whereas (at 0.125 mg/kg dose) it decreased L-Glu and L-Asp in the DS. The L-Glu was decreased, at 0.125 mg/kg, only in the VS of NRB rats. The DAT levels were decreased with the 0.125 mg/kg dose (in the PFC), and increased with the 0.250 mg/kg dose (in the VS), significantly for NHE rats. The basal NMDAR1 level was higher in the PFC of NHE than NRB rats; LP-211 treatment (at 0.125 mg/kg dose) decreased NMDAR1 in the VS of NRB rats. This study represents a starting point about the impact of developmental 5-HT7-R activation on neuro-physiology of attentive processes, executive functions and their neural substrates

Analysis of ACE inhibitor drugs by high performance liquid chromatography.

Seven angiotensin-converting-enzyme (ACE) inhibitors have been analyzed by HPLC. The influence of different organic modifiers and counter-ions in the eluent at different pH values have been investigated allowing the identification of the best experimental conditions for the analysis of these compounds. The sinergic effect of TEA and propanol in the eluents resulted as a critical factor to obtain satisfactory peak shapes and resolution

Interactions of nonsteroidal antiinflammatory drugs with phospholipids: comparison between octanol/buffer partition coefficients and chromatographic indexes on immobilized artificial membranes.

A set of seventeen nonsteroidal antiinflammatory drugs (NSAIDs), consisting of structurally unrelated carboxylic acids and piroxicam, was examined by high-performance liquid chromatography (HPLC) on an immobilized artificial membrane (IAM) column that is a solid-phase model of fluid membranes. The chromatographic capacity factors extrapolated to 100\% aqueous phase (log KWIAM) were compared with n-octanol/buffer lipophilicity parameters. The interactions with phospholipids were much better predicted from the intrinsic partition coefficient, log P, than from the apparent partition value, log D7.4, indicating that phospholipids can counteract the influence of electrically charged functions of analytes on lipophilic interactions. The log KWIAM and log P values for both NSAIDs and structurally unrelated neutral compounds result in unique scale if uniquely partition-based mechanisms take place. However, an electrostatic repulsion component was observed for the NSAIDs bearing the carboxylic function directly linked to the aromatic ring, and for ibuprofen. Hence, the IAM-derived scale is distinctive from the one obtained by lipophilic parameters. The IC50 values on cyclooxygenase 2 (COX-2) in intact cells determined by different authors have been successfully correlated with respective IAM parameters, whereas no correlation was found with COX-1 activity data. These results suggest that membrane affinity may represent an important prerequisite for the specific binding NSAIDs/COX-2

Chromatographic indexes on immobilized artificial membranes for local anesthetics: relationships with activity data on closed sodium channels.

PURPOSE: To elucidate the effectiveness of the different parameters for the prediction of biological activity, the n-octanol/buffer partition coefficients and theoretical calculated lipophilicity parameters of thirteen local anesthetic drugs (LAs), including two beta-blockers, were compared to the affinity values for phospholipids, calculated by a recent technique. METHODS: Interactions with phospholipids were measured by high performance liquid chromatography on a stationary phase made up of phospholipids, the so-called "Immobilized Artificial Membrane" (IAM). Reference lipophilicity parameters were measured by shake-flask method between n-octanol and buffer phases. RESULTS: Interactions with phospholipids were predicted from log P for all compounds except tocainide, which also showed additive polar extra-interactions. Moreover, when the retention on Immobilized Artificial Membrane (IAM) phase was mainly lipophilicity-based, a unique scale included the correlation between log kwIAM and log P values, for both LAs (bases) and the structurally unrelated (nonionizable and acidic) compounds previously studied. IAM interaction values for LAs were predictive of the partition measures on liposome membranes already reported in literature. The half-blocking doses for closed sodium channel, corrected for ionization at pH 7.4, were successfully correlated with the respective IAM values for eleven compounds while procaine and tetracaine, which are ester-linked compounds and have a p-amino group as well, gave more potent results than predicted by phospholipid interactions. CONCLUSIONS: The IAM chromatographic parameters were much more effective than reference lipophilicity values in describing partition on model membranes and in predicting pharmacological potency on closed sodium channels

Retention behaviour of anti-emetic serotonin antagonists in reversed phase high performance liquid chromatography.

Granisetron, Ondansetron and Tropisetron, three 5-HT3 antagonists showing anti-emetic activity, were analysed by HPLC on lipophilic stationary phases. The addition of an amine or quaternary ammonium salt to the eluents was a powerful tool in the analysis of these basic substances. The influence on chromatographic parameters of pH, ionic strength and various counter-ions in the aqueous phase as well as of different organic modifiers is discussed. Some of the proposed experimental conditions allow a more strictly partition-based separation mechanism and can produce chromatographic parameters suitable for structure-activity studies. These experimental conditions are also suitable for analysis of the considered compounds in pharmaceutical dosage forms or in biological fluids

Inclusion complexation of carbaryl and -cyclodextrin in solution and in the solid state

This study was carried out with the aim of investigating the interactions between beta-cyclodextrin and carbaryl, a carbamate pesticide, and their effect on some physico-chem. properties of carbaryl, such as aq. soly. and lipophilicity. The interactions between carbaryl and beta-cyclodextrin were thoroughly investigated both in soln. and in the solid state. The effect of beta-cyclodextrin on the aq. soly. of carbaryl was evaluated by the phase soly. method. The amt. of carbaryl dissolved increased linearly with the addn. of beta-cyclodextrin according to an AL type plot and without pptn. of the complex. The apparent stability const. of the complex was 289 +/- 21 M-1, assuming a 1:1 stoichiometry; this value was confirmed by a method based on CD measurements. Equimolar carbaryl/beta-cyclodextrin solid systems were prepd. by phys.-mixing and freeze-drying, and fully characterized by Differential Scanning Calorimetry, X-ray powder diffractometry and Fourier Transform Infra-Red anal. The results of the solid state study demonstrated that the freeze-drying method yields a system with a high degree of amorphization and yields an inclusion complex. The dissoln. profile of the pesticide was affected by the physico-chem. properties of each solid system, the freeze-dried form dissolving more rapidly. However, the phys. assocn. of beta-cyclodextrin and carbaryl enhanced the aq. soly. of the insecticide as well

Diclofenac/beta-cyclodextrin binary systems: a study in solution and in the solid state

This study was carried out with the aim to optimize the dissoln. properties of diclofenac (DIC) - a non-steroidal anti-inflammatory drug sparingly sol. in water - through assocn. with beta-cyclodextrin (betaCD). The effect of betaCD on the aq. soly. of DIC was evaluated by the phase soly. method. The amt. of DIC dissolved increased linearly with the addn. of betaCD according to an AL type plot and without pptn. of the complex. The apparent stability const. of the complex, calcd. supposing a 1:1 stoichiometry, was 295 M-1; this value was confirmed by CD anal. DIC/betaCD interactions were also studied in water by 1H and 13C NMR spectroscopy. Equimolar DIC/betaCD solid systems were prepd. by phys.-mixing, kneading, co-evapn. and freeze drying, and their properties in the solid state studied by differential scanning calorimetry, x-ray powder diffractometry and Fourier-Transform IR anal. For sake of comparison, the mixt. of DIC and betaCD sep. lyophilized was investigated too. The results demonstrated that the freeze-dried product had the highest degree of amorphization and they were in agreement with the existence of an inclusion complex in the solid state. The dissoln. profiles of the drug from each solid system were affected by its physico-chem. properties, the freeze-dried being the most rapidly dissolving forms