HPLC/UV/MS method application for the separation of obeticholic acid and its related compounds in development process and quality control.

An HPLC method with UV and electrospray ionization - mass spectrometry (ESI-MS) detection was developed for the separation and determination of obeticholic acid (OBE) and its related compounds in development process and quality control. OBE and its related compounds were classified into three major group based on the mass spectra profiles: (A) those containing a hydroxyl group at position 3 and 7, (B) those containing a hydroxyl group and/or carbonyl group at position 3, hydrogen, ethyl or ethylidene group at position 6 and a hydroxyl group and/or carbonyl group at position 7, and (C) those containing carbonyl groups at position 3 and 7. ESI-MS ionization of OBE and its related compounds often produced intense adduct ions [M+H+98] and/or [M+H+196] that were identified as the adduct ions of phosphoric acid ([M+H+HPO] and [M+H+2HPO]) originating from the mobile phase.

A novel approach for HPLC determination of 2-cynaoacetamide using derivatization procedure with 2-hydroxyacetophenone as a new useful derivatization reagent.

A novel and sensitive derivatization procedure for the determination of 2-cynaoacetamide in pharmaceutical samples using liquid chromatography with the fluorescence detection was discovered. The method is based on derivatization of 2-cynaoacetamide using 2-hydroxyacetophenone as a new derivatization reagent. The product of derivatization reaction was isolated and characterized using spectroscopic techniques namely LC-MS, NMR and IR.

A two-state model for the diffusion of the A2A adenosine receptor in hippocampal neurons: agonist-induced switch to slow mobility is modified by synapse-associated protein 102 (SAP102).

The A2A receptor is a class A/rhodopsin-like G protein-coupled receptor. Coupling to its cognate protein, Gs, occurs via restricted collision coupling and is contingent on the presence of cholesterol. Agonist activation slows diffusion of the A2A adenosine receptor in the lipid bilayer.

A single allele of Hdac2 but not Hdac1 is sufficient for normal mouse brain development in the absence of its paralog.

The histone deacetylases HDAC1 and HDAC2 are crucial regulators of chromatin structure and gene expression, thereby controlling important developmental processes. In the mouse brain, HDAC1 and HDAC2 exhibit different developmental stage- and lineage-specific expression patterns. To examine the individual contribution of these deacetylases during brain development, we deleted different combinations of Hdac1 and Hdac2 alleles in neural cells.

Mechanism of hERG channel block by the psychoactive indole alkaloid ibogaine.

Ibogaine is a psychoactive indole alkaloid. Its use as an antiaddictive agent has been accompanied by QT prolongation and cardiac arrhythmias, which are most likely caused by human ether a go-go-related gene (hERG) potassium channel inhibition. Therefore, we studied in detail the interaction of ibogaine with hERG channels heterologously expressed in mammalian kidney tsA-201 cells.

Reengineering the collision coupling and diffusion mode of the A2A-adenosine receptor: palmitoylation in helix 8 relieves confinement.

The A(2A)-adenosine receptor undergoes restricted collision coupling with its cognate G protein G(s) and lacks a palmitoylation site at the end of helix 8 in its intracellular C terminus. We explored the hypothesis that there was a causal link between the absence of a palmitoyl moiety and restricted collision coupling by introducing a palmitoylation site. The resulting mutant A(2A)-R309C receptor underwent palmitoylation as verified by both mass spectrometry and metabolic labeling.

Synthesis and pharmacological effects of the enantiomers of the N-phenethyl analogues of the ortho and para e- and f-oxide-bridged phenylmorphans.

The N-phenethyl analogues of (1R*,4aR*,9aS*)-2-phenethyl-1,3,4,9a-tetrahydro-2H-1,4a-propanobenzofuro[2,3-c]pyridin-6-ol and 8-ol and (1R*,4aR*,9aR*)-2-phenethyl-1,3,4,9a-tetrahydro-2H-1,4a-propanobenzofuro[2.3-c]pyridin-6-ol and 8-ol, the ortho- (43) and para-hydroxy e- (20), and f-oxide-bridged 5-phenylmorphans (53 and 26) were prepared in racemic and enantiomerically pure forms from a common precursor, the quaternary salt 12. Optical resolutions were accomplished by salt formation with suitable enantiomerically pure chiral acids or by preparative HPLC on a chiral support.

Profound inhibition of antigen-specific T-cell effector functions by dasatinib.

Purpose: The dual BCR-ABL/SRC kinase inhibitor dasatinib entered the clinic for the treatment of chronic myeloid leukemia and Ph+ acute lymphoblastic leukemia. Because SRC kinases are known to play an important role in physiologic T-cell activation, we analyzed the immunobiological effects of dasatinib on T-cell function. The effect of dasatinib on multiple T-cell effector functions was examined at clinically relevant doses (1-100 nmol/L); the promiscuous tyrosine kinase inhibitor staurosporine was used as a comparator.

Essential role of B-Raf in oligodendrocyte maturation and myelination during postnatal central nervous system development.

Mutations in the extracellular signal-regulated kinase (ERK) pathway, particularly in the mitogen-activated protein kinase/ERK kinase (MEK) activator B-Raf, are associated with human tumorigenesis and genetic disorders. Hence, B-Raf is a prime target for molecule-based therapies, and understanding its essential biological functions is crucial for their success. B-Raf is expressed preferentially in cells of neuronal origin.

The A(2A)-adenosine receptor: a GPCR with unique features?

The A(2A)-adenosine receptor is a prototypical G(s)-coupled receptor. However, the A(2A)-receptor has several structural and functional characteristics that make it unique. In contrast to the classical model of collision coupling described for the beta-adrenergic receptors, the A(2A)-receptor couples to adenylyl cyclase by restricted collision coupling and forms a tight complex with G(s).

Restricted collision coupling of the A2A receptor revisited: evidence for physical separation of two signaling cascades.

The A(2A)-adenosine receptor is a prototypical G(s) protein-coupled receptor but stimulates MAPK/ERK in a G(s)-independent way. The A(2A) receptor has long been known to undergo restricted collision coupling with G(s); the mechanistic basis for this mode of coupling has remained elusive. Here we visualized agonist-induced changes in mobility of the yellow fluorescent protein-tagged receptor by fluorescence recovery after photobleaching microscopy.

Heterotrimeric G protein-independent signaling of a G protein-coupled receptor. Direct binding of ARNO/cytohesin-2 to the carboxyl terminus of the A2A adenosine receptor is necessary for sustained activation of the ERK/MAP kinase pathway.

The A2A adenosine receptor is a prototypical G(s)-coupled receptor, but it also signals, e.g. to mitogen-activated protein (MAP) kinase, via a pathway that is independent of heterotrimeric G proteins.

p21cip1 is required for the differentiation of oligodendrocytes independently of cell cycle withdrawal.

Differentiation of most cell types requires both establishment of G1 arrest and the induction of a program related to achieving quiescence. We have chosen to study the differentiation of oligodendrocyte cells to determine the role of p27 and p21 in this process. Here we report that both p27 and p21 are required for the appropriate differentiation of these cells.

The cyclin-dependent kinase inhibitor p21cip1 mediates the growth inhibitory effect of phorbol esters in human venous endothelial cells.

Long-term application of the phorbol ester phorbol 12,13-dibutyrate (PDBu) inhibits the proliferation of human venous endothelial cells. The cyclin-dependent kinase inhibitor p21cip1 is a potential candidate mediating the PDBu-induced delayed entry of the cells into S-phase (by approximately 10 h when compared with cells stimulated with basic fibroblast growth factor (bFGF)). Levels of p21cip1 (protein and mRNA) rapidly rise (within approximately 2 h) in endothelial cells treated with the active isomer beta-PDBu, but not with alpha-PDBu; this effect is blocked by the mitogen-activated protein kinase kinase-1 (Mek1) inhibitor PD098059 and by the protein kinase C (PKC) antagonists GF109203X and rottlerin (selective for PKC-delta), but not Gö 6976 (selective for Ca2+-dependent PKC isoforms).

Interaction of allosteric ligands with GABAA receptors containing one, two, or three different subunits.

The presence of allosteric binding sites on recombinant GABAA receptors formed after transfection of human embryonic kidney (HEK) 293 cells with alpha 1-, beta 3-, or gamma 2-subunits, or with various combinations of these subunits, was systematically investigated. From all possible subunit combinations, high affinity [3H]muscimol binding sites were induced in cells transfected with alpha 1 beta 3- or alpha 1 beta 3 gamma 2-subunits only. GABAA receptor associated [3H]flunitrazepam binding sites were induced in cells after transfection with alpha 1 gamma 2- or alpha 1 beta 3, gamma 2-subunits, and [35S]r-butylbicyclophosphorothionate (TBPS) binding sites were found in cells transfected with beta 3-, beta 3 gamma 2-, alpha 1 beta 3-, or alpha 1 beta 3 gamma 2-subunits.

Allosteric modulation of [3H]flunitrazepam binding to recombinant GABAA receptors.

The allosteric modulation of [3H]flunitrazepam binding by gamma-aminobutyric acid (GABA), pentobarbital, (+)-etomidate, etazolate, alphaxalone, propofol and chlormethiazole was investigated in cerebellar membranes and membranes from human embryonic kidney (HEK) 193 cells transfected with alpha 1 beta 3 gamma 2 or alpha 1 gamma 2 subunits. Results obtained indicate that [3H]flunitrazepam binding to recombinant GABAA receptors consisting of alpha 1 beta 3 gamma 2 subunits could be modulated by these compounds in a way and with a potency similar to that observed in cerebellar membranes. In addition, it was demonstrated that not only receptors consisting of alpha 1 beta 3 gamma 3, but also those consisting of alpha 1 gamma 2 subunits exhibited [3H]flunitrazepam binding which could be stimulated by GABA.

Rat beta 3 subunits expressed in human embryonic kidney 293 cells form high affinity [35S]t-butylbicyclophosphorothionate binding sites modulated by several allosteric ligands of gamma-aminobutyric acid type A receptors.

Human embryonic kidney 293 cells transiently transfected with beta 3 subunits of gamma-aminobutyric acid type A receptors from the rat exhibited a specific high affinity binding for [35S]t-butylbicyclophosphorothionate (TBPS) that could be inhibited by pentobarbital, etazolate, (+)-etomidate, alphaxalone, propofol, chlormethiazole, and Ro 5-4864. The potency of these compounds for inhibition of [35S]TBPS binding was similar in membranes from beta 3 subunit-transfected human embryonic kidney 293 cells and in cerebellar membranes. In contrast to maximally inhibiting concentrations of unlabeled TBPS or picrotoxin, which caused a monophasic and rather slow dissociation of [35S]TBPS, maximally inhibiting concentrations of pentobarbital, etazolate, alphaxalone, propofol, chlormethiazole, and Ro 5-4864 accelerated the dissociation of [35S]TBPS from beta 3 subunit-containing membranes.

[3H]propyl-6-azido-beta-carboline-3-carboxylate: a new photoaffinity label for the GABAA-benzodiazepine receptor.

[3H]Propyl-6-azido-beta-carboline-3-carboxylate ([3H]ACCP) exhibited a high affinity for GABAA receptors affinity purified from the brains of adult rats, and binding of this compound could be inhibited by several ligands of the benzodiazepine binding site of GABAA receptors. On irradiation with UV light, [3H]ACCP, similarly to [3H]flunitrazepam, irreversibly labeled a protein with an apparent molecular weight of 51 kDa in affinity-purified GABAA receptors, and this labeling could be inhibited in the presence of diazepam. These data indicate that [3H]ACCP can be used as a photoaffinity label for GABAA receptors.

Endogenous [3H]flunitrazepam binding in human embryonic kidney cell line 293.

Specific endogenous [3H]flunitrazepam binding sites were identified and characterized in membranes from the human embryonic kidney (HEK) cell line 293. A large part of these binding sites exhibited an intermediate affinity for [3H]flunitrazepam and a microM affinity for diazepam, clonazepam, 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxam ide (PK 11195) or 4'-chlorodiazepam (Ro 5-4864). These sites, thus, resembled neither gamma-aminobutyric acidA (GABAA) receptor associated nor 'peripheral' benzodiazepine binding sites.

Cyclosporin A reduces skin collagen content in renal graft recipients.

Stimulated by the observation of a direct cytopathic effect of cyclosporin A on dermal fibroblasts we have examined total skin collagen content and collagenase activity in three groups of patients. Group 1 (controls) consisted of 16 patients without internal diseases, group 2 of 12 patients with renal transplantation on cyclosporin A therapy and group 3 of six patients with renal transplantation on corticosteroid/azathioprine therapy.Total skin collagen was measured by hydroxyproline/protein determination, collagenase activity according to the principle of Wünsch.

Separation of alpha 1, alpha 2 and alpha 3 subunits of the GABAA-benzodiazepine receptor complex by immunoaffinity chromatography.

Polyclonal antibodies raised to synthetic amino acid sequences of the rat gamma-aminobutyric acid (GABAA) receptor alpha 1-, alpha 2- and alpha 3-subunits selectively recognized single proteins with apparent molecular weight 51 kDa (P51), 53 kDa (P53) and 59 kDa (P59), respectively, in GABAA receptor preparations affinity purified from the brains of 5-10-day-old rats. The antibodies were coupled to Affigel 10, and the resulting immunoaffinity columns were used to isolate these proteins from affinity purified GABAA receptors.

Isolation of type I and type II GABAA-benzodiazepine receptors by immunoaffinity chromatography.

Anti-peptide alpha 1 (1-9) and anti-peptide alpha 3 (459-467) antibodies coupled to Affigel-10 were used for the isolation of GABAA receptors containing the alpha 1- or alpha 3-subunit, respectively. Both types of GABAA receptors exhibited a high affinity for [3H]flunitrazepam, and binding of [3H]flunitrazepam was stimulated in the presence of GABA. GABAA receptors eluted from the anti-peptide alpha 1 (1-9) immunoaffinity column exhibited a high affinity and those from the anti-peptide alpha 3 (459-467) columns a low affinity for the type I benzodiazepine receptor-selective ligand Cl 218872, indicating the enrichment of type I and type II GABAA-benzodiazepine receptors, respectively.