Automated dual two-dimensional liquid chromatography approach for fast acquisition of three-dimensional data using combinations of zwitterionic polymethacrylate and silica-based monolithic columns.

A monolithic sulfobetaine polymethacrylate micro-column BIGDMA-MEDSA designed in our laboratory, shows dual retention mechanism: In acetonitrile-rich mobile phase, hydrophilic interactions control the retention (HILIC system), whereas in more aqueous mobile phases the column shows essentially reversed-phase behavior with major role of hydrophobic interactions. The zwitterionic polymethacrylate micro-column can be used in the first dimension of two-dimensional LC in alternating reversed-phase (RP) and HILIC modes, coupled with an alkyl-bonded core-shell or silica-based monolithic column in the second dimension, for HILIC×RP and RP×RP comprehensive two-dimensional separations. During the HILIC×RP period, a gradient of decreasing acetonitrile gradient is used for separation in the first dimension, so that at the end of the gradient the polymeric monolithic micro-column is equilibrated with a highly aqueous mobile phase and is ready for repeated sample injection, this time for separation under reversed-phase gradient conditions with increasing concentration of acetonitrile in the first dimension.

Monoclonal 1- and 3-Phosphohistidine Antibodies: New Tools to Study Histidine Phosphorylation.

Histidine phosphorylation (pHis) is well studied in bacteria; however, its role in mammalian signaling remains largely unexplored due to the lack of pHis-specific antibodies and the lability of the phosphoramidate (P-N) bond. Both imidazole nitrogens can be phosphorylated, forming 1-phosphohistidine (1-pHis) or 3-phosphohistidine (3-pHis). We have developed monoclonal antibodies (mAbs) that specifically recognize 1-pHis or 3-pHis; they do not cross-react with phosphotyrosine or the other pHis isomer.

Monolithic and core-shell columns in comprehensive two-dimensional HPLC: a review.

The crucial point affecting the separation time in comprehensive two-dimensional liquid chromatography is the performance of the column used in the second dimension, which should allow highly efficient fast chromatographic separations in the short time available for the analysis of fractions transferred from the first to the second dimension (often 1 min or less). This can be accomplished on short columns packed with sub-2-μm particles, at the cost of very high operation pressure. Core-shell or silica monolithic columns have better permeability, and their use in the second dimension of comprehensive two-dimensional liquid chromatography with conventional liquid chromatography instrumentation is continuously increasing.

New zwitterionic polymethacrylate monolithic columns for one- and two-dimensional microliquid chromatography.

We prepared 0.53 and 0.32 mm id monolithic microcolumns by in situ copolymerization of a zwitterionic sulfobetaine functional monomer with bisphenol A glycerolate dimethacrylate (BIGDMA) and dioxyethylene dimetacrylate crosslinkers.

Cross-linker effects on the separation efficiency on (poly)methacrylate capillary monolithic columns. Part II. Aqueous normal-phase liquid chromatography.

We synthesized seven different polymethacrylate monolithic capillary columns using N,N-dimethyl-N-metacryloxyethyl-N-(3-sulfopropyl) ammonium betaine functional monomer (MEDSA) and various cross-linking monomers differing in the polarity and size. The efficiency of monolithic columns for polar low-molecular compounds in the aqueous normal-phase (HILIC) mode depends rather on the polarity, than on the size of the cross-linker molecules. Cross-linking molecules, which exhibit high polarity can produce poly(methacrylate) monoliths with an increase in pore sizes below 50nm.

An unusual conformation of γ-melanocyte-stimulating hormone analogues leads to a selective human melanocortin 1 receptor antagonist for targeting melanoma cells.

γ-MSH (γ-melanocyte-stimulating hormone, H-Tyr-Val-Met-Gly-His-Phe-Arg-Trp-Asp-Arg-Phe-Gly-OH), with its exquisite specificity and potency, has recently created much excitement as a drug lead. However, this peptide is like most peptides susceptible to proteolysis in vivo, which potentially decreases its beneficial activities. In our continued effort to design a proteolytically stable ligand with specific receptor binding, we have engineered peptides by cyclizing γ-MSH using a thioether bridge.

Cross-linker effects on the separation efficiency on (poly)methacrylate capillary monolithic columns. Part I. Reversed-phase liquid chromatography.

We synthesized 8 polymethacrylate monolithic capillary columns using laurylmethacrylate functional monomer and various cross-linking monomers differing in the polarity and size. The efficiency of monolithic columns for low-molecular compounds significantly improved with increasing number of repeat non-polar methylene groups in the cross-linker molecules, correlating with greater proportion of small pores with size less than 50 nm. The best efficiency with HETP=25 μm for alkylbenzenes was achieved for columns prepared using hexamethylene dimethacrylate (HEDMA).

Optimization of comprehensive two-dimensional gradient chromatography coupling in-line hydrophilic interaction and reversed phase liquid chromatography.

In-line coupled comprehensive HILIC×RP systems should offer larger selectivity differences and better two-dimensional orthogonality than coupled RP×RP systems. However, this may not apply for all systems. The HILIC selectivity depends on the mix of selective polar and non-polar interactions with the functional groups, but also with the matrix of polar columns and depends on the sample type.

Sex-specific mediation of opioid-induced hyperalgesia by the melanocortin-1 receptor.

Background: N-Methyl-D-aspartate receptor antagonists reverse hyperalgesia during morphine infusion in male mice only. Because the melanocortin-1 receptor can act as a female-specific counterpart to N-methyl-D-aspartate receptors in kappa-opioid analgesic mechanisms, the authors assessed the contribution of melanocortin-1 receptors to the sex-specific mechanisms underlying morphine hyperalgesia.

Methods: The tail-withdrawal test was used to compare the nociceptive responses of male and female C57BL/6J (B6) mice with those of C57BL/6J-Mc(1r(e/e)) mice, spontaneous mutants of the B6 background lacking functional melanocortin-1 receptors, during continuous morphine infusion (1.

Cell signaling and trafficking of human melanocortin receptors in real time using two-photon fluorescence and confocal laser microscopy: differentiation of agonists and antagonists.

Melanocortin hormones and neurotransmitters regulate a vast array of physiologic processes by interacting with five G-protein-coupled melanocortin receptor types. In the present study, we have systematically studied the regulation of individual human melanocortin receptor wild subtypes using a synthetic rhodamine-labeled human melanotropin agonist and antagonist, arrestins fused to green fluorescent protein in conjunction with two-photon fluorescence laser scanning microscopy and confocal microscopy. Stimulation of the melanocortin receptors by its cognate agonist triggered rapid arrestin recruitment and receptor internalization for all four human melanocortin receptors examined.

Design of novel melanotropin agonists and antagonists with high potency and selectivity for human melanocortin receptors.

alpha-MSH and gamma-MSH are the natural endogenous hormones for the human melanocortin-1, 3, 4 and 5 receptors (hMC1R, hMC3R, hMC4R and hMC5R). These and more potent, stable and prolonged acting analogues such as NDP-alpha-MSH, MT-II and SHU-9119 are not very receptor selective. To develop potent and selective agonist and antagonist ligands for the melanocortin receptors we have used state-of-the-art biophysical studies, computational chemistry, and design of conformational and topographical constraints with novel templates.

Novel 3D pharmacophore of alpha-MSH/gamma-MSH hybrids leads to selective human MC1R and MC3R analogues.

To further evaluate elements that could contribute to the 3D topographical structure of gamma-MSH, we have systematically designed a group of linear gamma-MSH analogues and evaluated their biological activities: without a N-terminal acetyl, with and without a C-terminal amide, with Nle(3), with l- or d-Phe(6) or d-Nal(2')(6), and with d-Trp(8) or d-Nal(2')(8). It was found that changing the C-terminal acid in gamma-MSH to an amide and replacing Met with Nle leads to increased binding affinities at all four subtypes of melanocortin receptors (10-100 fold). Substitution of Trp(8) with d-Nal(2')(8) and Phe(6) with d-Phe(6) in gamma-MSH-NH(2) forms a selective antagonist for the hMC3R, whereas, substitution of Phe(6) with d-Nal(2')(6) and replacing Trp(8) with d-Trp(8) at gamma-MSH-NH(2) yields a selective partial agonist for the hMC1R.

Real time differentiation of G-protein coupled receptor (GPCR) agonist and antagonist by two photon fluorescence laser microscopy.

Receptor-based signaling mechanisms are the primary source of cellular regulation. The superfamily of G protein-coupled receptors (GPCR) is the largest and most ubiquitous of the receptor-mediated processes. Desensitization of G-protein-coupled receptors is a fundamental mechanism regulating the cellular response to agonists.

The melanocortin-1 receptor gene mediates female-specific mechanisms of analgesia in mice and humans.

Sex specificity of neural mechanisms modulating nociceptive information has been demonstrated in rodents, and these qualitative sex differences appear to be relevant to analgesia from kappa-opioid receptor agonists, a drug class reported to be clinically effective only in women. Via quantitative trait locus mapping followed by a candidate gene strategy using both mutant mice and pharmacological tools, we now demonstrate that the melanocortin-1 receptor (Mc1r) gene mediates kappa-opioid analgesia in female mice only. This finding suggested that individuals with variants of the human MC1R gene, associated in our species with red hair and fair skin, might also display altered kappa-opioid analgesia.