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. The fast repeating reversed-phase gradients on a short silica-monolithic or core-shell column in the second dimension can be optimized independently of the actual running first-dimension gradient program. As the alternating HILIC and RP separations on the first-dimension zwitterionic methacrylate column are based on complementary retention mechanisms, the instrumental setup essentially represents two coupled two-dimensional systems. It is first time that such an automated dual LCxLC approach is reported. The novel system allows obtaining three-dimensional data in a relatively short time and can be applied not only to multidimensional gradient separations of flavones and related polyphenolic compounds.

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. Assays based on the isomer-specific autophosphorylation of NME1 and phosphoglycerate mutase were used with immunoblotting and sequencing IgG variable domains to screen, select, and characterize anti-1-pHis and anti-3-pHis mAbs. Their sequence independence was determined by blotting synthetic peptide arrays, and they have been tested for immunofluorescence staining and immunoaffinity purification, leading to putative identification of pHis-containing proteins. These reagents should be broadly useful for identification of pHis substrates and functional study of pHis using a variety of immunological, proteomic, and biological assays.

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. Monolithic columns based on organic polymer matrices offer a wide selection of stationary phase chemistries, including new hydrophilic interaction liquid chromatography materials, which can be used in the design of novel two-dimensional separations. Some organic polymer monolithic materials offer a dual retention mechanism (reversed-phase hydrophilic interaction liquid chromatography), so a single column can be used in alternating runs for highly orthogonal off-line two-dimensional and even three-dimensional separations. In the present work, the properties of core-shell and silica gel monolithic columns are briefly summarized and their applications in two-dimensional separations of peptides, proteins, oligomer surfactants, fats and oils, carotenoids, phenolic and flavone compounds in plant extracts, food, and beverages are reviewed.

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. The columns show a dual retention mechanism (hydrophilic-interaction mode) in acetonitrile-rich mobile phases and RP in highly aqueous mobile phases. The new 0.53 mm id columns provided excellent reproducibility, retention, and separation selectivity for phenolic acids and flavonoids. The new zwitterionic monolithic columns are highly orthogonal, with respect to alkyl silica stationary phases, not only in the hydrophilic-interaction mode but also in the RP mode. The optimized monolithic zwitterionic microcolumn of 0.53 mm id was employed in the first dimension, either in the aqueous normal-phase or in the RP mode, coupled with a short nonpolar core-shell column in the second dimension, for comprehensive 2D LC separations of phenolic and flavonoid compounds. When the 2D setup with the sulfobetaine-BIGDMA column was used for repeated sample analysis, with alternating gradients of decreasing (hydrophilic-interaction mode), and increasing (RP mode) concentration of acetonitrile on the sulfobetaine-BIGDMA column in the first dimension, useful complementary information on the sample could be obtained.

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. Columns prepared with pentaerythritol triacrylate or bisphenol A dimethacrylate cross-linkers show large inner pore (mesopore) porosity, and provide poor efficiency, whereas columns with trioxyethylene dimethacrylate (TriEDMA) and tetraoxyethylene dimethacrylate (TeEDMA) cross-linkers showed poor permeability. Columns prepared using dioxyethylene dimethacrylate (DiEDMA), and especially glycerolate dimethacrylate (BIGDMA) cross-linkers, showed best efficiency, with more 60,000-70,000 theoretical plates/m, almost twice in comparison to (poly)methylene dimethacrylate (HEDMA) in the HILIC mode. All columns show dual retention mechanism and can be used for separations of low-molcular compounds such as phenolic acids in the HILIC mode in acetonitrile-rich mobile phases and in the reversed-phase mode in mobile phases with higher concentrations of water. Some columns show broad pore distribution and can be used for size-exclusion chromatography of non-polar polymers in tetrahydrofuran.

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). Columns prepared with polar (poly)oxyethylene dimethacrylate cross-linkers show also improved efficiency with increasing chain length and generally better performance in comparison to the (poly)methylene dimethacrylate cross-linkers of comparable size, however with less apparent effects of the chain lengths on the pore distribution. The monolithic columns prepared with tetraoxyethylene dimethacrylate (TeEDMA) showed the best efficiency of all the columns tested, corresponding to HETP=15 µm (approx. 70,000 theoretical plates/m), show excellent column-to-column reproducibility with standard deviations of 2.5% in retention times, good permeability and low mass transfer resistance, so that is suitable for fast separation of low-molecular compounds in 2 min or less. By modification of the fused-silica capillary inner walls pre-treatment procedure, very good long-term stability was achieved even in 0.5 mm i.d. capillary format. The TeEDMA column can be also used for size-exclusion chromatography of lower non-polar synthetic polymers, whereas it is less suitable for separations of proteins than the HEDMA column.

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. A number of novel cyclic truncated γ-MSH analogues were designed and synthesized, in which a thioether bridge was incorporated between a cysteine side chain and an N-terminal bromoacyl group. One of these peptides, cyclo-[(CH(2))(3)CO-Gly(1)-His(2)-D-Phe(3)-Arg(4)-D-Trp(5)-Cys(S-)(6)]-Asp(7)-Arg(8)-Phe(9)-Gly(10)-NH(2), demonstrated potent antagonist activity and receptor selectivity for the human melanocortin 1 receptor (hMC1R) (IC(50) = 17 nM). This novel peptide is the most selective antagonist for the hMC1R to date. Further pharmacological studies have shown that this peptide can specifically target melanoma cells. The nuclear magnetic resonance analysis of this peptide in a membrane-like environment revealed a new turn structure, specific to the hMC1R antagonist, at the C-terminus, where the side chain and backbone conformation of D-Trp(5) and Phe(9) of the peptide contribute to hMC1R selectivity. Cyclization strategies represent an approach for stabilizing bioactive peptides while keeping their full potencies and should boost applications of peptide-based drugs in human medicine.

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. We synthesized a new polar monolithic sulfobetaine polymethacrylate capillary column with excellent efficiency for low-molecular compounds. When used in the first, HILIC dimension coupled to core-shell or monolithic RP columns in the second dimension, this column provides much improved orthogonality for two-dimensional separations of phenolic and flavonoid compounds, in comparison to silica-bonded Diol, Polyethylene glycol or Zwitterionic columns. We investigated the performance of 11 short 5 cm and 3 cm columns for fast (1-2 min) gradient second-dimension separations. Band broadening or distortion may occur in directly coupled comprehensive HILIC×RP systems, due to strong solvent-strength differences between the mobile phases used in the first and in the second dimension. To suppress this effect, low fraction volumes were collected from a 0.5mm I.D. capillary monolithic sulfobetaine column at the flow-rate of a few microliters per min, coupled in-line with various core-shell columns operated at the maximum flow-rate. This setup with simultaneous gradient elution in the HILIC and in the RP dimension provided successful separation of natural antioxidants.

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 (1.6 and 40.0 mgkg(-1) . 24 h(-1)). Separate groups of hyperalgesic B6 and outbred CD-1 mice were injected with MK-801 or MSG606, selective N-methyl-D-aspartate and melanocortin-1 receptor antagonists, respectively. RESULTS: Morphine infusion (40.0 mg . kg(-1) . 24 h(-1)) reduced baseline withdrawal latencies by 45-55% in B6 mice of both sexes, indicating hyperalgesia; this increased nociception was manifest in male e/e mice only. Although MK-801 reversed hyperalgesia in male mice only, increasing latencies by 72%, MSG606 increased latencies by approximately 60% exclusively in females. A lower morphine infusion dose (1.6 mg . kg(-1) . 24 h(-1)) reduced baseline withdrawal latencies by 45-52% in B6 and e/e mice of both sexes, which was reversed by MK-801, but not MSG606, in both male and female B6 mice. CONCLUSIONS: The data indicate the sex-specific mediation of high-dose morphine-induced hyperalgesia by N-methyl-d-aspartate and melanocortin-1 receptors in male and female mice, respectively, suggesting a broader relevance of this known sexual dimorphism. The data further indicate that the neural substrates contributing to hyperalgesia are morphine dose-dependent.

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. Antagonists-bound melanocortin receptors, on the other hand, did not recruit beta-arrestins, and remained in the cell membrane even after long-term (30 min) treatment. Agonist-mediated internalization of all melanocortin receptor subtypes was sensitive to inhibitors of clathrin-dependent endocytosis, but not to caveolae inhibitors. In summary, agonist-mediated internalization of all subtypes of melanocortin receptors are dependent upon beta-arrestin-mediated clathrin-coated pits, whereas, beta-arrestin-2 conjugated green fluorescence protein (beta-arrestin-2-GFP) recruitment is not dependent on protein kinase A activation. Real time two-photon fluorescence laser scanning microscopy is a most powerful tool to study the dynamic processes in living cells and tissues, without inflicting significant and often lethal damage to the specimen.

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. Finally, substitution of His(5) with Pro(5) and Trp(8) with d-Nal(2')(8) in gamma-MSH-NH(2) leads to a highly potent and selective agonist for the hMC1R. Molecular modeling showed that, at the C-terminal of Nle(3)-gamma-MSH-NH(2), there is a reverse-turn-like structure, suggesting that there might be a secondary binding site involved in ligand-receptor interaction for gamma-MSH analogues that may explain the enhanced binding affinities of the Nle(3)-gamma-MSH-NH(2) analogues. Our results indicate that increasing the hydrophobicity and replacing Phe(6) and Trp(8) with bulkier aromatic amino acid residues is very important for selectivity of alpha-MSH/gamma-MSH hybrids for hMCRs.

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. We have recently studied the agonist and antagonist of the human melanocortin receptors (hMC1, hMC3, hMC4, and hMC5 receptors), the human delta opioid receptor, and the human gluacagon receptor with the help of synthetic fluorescent labeled ligands and fluorescent protein-labeled beta-arrestin-receptors that shed new insight on cellular signaling and rapid screening of drugs in real time. It was demonstrated that stimulation of these receptors by the cognate agonist triggers the rapid internalization of ligand-receptor complexes, while the interaction of the receptor with antagonists does not follow this pathway. Furthermore, receptor internalization is dependent upon beta-arrestin, which has been shown to be responsible for the rapid desensitization of cAMP-signaling processes.

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. We found that women with two variant MC1R alleles displayed significantly greater analgesia from the kappa-opioid, pentazocine, than all other groups. This study demonstrates an unexpected role for the MC1R gene, verifies that pain modulation in the two sexes involves neurochemically distinct substrates, and represents an example of a direct translation of a pharmacogenetic finding from mouse to human.