Reversed-phase HPLC enantioseparation of pantoprazole using a teicoplanin aglycone stationary phase—Determination of the enantiomer elution order using HPLC-CD analyses

A direct HPLC method was developed for the enantioseparation of pantoprazole using macrocyclic glycopeptide-based chiral stationary phases, along with various methods to determine the elution order without isolation of the individual enantiomers. In the preliminary screening, four macrocyclic glycopeptide-based chiral stationary phases containing vancomycin (Chirobiotic V), ristocetin A (Chirobiotic R), teicoplanin (Chirobiotic T), and teicoplanin-aglycone (Chirobiotic TAG) were screened in polar organic and reversed-phase mode. Best results were achieved by using Chirobiotic TAG column and a methanol-water mixture as mobile phase. Further method optimization was performed using a face-centered central composite design to achieve the highest chiral resolution. Optimized parameters, offering baseline separation (resolution = 1.91 ± 0.03) were as follows: Chirobiotic TAG stationary phase, thermostated at 10°C, mobile phase consisting of methanol/20mM ammonium acetate 60:40 v/v, and 0.6 mL/min flow rate. Enantiomer elution order was determined using HPLC hyphenated with circular dichroism (CD) spectroscopy detection. The online CD signals of the separated pantoprazole enantiomers at selected wavelengths were compared with the structurally analogous esomeprazole enantiomer. For further verification, the inline rapid, multiscan CD signals were compared with the quantum chemically calculated CD spectra. Furthermore, docking calculations were used to investigate the enantiorecognition at molecular level. The molecular docking shows that the R-enantiomer binds stronger to the chiral selector than its antipode, which is in accordance with the determined elution order on the column—S- followed by the R-isomer. Thus, combined methods, HPLC-CD and theoretical calculations, are highly efficient in predicting the elution order of enantiomers.     

Accumulation of the hydroxyl free radical markers meta-, ortho-tyrosine and DOPA in cataractous lenses is accompanied by a lower protein and phenylalanine content of the water-soluble phase

Post-translational modifications of lens proteins play a crucial role in the formation of cataract during ageing. The aim of our study was to analyze protein composition of the cataractous lenses by electrophoretic and high-performance liquid chromatographic (HPLC) methods.

Samples were obtained after extracapsular cataract surgery performed by phacoemulsification technique from cataract patients with type 2 diabetes mellitus (DM CAT, n = 22) and cataract patients without diabetes (non-DM CAT, n = 20), while non-diabetic non-cataractous lenses obtained from cadaver eyes served as controls (CONTR, n = 17). Lens fragments were derived from the surgical medium by centrifugation. Samples were homogenized in a buffered medium containing protease inhibitor. Soluble and insoluble protein fractions were separated by centrifugation. The electrophoretic studies were performed according to Laemmli on equal amounts of proteins and were followed by silver intensification. Oxidized amino acid and Phe content of the samples were also analyzed by HPLC following acid hydrolysis of proteins.

Our results showed that soluble proteins represented a significantly lower portion of the total protein content in cataractous lenses in comparison with the control group (CONTR, 71.25%; non-DM CAT, 32.00%; DM CAT, 33.15%; p < 0.05 vs CONTR for both). Among the proteins, the crystallin-like proteins with low-molecular weight can be found both in the soluble and insoluble fractions, and high-molecular weight aggregates were found mainly in the total homogenates. In our HPLC analysis, oxidatively modified derivatives of phenylalanine were detected in cataractous samples. We found higher levels of m-Tyr, o-Tyr and DOPA in the total homogenates of cataractous samples compared to the supernatants. In all three groups, the median Phe/protein ratio of the total homogenates was also higher than that of the supernatants (total homogenates vs supernatants, in the CONTR group 1102 vs 633 μmol/g, in the DM CAT group 1187 vs 382 μmol/g and in the non-DM CAT group 967 vs 252 μmol/g; p < 0.05 for all).

In our study we found that oxidized amino acids accumulate in cataractous lenses, regardless of the origin of the cataract. The accumulation of the oxidized amino acids probably results from oxidation of Phe residues of the non-water soluble lens proteins. We found the presence of high-molecular weight protein aggregates in cataractous total homogenates, and a decrease of protein concentration in the water-soluble phase of cataractous lenses. The oxidation of lens proteins and the oxidative modification of Phe residues in key positions may lead to an altered interaction between protein and water molecules and thus contribute to lens opacification.     

Performance tradeoffs of energy-aware virtual machine consolidation

Increasing power consumption of IT infrastructures and growing electricity prices have led to the development of several energy-saving techniques in the last couple of years. Virtualization and consolidation of services is one of the key technologies in data centers to reduce overprovisioning and therefore increase energy savings. This paper shows that the energy-optimal allocation of virtualized services in a heterogeneous server infrastructure is NP-hard and can be modeled as a variant of the multidimensional vector packing problem. Furthermore, it proposes a model to predict the performance degradation of a service when it is consolidated with other services. The model allows considering the tradeoff between power consumption and service performance during service allocation. Finally, the paper presents two heuristics that approximate the energy-optimal and performance-aware resource allocation problem and shows that the allocations determined by the proposed heuristics are more energy-efficient than the widely applied maximum-density consolidation.     

Structural Mechanism for the Specific Assembly and Activation of the Extracellular Signal Regulated Kinase 5 (ERK5) Module

Mitogen-activated protein kinase (MAPK) activation depends on a linear binding motif found in all MAPK kinases (MKK). In addition, the PB1 (Phox and Bem1) domain of MKK5 is required for extracellular signal regulated kinase 5 (ERK5) activation. We present the crystal structure of ERK5 in complex with an MKK5 construct comprised of the PB1 domain and the linear binding motif. We show that ERK5 has distinct protein-protein interaction surfaces compared with ERK2, which is the closest ERK5 paralog. The two MAPKs have characteristically different physiological functions and their distinct protein-protein interaction surface topography enables them to bind different sets of activators and substrates. Structural and biochemical characterization revealed that the MKK5 PB1 domain cooperates with the MAPK binding linear motif to achieve substrate specific binding, and it also enables co-recruitment of the upstream activating enzyme and the downstream substrate into one signaling competent complex. Studies on present day MAPKs and MKKs hint on the way protein kinase networks may evolve. In particular, they suggest how paralogous enzymes with similar catalytic properties could acquire novel signaling roles by merely changing the way they make physical links to other proteins.     

Site-specific basicities regulate molecular recognition in receptor binding: in silico docking of thyroid hormones

Interactions between thyroid hormone α and β receptors and the eight protonation microspecies of each of the main thyroid hormones (thyroxine, liothyronine, and reverse liothyronine) were investigated and quantitated by molecular modeling. Flexible docking of the various protonation forms of thyroid hormones and high-affinity thyromimetics to the two thyroid receptors was carried out. In this method the role of the ionization state of each basic site could be studied in the composite process of molecular recognition. Our results quantitate at the molecular level how the ionization state and the charge distribution influence the protein binding. The anionic form of the carboxyl group (i.e., carboxylate site) is essential for protein binding, whereas the protonated form of amino group worsens the binding. The protonation state of the phenolate plays a less important role in the receptor affinity; its protonation, however, alters the electron density and the concomitant stacking propensity of the aromatic rings, resulting in a different binding score. The combined results of docking and microspeciation studies show that microspecies with the highest concentration at the pH of blood are not the strongest binding ones. The calculated binding free energy values can be well interpreted in terms of the interactions between the actual sites of the microspecies and the receptor amino acids. Our docking results were validated and compared with biological data from the literature. Since the thyroid hormone receptors influence several physiologic functions, such as metabolic rate, cholesterol and triglyceride levels, and heart frequency, our binding results provide a molecular basis for drug design and development in related therapeutic indications.     

Chiral Separation of Uncharged Pomalidomide Enantiomers Using Carboxymethyl‐β‐Cyclodextrin: A Validated Capillary Electrophoretic Method

The racemic mixture of pomalidomide (POM), a second‐generation immunomodulatory uncharged drug, was separated into enantiomers by capillary zone electrophoresis for the first time. Seven different chargeable cyclodextrin (CD) derivatives were screened as complexing agents and chiral selectors, investigating the stability of the POM‐CD inclusion complexes and their enantiodiscriminating capacities. Based on preliminary experiments, carboxymethyl‐β‐CD (CM‐β‐CD) was found to be the most effective chiral selector. Factors influencing enantioseparation were systematically optimized, using an orthogonal experimental design. Optimal parameters (background electrolyte [BGE]: 50 mM Tris‐acetate buffer, pH 6.5, containing 15 mM CM‐β‐CD; capillary temperature: 20°C; voltage applied +15 kV) allowed baseline separation of POM enantiomers with a resolution as high as 4.87. The developed method was validated, in terms of sensitivity (limit of detection and limit of quantification), linearity, accuracy, repeatability, and intermediate precision. Chirality 28:199–203, 2016. © 2015 Wiley Periodicals, Inc.     

Measurement of Inositol 1,4,5-Trisphosphate in Living Cells Using an Improved Set of Resonance Energy Transfer-Based Biosensors

Improved versions of inositol-1,4,5-trisphosphate (InsP₃) sensors were created to follow intracellular InsP₃ changes in single living cells and in cell populations. Similar to previous InsP₃ sensors the new sensors are based on the ligand binding domain of the human type-I InsP₃ receptor (InsP₃R-LBD), but contain a mutation of either R265K or R269K to lower their InsP₃ binding affinity. Tagging the InsP₃R-LBD with N-terminal Cerulean and C-terminal Venus allowed measurement of InsP₃ in single-cell FRET experiments. Replacing Cerulean with a Luciferase enzyme allowed experiments in multi-cell format by measuring the change in the BRET signal upon stimulation. These sensors faithfully followed the agonist-induced increase in InsP₃ concentration in HEK 293T cells expressing the Gq-coupled AT1 angiotensin receptor detecting a response to agonist concentration as low as 10 pmol/L. Compared to the wild type InsP₃ sensor, the mutant sensors showed an improved off-rate, enabling a more rapid and complete return of the signal to the resting value of InsP₃ after termination of M3 muscarinic receptor stimulation by atropine. For parallel measurements of intracellular InsP₃ and Ca²⁺ levels in BRET experiments, the Cameleon D3 Ca²⁺ sensor was modified by replacing its CFP with luciferase. In these experiments depletion of plasma membrane PtdIns(4,5)P₂ resulted in the fall of InsP₃ level, followed by the decrease of the Ca²⁺-signal evoked by the stimulation of the AT1 receptor. In contrast, when type-III PI 4-kinases were inhibited with a high concentration of wortmannin or a more specific inhibitor, A1, the decrease of the Ca²⁺-signal preceded the fall of InsP₃ level indicating an InsP₃-, independent, direct regulation of capacitative Ca²⁺ influx by plasma membrane inositol lipids. Taken together, our results indicate that the improved InsP₃ sensor can be used to monitor both the increase and decrease of InsP₃ levels in live cells suitable for high-throughput BRET applications.     

Full-length L1CAM and not its Δ2Δ27 splice variant promotes metastasis through induction of gelatinase expression

Tumour-specific splicing is known to contribute to cancer progression. In the case of the L1 cell adhesion molecule (L1CAM), which is expressed in many human tumours and often linked to bad prognosis, alternative splicing results in a full-length form (FL-L1CAM) and a splice variant lacking exons 2 and 27 (SV-L1CAM). It has not been elucidated so far whether SV-L1CAM, classically considered as tumour-associated, or whether FL-L1CAM is the metastasis-promoting isoform. Here, we show that both variants were expressed in human ovarian carcinoma and that exposure of tumour cells to pro-metastatic factors led to an exclusive increase of FL-L1CAM expression. Selective overexpression of one isoform in different tumour cells revealed that only FL-L1CAM promoted experimental lung and/or liver metastasis in mice. In addition, metastasis formation upon up-regulation of FL-L1CAM correlated with increased invasive potential and elevated Matrix metalloproteinase (MMP)-2 and -9 expression and activity in vitro as well as enhanced gelatinolytic activity in vivo. In conclusion, we identified FL-L1CAM as the metastasis-promoting isoform, thereby exemplifying that high expression of a so-called tumour-associated variant, here SV-L1CAM, is not per se equivalent to a decisive role of this isoform in tumour progression.     

Synthesis, characterization and biological evaluation of some novel 17-isoxazoles in the estrone series

Regioselective 1,3-dipolar cycloadditions of different aryl nitrile oxides to mestranol were carried out to furnish novel steroidal 17α-isoxazoles in good to excellent yields. Copper(I) was found to be an efficient catalyst, accelerating the intermolecular ring-closures and leading exclusively to 3,5-disubstituted isoxazoles. The yields of the cycloadducts, however, were influenced by the substituents on the aromatic moiety of the 1,3-dipoles. Moreover, dehydration of the primary products resulted in the corresponding Δ(16,17)exo-heterocyclic derivatives. All the synthesized compounds were subjected to in vitro pharmacological studies of their antiproliferative effects relative to three human malignant cell lines (HeLa, MCF7 and A2780).