Transferred cross-relaxation and cross-correlation in NMR: effects of intermediate exchange on the determination of the conformation of bound ligands

Exchange transferred effects in solution-state NMR experiments allow one to determine the conformation of ligands that are weakly bound to macromolecules. Exchange-transferred nuclear Overhauser effect spectroscopy ('TR-NOESY') provides information about internuclear distances in a ligand in the bound state. Recently the possibility of obtaining dihedral angle information from a ligand in the bound state by exchange-transferred cross-correlation spectroscopy ('TR-CCSY') has been reported. In both cases the analysis of the signal amplitudes is usually based on the assumption that rapid exchange occurs between the free and bound forms of the ligand. In this paper we show that the fast exchange condition is not easily attained for observing exchange-transferred cross-correlation effects even in systems where exchange-transferred NOE can be observed. Extensive simulations based on analytical expressions for signal intensities corresponding to fast, intermediate, and slow chemical exchange have been carried out on a test system to determine the exchange regimes in which the fast exchange condition can be fulfilled for successfully implementing TR-NOESY and TR-CCSY.     

Effects of prey refuge on a ratio-dependent predator–prey model with stage-structure of prey population

In this paper, a stage-structured predator–prey model is proposed and analyzed to study how the type of refuges used by prey population influences the dynamic behavior of the model. Two types of refuges: those that protect a fixed number of prey and those that protect a constant proportion of prey are considered. Mathematical analyses with regard to positivity, boundedness, equilibria and their stabilities, and bifurcation are carried out. Persistence condition which brings out the useful relationship between prey refuge parameter and maturation time delay is established. Comparing the conclusions obtained from analyzing properties of two types of refuges using by prey, we observe that value of maturation time at which the prey population and hence predator population go extinct is greater in case of refuges which protect a constant proportion of prey.     

Conformationally constrained aliphatic-aromatic amino-acid-conjugated hybrid foldamers with periodic beta-turn motifs

In this note, we describe the design, synthesis, and structural studies of novel hybrid foldamers derived from Aib-Pro-Adb building blocks that display repeat beta-turn structure motif. The foldamer having a conformationally constrained aliphatic-aromatic amino acid conjugate adopts a well-defined, compact, three-dimensional structure, governed by a combined conformational restriction imposed by the individual amino acids with which it is made of. Conformational investigations by single-crystal X-ray and solution-state NMR studies were undertaken to investigate the conformational preference of these foldamers with a hetero-backbone. Our findings suggest that constrained aliphatic-aromatic amino acid conjugates would offer new avenues for the de novo design of hybrid foldamers with distinctive structural architectures.     

Rapid,single-step nucleic acid detection

A rapid detection method for nucleic acid based on bioluminescence resonance energy transfer (BRET) from the luminescence donor Renilla luciferase to an acceptor quantum dot upon oligonucleotide probe hybridization has been developed. Utilizing a competitive assay, we detected the target nucleic acid by correlating the BRET signal with the amount of target present in the sample. This method allows for the detection of as little as 4 pmol (20 nM) of nucleic acid in a single-step, homogeneous format both in vitro in a buffer matrix as well as in a cellular matrix. Using this method, one may perform nucleic acid detection in as little as 30 min, showing much improvement over time-consuming blotting methods and solid-phase methods which require multiple wash steps to remove unbound probe. This is the first report on the use of quantum dots as a BRET acceptor in the development of a nucleic acid hybridization assay. An erratum to this article can be found at     

A whole-cell assay for the high throughput screening of calmodulin antagonists

Cell-based screening systems for pharmaceuticals are desired over molecular biosensing systems because of the information they provide on toxicity and bioavailability. However, the majority of sensing systems developed are molecular biosensing type screening systems and cannot be easily adapted to cell-based screening. In this study, we demonstrate that protein-based molecular sensing systems that employ a fluorescent protein as a signal transducer are amenable to cell-based sensing by expressing the protein molecular sensing system in the cell and employing these cells for screening of desired molecules. To achieve this, we expressed a molecular sensing system based on the fusion protein of calmodulin (CaM) and enhanced green fluorescent protein (EGFP) in bacterial cells, and utilized these cells for the screening of CaM antagonists. In the presence of Ca²⁺, CaM undergoes a conformational change exposing a hydrophobic pocket that interacts with CaM-binding proteins, peptides, and drugs. This conformational change induced in CaM leads to a change in the microenvironment of EGFP, resulting in a change in its fluorescence intensity. The observed change in fluorescence intensity of EGFP can be correlated to the concentration of the analyte present in the sample. Dose-response curves for various tricyclic antidepressants were generated using cells containing CaM-EGFP fusion protein. Additionally, we demonstrate the versatility of our system for studying protein-protein interactions by using cells to study the binding of a peptide to CaM. The study showed that the CaM-EGFP fusion protein within the intact cells responds similarly to that of the isolated fusion protein, hence eliminating the need for any isolation and purification steps. We have demonstrated that this system can be used for the rapid screening of various CaM antagonists that are potential antipsychotic drugs.     

Analysis of the wave properties of a new two-lane continuum model with the coupling effect

A multilane extension of the single-lane anisotropic continuum model (GK model) developed by Gupta and Katiyar for traffic flow is discussed with the consideration of the coupling effect between the vehicles of different lanes in the instantaneous traffic situation and the lane-changing effect. The conditions for securing the linear stability of the new model are presented. The shock and the rarefaction waves, the local cluster effect and the phase transition are investigated through simulation experiments with the new model and are found to be consistent with the diverse nonlinear dynamical phenomena observed in a real traffic flow. The analysis also focuses on empirically observed two-lane phenomena, such as lane usage inversion and the density dependence of the number of lane changes. It is shown that single-lane dynamics can be extended to multilane cases without changing the basic properties of the single-lane model. The results show that the new multilane model is capable of explaining some particular traffic phenomena and is in accordance with real traffic flow.     

Synthesis of β-amino alcohols catalyzed by poly(vinyl chloride)-supported Schiff base metal complexes

Abstract  

Eight transition metal complexes of various Schiff bases supported on poly(vinyl chloride) (PVC) were prepared and characterized. These metal complexes were screened as heterogeneous catalysts in the synthesis of β-amino alcohols by ring opening of epoxides with amines. The best catalyst was identified as a Ni(II) complex of PVC-supported 2-[(2-aminoethylimino)methyl]phenol and it was used in the synthesis of a number of different β-amino alcohols. The catalyst was found to be reusable for up to five cycles.     

Liquid chromatography/electrospray ionization tandem mass spectrometry validated method for the simultaneous quantification of sibutramine and its primary and secondary amine metabolites in human plasma and its application to a bioequivalence study

A high-throughput and sensitive bioanalytical method using liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) has been developed for the estimation of sibutramine and its two metabolites (M1 and M2). The extraction of sibutramine, its metabolites and imipramine (internal standard (IS)) from the plasma involved treatment with phosphoric acid followed by solid-phase extraction (SPE) using a hydrophilic-lipophilic balanced HLB cartridge. The SPE eluate without drying and reconstitution was analyzed by LC/MS/MS, equipped with a with turbo ion spray (TIS) source, operating in the positive and multiple reaction monitoring (MRM) acquisition mode. Sample preparation by this method yielded extremely clean extracts with quantitative and consistent mean recoveries; 95.12% for sibutramine, 92.74% for M1, 95.97% for M2 and 96.60% for the IS. The total chromatographic run time was 3.0 min with retention times of 2.51, 2.13, 2.09 min for sibutramine, M1, M2 and imipramine, respectively. The developed method was validated in human plasma matrix, with a sensitivity of 0.1 ng/mL (coefficient of variance (CV), 2.07%) for sibutramine, 0.1 ng/mL (CV, 3.59%) for M1 and 0.2 ng/mL (CV, 4.93%) for M2. Validation of the method for its accuracy, precision, recovery, matrix effect and stability was carried out especially with regard to real subject sample analysis. The response was linear over the dynamic range 0.1 to 8.0 ng/mL for sibutramine and M1, and 0.2 to 16.0 ng/mL for M2 with correlation coefficients of r > or = 0.9959 (sibutramine), 0.9935 (M1) and 0.9943 (M2). The method was successfully applied for bioequivalence studies in 40 human subjects with 15 mg capsule formulations.