In vitro regeneration from immature cotyledon explant and protein profile changes during organogenesis in groundnut (Arachis hypogaea L.)

Complete plants were regenerated from in vitro cultured immature cotyledon segments of groundnut (Arachis hypogaea L. cv. TMV-7) by organogenesis. Callus cultures were best Initiated from immature cotyledon segments on MS (Murashige and Skoog) salts containing B5 vitamins supplemented with indole-3-acetic acid (IAA) and alpha -naphthalene acetic acid (NAA; 4.0 mg L-1) and kinetin (KIN; 0.5 L-1). Calluses were transferred to a medium containing KIN (2.0 mg L-1) and IAA and NAA (0.5 mg L-1) for shoot Initiation. The regenerated shoots were transferred to a medium containing Indole-3-butyric acid (IBA; 2.0 mg L-1) and KIN (0.2 mg L-1) for developing roots. In vitro produced plantlets developed sucessfully, matured, and set seed. The protein profiles [sodium dodecyl sulphate - polyacrylamide gel electrophoresis (SDS-PAGE)] of callus, callus with shoot, and callus with shoot and root showed differences.

NMR in liquid crystals spinning at and near magic angle

NMR spectra of liquid crystalline phases and the molecules dissolved therein, spinning at and near the magic angle provide information on the director dynamics and the order parameter. The studies on the dynamics of the liquid crystal director for sample spinning near magic angle in mesophases with positive and negative diamagnetic susceptibility anisotropies (Delta chi) and their mixtures with near-zero macroscopic diamagnetic susceptibility anisotropies have been reported. In systems with weakly positive Delta chi, the director has been observed to switch from an orientation parallel to the spinning axis at low rotational speeds to one perpendicular to the spinning axis at high rotational speeds, when the angle theta, the axis of rotation makes with the magnetic field is smaller than the magic angle theta(m). For systems with a small negative Delta chi, similar director behaviour has been observed for theta greater than theta(m). At magic angle, the spectra under slow spinning speeds exhibit a centre band and side bands at integral values of the spinning speeds. The intensities of the spinning side bands have been shown to contain information on the sign and the magnitude of the order parameter(s). The results are discussed with illustrative examples. Results on the orientation of the chemical shielding tensor obtained from a combination of the NMR studies in the solid and the liquid crystalline states, have been described.

Angelica archengelica extract induced perturbation of rat skin and tight junctional protein (ZO-1) of HaCaT cells

Background and purpose of the study: Herbal enhancers compared to the synthetic ones have shown less toxis effects. Coumarins have been shown at concentrations inhibiting phospoliphase C-Y (Phc-Y) are able to enhance tight junction (TJ) permeability due to hyperpoalation of Zonolous Occludense-1 (ZO-1) proteins. The purpose of this study was to evaluate the influence of ethanolic extract of Angelica archengelica (AA-E) which contain coumarin on permeation of repaglinide across rat epidermis and on the tight junction plaque protein ZO-1 in HaCaT cells. Methods: Transepidermal water loss (TEWL) from the rat skin treated with different concentrations of AA-E was assessed by Tewameter. Scanning and Transmission Electron Microscopy (TEM) on were performed on AA-E treated rat skin portions. The possibility of AA-E influence on the architecture of tight junctions by adverse effect on the cytoplasmic ZO-1 in HaCaT cells was investigated. Finally, the systemic delivery of repaglinide from the optimized transdermal formulation was investigated in rats. Results: The permeation of repaglinide across excised rat epidermis was 7-fold higher in the presence of AA-E (5% w/v) as compared to propylene glycol:ethanol (7:3) mixture. The extract was found to perturb the lipid microconstituents in both excised and viable rat skin, although, the effect was less intense in the later. The enhanced permeation of repaglinide across rat epidermis excised after treatment with AA-E (5% w/v) for different periods was in concordance with the high TEWL values of similarly treated viable rat skin. Further, the observed increase in intercellular space, disordering of lipid structure and corneocyte detachment indicated considerable effect on the ultrastructure of rat epidermis. Treatment of HaCaT cell line with AA-E (0.16% w/v) for 6 hrs influenced ZO-1 as evidenced by reduced immunofluorescence of anti-TJP1 (ZO-1) antibody in Confocal Laser Scanning Microscopy studies (CLSM) studies. The plasma concentration of repaglinide from transdermal formulation was maintained higher and for longer time as compared to oral administration of repaglinide. Major conclusion: Results suggest the overwhelming influence of Angelica archengelica in enhancing the percutaneous permeation of repaglinide to be mediated through perturbation of skin lipids and tight junction protein (ZO-1).

Structural and conformational study of peptides containing glycine and alanine residues by C-13 CPMAS NMR spectroscopy in solid state

The results of the structural and conformational studies carried out using C-13 CPMAS NMR technique on several glycine and alanine containing peptides in the solid state are reported. The study demonstrates the effects of variations in C-13 chemical shifts due to conformation and hydrogen bonding. The possibility of applying this technique to obtain insight into the conformational characteristics of peptides of unknown structures is discussed.

Quantum clustering and network analysis of MD simulation trajectories to probe the conformational ensembles of protein-ligand interactions

In this article, we present a novel application of a quantum clustering (QC) technique to objectively cluster the conformations, sampled by molecular dynamics simulations performed on different ligand bound structures of the protein. We further portray each conformational population in terms of dynamically stable network parameters which beautifully capture the ligand induced variations in the ensemble in atomistic detail. The conformational populations thus identified by the QC method and verified by network parameters are evaluated for different ligand bound states of the protein pyrrolysyl-tRNA synthetase (DhPylRS) from D. hafniense. The ligand/environment induced re-distribution of protein conformational ensembles forms the basis for understanding several important biological phenomena such as allostery and enzyme catalysis. The atomistic level characterization of each population in the conformational ensemble in terms of the re-orchestrated networks of amino acids is a challenging problem, especially when the changes are minimal at the backbone level. Here we demonstrate that the QC method is sensitive to such subtle changes and is able to cluster MD snapshots which are similar at the side-chain interaction level. Although we have applied these methods on simulation trajectories of a modest time scale (20 ns each), we emphasize that our methodology provides a general approach towards an objective clustering of large-scale MD simulation data and may be applied to probe multistate equilibria at higher time scales, and to problems related to protein folding for any protein or protein-protein/RNA/DNA complex of interest with a known structure.

Water-mediated ionic interactions in protein structures

It is well known that water molecules play an indispensable role in the structure and function of biological macromolecules. The water-mediated ionic interactions between the charged residues provide stability and plasticity and in turn address the function of the protein structures. Thus, this study specifically addresses the number of possible water-mediated ionic interactions, their occurrence, distribution and nature found in 90% non-redundant protein chains. Further, it provides a statistical report of different charged residue pairs that are mediated by surface or buried water molecules to form the interactions. Also, it discusses its contributions in stabilizing various secondary structural elements of the protein. Thus, the present study shows the ubiquitous nature of the interactions that imparts plasticity and flexibility to a protein molecule.

Structural characterization and chain dynamics of poly(alpha-methylstyrene peroxide) by NMR spectroscopy

Poly(alpha-methylstyrene peroxide) has been synthesized and characterized spectroscopically. The H-1 and C-13 NMR spectra are shown to reveal the stereochemical features and the endgroups in the peroxide chain. The preliminary studies on the chain dynamics of the polyperoxide chain has been done by measuring the spin-lattice relaxation times (T-1) of the main chain as well as the side chain carbons. It has been shown from the dependence of the spin-lattice relaxation times that the polyperoxide chain is more flexible compared to the corresponding hydrocarbon-backbone analog.

Stochastic dynamics modeling of the protein sequence length distribution in genomes: implications for microbial evolution

In this paper, we report an analysis of the protein sequence length distribution for 13 bacteria, four archaea and one eukaryote whose genomes have been completely sequenced, The frequency distribution of protein sequence length for all the 18 organisms are remarkably similar, independent of genome size and can be described in terms of a lognormal probability distribution function. A simple stochastic model based on multiplicative processes has been proposed to explain the sequence length distribution. The stochastic model supports the random-origin hypothesis of protein sequences in genomes. Distributions of large proteins deviate from the overall lognormal behavior. Their cumulative distribution follows a power-law analogous to Pareto's law used to describe the income distribution of the wealthy. The protein sequence length distribution in genomes of organisms has important implications for microbial evolution and applications. (C) 1999 Elsevier Science B.V. All rights reserved.

Structural characterisation of a uracil containing hairpin DNA by NMR and molecular dynamics

Three-dimensional (3D) structure of a hairpin DNA d-CTAGAGGATCCTTTUGGATCCT (22mer; abbreviated as U4-hairpin), which has a uracil nucleotide unit at the fourth position from the 5' end of the tetra-loop has been solved by NMR spectroscopy. The H-1 resonances of this hairpin have been assigned almost completely. NMR restrained molecular dynamics and energy minimisation procedures have been used to describe the 3D structure of the U4 hairpin. This study establishes that the stem of the hairpin adopts a right handed B-DNA conformation while the T-12 and U-15 nucleotide stack upon 3' and 5' ends of the stem, respectively. Further, T-14 stacks upon both T-12 and U-15 while T-13 partially stacks upon T-14. Very weak stacking interaction is observed between T-13 and T-12. All the individual nucleotide bases adopt 'anti' conformation with respect to their sugar moiety. The turning phosphate in the loop is located between T-13 and T-14. The stereochemistry of U-15 mimics the situation where uracil would stack in a B-DNA conformation. This could be the reason as to why the U4-hairpin is found to be the best substrate for its interaction with uracil DNA glycosylase (UDG) compared to the other substrates in which the uracil is at the first, second and third positions of the tetra-loop from its 5' end, as reported previously.

Structure of 2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9-hexadecafluorodecyl 1,10-ditosylate by X-ray crystallography and F-19-NMR spectroscopy

2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9-hexadecafluorodecyl 1,10-ditosylate and its precursors were synthesized and characterized by H-1- and F-19-NMR spectroscopic methods and X-ray crystallography. These compounds are building blocks for the syntheses of the surfactants containing polyperfluoromethylene spacer. The molecule has extended all-trans conformation with molecular symmetry (1) over bar (C-i). There is a reasonably strong C-H ... O interaction in the crystal and there are two F ... F intermolecular contact distances less than the sum of van der Waals radii. (C) 1999 Elsevier Science B.V. All rights reserved.

Essential dynamics and sidechain hydrogen bond cluster studies on eosinophil cationic protein

Eosinophil Cationic Protein (ECP) is a member of RNase A superfamily which carries out the obligatory catalytic role of cleaving RNA. It is involved in a variety of biological functions. Molecular dynamics simulations followed by essential dynamics analysis on this protein are carried out with the goal of gaining insights into the dynamical properties at atomic level. The top essential modes contribute to subspaces and to the transition phase. Further, the sidechain-sidechain/sidechain-mainchain hydrogen bond clusters are analyzed in the top modes, and compared with those of crystal structure. The role of residues identified by these methods is discussed in the context of concerted motion, structure and stability of the protein.

iPBA: a tool for protein structure comparison using sequence alignment strategies

With the immense growth in the number of available protein structures, fast and accurate structure comparison has been essential. We propose an efficient method for structure comparison, based on a structural alphabet. Protein Blocks (PBs) is a widely used structural alphabet with 16 pentapeptide conformations that can fairly approximate a complete protein chain. Thus a 3D structure can be translated into a 1D sequence of PBs. With a simple Needleman-Wunsch approach and a raw PB substitution matrix, PB-based structural alignments were better than many popular methods. iPBA web server presents an improved alignment approach using (i) specialized PB Substitution Matrices (SM) and (ii) anchor-based alignment methodology. With these developments, the quality of similar to 88% of alignments was improved. iPBA alignments were also better than DALI, MUSTANG and GANGSTA(+) in > 80% of the cases. The webserver is designed to for both pairwise comparisons and database searches. Outputs are given as sequence alignment and superposed 3D structures displayed using PyMol and Jmol. A local alignment option for detecting subs-structural similarity is also embedded. As a fast and efficient `sequence-based' structure comparison tool, we believe that it will be quite useful to the scientific community. iPBA can be accessed at http://www.dsimb.inserm.fr/dsimb_tools/ipba/.

PRUNE and PROBE-two modular web services for protein-protein docking

The protein-protein docking programs typically perform four major tasks: (i) generation of docking poses, (ii) selecting a subset of poses, (iii) their structural refinement and (iv) scoring, ranking for the final assessment of the true quaternary structure. Although the tasks can be integrated or performed in a serial order, they are by nature modular, allowing an opportunity to substitute one algorithm with another. We have implemented two modular web services, (i) PRUNE: to select a subset of docking poses generated during sampling search (http://pallab.serc.iisc.ernet.in/prune) and (ii) PROBE: to refine, score and rank them (http://pallab.serc.iisc.ernet.in/probe). The former uses a new interface area based edge-scoring function to eliminate > 95% of the poses generated during docking search. In contrast to other multi-parameter-based screening functions, this single parameter based elimination reduces the computational time significantly, in addition to increasing the chances of selecting native-like models in the top rank list. The PROBE server performs ranking of pruned poses, after structure refinement and scoring using a regression model for geometric compatibility, and normalized interaction energy. While web-service similar to PROBE is infrequent, no web-service akin to PRUNE has been described before. Both the servers are publicly accessible and free for use.

Half-sandwich (eta(6)-arene)ruthenium(II) chiral Schiff base complexes: Analysis of the diastereomeric mixtures in solution by 2D-NMR spectroscopy

2D NMR spectroscopy has been used to determine the metal configuration in solution of three complexes, viz. [(eta(6)-p-cymene)Ru(L*)Cl] (1) and [(eta(6)-p-cymene)Ru(L*)(L')] (ClO4) (L' = H2O, 2; PPh3, 3), where L* is the anion of (S)-(1-phenylethyl)salicylaldimine. The complexes exist in two diastereomeric forms in solution. Both the (R-Ru,S-C)- and (S-Ru,S-C)-diastereomers display the presence of attractive, CH/pi interaction involving the phenyl group attached to the chiral carbon and the cymene ring hydrogens. This interaction restricts the rotation of the C*-N single bond and, as a result, two structural types with either the hydrogen atom attached to the chiral carbon (C*) or the methyl group attached to C* in close proximity of the cymene ring protons get stabilized. Using 2D NMR spectroscopy as a tool, the spatial interaction involving these protons are studied in order to obtain the metal configuration(s) of the diastereomeric complexes in solution. This technique has enabled us to determine the metal configuration as (R-Ru,S-C) for the major isomers of 1-3 in solution.