Fusion of a novel genetically engineered chitosan affinity protein and green fluorescent protein for specific detection of chitosan in vitro and in situ

Chitin is the second most abundant polysaccharide, present, e.g., in insect and arthropod exoskeletons and fungal cell walls. In some species or under specific conditions, chitin appears to be enzymatically de-N-acetylated to chitosan-e.g., when pathogenic fungi invade their host tissues. Here, the deacetylation of chitin is assumed to represent a pathogenicity mechanism protecting the fungus from the host's chitin-driven immune response. While highly specific chitin binding lectins are well known and easily available, this is not the case for chitosan-specific probes. This is partly due to the poor antigenicity of chitosan so that producing high-affinity, specific antibodies is difficult. Also, lectins with specificity to chitosan have been described but are not commercially available, and our attempts to reproduce the findings were not successful. We have, therefore, generated a fusion protein between a chitosanase inactivated by site-directed mutagenesis, the green fluorescent protein (GFP), and StrepII, as well as His(6) tags for purification and detection. The recombinant chitosan affinity protein (CAP) expressed in Escherichia coli was shown to specifically bind to chitosan, but not to chitin, and the affinity increased with decreasing degree of acetylation. In vitro, CAP detection was possible either based on GFP fluorescence or using Strep-Tactin conjugates or anti-His(5) antibodies. CAP fluorescence microscopy revealed binding to the chitosan exposing endophytic infection structures of the wheat stem rust fungus, but not the chitin exposing ectophytic infection structures, verifying its suitability for in situ chitosan staining.     

De-intercalation of ethidium bromide and acridine orange by xanthine derivatives and their modulatory effect on anticancer agents: a study of DNA-directed toxicity enlightened by time correlated single photon counting

Time Correlated Single Photon Counting (TCSPC) was used for the first time to analyze the effect/changes in the mode of intercalation of ethidium bromide (EtBr) and acridine orange (AO) to calf thymus DNA brought about due to interaction of naturally occurring methylxanthines such as theophylline (X1), theobromine (X2) and caffeine (X3). UV absorption and fluorescence studies were also carried to observe the behaviour of these xanthines on the modulation of the binding mode of anticancer agents (cisplatin, novantrone, and actinomycin D) and certain intercalating dyes (EtBr and AO) to DNA. In TCSPC analysis we found that when the concentration of the drugs (X1, X2 and X3) increased from 0.025 mM to 2 mM i.e. P/D 2.4 to P/D 0.03 reduction in intercalation of EtBr and AO was observed, suggesting that xanthine derivatives could play very important role in reducing the DNA-directed toxicity in a dose dependent manner. In TCSPC, the amplitude of smaller lifetime component A(1) and higher lifetime component A(2) are attributed to free and intercalated dye concentration and their variation could indicate the process of intercalation or reduced intercalation of EtBr and AO by xanthine derivatives. We found that at the maximum drug concentration the smaller lifetime component A(1) was increased by 7-8% and 17-37% in EtBr and AO intercalated complex respectively. Also the changes in lifetime and fluorescence decay profile were observed for the DNA-intercalated dyes before and after treatment with xanthines. Especially, at maximum P/D 0.03 the lifetime of DNA-intercalated EtBr and AO reduced by 1-2 ns. The present analysis reveals that xanthines are able to interact with free dyes and also with intercalated dyes, suggesting that when they interact with free dyes they might inhibit the further intercalation of dye molecules to DNA and the interaction with intercalated dyes might lead to displacement of the dyes resulting in de-intercalation. The results obtained from UV and fluorescence spectroscopy also support the present investigation of probable interaction of xanthines with the DNA damaging agents in modulating/reducing the DNA-directed toxicity.     

RNA binding efficacy of theophylline,theobromine and caffeine

The binding of naturally occurring methylxanthines such as theophylline, theobromine and caffeine to nucleic acids are reckoned to be pivotal as they are able to modulate the cellular activities. We explore the interaction of yeast RNA binding efficacy of the above xanthine derivatives by using UV absorption differential spectroscopy and Fourier Transform Infrared (FTIR) spectroscopy. Both the analyses show discrimination in their binding affinity to RNA. The differential UV-spectrum at P/D 3.3 reveals the greater RNA binding activity for theophylline (85 +/- 5%), whereas moderate and comparatively less binding activity for theobromine (45 +/- 5%) and caffeine (30 +/- 5%) and the binding activity was found to depend on concentration of the drugs. In FTIR analysis we observed changes in the amino group (NH) of RNA complexed by drugs, where the NH band is found to become very broad, indicating hydrogen bonding (H-bonding) with theophylline (3343.4 cm(-1)), theobromine (3379.8 cm(-1)) and caffeine (3343 cm(-1)) as compared to the free RNA (3341.6 cm(-1)). Furthermore in RNA-theophylline complex, it is observed that the carbonyl (C=O) vibration frequency (nu(C=O)) of both drug (nu(C=O)=1718, 1666 cm(-1)) as well as RNA (nu(C=O)=1699, 1658 cm(-1)) disappeared and a new vibration band appeared around 1703 cm(-1), indicating that the C=O and NH groups of drug and RNA are effectively involved in H-bonding. Whereas in RNA-theobromine and RNA-caffeine complexes, we found very little changes in C=O frequency and only broadening of the NH band of RNA due to complexation is observed in these groups. The changes in the vibrations of G-C/A-U bands and other bending frequencies are discussed. Thus the discrimination in the binding affinity of methylxanthines with RNA molecule shows that strong RNA binding drugs like theophylline can selectively be delivered to RNA targets of microbial pathogens having the mechanism of RNA catalysis.     

Mutagenesis of the putative sterol-sensing domain of yeast Niemann Pick C-related protein reveals a primordial role in subcellular sphingolipid distribution

Lipid movement between organelles is a critical component of eukaryotic membrane homeostasis. Niemann Pick type C (NP-C) disease is a fatal neurodegenerative disorder typified by lysosomal accumulation of cholesterol and sphingolipids. Expression of yeast NP-C-related gene 1 (NCR1), the orthologue of the human NP-C gene 1 (NPC1) defective in the disease, in Chinese hamster ovary NPC1 mutant cells suppressed lipid accumulation. Deletion of NCR1, encoding a transmembrane glycoprotein predominantly residing in the vacuole of normal yeast, gave no phenotype. However, a dominant mutation in the putative sterol-sensing domain of Ncr1p conferred temperature and polyene antibiotic sensitivity without changes in sterol metabolism. Instead, the mutant cells were resistant to inhibitors of sphingolipid biosynthesis and super sensitive to sphingosine and C2-ceramide. Moreover, plasma membrane sphingolipids accumulated and redistributed to the vacuole and other subcellular membranes of the mutant cells. We propose that the primordial function of these proteins is to recycle sphingolipids and that defects in this process in higher eukaryotes secondarily result in cholesterol accumulation.     

Bioinformatics approaches for new drug discovery: a review

Prolonged antibiotic therapy for the bacterial infections has resulted in high levels of antibiotic resistance. Initially, bacteria are susceptible to the antibiotics, but can gradually develop resistance. Treating such drug-resistant bacteria remains difficult or even impossible. Hence, there is a need to develop effective drugs against bacterial pathogens. The drug discovery process is time-consuming, expensive and laborious. The traditionally available drug discovery process initiates with the identification of target as well as the most promising drug molecule, followed by the optimization of this, in-vitro, in-vivo and in pre-clinical studies to decide whether the compound has the potential to be developed as a drug molecule. Drug discovery, drug development and commercialization are complicated processes. To overcome some of these problems, there are many computational tools available for new drug discovery, which could be cost effective and less time-consuming. In-silico approaches can reduce the number of potential compounds from hundreds of thousands to the tens of thousands which could be studied for drug discovery and this results in savings of time, money and human resources. Our review is on the various computational methods employed in new drug discovery processes.     

Random coil conformations of polynucleotides: a study incorporating long-range correlations due to dynamics of the sugar residue

A novel virtual bond scheme joining the atoms P-C4′ and C4′-P of a nucleotide repeat, consistent with the stereochemistry of polynucleotide chains, has been developed. The scheme, with its inherent feature to account for all the major sources of flexibility, could also consider effectively the short range and long range interactions, thereby simplifying the analysis of random coil and ordered structures. Using this scheme, unperturbed end-to-end dimensions and persistence lengths of polynucleotide chains have been computed incorporating the dynamical aspects of the sugar ring as well as the C4′ C5′ bond and the correlated changes in phosphodiester conformations. Calculated unperturbed dimensions are in excellent agreement with experimental values. Results show that the random coil is characterized by a large proportion of helical segments of the A-form.     

Polynucleotide folding in yeast tRNAPhe: elucidation of short-, medium-, and long-range interactions of sugar-phosphate-sugar backbone and base using a "blocked" nucleotide probe

It has recently been proposed that the repeating backbone nucleotide may be regarded as consisting of two blocks of equal magnitude representable by two virtual bonds. Implicit consideration of the nucleotide (ψ,ψ) and internucleotide (ω′,ω) geometry that generate variety in polynucleotide conformations, and of the constancy of the repeating structural moieties (P-C4′ and C4′-P) independent of the above rotations, has enabled us to utilize this scheme in the study of ordered structures such as di-, oligonucleotides and, most significantly, tRNA. The polynucleotide folding dictated by short-, intermediate-, and long-range interactions in the monoclinic and orthorhombic forms is described and compared through circular plots depicting the virtual bond torsions and distance plots constructed independently for backbone as well as bases. The torsions and the bond angles associated with the virtual bonds afford a clear distinction between ordered helical segments from loops and bends of tRNA. Lower virtual bond torsions (?60° to 60°) concomitant with higher values of virtual bond angles characterize various bend regions, while torsions around 160°–210° typify ordered helical strands. The distance plot elucidates the type of interaction associated with various sub-structures (helix–helix, helix–loop, and loop–loop) that form the constituents of different structural domains. Several other features such as the manifestation of the P₁₀ loop and the approximate twofold symmetry in the tRNA molecule are conspicuous on the distance plot.