Molecular profiling in relation to drought tolerance in advance breeding lines of rice using microsatellite markers

Using agro-morphological characters and microsatellite markers, advance breeding lines of rice were discriminated for their ability to tolerate drought stress at reproductive stage. Experimental materials consisting of 17 advance breeding lines and a check were evaluated in randomized block design with three replications under irrigated condition and drought condition created under rainout shelter during three consecutive years. An analysis of variance revealed significant differences among the genotypes for all the ten agro-morphological characters evaluated under both the conditions across the years. Principal component analysis showed the relative importance of root length, number of tillers per plant, number of grains per panicle, harvest index and grain yield per plant among agro-morphological characters and stress tolerance level, stress susceptibility index, stress tolerance index and drought tolerance efficiency among drought tolerance indices as the important classification variables. Relative mean performance in respect of grain yield as well as drought tolerance indices reflected remarkably greater degree of drought tolerance in 11 advance breeding lines and the check, discriminating them from remaining entries under evaluation. Utilizing a panel of 32 microsatellite primers, selective amplification of targeted genomic regions revealed that the primers RM 72, RM 163, RM 212, RM 225, RM 231, RM 302, RM 327, RM 518, RM 521, RM 555, RM 1349, RM 3549 and RM 5443 were highly informative with greater gene diversity and discrimination ability. Hierarchical cluster analysis based on molecular profiles discriminated the entries into five genotypic groups and drought tolerant entries were accommodated into three distinct groups with remarkably greater efficiency (85.7%). Principal coordinate analysis based two dimensional plots of microsatellites dependent genetic profiles displayed a very close correspondence with the genotypic clustering pattern revealed from a perusal of dendrogram. Sequential exclusion of primers in cluster analysis led to identification of RM 212, RM 231, RM 324, RM 431, RM 521, RM 3549 and RM 6374 as the most useful primers for discrimination of drought tolerant and susceptible lines of rice. Molecular profiling based on these markers can be utilized as efficient tools for discrimination and identification of drought tolerant lines.

     

Regulation of Opioid Receptors by Their Endogenous Opioid Peptides

Cellular and Molecular Neurobiology - Activation of μ, δ, and κ opioid receptors by endogenous opioid peptides leads to the regulation of many emotional and physiological responses....     

Cryopreservation of Cymbidium eburneum Lindl. and C. hookerianum Rchb. f., two threatened and vulnerable orchids via encapsulation–dehydration

A successful cryopreservation protocol for the long-term conservation of protocorms of two threatened and vulnerable orchids, Cymbidium eburneum Lindl. and Cymbidium hookerianum Rchb. f., was developed using encapsulation–dehydration. Protocorms were osmoprotected in liquid Murashige and Skoog medium (MS) containing 0.7 M sucrose for 20 h at 25?±?2°C on a rotary shaker, and incorporated into an encapsulation matrix [consisting of 3% (w/v) sodium alginate and 100 mM CaCl₂]. The encapsulated protocorms, which were desiccated in a laminar airflow cabinet for 6 h, were able to withstand cryostorage in liquid nitrogen. Maximum regeneration into complete plantlets (72% for C. eburneum and 70% for C. hookerianum) of the cryostored, encapsulated protocorms was obtained using MS medium containing 3% sucrose and 0.8% agar. Using this protocol of cryopreservation, long-term preservation for ex situ conservation of these two threatened orchids can be accomplished.     

Ratiometric Ca+2 measurement in human recombinant muscarinic receptor subtypes using the Flexstation scanning fluorometer

In modern drug discovery, numerous assay formats are available to screen and quantitate receptor-ligand interactions. Radioactive assays are “gold standard” because they are fast, easy, and reproducible; however, they are hazardous, produce radioactive waste, require special lab conditions, and are expensive on a large scale. Thus, it provides a lot of importance to the “mix & measure” assays that have an optical readout. Fluorescence techniques are likely to be among the most important detection approaches used for high throughput screening due to their high sensitivity and amenability to automation. The aim of the present study was to determine the functional antagonistic affinities of standard muscarinic antagonists in CHO cells over expressing m1, m3, and m5 receptors and to compare them with the respective binding affinities. This study was further extended to elucidate that Ca⁺² measurement assays can serve as a functional screening tool for GPCRs. For this purpose, standard muscarinic receptor antagonists, namely, tolterodine, oxybutynin, and atropine were used. We determined and compard the IC₅₀ values of these three standard inhibitors in fura 2 AM loaded m1, m3, and m5 overexpressing CHO cells and in radioligand binding assay. Both the assays exhibited comparable rank order potencies of the standard inhibitors. This study suggests that Ca⁺² mobilization assays can be an alternate to radioligand binding assays.     

In silico insights into the identification of potential novel angiogenic inhibitors against human VEGFR-2: a new SAR-based hierarchical clustering approach

Abstract

In this study, binding efficiency of new pyrrolopyrimidine structural analogs against human vascular endothelial growth factor receptor-2 (VEGFR-2) were elucidated using integrated in silico methods. Optimized high-resolution model of VEGFR-2 was generated and adopted for structure-based virtual screening approaches. Pyrrolopyrimidine inhibitor (CP15) associated compounds were screened from PubChem database and subjected to virtual screening and comparative docking methods against the receptor ligand-binding domain. Accordingly, high efficient compounds were clustered with similarity indices through PubChem structure cluster module using single-linkage algorithm. Moreover, pharmacokinetics including drug metabolism activities of high-binding leads under investigation was portrayed using ADMET and similarity ensemble analysis. Optimal energy orientations of the selected protein model have been shown to be reliable, and highly recommended for screening and docking studies. Docking and clustering strategies were shown that nineteen candidates as most effective binders for VEGFR-2 than CP15, and are grouped as three classes. Lys⁸⁶⁸, Glu⁸⁸⁵, Cys⁹¹⁹, His¹⁰²⁶, Arg¹⁰²⁷, Asp¹⁰⁴⁶, and Gly¹⁰⁴⁸ residues were predicted as novel hotspot residues, and participate in H-bonds, π-cation, π-stacking, halogen bonds, and salt-bridges formation with ligands. These additional bonds are contributing extent stability that holds the receptor structure at flexible state, this make difficult to any further conformational changes for evoking angiogenic signals. The ADMET and similarity ensemble analysis results were strongly indicated that thirteen candidates as best ligands for angiogenesis targets. Altogether, these findings indicate potential angiogenic templates and their binding levels with VEGFR-2; sorted viewpoints could be useful as a promising way to describe potential angiogenesis inhibitors with related molecular targets.     

Structural and Functional Studies of a Phosphatidic Acid-Binding Antifungal Plant Defensin MtDef4: Identification of an RGFRRR Motif Governing Fungal Cell Entry

MtDef4 is a 47-amino acid cysteine-rich evolutionary conserved defensin from a model legume Medicago truncatula. It is an apoplast-localized plant defense protein that inhibits the growth of the ascomycetous fungal pathogen Fusarium graminearum in vitro at micromolar concentrations. Little is known about the mechanisms by which MtDef4 mediates its antifungal activity. In this study, we show that MtDef4 rapidly permeabilizes fungal plasma membrane and is internalized by the fungal cells where it accumulates in the cytoplasm. Furthermore, analysis of the structure of MtDef4 reveals the presence of a positively charged γ-core motif composed of β₂ and β₃ strands connected by a positively charged RGFRRR loop. Replacement of the RGFRRR sequence with AAAARR or RGFRAA abolishes the ability of MtDef4 to enter fungal cells, suggesting that the RGFRRR loop is a translocation signal required for the internalization of the protein. MtDef4 binds to phosphatidic acid (PA), a precursor for the biosynthesis of membrane phospholipids and a signaling lipid known to recruit cytosolic proteins to membranes. Amino acid substitutions in the RGFRRR sequence which abolish the ability of MtDef4 to enter fungal cells also impair its ability to bind PA. These findings suggest that MtDef4 is a novel antifungal plant defensin capable of entering into fungal cells and affecting intracellular targets and that these processes are mediated by the highly conserved cationic RGFRRR loop via its interaction with PA.     

Fabrication of an amperometric d-amino acid biosensor based on nickel hexacyanoferrate polypyrrole hybrid film deposited on glassy carbon electrode

A nickel hexacyanoferrate polypyrrole film was synthesized through an electrochemical two-step methodology leading to a very stable and homogenous robust hybrid film. A highly sensitive, specific and rapid amperometric d-amino acid biosensor was constructed by immobilizing d-amino acid oxidase on this film deposited over the surface of a glassy carbon electrode. The modified electrode was characterized by scanning electron microscopy, electrochemical impedance spectroscopy and Fourier transform infrared spectrophotometry. The biosensor showed optimum response within 1 s, when operated at 50 mV s^?1 in 0.01 M Tris HCl buffer, pH 7.0 at 30 °C. The biosensor exhibited excellent sensitivity with a detection limit of 1.5 µM (S/N = 3) for d-amino acids and wider linear range, 20–500 µM. Analytical recovery of added d-alanine (5 and 10 mM) in serum samples was 98.00 and 98.80 %, respectively. Within-batch and between-batch coefficients of variation in serum samples were 1.36 and 2.77 %, respectively. The enzyme electrode was used more than 50 times over 2 months, when stored at 4 °C. The proposed modified electrode exhibited sufficient mechanical and electrochemical stability and high sensitivity compared to earlier electrochemical d-amino acid biosensors. Interference by ascorbic acid and uric acid, the main interfering species in the biological samples, was negligible.