The first draft of the pigeonpea genome sequence

Pigeonpea (Cajanus cajan) is an important grain legume of the Indian subcontinent, South-East Asia and East Africa. More than eighty five percent of the world pigeonpea is produced and consumed in India where it is a key crop for food and nutritional security of the people. Here we present the first draft of the genome sequence of a popular pigeonpea variety ??Asha??. The genome was assembled using long sequence reads of 454 GS-FLX sequencing chemistry with mean read lengths of >550?bp and >10-fold genome coverage, resulting in 510,809,477?bp of high quality sequence. Total 47,004 protein coding genes and 12,511 transposable elements related genes were predicted. We identified 1,213 disease resistance/defense response genes and 152 abiotic stress tolerance genes in the pigeonpea genome that make it a hardy crop. In comparison to soybean, pigeonpea has relatively fewer number of genes for lipid biosynthesis and larger number of genes for cellulose synthesis. The sequence contigs were arranged in to 59,681 scaffolds, which were anchored to eleven chromosomes of pigeonpea with 347 genic-SNP markers of an intra-species reference genetic map. Eleven pigeonpea chromosomes showed low but significant synteny with the twenty chromosomes of soybean. The genome sequence was used to identify large number of hypervariable ??Arhar?? simple sequence repeat (HASSR) markers, 437 of which were experimentally validated for PCR amplification and high rate of polymorphism among pigeonpea varieties. These markers will be useful for fingerprinting and diversity analysis of pigeonpea germplasm and molecular breeding applications. This is the first plant genome sequence completed entirely through a network of Indian institutions led by the Indian Council of Agricultural Research and provides a valuable resource for the pigeonpea variety improvement.     

Therapeutic associated with occupational exposure to silica

Occupational exposure to silica dust has been increasing the possible risk of varieties of pathologies. The aim of this study was to evaluate the protective activity of ethanolic extract of Glycyrrhiza glabra roots at doses of 500 and 1000 mg/kg, p.o., given for 7 days against the toxicity of SiO(2) nanoparticles (50mg/kg intraperitoneal for 6 weeks) in rats. Exposure to silica altered various respiratory and biochemical variables, including ALT, AST, albumin, urea, uric acid, creatinine, catalase, LPO and GSH. Treatments with G. glabra extract significantly improved antioxidant status towards control. Stone workers in the Gwalior region exposed to silica dust had higher prevalence of cough, wheezing and shortness of breath. Increased serum ACE level was noted in the silica exposed group. It is of immense need to monitor this problem for betterment of worker's health.     

An empirical phase diagram approach to investigate conformational stability of "second-generation" functional mutants of acidic fibroblast growth factor-1

Acidic fibroblast growth factor-1 (FGF-1) is an angiogenic protein which requires binding to a polyanion such as heparin for its mitogenic activity and physicochemical stability. To evaluate the extent to which this heparin dependence on solution stability could be reduced or eliminated, the structural integrity and conformational stability of 10 selected FGF-1 mutants were examined as a function of solution pH and temperature by a series of spectroscopic methods including circular dichroism, intrinsic and extrinsic fluorescence spectroscopy and static light scattering. The biophysical data were summarized in the form of colored empirical phase diagrams (EPDs). FGF-1 mutants were identified with stability profiles in the absence of heparin comparable to that of wild-type FGF-1 in the presence of heparin while still retaining their biological activity. In addition, a revised version of the EPD methodology was found to provide an information rich, high throughput approach to compare the effects of mutations on the overall conformational stability of proteins in terms of their response to environmental stresses such as pH and temperature.     

Improved data visualization techniques for analyzing macromolecule structural changes

The empirical phase diagram (EPD) is a colored representation of overall structural integrity and conformational stability of macromolecules in response to various environmental perturbations. Numerous proteins and macromolecular complexes have been analyzed by EPDs to summarize results from large data sets from multiple biophysical techniques. The current EPD method suffers from a number of deficiencies including lack of a meaningful relationship between color and actual molecular features, difficulties in identifying contributions from individual techniques, and a limited ability to be interpreted by color‐blind individuals. In this work, three improved data visualization approaches are proposed as techniques complementary to the EPD. The secondary, tertiary, and quaternary structural changes of multiple proteins as a function of environmental stress were first measured using circular dichroism, intrinsic fluorescence spectroscopy, and static light scattering, respectively. Data sets were then visualized as (1) RGB colors using three‐index EPDs, (2) equiangular polygons using radar charts, and (3) human facial features using Chernoff face diagrams. Data as a function of temperature and pH for bovine serum albumin, aldolase, and chymotrypsin as well as candidate protein vaccine antigens including a serine threonine kinase protein (SP1732) and surface antigen A (SP1650) from S. pneumoniae and hemagglutinin from an H1N1 influenza virus are used to illustrate the advantages and disadvantages of each type of data visualization technique.     

Towards irreversible HIV inactivation: stable gp120 binding by nucleophilic antibodies

Conventional antibodies react with antigens reversibly. We report the formation of unusually stable complexes of HIV gp120 and nucleophilic antibodies raised by immunization with an electrophilic HIV gp120 analog (E-gp120). The stability of the complexes was evident from their very slow dissociation in a nondenaturing solvent (approximate t(1/2) 18.5 days) and their resistance to dissociation by a denaturant commonly employed to disrupt noncovalent protein-protein binding (sodium dodecyl sulfate). Kinetic studies indicated time-dependent and virtually complete progression of the antibody-gp120 complexes from the initial noncovalent state to a poorly dissociable state. The antibodies to E-gp120 displayed improved covalent reactivity with an electrophilic phosphonate probe compared to control antibodies, suggesting their enhanced nucleophilicity. One of the stably binding antibodies neutralized the infectivity of CCR5-dependent primary HIV strains belonging to clades B and C. These findings suggest the feasibility of raising antibodies capable of long-lasting inactivation of antigens by electrophilic immunization.     

Engineering tissues for in vitro applications

Engineered tissues can be employed for studies on the fundamental mechanisms of embryology and adult physiology and for investigating the evolution of disease processes. They also provide platforms to evaluate the behavior of new chemical entities in drug development. The recent development of three specific technologies has greatly facilitated the engineering of tissues for in vitro applications: the microfabrication tools that serve to both define the cellular microenvironment and enable parallelization of cell-based assays; synthetic, tunable hydrogels to create three-dimensional microenvironments; and bioreactors to control nutrient transport and fluid shear stress. Furthermore, convergence of these tools is providing investigators with the opportunity to construct and study tissues in vitro with unprecedented levels of sophistication.     

Probing protein structure and dynamics by second-derivative ultraviolet absorption analysis of cation-{pi} interactions

We describe an alternate approach for studying protein structure using the detection of ultraviolet (UV) absorbance peak shifts of aromatic amino acid side chains induced by the presence of salts. The method is based on the hypothesis that salt cations (Li+, Na+, and Cs+) of varying sizes can differentially diffuse through protein matrices and interact with benzyl, phenyl, and indole groups through cation-pi interactions. We have investigated the potential of this method to probe protein dynamics by measuring high resolution second-derivative UV spectra as a function of salt concentration for eight proteins of varying physical and chemical properties and the N-acetylated C-ethyl esterified amino acids to represent totally exposed side chains. We show that small shifts in the wavelength maxima for Phe, Tyr, and Trp in the presence of high salt concentrations can be reliably measured and that the magnitude and direction of the peak shifts are influenced by several factors, including protein size, charge, and the local environment and solvent accessibility of the aromatic groups. Evaluating the empirical UV spectral data in light of known protein structural information shows that probing cation-pi interactions in proteins reveals unique information about the influence of structure on aromatic side chain spectroscopic behavior.     

The synthesis and high-level expression of a beta2-adrenergic receptor gene in a tetracycline-inducible stable mammalian cell line

High-level expression of G-protein-coupled receptors (GPCRs) in functional form is required for structure-function studies. The main goal of the present work was to improve expression levels of beta2-adrenergic receptor (beta2-AR) so that biophysical studies involving EPR, NMR, and crystallography can be pursued. Toward this objective, the total synthesis of a codon-optimized hamster beta2-AR gene suitable for high-level expression in mammalian systems has been accomplished. Transient expression of the gene in COS-1 cells resulted in 18 +/- 3 pmol beta2-AR/mg of membrane protein, as measured by saturation binding assay using the beta2-AR antagonist [3H] dihydroalprenolol. Previously, we reported the development of an HEK293S tetracycline-inducible system for high-level expression of rhodopsin. Here, we describe construction of beta2-AR stable cell lines using the HEK293S-TetR-inducible system, which, after induction, express wild-type beta2-AR at levels of 220 +/- 40 pmol/mg of membrane protein corresponding to 50 +/- 8 microg/15-cm plate. This level of expression is the highest reported so far for any wild-type GPCR, other than rhodopsin. The yield of functional receptor using the single-step affinity purification is 12 +/- 3 microg/15-cm plate. This level of expression now makes it feasible to pursue structure-function studies using EPR. Furthermore, scale-up of beta2-AR expression using suspension cultures in a bioreactor should now allow production of enough beta2-AR for the application of biophysical techniques such as NMR spectroscopy and crystallography.     

Kolaviron inhibits dimethyl nitrosamine-induced liver injury by suppressing COX-2 and iNOS expression via NF-κB and AP-1

AimsKolaviron, a bioflavonoid isolated from the seeds of Garcinia kola has been reported to possess anti-inflammatory, antioxidant, antigenotoxic and hepatoprotective activities in model systems via multiple biochemical mechanisms. The present study investigated the possible molecular mechanisms underlying the hepatoprotective effects of kolaviron.Main methodsBiomarkers of hepatic oxidative injury, histological and immunohistochemical techniques were used. In addition, the protein expression levels of cyclooxygenase (COX-2) and inducible nitric oxide synthase (iNOS) were evaluated by western blotting while DNA-binding activities of nuclear factor kappa B (NF-κB) and activator protein-1 (AP-1) were determined by electrophoretic mobility shift assay.Key findingsKolaviron administered orally at doses of 100 and 200 mg/kg for 7 days significantly lowered the activities of serum transaminases and γ-glutamyl tranferase induced by single intraperitoneal administration of dimethyl nitrosamine (DMN) (20 mg/kg) and preserved the integrity of the hepatocytes. Also, kolaviron at both doses reduced the DMN induced elevated hepatic levels of malondialdehyde and reversed DMN mediated decrease in hepatic glutathione. The hepatoprotective effect of kolaviron was compared to that of curcumin, an established hepatoprotective agent. Kolaviron inhibited the DMN induced expression of COX-2 and iNOS. Immunohistochemical staining of rat liver verified the inhibitory effect of kolaviron on DMN-induced hepatic COX-2 expression. Furthermore, kolaviron abrogated DMN induced binding activity of NF-κB as well as AP-1.SignificanceThe ability of kolaviron to inhibit COX-2 and iNOS expression through down regulation of NF-κB and AP-1 DNA binding activities could be a mechanism for the hepatoprotective properties of kolaviron.