Microsatellite marker-based genetic diversity analysis of elite lentil lines differing in grain iron and zinc concentration

Trial comprising fifty lentil genotypes was conducted at three locations in India during year 2013–2014 for estimation of grain Fe and Zn concentrations and to analyze G × E interactions by employing the additive main effect and multiplication interaction model. The study revealed significant effects for genotype, environment and genotype by environment interaction for both grain Fe and Zn concentration. In this model genotypes with the IPCA score of nearly zero and mean greater than grand mean are considered as adaptable to the studied environments. For grain Fe concentration genotypes P13143, P13135, ILL2581, P2130, LL147, L4603, PL101 and Globe mutant and for grain Zn concentration genotypes P13122, P2239, P3204 and L11-245 were found stable. The diversity analysis using 20 genomic and 54 EST-SSR markers indicated that the studied genotypes were diverse. The EST-SSRs revealed lower polymorphism as compare to genomic SSRs. Two major clusters were identified with 37 % similarity. Indigenous genotypes were grouped in cluster I and all exotic accessions were grouped in cluster II indicating the role of geographic origin in diversity. Based on multi location evaluation for grain Fe and Zn concentrations and molecular characterization crosses are proposed for development of grain Fe and Zn rich varieties (P3220 × L4649 and VL103 × P2130) and development of mapping populations (P13122 × L11-287 and LL931 × P3220) for study of genetics and mapping of QTLs/gene(s).     

A DSS for job scheduling under process interruptions

The primary objective of this research is to solve the job-shop scheduling problems (JSSPs), by minimizing the makespan, with and without process interruptions. In this paper, we first developed a genetic algorithm for solving JSSPs, and then improved the algorithm by integrating it with two simple priority rules and a hybrid rule. The performance of the developed algorithm was tested by solving 40 benchmark problems and comparing their results with that of a number of well-known algorithms. In addition, we have studied the job-shop scheduling under process interruptions such as machine unavailability and breakdown. For convenience of implementation, we have developed a decision support system (DSS). In the DSS, we built a graphical user interface for user friendly data inputs, model choices, and output generation. An overview of the DSS and an analysis of the experimental results are provided. The incorporation of priority rules, and a hybrid rule, not only improves the solutions but also reduces the overall computational time. The experimental results show that when the machine unavailability information is known in advance, the effect on the schedule is very little compared to the sudden machine breakdown scenario.     

Cyclic GMP Kinase II (cGKII) Inhibits NHE3 by Altering Its Trafficking and Phosphorylating NHE3 at Three Required Sites: IDENTIFICATION OF A MULTIFUNCTIONAL PHOSPHORYLATION SITE*

The epithelial brush-border Na⁺/H⁺ exchanger NHE3 is acutely inhibited by cGKII/cGMP, but how cGKII inhibits NHE3 is unknown. This study tested the hypothesis that cGMP inhibits NHE3 by phosphorylating it and altering its membrane trafficking. Studies were carried out in PS120/NHERF2 and in Caco-2/Bbe cells overexpressing HA-NHE3 and cGKII, and in mouse ileum. NHE3 activity was measured with 2′,7′-bis(carboxyethyl)-S-(and 6)carboxyfluorescein acetoxy methylester/fluorometry. Surface NHE3 was determined by cell surface biotinylation. Identification of NHE3 phosphorylation sites was by iTRAQ/LC-MS/MS with TiO₂ enrichment and immunoblotting with specific anti-phospho-NHE3 antibodies. cGMP/cGKII rapidly inhibited NHE3, which was associated with reduced surface NHE3. cGMP/cGKII increased NHE3 phosphorylation at three sites (rabbit Ser⁵⁵⁴, Ser⁶⁰⁷, and Ser⁶⁶³, equivalent to mouse Ser⁵⁵², Ser⁶⁰⁵, and Ser⁶⁵⁹), all of which had to be present at the same time for cGMP to inhibit NHE3. NHE3-Ser⁶⁶³ phosphorylation was not necessary for cAMP inhibition of NHE3. Dexamethasone (4 h) stimulated wild type NHE3 activity and increased surface expression but failed to stimulate NHE3 activity or increase surface expression when NHE3 was mutated to either S663A or S663D. We conclude that 1) cGMP inhibition of NHE3 is associated with phosphorylation of NHE3 at Ser⁵⁵⁴, Ser⁶⁰⁷, and Ser⁶⁶³, all of which are necessary for cGMP/cGKII to inhibit NHE3. 2) Dexamethasone stimulates NHE3 by phosphorylation of a single site, Ser⁶⁶³. The requirement for three phosphorylation sites in NHE3 for cGKII inhibition, and for phosphorylation of one of these sites for dexamethasone stimulation of NHE3, is a unique example of regulation by phosphorylation.     

Functions and Roles of Proteins: Diabetes as a Paradigm

Molecular and cellular biology has moved towards complete and accurate knowledge of how molecules behave in space and time. Protein is considered as the primary group of molecules responsible for mediating most physiological processes. Changes in the levels of proteins may lead to the altered function and are responsible for many diseases. This review provides a partial molecular explanation of biological force-ratio generation that may act to split protein into branches, and shows molecular functional divergence. Developing a non-reductionist theory of the cellular function in medicine is clearly not sufficient. Finding effective parameters of the models by characterizing molecular interactions becomes necessary. Protein interactivity and stability provides a basis for an integrated understanding of pathologies such diabetes. One example of how a mechanistic analysis of such physiological processes can be of value is the time-delay between mRNA and translation that can act as a fork allowing a slowdown in gene expression.     

Soil fertility promotes decomposition rate of nutrient poor,but not nutrient rich litter through nitrogen transfer

Plant and Soil - Litter decomposition is a critical process in terrestrial ecosystems and understanding the effects of soil fertility on the litter decay rate is of great ecological relevance. Here...     

Structural and Functional Characterization of the PaaI Thioesterase from Streptococcus pneumoniae Reveals a Dual Specificity for Phenylacetyl-CoA and Medium-chain Fatty Acyl-CoAs and a Novel CoA-induced Fit Mechanism

PaaI thioesterases are members of the TE13 thioesterase family that catalyze the hydrolysis of thioester bonds between coenzyme A and phenylacetyl-CoA. In this study we characterize the PaaI thioesterase from Streptococcus pneumoniae (SpPaaI), including structural analysis based on crystal diffraction data to 1.8-Å resolution, to reveal two double hotdog domains arranged in a back to back configuration. Consistent with the crystallography data, both size exclusion chromatography and small angle x-ray scattering data support a tetrameric arrangement of thioesterase domains in solution. Assessment of SpPaaI activity against a range of acyl-CoA substrates showed activity for both phenylacetyl-CoA and medium-chain fatty-acyl CoA substrates. Mutagenesis of putative active site residues reveals Asn³⁷, Asp⁵², and Thr⁶⁸ are important for catalysis, and size exclusion chromatography analysis and x-ray crystallography confirm that these mutants retain the same tertiary and quaternary structures, establishing that the reduced activity is not a result of structural perturbations. Interestingly, the structure of SpPaaI in the presence of CoA provides a structural basis for the observed substrate specificity, accommodating a 10-carbon fatty acid chain, and a large conformational change of up to 38 Å in the N terminus, and a loop region involving Tyr³⁸-Tyr³⁹. This is the first time PaaI thioesterases have displayed a dual specificity for medium-chain acyl-CoAs substrates and phenylacetyl-CoA substrates, and we provide a structural basis for this specificity, highlighting a novel induced fit mechanism that is likely to be conserved within members of this enzyme family.     

Screening for natural inhibitors of human topoisomerases from medicinal plants with bio-affinity ultrafiltration and LC–MS

Phytochemistry Reviews - The continuing rise in cancer morbidity and mortality all over the world makes cancer the No. 2 cause of death in humans, posing a major public health challenge. Currently,...     

Affinities of phosphorylated substrates for the E. coli tryptophan synthase α-subunit: Roles of Ser-235 and helix-8′ dipole

The roles of Ser-235 and helix-8′ (residues 235–242) in the functional binding and turnover of phosphorylated substrates by the α-subunit of the E. coli tryptophan synthase (TSase) α₂β₂-holoenzyme complex are examined. Previous crystallographic analyses indicated that this region was one of several near the phosphate moiety of the physiological substrate, indole-3-glycerol phosphate (IGP). The peptidyl amido group of Ser-235 was suggested to H-bond to the phosphate group; a helix macrodipole binding role was suggested for helix-8′. The activities and substrate K_ms of mutant α-subunits altered in this region by site-specific mutagenesis are reported here. Substitutions at Ser-235 by an acidic (glutamic acid, mutant SE235), basic (lysine, mutant SK235), or a nonpeptidyl amido-containing residue (proline, mutant SP235) exhibit 40- to 180-fold K_m increases for IGP and D -glyceraldehyde-3-phosphate; no K_m defects for indole were observed. k_cat values for SP235, SE235, and SK235 are 100, 70, and 40%, respectively, of the wild-type value. Steric considerations may explain the results with the SE235 and SK235 mutant α-subunits; however, the SP235 results are consistent with the suggested phosphate binding role for the Ser-235 peptidyl amide group during catalysis. A helix-8′ dipole role was explored following proline substitutions separately at the first six (of eight) residues. Proline substitutions at positions-1 through -4 in helix-8′ have normal indole K_ms and catalytic activities in all four TSase reactions, suggesting no major global structural changes in these proteins. By these criteria, substitutions at positions-5 and -6 lead to significant structural alterations. K_m increases for phosphorylated substrates are substantial (up to 40-fold) and are dependent upon the presence of L -serine at the β-subunit active site. In the absence of L -serine, substitution only at the first position results in binding defects; in the presence of L -serine, substitutions at the first, second and third positions show binding defects of decreasing magnitude, sequentially. Substitutions at the fourth and fifth position have no effect on substrate binding. It is suggested that during catalysis a helix dipole effect on binding may be exerted but only via inter-subunit-induced conformational changes due to ligand (L -serine) binding to the β-subunit. © 1995 Wiley-Liss, Inc.     

Electroacoustic Waves in a Collision-Free Magnetized Superthermal Bi-Ion Plasma

Plasma Physics Reports - The electroacoustic waves, particularly ion-acoustic waves (IAWs), and their expansion in the medium of a magnetized collision-free plasma system has been investigated...