Oxidoreductase activities of polyclonal IgGs from the sera of Wistar rats are better activated by combinations of different metal ions

It was shown that IgGs purified from the sera of healthy Wistar rats contain several different bound Me2+ ions and oxidize 3,3'-diaminobenzidine through a H2O2-dependent peroxidase and H2O2-independent oxidoreductase activity. IgGs have lost these activities after removing the internal metal ions by dialysis against EDTA. External Cu2+ or Fe2+ activated significantly both activities of non-dialysed IgGs containing different internal metals (Fe > or = Pb > or = Zn > or = Cu > or = Al > or = Ca > or = Ni > or = Mn > Co > or = Mg) showing pronounced biphasic dependencies corresponding to approximately 0.1-2 and approximately 2-5 mM of Me2+, while the curves for Mn2+ were nearly linear. Cu2+ alone significantly stimulated both the peroxidase and oxidoreductase activities of dialysed IgGs only at high concentration (> or = 2 mM), while Mn2+ weakly activated peroxidase activity at concentration >3 mM but was active in the oxidoreductase oxidation at a low concentration (<1 mM). Fe2+-dependent peroxidase activity of dialysed IgGs was observed at 0.1-5 mM, but Fe2+ was completely inactive in the oxidoreductase reaction. Mg2+, Ca2+, Zn2+, Al2+ and especially Co2+ and Ni2+ were not able to activate dialysed IgGs, but slightly activated non-dialysed IgGs. The use of the combinations of Cu2+ + Mn2+, Cu2+ + Zn2+, Fe2+ + Mn2+, Fe2+ + Zn2+ led to a conversion of the biphasic curves to hyperbolic ones and in parallel to a significant increase in the activity as compared with Cu2+, Fe2+ or Mn2+ ions taken separately; the rates of the oxidation reactions, catalysed by non-dialysed and dialysed IgGs, became comparable. Mg2+, Co2+ and Ni2+ markedly activated the Cu2+-dependent oxidation reactions catalysed by dialysed IgGs, while Ca2+ inhibited these reactions. A possible role of the second metal in the oxidation reactions is discussed.     

An EPR spin probe study of liposomes from sunflower and soybean phospholipids

Comparative properties of lecithin-based liposomes prepared from the mixed phospholipids of sunflower seeds, soybean and egg yolk were investigated by electron paramagnetic resonance (EPR) spectroscopy. For these investigations, stable nitroxide radicals, 1-oxyl-2,2,6,6-tetramethylpiperidin-4-yl 5,7-dimethyladamantane-1-carboxylate (DMAC-TEMPO), 5-doxylstearic acid (5-DSA) and 16-doxylstearic acid (16-DSA) were used as spin probes. Binding of the spin probes to the liposome membranes resulted in a substantial increase of the apparent rotational diffusion correlation times. The EPR spectra of the incorporated nitroxides underwent temperature-dependent changes. For every spin probe, values of apparent enthalpy and entropy of activation were calculated from the temperature dependence of rotational diffusion correlation times via Arrhenius equation. In case of DMAC-TEMPO, the data point to differences between the phospholipid bilayer of liposomes derived from sunflower and soy lecithin, and some similarity between the sunflower and egg yolk liposomes. Anisotropic hyperfine interaction constants of DMAC-TEMPO and 16-DSA included in the liposomes have been analyzed and attributed to different micropolarity of the surroundings of the spin probes. The kinetics of EPR signal decay of DMAC-TEMPO in the presence of 2,2′-azobis(2-amidinopropane) suggest the better stability of the sunflower liposomes to lipid peroxidation as compared to the liposomes prepared from soy lecithin.     

Open reading frame ssr2016 is required for antimycin A-sensitive photosystem I-driven cyclic electron flow in the cyanobacterium Synechocystis sp. PCC 6803

Open reading frame ssr2016 encodes a protein with substantial sequence similarities to PGR5 identified as a component of the antimycin A-sensitive ferredoxin:plastoquinone reductase (FQR) in PSI cyclic photophosphorylation in Arabidopsis thaliana. We studied cyclic electron flow in Synechocystis sp. PCC 6803 in vivo in ssr2016 deletion mutants generated either in a wild-type background or in a ndhB deletion mutant. Our results indicate that ssr2016 is required for FQR and that it operates in a parallel pathway to the NDH1 complex. The ssr2016 deletion mutants are high light sensitive, suggesting that FQR might be important in controlling redox poise under adverse conditions.     

A SNP-based genetic dissection of versatile traits in bread wheat (Triticum aestivum L.)

The continuous increase in global population prompts increased wheat production. Future wheat (Triticum aestivum L.) breeding will heavily rely on dissecting molecular and genetic bases of wheat yield and related traits which is possible through the discovery of quantitative trait loci (QTLs) in constructed populations, such as recombinant inbred lines (RILs). Here, we present an evaluation of 92 RILs in a bi-parental RIL mapping population (the International Triticeae Mapping Initiative Mapping Population [ITMI/MP]) using newly generated phenotypic data in 3-year experiments (2015), older phenotypic data (1997–2009), and newly created single nucleotide polymorphism (SNP) marker data based on 92 of the original RILs to search for novel and stable QTLs. Our analyses of more than 15 unique traits observed in multiple experiments included analyses of 46 traits in three environments in the USA, 69 traits in eight environments in Germany, 149 traits in 10 environments in Russia, and 28 traits in four environments in India (292 traits in 25 environments) with 7584 SNPs (292 × 7584 = 2 214 528 data points). A total of 874 QTLs were detected with limit of detection (LOD) scores of 2.01–3.0 and 432 QTLs were detected with LOD > 3.0. Moreover, 769 QTLs could be assigned to 183 clusters based on the common markers and relative proximity of related QTLs, indicating gene-rich regions throughout the A, B, and D genomes of common wheat. This upgraded genotype–phenotype information of ITMI/MP can assist breeders and geneticists who can make crosses with suitable RILs to improve or investigate traits of interest.     

N-Terminal segment of potato virus X coat protein subunits is glycosylated and mediates formation of a bound water shell on the virion surface.

The primary structures of N-terminal 19-mer peptides, released by limited trypsin treatment of coat protein (CP) subunits in intact virions of three potato virus X (PVX) isolates, were analyzed. Two wild-type PVX strains, Russian (Ru) and British (UK3), were used and also the ST mutant of UK3 in which all 12 serine and threonine residues in the CP N-terminal segment were replaced by glycine or alanine. With the help of direct carbohydrate analysis and MS, it was found that the acetylated N-terminal peptides of both wild-type strains are glycosylated by a single monosaccharide residue (galactose or fucose) at NAcSer in the first position of the CP sequence, whereas the acetylated N-terminal segment of the ST mutant CP is unglycosylated. Fourier transform infrared spectra in the 1000-4000 cm(-1) region were measured for films of the intact and in situ trypsin-degraded PVX preparations at low and high humidity. These spectra revealed the presence of a broad-band in the region of valent vibrations of OH bonds (3100-3700 cm(-1)), which can be represented by superposition of three bands corresponding to tightly bound, weakly bound, and free OH groups. On calculating difference ('wet' minus 'dry') spectra, it was found that the intact wild-type PVX virions are characterized by high water-absorbing capacity and the ability to order a large number of water molecules on the virus particle. This effect was much weaker for the ST mutant and completely absent in the trypsin-treated PVX. It is proposed that the surface-located and glycosylated N-terminal CP segments of intact PVX virions induce the formation of a columnar-type shell from bound water molecules around the virions, which probably play a major role in maintaining the virion surface structure.     

Effect of 6-Benzoyl-benzothiazol-2-one scaffold on the pharmacological profile of α-alkoxyphenylpropionic acid derived PPAR agonists

Abstract

A series of nitrogen heterocycles containing α–ethoxyphenylpropionic acid derivatives were designed as dual PPARα/γ agonist ligands for the treatment of type 2 diabetes (T2D) and its complications. 6-Benzoyl-benzothiazol-2-one was the most tolerant of the tested heterocycles in which incorporation of O-methyl oxime ether and trifluoroethoxy group followed by enantiomeric resolution led to the (S)-stereoisomer 44?b displaying the best in vitro pharmacological profile. Compound 44?b acted as a very potent full PPARγ agonist and a weak partial agonist on the PPARα receptor subtype. Compound 44?b showed high efficacy in an ob/ob mice model with significant decreases in serum triglyceride, glucose and insulin levels but mostly with limited body-weight gain and could be considered as a selective PPARγ modulator (SPPARγM).     

Thermodynamic,kinetic, and structural basis for recognition and repair of 8-oxoguanine in DNA by Fpg protein from Escherichia coli

X-ray analysis does not provide quantitative estimates of the relative importance of the molecular contacts it reveals or of the relative contributions of specific and nonspecific interactions to the total affinity of specific DNA to enzymes. Stepwise increase of DNA ligand complexity has been used to estimate the relative contributions of virtually every nucleotide unit of 8-oxoguanine-containing DNA to its total affinity for Escherichia coli 8-oxoguanine DNA glycosylase (Fpg protein). Fpg protein can interact with up to 13 nucleotide units or base pairs of single- and double-stranded ribo- and deoxyribo-oligonucleotides of different lengths and sequences through weak additive contacts with their internucleotide phosphate groups. Bindings of both single-stranded and double-stranded oligonucleotides follow similar algorithms, with additive contributions to the free energy of binding of the structural components (phosphate, sugar, and base). Thermodynamic models are provided for both specific and nonspecific DNA sequences with Fpg protein. Fpg protein interacts nonspecifically with virtually all of the base-pair units within its DNA-binding cleft: this provides approximately 7 orders of magnitude of affinity (Delta G degrees approximately equal to -9.8 kcal/mol) for DNA. In contrast, the relative contribution of the 8-oxoguanine unit of the substrate (Delta G degrees approximately equal to -0.90 kcal/mol) together with other specific interactions is <2 orders of magnitude (Delta G degrees approximately equal to -2.8 kcal/mol). Michaelis complex formation of Fpg protein with DNA containing 8-oxoguanine cannot of itself provide the major part of the enzyme specificity, which lies in the k(cat) term; the rate is increased by 6-8 orders of magnitude on going from nonspecific to specific oligodeoxynucleotides.     

A Model of the Membrane-bound Cytochrome b5-Cytochrome P450 Complex from NMR and Mutagenesis Data

Microsomal cytochrome b₅ (cytb₅) is a membrane-bound protein that modulates the catalytic activity of its redox partner, cytochrome P4502B4 (cytP450). Here, we report the first structure of full-length rabbit ferric microsomal cytb₅ (16 kDa), incorporated in two different membrane mimetics (detergent micelles and lipid bicelles). Differential line broadening of the cytb₅ NMR resonances and site-directed mutagenesis data were used to characterize the cytb₅ interaction epitope recognized by ferric microsomal cytP450 (56 kDa). Subsequently, a data-driven docking algorithm, HADDOCK (high ambiguity driven biomolecular docking), was used to generate the structure of the complex between cytP4502B4 and cytb₅ using experimentally derived restraints from NMR, mutagenesis, and the double mutant cycle data obtained on the full-length proteins. Our docking and experimental results point to the formation of a dynamic electron transfer complex between the acidic convex surface of cytb₅ and the concave basic proximal surface of cytP4502B4. The majority of the binding energy for the complex is provided by interactions between residues on the C-helix and β-bulge of cytP450 and residues at the end of helix α4 of cytb₅. The structure of the complex allows us to propose an interprotein electron transfer pathway involving the highly conserved Arg-125 on cytP450 serving as a salt bridge between the heme propionates of cytP450 and cytb₅. We have also shown that the addition of a substrate to cytP450 likely strengthens the cytb₅-cytP450 interaction. This study paves the way to obtaining valuable structural, functional, and dynamic information on membrane-bound complexes.