Biochemical identification of a hydroperoxide derivative of the free 8-oxo-7,8-dihydroguanine base

8-Oxo-7,8-dihydroguanine is one the most abundant base lesions in pro- and eukaryotic DNA. In mammalian cells, it is excised by the 8-oxoguanine DNA glycosylase (OGG1) during DNA base-excision repair, and the generated free 8-oxoG base is one of the DNA-derived biomarkers of oxidative stress in biological samples. The modification of 8-oxoG in the context of nucleoside and DNA has been the subject of many studies; however, the oxidative transformation of the free 8-oxoG base has not been described. By using biochemical and cell biological assays, we show that in the presence of molecular oxygen, the free 8-oxoG base transforms to a highly reactive hydroperoxide (8-oxoG*). Specifically, 8-oxoG* oxidizes Amplex red to resorufin, H(2)DCF to DCF, Fe(2+) to Fe(3+), and GSH to GSSG. This property of 8-oxoG* was diminished by treatment with catalase and glutathione peroxidase, but not superoxide dismutase. 8-OxoG* formation was prevented by reducing agents or nitrogen atmosphere. Its addition to CM-H(2)DCF-DA-loaded cells rapidly increased intracellular DCF fluorescence. There were no such properties observed for 8-oxodeoxyguanosine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, 2'-deoxyguanosine, guanine, adenine, guanosine, and 8-hydroxyadenine. These data imply that a free 8-oxoG base is more susceptible to oxidation than is its nucleoside form and, consequently, it stands as unique among intact and oxidatively modified purines.     

Identification of loci governing eight agronomic traits using a GBS‐GWAS approach and validation by QTL mapping in soya bean

Soya bean is a major source of edible oil and protein for human consumption as well as animal feed. Understanding the genetic basis of different traits in soya bean will provide important insights for improving breeding strategies for this crop. A genome‐wide association study (GWAS) was conducted to accelerate molecular breeding for the improvement of agronomic traits in soya bean. A genotyping‐by‐sequencing (GBS) approach was used to provide dense genome‐wide marker coverage (>47 000 SNPs) for a panel of 304 short‐season soya bean lines. A subset of 139 lines, representative of the diversity among these, was characterized phenotypically for eight traits under six environments (3 sites × 2 years). Marker coverage proved sufficient to ensure highly significant associations between the genes known to control simple traits (flower, hilum and pubescence colour) and flanking SNPs. Between one and eight genomic loci associated with more complex traits (maturity, plant height, seed weight, seed oil and protein) were also identified. Importantly, most of these GWAS loci were located within genomic regions identified by previously reported quantitative trait locus (QTL) for these traits. In some cases, the reported QTLs were also successfully validated by additional QTL mapping in a biparental population. This study demonstrates that integrating GBS and GWAS can be used as a powerful complementary approach to classical biparental mapping for dissecting complex traits in soya bean.     

Xenon reduces activation of transient receptor potential vanilloid type 1 (TRPV1) in rat dorsal root ganglion cells and in human TRPV1-expressing HEK293 cells

AimsXenon provides effective analgesia in several pain states at sub-anaesthetic doses. Our aim was to examine whether xenon may mediate its analgesic effect, in part, through reducing the activity of transient receptor potential vanilloid type 1 (TRPV1), a receptor known to be involved in certain inflammatory pain conditions.Main methodsWe studied the effect of xenon on capsaicin-evoked cobalt uptake in rat cultured primary sensory neurons and in human TRPV1 (hTRPV1)-expressing human embryonic kidney 293 (HEK293) cells. We also examined xenon's effect on the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) in the rat spinal dorsal horn evoked by hind-paw injection of capsaicin.Key findingsXenon (75%) reduced the number of primary sensory neurons responding to the TRPV1 agonist, capsaicin (100 nM–1 μM) by ~ 25% to ~ 50%. Xenon reduced the number of heterologously-expressed hTRPV1 activated by 300 nM capsaicin by ~ 50%. Xenon (80%) reduced by ~ 40% the number of phosporylated ERK1/2-expressing neurons in rat spinal dorsal horn resulting from hind-paw capsaicin injection.SignificanceXenon substantially reduces the activity of TRPV1 in response to noxious stimulation by the specific TRPV1 agonist, capsaicin, suggesting a possible role for xenon as an adjunct analgesic where hTRPV1 is an active contributor to the excitation of primary afferents which initiates the pain sensation.     

Antigen-specific CD8+ T cells mediate a peptide-induced fatal syndrome

Peptide immunotherapy both activates and suppresses the T cell response against known peptide Ags. Although pretreatment with VP2(121-130) peptide inhibits the development of antiviral CTL specific for the immunodominant D(b):VP2(121-130) epitope expressed during acute Theiler's murine encephalomyelitis virus infection, i.v. injection of this same peptide or MHC tetramers containing the peptide during an ongoing antiviral CTL response results in a peptide-induced fatal syndrome (PIFS) within 48 h. Susceptibility to PIFS is dependent on peptide-specific CD8(+) T cells, varies among inbred strains of mice, and is not mediated by traditionally defined mechanisms of shock. Analyses using bone marrow chimeras and mutant mice demonstrate that susceptibility to PIFS is determined by the genotype of bone marrow-derived cells and requires the expression of perforin. Animals responding to peptide treatment with PIFS develop classical stress responses in the brain. These findings raise important considerations for the development of peptide therapies for active diseases to modify immune responses involving expanded populations of T cells. In summary, treatment with peptides or MHC-tetramers during a peptide-specific immune response can result in a fatal shock-like syndrome. Susceptibility to the syndrome is genetically determined, is mediated by CD8(+) T cells, and requires expression of perforin. These findings raise concerns about the use of peptides and MHC tetramers in therapeutic schemes.     

Perforin Competent CD8 T Cells Are Sufficient to Cause Immune-Mediated Blood-Brain Barrier Disruption

Numerous neurological disorders are characterized by central nervous system (CNS) vascular permeability. However, the underlying contribution of inflammatory-derived factors leading to pathology associated with blood-brain barrier (BBB) disruption remains poorly understood. In order to address this, we developed an inducible model of BBB disruption using a variation of the Theiler''s murine encephalomyelitis virus (TMEV) model of multiple sclerosis. This peptide induced fatal syndrome (PIFS) model is initiated by virus-specific CD8 T cells and results in severe CNS vascular permeability and death in the C57BL/6 mouse strain. While perforin is required for BBB disruption, the cellular source of perforin has remained unidentified. In addition to CD8 T cells, various innate immune cells also express perforin and therefore could also contribute to BBB disruption. To investigate this, we isolated the CD8 T cell as the sole perforin-expressing cell type in the PIFS model through adoptive transfer techniques. We determined that C57BL/6 perforin^−/− mice reconstituted with perforin competent CD8 T cells and induced to undergo PIFS exhibited: 1) heightened CNS vascular permeability, 2) increased astrocyte activation as measured by GFAP expression, and 3) loss of linear organization of BBB tight junction proteins claudin-5 and occludin in areas of CNS vascular permeability when compared to mock-treated controls. These results are consistent with the characteristics associated with PIFS in perforin competent mice. Therefore, CD8 T cells are sufficient as a sole perforin-expressing cell type to cause BBB disruption in the PIFS model.     

Crystal structures of the semireduced and inhibitor-bound forms of cyclic nucleotide phosphodiesterase from Arabidopsis thaliana.

The crystal structure of the semireduced form of cyclic nucleotide phosphodiesterase (CPDase) from Arabidopsis thaliana has been solved by molecular replacement and refined at the resolution of 1.8 A. We have previously reported the crystal structure of the native form of this enzyme, whose main target is ADP-ribose 1",2"-cyclic phosphate, a product of the tRNA splicing reaction. CPDase possesses six cysteine residues, four of which are involved in forming two intra-molecular disulfide bridges. One of these bridges, between Cys-104 and Cys-110, is opened in the semireduced CPDase, whereas the other remains intact. This change of the redox state leads to a conformational rearrangement in the loop covering the active site of the protein. While the native structure shows this partially disordered loop in a coil conformation, in the semireduced enzyme the N-terminal lobe of this loop winds up and elongates the preceding alpha-helix. The semireduced state of CPDase also enabled co-crystallization with a putative inhibitor of its enzymatic activity, 2',3'-cyclic uridine vanadate. The ligand is bound within the active site, and the mode of binding is in agreement with the previously proposed enzymatic mechanism. Selected biophysical properties of the oxidized and the semireduced CPDase are also discussed.     

Membrane Curvature Revisited—the Archetype of Rhodopsin Studied by Time-Resolved Electronic Spectroscopy

G-protein-coupled receptors (GPCRs) comprise the largest and most pharmacologically targeted membrane protein family. Here, we used the visual receptor rhodopsin as an archetype for understanding membrane lipid influences on conformational changes involved in GPCR activation. Visual rhodopsin was recombined with lipids varying in their degree of acyl chain unsaturation and polar headgroup size using 1-palmitoyl-2-oleoyl-sn-glycero- and 1,2-dioleoyl-sn-glycerophospholipids with phosphocholine (PC) or phosphoethanolamine (PE) substituents. The receptor activation profile after light excitation was measured using time-resolved ultraviolet-visible spectroscopy. We discovered that more saturated POPC lipids back shifted the equilibrium to the inactive state, whereas the small-headgroup, highly unsaturated DOPE lipids favored the active state. Increasing unsaturation and decreasing headgroup size have similar effects that combine to yield control of rhodopsin activation, and necessitate factors beyond proteolipid solvation energy and bilayer surface electrostatics. Hence, we consider a balance of curvature free energy with hydrophobic matching and demonstrate how our data support a flexible surface model (FSM) for the coupling between proteins and lipids. The FSM is based on the Helfrich formulation of membrane bending energy as we previously first applied to lipid-protein interactions. Membrane elasticity and curvature strain are induced by lateral pressure imbalances between the constituent lipids and drive key physiological processes at the membrane level. Spontaneous negative monolayer curvature toward water is mediated by unsaturated, small-headgroup lipids and couples directly to GPCR activation upon light absorption by rhodopsin. For the first time to our knowledge, we demonstrate this modulation in both the equilibrium and pre-equilibrium evolving states using a time-resolved approach.     

Binding of small molecules to cavity forming mutants of a de novo designed protein

A central goal of protein design is to devise novel proteins for applications in biotechnology and medicine. Many applications, including those focused on sensing and catalysis will require proteins that recognize and bind to small molecules. Here, we show that stably folded α-helical proteins isolated from a binary patterned library of designed sequences can be mutated to produce binding sites capable of binding a range of small aromatic compounds. Specifically, we mutated two phenylalanine side chains to alanine in the known structure of de novo protein S-824 to create buried cavities in the core of this four-helix bundle. The parental protein and the Phe→Ala variants were exposed to mixtures of compounds, and selective binding was assessed by saturation transfer difference NMR. The affinities of benzene and a number of its derivatives were determined by pulse field gradient spin echo NMR, and several of the compounds were shown to bind the mutated protein with micromolar dissociation constants. These studies suggest that stably folded de novo proteins from binary patterned libraries are well-suited as scaffolds for the design of binding sites.     

Age-related loss of stress-induced nuclear proteasome activation is due to low PARP-1 activity

Changes in protein turnover are among the dominant metabolic changes during aging. Of special importance is the maintenance of nuclear protein homeostasis to ensure a coordinated cellular metabolism. Therefore, in the nucleus a special PARP-1-mediated mechanism of proteasomal activation exists to ensure a rapid degradation of oxidized nuclear proteins. It was already demonstrated earlier that the cytosolic proteasomal system declines dramatically with aging, whereas the nuclear proteasome remains less affected. We demonstrate here that the stress-mediated proteasomal activation in the nucleus declines during replicative senescence of human fibroblasts. Furthermore, we clearly show that this decline in the PARP-1-mediated proteasomal activation is due to a decline in the expression and activity of PARP-1 in senescent fibroblasts. In a final study we show that this process also happens in vivo, because the protein expression level of PARP-1 is significantly lower in the skin of aged donors compared to that of young ones. Therefore, we conclude that the rate-limiting factor in poly(ADP-ribose)-mediated proteasomal activation in oxidative stress is PARP-1 and not the nuclear proteasome itself.     

Polymorphic DNA haplotypes at the phenylalanine hydroxylase (PAH) locus in European families with phenylketonuria (PKU)

DNA haplotype data from the phenylalanine hydroxylase (PAH) locus are available from a number of European populations as a result of RFLP testing for genetic counseling in families with phenylketonuria (PKU). We have analyzed data from Hungary and Czechoslovakia together with published data from five additional countries--Denmark, Switzerland, Scotland, Germany, and France--representing a broad geographic and ethnographic range. The data include 686 complete chromosomal haplotypes for eight RFLP sites assayed in 202 unrelated Caucasian families with PKU. Forty-six distinct RFLP haplotypes have been observed to date, 10 unique to PKU-bearing chromosomes, 12 unique to non-PKU chromosomes, and the remainder found in association with both types. Despite the large number of haplotypes observed (still much less than the theoretical maximum of 384), five haplotypes alone account for more than 76% of normal European chromosomes and four haplotypes alone account for more than 80% of PKU-bearing chromosomes. We evaluated the distribution of haplotypes and alleles within these populations and calculated pairwise disequilibrium values between RFLP sites and between these sites and a hypothetical PKU "locus." These are statistically significant differences between European populations in the frequencies of non-PKU chromosomal haplotypes (P = .025) and PKU chromosomal haplotypes (P much less than .001). Haplotype frequencies of the PKU and non-PKU chromosomes also differ significantly (P much less than .001. Disequilibrium values are consistent with the PAH physical map and support the molecular evidence for multiple, independent PKU mutations in Caucasians. However, the data do not support a single geographic origin for these mutations.(ABSTRACT TRUNCATED AT 250 WORDS)