EPR, ENDOR, and TRIPLE resonance spectroscopy on the neutral flavin radical in Escherichia coli DNA photolyase

Ultraviolet radiation promotes the formation of a cyclobutane ring between adjacent pyrimidine residues on the same DNA strand to form a pyrimidine dimer. Such dimers may be restored to their monomeric forms through the action of a light-absorbing enzyme named DNA photolyase. The redox-active cofactor involved in the light-induced electron transfer reactions of DNA repair and enzyme photoactivation is a noncovalently bound FAD. In this paper, the FAD cofactor of Escherichia coli DNA photolyase was characterized as the neutral flavin semiquinone by EPR spectroscopy at 9.68 and 94.5 GHz. From the high-frequency/high-field EPR spectrum, the principal values of the axially symmetric g-matrix of FADH(*) were extracted. Both EPR spectra show an emerging hyperfine splitting of 0.85 mT that could be assigned to the isotropic hyperfine coupling constant (hfc) of the proton at N(5). To obtain more information about the electron spin density distribution ENDOR and TRIPLE resonance spectroscopies were applied. All major proton hfc's could be measured and unambiguously assigned to molecular positions at the isoalloxazin moiety of FAD. The isotropic hfc's of the protons at C(8alpha) and C(6) are among the smallest values reported for protein-bound neutral flavin semiquinones so far, suggesting a highly restricted delocalization of the unpaired electron spin on the isoalloxazin moiety. Two further hfc's have been detected and assigned to the inequivalent protons at C(1'). Some conclusions about the geometrical arrangement of the ribityl side chain with respect to the isoalloxazin ring could be drawn: Assuming tetrahedral angles at C(1') the dihedral angle between the C(1')-C(2') bond and the 2p(z)() orbital at N(10) has been estimated to be 170.4 degrees +/- 1 degrees.     

Exploring gene-environment interactions in Parkinson’s disease

The objective of this study was to explore combined effects of four candidate susceptibility genes and two exposures on Parkinson’s disease (PD) risk; namely, α-synuclein (SNCA) promoter polymorphism REP1, microtubule-associated protein tau (MAPT) H1/H2 haplotypes, apolipoprotein E (APOE) ε2/ε3/ε4 polymorphism, ubiquitin carboxy-terminal esterase L1 (UCHL1) S18Y variant, cigarette smoking and caffeinated coffee consumption. 932 PD patients and 664 control subjects from the NeuroGenetics Research Consortium, with complete data on all six factors, were studied. Uniform protocols were used for diagnosis, recruitment, data collection and genotyping. A logistic regression model which included gene-exposure interactions was applied. Likelihood ratio tests (LRTs) were used for significance testing and Bayesian inference was used to estimate odds ratios (ORs). MAPT (P = 0.007), SNCA REP1 (P = 0.012), smoking (P = 0.001), and coffee (P = 0.011) were associated with PD risk. Two novel interactions were detected: APOE with coffee (P = 0.005), and REP1 with smoking (P = 0.021). While the individual main effects were modest, each yielding OR < 1.6, the effects were cumulative, with some combinations reaching OR = 12.6 (95% CI: 5.9–26.8). This study provides evidence for the long-held notion that PD risk is modulated by cumulative and interactive effects of genes and exposures. Furthermore, the study demonstrates that while interaction studies are useful for exploring risk relationships that might otherwise go undetected, results should be interpreted with caution because of the inherent loss of power due to multiple testing. The novel findings of this study that warrant replication are the evidence for interaction of coffee with APOE, and of smoking with REP1 on PD risk. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The lys5 mutations of barley reveal the nature and importance of plastidial ADP-Glc transporters for starch synthesis in cereal endosperm

Much of the ADP-Glc required for starch synthesis in the plastids of cereal endosperm is synthesized in the cytosol and transported across the plastid envelope. To provide information on the nature and role of the plastidial ADP-Glc transporter in barley (Hordeum vulgare), we screened a collection of low-starch mutants for lines with abnormally high levels of ADP-Glc in the developing endosperm. Three independent mutants were discovered, all of which carried mutations at the lys5 locus. Plastids isolated from the lys5 mutants were able to synthesize starch at normal rates from Glc-1-P but not from ADP-Glc, suggesting a specific lesion in the transport of ADP-Glc across the plastid envelope. The major plastidial envelope protein was purified, and its sequence showed it to be homologous to the maize (Zea mays) ADP-Glc transporter BRITTLE1. The gene encoding this protein in barley, Hv.Nst1, was cloned, sequenced, and mapped. Like lys5, Hv.Nst1 lies on chromosome 6(6H), and all three of the lys5 alleles that were examined were shown to carry lesions in Hv.Nst1. Two of the identified mutations in Hv.Nst1 lead to amino acid substitutions in a domain that is conserved in all members of the family of carrier proteins to which Hv.NST1 belongs. This strongly suggests that Hv.Nst1 lies at the Lys5 locus and encodes a plastidial ADP-Glc transporter. The low-starch phenotype of the lys5 mutants shows that the ADP-Glc transporter is required for normal rates of starch synthesis. This work on Hv.NST1, together with the earlier work on BRITTLE1, suggests that homologous transporters are probably present in the endosperm of all cereals.     

DJ-1 Protects Pancreatic Beta Cells from Cytokine- and Streptozotocin-Mediated Cell Death

A hallmark feature of type 1 and type 2 diabetes mellitus is the progressive dysfunction and loss of insulin-producing pancreatic beta cells, and inflammatory cytokines are known to trigger beta cell death. Here we asked whether the anti-oxidant protein DJ-1 encoded by the Parkinson’s disease gene PARK7 protects islet cells from cytokine- and streptozotocin-mediated cell death. Wild type and DJ-1 knockout mice (KO) were treated with multiple low doses of streptozotocin (MLDS) to induce inflammatory beta cell stress and cell death. Subsequently, glucose tolerance tests were performed, and plasma insulin as well as fasting and random blood glucose concentrations were monitored. Mitochondrial morphology and number of insulin granules were quantified in beta cells. Moreover, islet cell damage was determined in vitro after streptozotocin and cytokine treatment of isolated wild type and DJ-1 KO islets using calcein AM/ethidium homodimer-1 staining and TUNEL staining. Compared to wild type mice, DJ-1 KO mice became diabetic following MLDS treatment. Insulin concentrations were substantially reduced, and fasting blood glucose concentrations were significantly higher in MLDS-treated DJ-1 KO mice compared to equally treated wild type mice. Rates of beta cell apoptosis upon MLDS treatment were twofold higher in DJ-1 KO mice compared to wild type mice, and in vitro inflammatory cytokines led to twice as much beta cell death in pancreatic islets from DJ-1 KO mice versus those of wild type mice. In conclusion, this study identified the anti-oxidant protein DJ-1 as being capable of protecting pancreatic islet cells from cell death induced by an inflammatory and cytotoxic setting.     

Seasonal changes in adiposity: the roles of the photoperiod,melatonin and other hormones,and sympathetic nervous system

It appears advantageous for many non-human animals to store energy body fat extensively and efficiently because their food supply is more labile and less abundant than in their human counterparts. The level of adiposity in many of these species often shows predictable increases and decreases with changes in the season. These cyclic changes in seasonal adiposity in some species are triggered by changes in the photoperiod that are faithfully transduced into a biochemical signal through the nightly secretion of melatonin (MEL) via the pineal gland. Here, we focus primarily on the findings from the most commonly studied species showing seasonal changes in adiposity-Siberian and Syrian hamsters. The data to date are not compelling for a direct effect of MEL on white adipose tissue (WAT) and brown adipose tissue (BAT) despite some recent data to the contrary. Thus far, none of the possible hormonal intermediaries for the effects of MEL on seasonal adiposity appear likely as a mechanism by which MEL affects the photoperiodic control of body fat levels indirectly. We also provide evidence pointing toward the sympathetic nervous system as a likely mediator of the effects of MEL on short day-induced body fat decreases in Siberian hamsters through increases in sympathetic drive on WAT and BAT. We speculate that decreases in the SNS drive to these tissues may underlie the photoperiod-induced seasonal increases in body fat of species such as Syrian hamsters. Clearly, we need to deepen our understanding of seasonal adiposity, although, to our knowledge, this is the only form of environmentally induced changes in body fat where the key elements of its external trigger have been identified and can be traced to and through their transduction into a physiological stimulus that ultimately affects identified responses of white adipocyte physiology and cellularity. Finally, the comparative physiological approach to the study of seasonal adiposity seems likely to continue to yield significant insights into the mechanisms underlying this phenomenon and for understanding obesity and its reversal in general.     

Brain mitochondrial cytochrome P-450scc: spectral and catalytic properties

The cytochrome P-450-dependent cholesterol side chain cleavage system of the brain has been studied using nonsynaptic mitochondria as the source of enzymatic activity. The system has been found to bind cholesterol and 11-deoxycorticosterone, producing type I difference spectra, whereas the binding of pregnenolone induced a reverse type I difference spectrum. Inhibitors of cytochrome P-450-linked monooxygenase activities produced type II spectra. The formation of labeled pregnenolone after incubation of brain mitochondria with [4-14C]cholesterol has been obtained, and this formation was inhibited by glutethimide, a specific inhibitor of cytochrome P-450scc. The functional significance of this enzymatic activity is discussed.     

Episomal persistence of recombinant adenoviral vector genomes during the cell cycle in vivo

Previously we showed that recombinant adenoviral helper-dependent (HD) vectors result in long-term transgene expression levels in vivo which slowly declined by 95% over a period of 1 year. In this study, we further establish that this was not predominantly immune mediated. To determine if cell turnover was responsible for the loss of transgene expression, we induced rapid hepatocyte cell cycling in mouse liver, by performing a surgical two-thirds partial hepatectomy. We observed a 55 and 65% reduction in transgene expression levels and a 50 and 71% loss of vector genomes for the HD vector and the first-generation adenoviral vector. In sharp contrast, in nonviral, episomal plasmid DNA-injected mice, transgene expression levels and DNA copy numbers decreased by 95 and 99%, respectively. These findings suggest that cell division alone was not the primary reason for the slow decrease in transgene expression levels and that recombinant adenoviral vectors have a more robust mechanism for maintaining persistence during cell cycling. Several potential mechanisms are proposed.     

Strong coupling effects during <Emphasis Type="Italic">X</Emphasis>-pulse CPMG experiments recorded on heteronuclear <Emphasis Type="Italic">ABX</Emphasis> spin systems: artifacts and a simple solution

Simulation and experiment have been used to establish that significant artifacts can be generated in X-pulse CPMG relaxation dispersion experiments recorded on heteronuclear ABX spin-systems, such as ¹³C _i –¹³C _j –¹H, where ¹³C _i and ¹³C _j are strongly coupled. A qualitative explanation of the origin of these artifacts is presented along with a simple method to significantly reduce them. An application to the measurement of ¹H CPMG relaxation dispersion profiles in an HIV-2 TAR RNA molecule where all ribose sugars are protonated at the 2′ position, deuterated at all other sugar positions and ¹³C labeled at all sugar carbons is presented to illustrate the problems that strong ¹³C–¹³C coupling introduces and a simple solution is proposed.     

Regulation of RasGRP via a Phorbol Ester-Responsive C1 Domain

As part of a cDNA library screen for clones that induce transformation of NIH 3T3 fibroblasts, we have isolated a cDNA encoding the murine homolog of the guanine nucleotide exchange factor RasGRP. A point mutation predicted to prevent interaction with Ras abolished the ability of murine RasGRP (mRasGRP) to transform fibroblasts and to activate mitogen-activated protein kinases (MAP kinases). MAP kinase activation via mRasGRP was enhanced by coexpression of H-, K-, and N-Ras and was partially suppressed by coexpression of dominant negative forms of H- and K-Ras. The C terminus of mRasGRP contains a pair of EF hands and a C1 domain which is very similar to the phorbol ester- and diacylglycerol-binding C1 domains of protein kinase Cs. The EF hands could be deleted without affecting the ability of mRasGRP to transform NIH 3T3 cells. In contrast, deletion of the C1 domain or an adjacent cluster of basic amino acids eliminated the transforming activity of mRasGRP. Transformation and MAP kinase activation via mRasGRP were restored if the deleted C1 domain was replaced either by a membrane-localizing prenylation signal or by a diacylglycerol- and phorbol ester-binding C1 domain of protein kinase C. The transforming activity of mRasGRP could be regulated by phorbol ester when serum concentrations were low, and this effect of phorbol ester was dependent on the C1 domain of mRasGRP. The C1 domain could also confer phorbol myristate acetate-regulated transforming activity on a prenylation-defective mutant of K-Ras. The C1 domain mediated the translocation of mRasGRP to cell membranes in response to either phorbol ester or serum stimulation. These results suggest that the primary mechanism of activation of mRasGRP in fibroblasts is through its recruitment to diacylglycerol-enriched membranes. mRasGRP is expressed in lymphoid tissues and the brain, as well as in some lymphoid cell lines. In these cells, RasGRP has the potential to serve as a direct link between receptors which stimulate diacylglycerol-generating phospholipase Cs and the activation of Ras.     

TROSY-based HNCO pulse sequences for the measurement of 1HN-15N, 15N-13CO, 1HN-13CO, 13CO-13Cα and 1HN-13Cα dipolar couplings in 15N, 13C, 2H-labeled proteins

HNCO-based 3D pulse schemes are presented for measuring ¹HN-¹⁵N,¹⁵N-¹³CO, ¹HN-¹³CO,¹³CO-¹³C and ¹HN-¹³C dipolar couplings in ¹⁵N,¹³C,²-labeled proteins. The experiments are based on recently developed TROSY methodology for improving spectral resolution and sensitivity. Data sets recorded on a complex of Val, Leu, Ile (1 only) methyl protonated ¹⁵N,¹³C,²H-labeled maltose binding protein and -cyclodextrin as well as ¹⁵N,¹³C,²H-labeled human carbonic anhydrase II demonstrate that precise dipolar couplings can be obtained on proteins in the 30–40 kDa molecular weight range. These couplings will serve as powerful restraints for obtaining global folds of highly deuterated proteins.