Transient radical pairs studied by time-resolved EPR

Photogenerated short-lived radical pairs (RP) are common in biological photoprocesses such as photosynthesis and enzymatic DNA repair. They can be favorably probed by time-resolved electron paramagnetic resonance (EPR) methods with adequate time resolution. Two EPR techniques have proven to be particularly useful to extract information on the working states of photoinduced biological processes that is only difficult or sometimes even impossible to obtain by other types of spectroscopy. Firstly, transient EPR yields crucial information on the chemical nature and the geometry of the individual RP halves in a doublet-spin pair generated by a short laser pulse. This time-resolved method is applicable in all magnetic field/microwave frequency regimes that are used for continuous-wave EPR, and is nowadays routinely utilized with a time resolution reaching about 10 ns. Secondly, a pulsed EPR method named out-of-phase electron spin echo envelope modulation (OOP-ESEEM) is increasingly becoming popular. By this pulsed technique, the mutual spin-spin interaction between the RP halves in a doublet-spin pair manifests itself as an echo modulation detected as a function of the microwave-pulse spacing of a two-pulse echo sequence subsequent to a laser pulse. From the dipolar coupling, the distance between the radicals is readily derived. Since the spin-spin interaction parameters are typically not observable by transient EPR, the two techniques complement each other favorably. Both EPR methods have recently been applied to a variety of light-induced RPs in photobiology. This review summarizes the results obtained from such studies in the fields of plant and bacterial photosynthesis and DNA repair mediated by the enzyme DNA photolyase.     

Structural characterization of the interaction of the delta and alpha subunits of the Escherichia coli F1F0-ATP synthase by NMR spectroscopy

A critical point of interaction between F(1) and F(0) in the bacterial F(1)F(0)-ATP synthase is formed by the alpha and delta subunits. Previous work has shown that the N-terminal domain (residues 3-105) of the delta subunit forms a 6 alpha-helix bundle [Wilkens, S., Dunn, S. D., Chandler, J., Dahlquist, F. W., and Capaldi, R. A. (1997) Nat. Struct. Biol. 4, 198-201] and that the majority of the binding energy between delta and F(1) is provided by the interaction between the N-terminal 22 residues of the alpha- and N-terminal domain of the delta subunit [Weber, J., Muharemagic, A., Wilke-Mounts, S., and Senior, A. E. (2003) J. Biol. Chem. 278, 13623-13626]. We have now analyzed a 1:1 complex of the delta-subunit N-terminal domain and a peptide comprising the N-terminal 22 residues of the alpha subunit by heteronuclear protein NMR spectroscopy. A comparison of the chemical-shift values of delta-subunit residues with and without alpha N-terminal peptide bound indicates that the binding interface on the N-terminal domain of the delta subunit is formed by alpha helices I and V. NOE cross-peak patterns in 2D (12)C/(12)C-filtered NOESY spectra of the (13)C-labeled delta-subunit N-terminal domain in complex with unlabeled peptide verify that residues 8-18 in the alpha-subunit N-terminal peptide are folded as an alpha helix when bound to delta N-terminal domain. On the basis of intermolecular contacts observed in (12)C/(13)C-filtered NOESY experiments, we describe structural details of the interaction of the delta-subunit N-terminal domain with the alpha-subunit N-terminal alpha helix.     

Solution structure of zinc- and calcium-bound rat S100B as determined by nuclear magnetic resonance spectroscopy

The EF-hand calcium-binding protein S100B also binds one zinc ion per subunit with a relatively high affinity (K(d) approximately 90 nM) [Wilder et al., (2003) Biochemistry 42, 13410-13421]. In this study, the structural characterization of zinc binding to calcium-loaded S100B was examined using high-resolution NMR techniques, including structural characterization of this complex in solution at atomic resolution. As with other S100 protein structures, the quaternary structure of Zn(2+)-Ca(2+)-bound S100B was found to be dimeric with helices H1, H1', H4, and H4' forming an X-type four-helix bundle at the dimer interface. NMR data together with mutational analyses are consistent with Zn(2+) coordination arising from His-15 and His-25 of one S100B subunit and from His-85 and Glu-89 of the other subunit. The addition of Zn(2+) was also found to extend helices H4 and H4' three to four residues similar to what was previously observed with the binding of target proteins to S100B. Furthermore, a kink in helix 4 was observed in Zn(2+)-Ca(2+)-bound S100B that is not in Ca(2+)-bound S100B. These structural changes upon Zn(2+)-binding could explain the 5-fold increase in affinity that Zn(2+)-Ca(2+)-bound S100B has for peptide targets such as the TRTK peptide versus Ca(2+)-bound S100B. There are also changes in the relative positioning of the two EF-hand calcium-binding domains and the respective helices comprising these EF-hands. Changes in conformation such as these could contribute to the order of magnitude higher affinity that S100B has for calcium in the presence of Zn(2+).     

Retroviral display of urokinase-binding domain fused to amphotropic envelope protein

Tumors frequently express urokinase (uPA) receptor (uPAR). To investigate whether uPAR can efficiently target cancerous cells using amphotropic retroviral vectors, we generated a retrovirus displaying the amino-terminal fragment (ATF) of uPA as an N-terminal extension of viral envelope protein. We also made use of a "two-step strategy" by inserting a uPA cleavage site between the ATF moiety and the envelope. We measured the ability of ATF-bearing chimeric envelopes to infect huPAR-overexpressing Madin-Darby canine kidney (MDCK) and control MDCK II cells. The ATF-viruses infected both MDCK cell lines with an equivalent efficiency, suggesting that the chimeric viruses were not sequestered by uPAR and infect cells preferentially via the Pit-2 receptor. The addition of a uPA cleavage site increased the infection level of huPAR-MDCK cells by 2-fold when uPA was present in the infection medium. Surprisingly, ATF-env viruses infected huPAR-MDCK cells 5.5-fold more efficiently in the presence of exogenous uPA. This stimulatory effect of uPA on infection of huPAR-MDCK cells by the ATF-env virus was completely abolished by methyl-beta-cyclodextrin, suggesting that this effect involves the caveolar endocytosis pathway.     

A Late Devonian isoetalean lycopsid, Otzinachsonia Beerboweri, gen. et sp. nov., from north-central Pennsylvania, USA

Compressions and impressions of an isoetalean lycopsid, comprising lower portions of stems, lobed bases, attached rootlets, and rounded rootlet scars, discovered in Late Devonian (Famennian) rocks of Clinton County, north-central Pennsylvania, Appalachian Basin, USA, are here described as Otzinachsonia beerboweri, gen. et sp. nov. These specimens demonstrate unequivocally the existence of the isoetalean lobe-and-furrow rhizomorphic growth pattern as early as the Late Devonian. They were found in an Archaeopteris- and Rhacophyton-dominated flora at Red Hill, an outcrop of the Duncannon Member of the Catskill Formation. The fossils were found in a dark-gray to greenish-gray lenticular siltstone layer that has an average thickness of 1.0 m. This deposit is interpreted as a floodplain pond. The low-energy nature of the deposit and the fine preservation of the intact rootlets of the specimens imply little or no transport. The plants were probably growing along the edge of the floodplain pond with their lower portions submerged for at least part of the year.     

The Y deletion gr/gr and susceptibility to testicular germ cell tumor

Testicular germ cell tumor (TGCT) is the most common cancer in young men. Despite a considerable familial component to TGCT risk, no genetic change that confers increased risk has been substantiated to date. The human Y chromosome carries a number of genes specifically involved in male germ cell development, and deletion of the AZFc region at Yq11 is the most common known genetic cause of infertility. Recently, a 1.6-Mb deletion of the Y chromosome that removes part of the AZFc region—known as the “gr/gr” deletion—has been associated with infertility. In epidemiological studies, male infertility has shown an association with TGCT that is out of proportion with what can be explained by tumor effects. Thus, we hypothesized that the gr/gr deletion may be associated with TGCT. Using logistic modeling, we analyzed this deletion in a large series of TGCT cases with and without a family history of TGCT. The gr/gr deletion was present in 3.0% (13/431) of TGCT cases with a family history, 2% (28/1,376) of TGCT cases without a family history, and 1.3% (33/2,599) of unaffected males. Presence of the gr/gr deletion was associated with a twofold increased risk of TGCT (adjusted odds ratio [aOR] 2.1; 95% confidence interval [CI] 1.3–3.6; P = .005) and a threefold increased risk of TGCT among patients with a positive family history (aOR 3.2; 95% CI 1.5–6.7; P = .0027). The gr/gr deletion was more strongly associated with seminoma (aOR 3.0; 95% CI 1.6–5.4; P = .0004) than with nonseminoma TGCT (aOR 1.5; 95% CI 0.72–3.0; P = .29). These data indicate that the Y microdeletion gr/gr is a rare, low-penetrance allele that confers susceptibility to TGCT.     

Endosomal translocation of vertebrate DNA activates dendritic cells via TLR9-dependent and -independent pathways

TLRs discriminate foreign from self via their specificity for pathogen-derived invariant ligands, an example being TLR9 recognizing bacterial unmethylated CpG motifs. In this study we report that endosomal translocation of CpG DNA via the natural endocytotic pathway is inefficient and highly saturable, whereas endosomal translocation of DNA complexed to the cationic lipid N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP) is not. Interestingly, DOTAP-mediated enhanced endosomal translocation of otherwise nonstimulatory vertebrate DNA or of certain noncanonical CpG motifs triggers robust dendritic cell activation in terms of both up-regulation of CD40/CD69 and cytokine production, such as type I IFN and IL-6. We report that the stimulatory activity of phosphorothioated noncanonical CpG oligodeoxynucleotides is TLR9 dependent, whereas phosphodiester DNA, such as vertebrate DNA, in addition trigger TLR9-independent pathways. We propose that the inefficiency of the natural route for DNA internalization hinders low affinity TLR9 ligands in endosomes to reach threshold concentrations required for TLR9 activation. Endosomal compartmentalization of TLR9 may thus reflect an evolutionary strategy to avoid TLR9 activation by self-DNA.     

Differential processing of autoantigens in lysosomes from human monocyte-derived and peripheral blood dendritic cells

Dendritic cells (DC) initiate immunity and maintain tolerance. Although in vitro-generated DC, usually derived from peripheral blood monocytes (MO-DC), serve as prototype DC to analyze the biology and biochemistry of DC, phenotypically distinct primary types of DC, including CD1c-DC, are present in peripheral blood (PB-DC). The composition of lysosomal proteases in PB-DC and the way their MHC class II-associated Ag-processing machinery handles a clinically relevant Ag are unknown. We show that CD1c-DC lack significant amounts of active cathepsins (Cat) S, L, and B as well as the asparagine-specific endopeptidase, the major enzymes believed to mediate MHC class II-associated Ag processing. However, at a functional level, lysosomal extracts from CD1c-DC processed the multiple sclerosis-associated autoantigens myelin basic protein and myelin oligodendrocyte glycoprotein in vitro more effectively than MO-DC. Although processing was dominated by CatS, CatD, and asparagine-specific endopeptidase in MO-DC, it was dominated by CatG in CD1c-DC. Thus, human MO-DC and PB-DC significantly differ with respect to their repertoire of active endocytic proteases, so that both proteolytic machineries process a given autoantigen via different proteolytic pathways.     

CCL1-CCR8 interactions: an axis mediating the recruitment of T cells and Langerhans-type dendritic cells to sites of atopic skin inflammation

Atopic dermatitis represents a chronically relapsing skin disease with a steadily increasing prevalence of 10-20% in children. Skin-infiltrating T cells, dendritic cells (DC), and mast cells are thought to play a crucial role in its pathogenesis. We report that the expression of the CC chemokine CCL1 (I-309) is significantly and selectively up-regulated in atopic dermatitis in comparison to psoriasis, cutaneous lupus erythematosus, or normal skin. CCL1 serum levels of atopic dermatitis patients are significantly higher than levels in healthy individuals. DC, mast cells, and dermal endothelial cells are abundant sources of CCL1 during atopic skin inflammation and allergen challenge, and Staphylococcus aureus-derived products induce its production. In vitro, binding and cross-linking of IgE on mast cells resulted in a significant up-regulation of this inflammatory chemokine. Its specific receptor, CCR8, is expressed on a small subset of circulating T cells and is abundantly expressed on interstitial DC, Langerhans cells generated in vitro, and their monocytic precursors. Although DC maintain their CCR8+ status during maturation, brief activation of circulating T cells recruits CCR8 from intracytoplamic stores to the cell surface. Moreover, the inflammatory and atopy-associated chemokine CCL1 synergizes with the homeostatic chemokine CXCL12 (SDF-1alpha) resulting in the recruitment of T cell and Langerhans cell-like DC. Taken together, these findings suggest that the axis CCL1-CCR8 links adaptive and innate immune functions that play a role in the initiation and amplification of atopic skin inflammation.