Quinone reduction via secondary B-branch electron transfer in mutant bacterial reaction centers

Symmetry-related branches of electron-transfer cofactors-initiating with a primary electron donor (P) and terminating in quinone acceptors (Q)-are common features of photosynthetic reaction centers (RC). Experimental observations show activity of only one of them-the A branch-in wild-type bacterial RCs. In a mutant RC, we now demonstrate that electron transfer can occur along the entire, normally inactive B-branch pathway to reduce the terminal acceptor Q(B) on the time scale of nanoseconds. The transmembrane charge-separated state P(+)Q(B)(-) is created in this manner in a Rhodobacter capsulatus RC containing the F(L181)Y-Y(M208)F-L(M212)H-W(M250)V mutations (YFHV). The W(M250)V mutation quantitatively blocks binding of Q(A), thereby eliminating Q(B) reduction via the normal A-branch pathway. Full occupancy of the Q(B) site by the native UQ(10) is ensured (without the necessity of reconstitution by exogenous quinone) by purification of RCs with the mild detergent, Deriphat 160-C. The lifetime of P(+)Q(B)(-) in the YFHV mutant RC is >6 s (at pH 8.0, 298 K). This charge-separated state is not formed upon addition of competitive inhibitors of Q(B) binding (terbutryn or stigmatellin). Furthermore, this lifetime is much longer than the value of approximately 1-1.5 s found when P(+)Q(B)(-) is produced in the wild-type RC by A-side activity alone. Collectively, these results demonstrate that P(+)Q(B)(-) is formed solely by activity of the B-branch carriers in the YFHV RC. In comparison, P(+)Q(B)(-) can form by either the A or B branches in the YFH RC, as indicated by the biexponential lifetimes of approximately 1 and approximately 6-10 s. These findings suggest that P(+)Q(B)(-) states formed via the two branches are distinct and that P(+)Q(B)(-) formed by the B side does not decay via the normal (indirect) pathway that utilizes the A-side cofactors when present. These differences may report on structural and energetic factors that further distinguish the functional asymmetry of the two cofactor branches.     

A linkage map for the hybridising toads Bombina bombina and B. variegata (Anura: Discoglossidae)

Stable hybrid zones in which ecologically divergent taxa give rise to a range of recombinants are natural laboratories in which the genetic basis of adaptation and reproductive isolation can be unraveled. One such hybrid zone is formed by the fire-bellied toads Bombina bombina and B. variegata (Anura: Discoglossidae). Adaptations to permanent and ephemeral breeding habitats, respectively, have shaped numerous phenotypic differences between the taxa. All of these are, in principle, candidates for a genetic dissection via QTL mapping. We present here a linkage map of 28 codominant and 10 dominant markers in the Bombina genome. In an F2 cross, markers that were mainly microsatellites, SSCPs or allozymes were mapped to 20 linkage groups. Among the 40 isolated CA microsatellites, we noted a preponderance of compound and frequently interleaved CA-TA repeats as well as a striking polarity at the 5' end of the repeats.     

Residues Phe342-Asn346 of activated coagulation factor IX contribute to the interaction with low density lipoprotein receptor-related protein

When blood coagulation factor IX is converted to activated factor IX (factor IXa), it develops enzymatic activity and exposes the binding sites for both activated factor VIII and the endocytic receptor low density lipoprotein receptor-related protein (LRP). In the present study we investigated the interaction between factor IXa and LRP in more detail, using an affinity-purified soluble form of LRP (sLRP). Purified sLRP and full-length LRP displayed similar binding to factor IXa. An anti-factor IX monoclonal antibody CLB-FIX 13 inhibited factor IXa.sLRP complex formation. Both the antibody and a soluble recombinant fragment of LRP (i.e. cluster IV) interfered with factor IXa amidolytic activity, suggesting that the antibody and LRP share similar binding regions near the active site of factor IXa. Next, a panel of recombinant factor IXa variants with amino acid replacements in the surface loops bordering the active site was tested for binding to antibody CLB-FIX 13 and sLRP in a solid phase binding assay. Factor IXa variants with mutations in the region Phe(342)-Asn(346), located between the active site of factor IXa and factor VIII binding helix, showed reduced binding to both antibody CLB-FIX 13 and sLRP. Surface plasmon resonance analysis revealed that the variant with Asn(346) replaced by Asp displayed slower association to sLRP, whereas the variant with residues Phe(342)-Tyr(345) replaced by the corresponding residues of thrombin showed faster dissociation. Recombinant soluble LRP fragment cluster IV inhibited factor IXa-mediated activation of factor X with IC(50) values of 5 and 40 nm in the presence and absence of factor VIII, respectively. This inhibition thus seems to occur via two mechanisms: by interference with factor IXa.factor VIIIa complex assembly and by direct inhibition of factor IXa enzymatic activity. Accordingly, we propose that LRP may function as a regulator of blood coagulation.     

Effect of endothelial cell polarity on beta-amyloid-induced migration of monocytes across normal and AD endothelium

During normal aging and amyloid beta-peptide (Abeta) disorders such as Alzheimer's disease (AD), one finds increased deposition of Abeta and activated monocytes/microglial cells in the brain. Our previous studies show that Abeta interaction with a monolayer of normal human brain microvascular endothelial cells results in increased adherence and transmigration of monocytes. Relatively little is known of the role of Abeta accumulated in the AD brain in mediating trafficking of peripheral blood monocytes (PBM) across the blood-brain barrier (BBB) and concomitant accumulation of monocytes/microglia in the AD brain. In this study, we showed that interaction of Abeta(1--40) with apical surface of monolayer of brain endothelial cells (BEC), derived either from normal or AD individuals, resulted in increased transendothelial migration of monocytic cells (HL-60 and THP-1) and PBM. However, transmigration of monocytes across the BEC monolayer cultivated in a Transwell chamber was increased 2.5-fold when Abeta was added to the basolateral side of AD compared with normal individual BEC. The Abeta-induced transmigration of monocytes was inhibited in both normal and AD-BEC by antibodies to the putative Abeta receptor, receptor for advanced glycation end products (RAGE), and to the endothelial cell junction molecule, platelet-endothelial cell adhesion molecule-1 (PECAM-1). We conclude that interaction of Abeta with the basolateral surface of AD-BEC induces cellular signaling, promoting transmigration of monocytes from the apical to basolateral direction. We suggest that Abeta in the AD brain parenchyma or cerebrovasculature initiates cellular signaling that induces PBM to transmigrate across the BBB and accumulate in the brain.     

Significance of 14-3-3 self-dimerization for phosphorylation-dependent target binding

14-3-3 proteins via binding serine/threonine-phosphorylated proteins regulate diverse intracellular processes in all eukaryotic organisms. Here, we examine the role of 14-3-3 self-dimerization in target binding, and in the susceptibility of 14-3-3 to undergo phosphorylation. Using a phospho-specific antibody developed against a degenerated mode-1 14-3-3 binding motif (RSxpSxP), we demonstrate that most of the 14-3-3-associated proteins in COS-7 cells are phosphorylated on sites that react with this antibody. The binding of these phosphoproteins depends on 14-3-3 dimerization, inasmuch as proteins associated in vivo with a monomeric 14-3-3 form are not recognized by the phospho-specific antibody. The role of 14-3-3 dimerization in the phosphorylation-dependent target binding is further exemplified with two well-defined 14-3-3 targets, Raf and DAF-16. Raf and DAF-16 can bind both monomeric and dimeric 14-3-3; however, whereas phosphorylation of specific Raf and DAF-16 sites is required for binding to dimeric 14-3-3, binding to monomeric 14-3-3 forms is entirely independent of Raf and DAF-16 phosphorylation. We also find that dimerization diminishes 14-3-3 susceptibility to phosphorylation. These findings establish a significant role of 14-3-3 dimerization in its ability to bind targets in a phosphorylation-dependent manner and point to a mechanism in which 14-3-3 phosphorylation and dimerization counterregulate each other.     

RAGE mediates amyloid-beta peptide transport across the blood-brain barrier and accumulation in brain

Amyloid-beta peptide (Abeta) interacts with the vasculature to influence Abeta levels in the brain and cerebral blood flow, providing a means of amplifying the Abeta-induced cellular stress underlying neuronal dysfunction and dementia. Systemic Abeta infusion and studies in genetically manipulated mice show that Abeta interaction with receptor for advanced glycation end products (RAGE)-bearing cells in the vessel wall results in transport of Abeta across the blood-brain barrier (BBB) and expression of proinflammatory cytokines and endothelin-1 (ET-1), the latter mediating Abeta-induced vasoconstriction. Inhibition of RAGE-ligand interaction suppresses accumulation of Abeta in brain parenchyma in a mouse transgenic model. These findings suggest that vascular RAGE is a target for inhibiting pathogenic consequences of Abeta-vascular interactions, including development of cerebral amyloidosis.     

Superoxide prevents nitric oxide-mediated suppression of helper T lymphocytes: decreased autoimmune encephalomyelitis in nicotinamide adenine dinucleotide phosphate oxidase knockout mice

NO, which suppresses T cell proliferation, may be inactivated by superoxide (O2-) due to their strong mutual affinity. To examine this possibility, preactivated Th clones were cocultured with stimulated macrophages. PMA neutralized the inhibitory activity of NO, which was dependent on extracellular O2- production. In contrast, macrophages from p47phox -/- (pKO) mice, which lack functional NADPH oxidase, retained their NO-dependent inhibition of T cell proliferation upon stimulation with PMA, indicating that NADPH oxidase is the major source of NO-inactivating O2- in this system. To examine the NO-O2- interaction in vivo, the role of NADPH oxidase in experimental autoimmune encephalomyelitis was studied in pKO mice. No clinical or histological signs were observed in the pKO mice. Neither a bias in Th subsets nor a reduced intensity of T cell responses could account for the disease resistance. Although spleen cells from pKO mice proliferated poorly in response to the immunogen, inhibition of NO synthase uncovered a normal proliferative response. These results indicate that NO activity may play a critical role in T cell responses in pKO mice and that in normal spleens inhibition of T cell proliferation by NO may be prevented by simultaneous NADPH oxidase activity.     

Early expression of beta- and gamma-subunits of epithelial sodium channel during human airway development

The amiloride-sensitive epithelial Na(+) channel (ENaC) is an apical membrane protein complex involved in active Na(+) absorption and in control of fluid composition in airways. There are no data reporting the distribution of its pore-forming alpha-, beta-, and gamma-subunits in the developing human lung. With use of two different rabbit polyclonal antisera raised against beta- and gamma-ENaC, immunohistochemical localization of the channel was performed in fetal (10-35 wk) and in adult human airways. Both subunits were detected after 17 wk of gestation on the apical domain of bronchial ciliated cells, in glandular ducts, and in bronchiolar ciliated and Clara cells. After 30 wk, the distribution of beta- and gamma-subunits was similar in fetal and adult airways. In large airways, the two subunits were detected in ciliated cells, in cells lining glandular ducts, and in the serous gland cells. In the distal bronchioles, beta- and gamma-subunits were identified in ciliated and Clara cells. Ultrastructural immunogold labeling confirmed the identification of beta- and gamma-ENaC proteins in submucosal serous cells and bronchiolar Clara cells. Early expression of ENaC proteins in human fetal airways suggests that Na(+) absorption might begin significantly before birth, even if secretion is still dominant.     

Genetic association of apolipoprotein E with age-related macular degeneration.

Age-related macular degeneration (AMD) is the most common geriatric eye disorder leading to blindness and is characterized by degeneration of the neuroepithelium in the macular area of the eye. Apolipoprotein E (apoE), the major apolipoprotein of the CNS and an important regulator of cholesterol and lipid transport, appears to be associated with neurodegeneration. The apoE gene (APOE) polymorphism is a strong risk factor for various neurodegenerative diseases, and the apoE protein has been demonstrated in disease-associated lesions of these disorders. Hypothesizing that variants of APOE act as a potential risk factor for AMD, we performed a genetic-association study among 88 AMD cases and 901 controls derived from the population-based Rotterdam Study in the Netherlands. The APOE polymorphism showed a significant association with the risk for AMD; the APOE epsilon4 allele was associated with a decreased risk (odds ratio 0.43 [95% confidence interval 0.21-0. 88]), and the epsilon2 allele was associated with a slightly increased risk of AMD (odds ratio 1.5 [95% confidence interval 0.8-2. 82]). To investigate whether apoE is directly involved in the pathogenesis of AMD, we studied apoE immunoreactivity in 15 AMD and 10 control maculae and found that apoE staining was consistently present in the disease-associated deposits in AMD-maculae-that is, drusen and basal laminar deposit. Our results suggest that APOE is a susceptibility gene for AMD.