Activation of thromboxane receptor modulates interleukin-1β-induced monocyte adhesion--a novel role of Nox1

Activation of thromboxane receptors (TPr) may promote atherosclerosis by enhancing oxidative stress and inflammation. This study examined the role of Nox1, an NADPH-oxidase subunit, in the enhancement of interleukin (IL)-1β-induced monocyte adhesion by TPr. In cultured rat aortic vascular smooth muscle cells (VSMCs), U46619, a stable thromboxane A(2) mimetic, together with interleukin-1β significantly enhanced Nox1 mRNA expression, as well as adhesion of THP-1 monocytes. Activation of TPr also enhanced IL-1β-induced vascular cell adhesion molecule (VCAM)-1 expression, but inhibited inducible nitric oxide synthase (iNOS) expression. Silencing Nox1 expression by siRNA prevented the U46619 enhancement of IL-1β-induced monocyte adhesion, but had no significant effect on VCAM-1 or iNOS expression. Furthermore, monocyte adhesion was inhibited by superoxide dismutase, enhanced by a specific iNOS inhibitor, l-N(6)-(1-iminoethyl)-lysine, but not influenced by catalase. U46619 inhibited IL-1β-induced cyclic GMP production, and the inhibition was partially prevented by superoxide dismutase. In conclusion, activation of TPr enhances IL-1β-induced Nox1 expression in VSMCs, which is responsible for the up-regulation of monocyte adhesion. The effect of Nox1 is independent of the changes in VCAM-1 and iNOS expression, but depends on the inactivation of nitric oxide via generation of superoxide anion.     

Deconstructing the neuropathic pain phenotype to reveal neural mechanisms

After nerve injury maladaptive changes can occur in injured sensory neurons and along the entire nociceptive pathway within the CNS, which may lead to spontaneous pain or pain hypersensitivity. The resulting neuropathic pain syndromes present as a complex combination of negative and positive symptoms, which vary enormously from individual to individual. This variation depends on a diversity of underlying pathophysiological changes resulting from the convergence of etiological, genotypic, and environmental factors. The pain phenotype can serve therefore, as a window on underlying pathophysiological neural mechanisms and as a guide for developing personalized pain medicine.     

Hypoxia induces Kv channel current inhibition by increased NADPH oxidase-derived reactive oxygen species

There is current discussion whether reactive oxygen species are up- or downregulated in the pulmonary circulation during hypoxia, from which sources (i.e., mitochondria or NADPH oxidases) they are derived, and what the downstream targets of ROS are. We recently showed that the NADPH oxidase homolog NOX4 is upregulated in hypoxia-induced pulmonary hypertension in mice and contributes to the vascular remodeling in pulmonary hypertension. We here tested the hypothesis that NOX4 regulates K(v) channels via an increased ROS formation after prolonged hypoxia. We showed that (1) NOX4 is upregulated in hypoxia-induced pulmonary hypertension in rats and isolated rat pulmonary arterial smooth muscle cells (PASMC) after 3days of hypoxia, and (2) that NOX4 is a major contributor to increased reactive oxygen species (ROS) after hypoxia. Our data indicate colocalization of K(v)1.5 and NOX4 in isolated PASMC. The NADPH oxidase inhibitor and ROS scavenger apocynin as well as NOX4 siRNA reversed the hypoxia-induced decrease in K(v) current density whereas the protein levels of the channels remain unaffected by siNOX4 treatment. Determination of cysteine oxidation revealed increased NOX4-mediated K(v)1.5 channel oxidation. We conclude that sustained hypoxia decreases K(v) channel currents by a direct effect of a NOX4-derived increase in ROS.     

Multi locus analysis of Pristionchus pacificus on La Réunion Island reveals an evolutionary history shaped by multiple introductions, constrained dispersal events and rare out-crossing

Pristionchus pacificus, recently established as a model organism in evolutionary biology, is a cosmopolitan nematode that has a necromenic association with scarab beetles. The diverse array of host beetle species and habitat types occupied by P. pacificus make it a good model for investigating local adaptation to novel environments. Presence of P. pacificus on La Réunion Island, a young volcanic island with a dynamic geological history and a wide variety of ecozones, facilitates such investigation in an island biogeographic setting. Microsatellite data from 20 markers and 223 strains and mitochondrial sequence data from 272 strains reveal rich genetic diversity among La Réunion P. pacificus isolates, shaped by differentially timed introductions from diverse sources and in association with different beetle species. Distinctions between volcanic zones and between arid western and wet eastern climatic zones have likely limited westward dispersal of recently colonized lineages and maintained a genetic distinction between eastern and western clades. The highly selfing lifestyle of P. pacificus contributes to the strong fine‐scale population structure detected, with each beetle host harbouring strongly differentiated assemblages of strains. Periodic out‐crossing generates admixture between genetically diverse lineages, creating a diverse array of allelic combinations likely to increase the evolutionary potential of the species and facilitate adaptation to local environments and beetle hosts.     

High density lipoprotein inhibits hepatitis C virus-neutralizing antibodies by stimulating cell entry via activation of the scavenger receptor BI

Hepatitis C virus (HCV) exploits serum-dependent mechanisms that inhibit neutralizing antibodies. Here we demonstrate that high density lipoprotein (HDL) is a key serum factor that attenuates neutralization by monoclonal and HCV patient-derived polyclonal antibodies of infectious pseudo-particles (HCVpp) harboring authentic E1E2 glycoproteins and cell culture-grown genuine HCV (HCVcc). Over 10-fold higher antibody concentrations are required to neutralize either HCV-enveloped particles in the presence of HDL or human serum, and less than 3-5-fold reduction of infectious titers are obtained at saturating antibody concentrations, in contrast to complete inhibition in serum-free conditions. We show that HDL interaction with the scavenger receptor BI (SR-BI), a proposed cell entry co-factor of HCV and a receptor mediating lipid transfer with HDL, strongly reduces neutralization of HCVpp and HCVcc. We found that HDL activation of target cells strongly stimulates cell entry of viral particles by accelerating their endocytosis, thereby suppressing a 1-h time lag during which cell-bound virions are not internalized and can be targeted by antibodies. Compounds that inhibit lipid transfer functions of SR-BI fully restore neutralization by antibodies in human serum. We demonstrate that this functional HDL/SR-BI interaction only interferes with antibodies blocking HCV-E2 binding to CD81, a major HCV receptor, reflecting its prominent role during the cell entry process. Moreover, we identify monoclonal antibodies targeted to epitopes in the E1E2 complex that are not inhibited by HDL. Consistently, we show that antibodies targeted to HCV-E1 efficiently neutralize HCVpp and HCVcc in the presence of human serum.     

Saposin A mobilizes lipids from low cholesterol and high bis(monoacylglycerol)phosphate-containing membranes: patient variant Saposin A lacks lipid extraction capacity

Saposin A (Sap-A) is one of five known sphingolipid activator proteins required for the lysosomal degradation of sphingolipids and for the loading of lipid antigens onto antigen-presenting molecules of the CD1 type. Sap-A assists in the degradation of galactosylceramide by galactosylceramide-beta-galactosidase in vivo, which takes place at the surface of intraendosomal/intralysosomal vesicles. Sap-A is believed to mediate the interaction between the enzyme and its membrane-bound substrate. Its dysfunction causes a variant form of Krabbe disease. In the present study we prepared glycosylated Sap-A free of other Saps, taking advantage of the Pichia pastoris expression system. Using liposomes and surface plasmon resonance spectroscopy, we tested the binding and lipid mobilization capacity of Sap-A under different conditions. Along the endocytic pathway, the pH value decreases, and the lipid composition of intraendosomal and intralysosomal membranes changes drastically. In the inner membranes the cholesterol concentration decreases, and that of the anionic phospholipid bis(monoacylglycero)phosphate increases. Here, we show that Sap-A is able to bind to liposomes and to mobilize lipids out of them at acidic pH values below pH 4.7. Low cholesterol levels and increasing concentrations of bis(monoacylglycero)phosphate favor lipid extraction significantly. Galactosylceramide as a bilayer component is not essential for lipid mobilization by Sap-A, which requires intact disulfide bridges for activity. We also show for the first time that glycosylation of Sap-A is essential for its lipid extraction activity. Variant Sap-A proteins, which cause storage of galactosylceramide in humans (Krabbe disease, Spiegel, R., Bach, G., Sury, V., Mengistu, G., Meidan, B., Shalev, S., Shneor, Y., Mandel, H., and Zeigler, M. (2005) Mol. Genet. Metab. 84, 160-166) and in mutant mice (Matsuda, J., Vanier, M. T., Saito, Y., Tohyama, J., and Suzuki, K. (2001) Hum. Mol. Genet. 10, 1191-1199) are deficient in lipid extraction capacity.     

Membrane association of the cycling peroxisome import receptor Pex5p

Peroxisomal proteins carrying a peroxisome targeting signal type 1 (PTS1) are recognized in the cytosol by the cycling import receptor Pex5p. The receptor-cargo complex docks at the peroxisomal membrane where it associates with multimeric protein complexes, referred to as the docking and RING finger complexes. Here we have identified regions within the Saccharomyces cerevisiae Pex5p sequence that interconnect the receptor-cargo complex with the docking complex. Site-directed mutagenesis of the conserved tryptophan residue within a reverse WXXXF motif abolished two-hybrid binding with the N-terminal half of Pex14p. In combination with an additional mutation introduced into the Pex13p-binding site, we generated a Pex5p mutant defective in a stable association not only with the docking complex but also with the RING finger peroxins at the membrane. Surprisingly, PTS1 proteins are still imported into peroxisomes in these mutant cells. Because these mutations had no significant effect on the membrane binding properties of Pex5p, we examined yeast and human Pex5p for intrinsic lipid binding activity. In vitro analyses demonstrated that both proteins have the potential to insert spontaneously into phospholipid membranes. Altogether, these data strongly suggest that a translocation-competent state of the PTS1 receptor enters the membrane via protein-lipid interactions before it tightly associates with other peroxins.     

The N-terminal domains of neuregulin 1 confer signal attenuation

Degradation of activated ERBB receptors is an important mechanism for signal attenuation. However, compared with epidermal growth factor (EGF) receptor, the ERBB2/ERBB3 signaling pair is considered to be attenuation-deficient. The ERBB2/ERBB3 ligands of the neuregulin family rely on an EGF-like domain for signaling and are generated from larger membrane-bound precursors. In contrast to EGF, which is processed to yield a 6-kDa peptide ligand, mature neuregulins retain a variety of segments N-terminal to the EGF-like domain. Here we evaluate the role of the N-terminal domain of neuregulin 1 in signaling and turnover of ERBB2/ERBB3. Our data suggest that whereas the EGF-like domain of neuregulin 1 is required and sufficient for the formation of active receptor heterodimers, the presence of the N-terminal Ig-like domain is required for efficient signal attenuation. This manifests itself for both ERBB2 and ERBB3 but is more pronounced and coupled directly to degradation for ERBB3. When stimulated with only the EGF-like domain, ERBB3 shows degradation rates comparable with constitutive turnover, but stimulation with full-length neuregulin 1 resulted in receptor degradation at rates that are comparable with activated EGF receptor. Most of the enhancement in down-regulation was maintained after replacing the Ig-like domain with a thioredoxin protein of comparable size but different amino acid composition, suggesting that the physical presence but not specific properties of the Ig-like domain are needed. This sequence-independent effect of the N-terminal domain correlates with an enhanced ability of full-size neuregulin 1 to disrupt higher order oligomers of the ERBB3 extracellular domains in vitro.     

Reduced peripheral and mucosal Tropheryma whipplei-specific Th1 response in patients with Whipple's disease

Whipple's disease is a rare infectious disorder caused by Tropheryma whipplei. Major symptoms are arthropathy, weight loss, and diarrhea, but the CNS and other organs may be affected, too. The incidence of Whipple's disease is very low despite the ubiquitous presence of T. whipplei in the environment. Therefore, it has been suggested that host factors indicated by immune deficiencies are responsible for the development of Whipple's disease. However, T. whipplei-specific T cell responses could not be studied until now, because cultivation of the bacteria was established only recently. Thus, the availability of T. whipplei Twist-Marseille(T) has enabled the first analysis of T. whipplei-specific reactivity of CD4(+) T cells. A robust T. whipplei-specific CD4(+) Th1 reactivity and activation (expression of CD154) was detected in peripheral and duodenal lymphocytes of all healthy (16 young, 27 age-matched, 11 triathletes) and disease controls (17 patients with tuberculosis) tested. However, 32 Whipple's disease patients showed reduced or absent T. whipplei-specific Th1 responses, whereas their capacity to react to other common Ags like tetanus toxoid, tuberculin, actinomycetes, Giardia lamblia, or CMV was not reduced compared with controls. Hence, we conclude that an insufficient T. whipplei-specific Th1 response may be responsible for an impaired immunological clearance of T. whipplei in Whipple's disease patients and may contribute to the fatal natural course of the disease.