Oxidized phospholipids are more potent antagonists of lipopolysaccharide than inducers of inflammation

Polyunsaturated fatty acids are precursors of multiple pro- and anti-inflammatory molecules generated by enzymatic stereospecific and positionally specific insertion of oxygen, which is a prerequisite for recognition of these mediators by cellular receptors. However, nonenzymatically oxidized free and esterified polyunsaturated fatty acids also demonstrate activities relevant to inflammation. In particular, phospholipids containing oxidized fatty acid residues (oxidized phospholipids; OxPLs) were shown to induce proinflammatory changes in endothelial cells but paradoxically also to inhibit inflammation induced via TLR4. In this study, we show that half-maximal inhibition of LPS-induced elevation of E-selectin mRNA in endothelial cells developed at concentrations of oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC) 10-fold lower than those required to induce proinflammatory response. Similar concentration difference was observed for other classes and molecular species of OxPLs. Upon injection into mice, OxPAPC did not elevate plasma levels of IL-6 and keratinocyte chemoattractant but strongly inhibited LPS-induced upregulation of these inflammatory cytokines. Thus, both in vitro and in vivo, anti-LPS effects of OxPLs are observed at lower concentrations than those required for their proinflammatory action. Quantification of the most abundant oxidized phosphatidylcholines by HPLC/tandem mass spectrometry showed that circulating concentrations of total oxidized phosphatidylcholine species are close to the range where they demonstrate anti-LPS activity but significantly lower than that required for induction of inflammation. We hypothesize that low levels of OxPLs in circulation serve mostly anti-LPS function and protect from excessive systemic response to TLR4 ligands, whereas proinflammatory effects of OxPLs are more likely to develop locally at sites of tissue deposition of OxPLs (e.g., in atherosclerotic vessels).     

Repositioning of Transmembrane α-Helices during Membrane Protein Folding

We have determined the optimal placement of individual transmembrane helices in the Pyrococcus horikoshii Glt_Ph glutamate transporter homolog in the membrane. The results are in close agreement with theoretical predictions based on hydrophobicity, but do not, in general, match the known three-dimensional structure, suggesting that transmembrane helices can be repositioned relative to the membrane during folding and oligomerization. Theoretical analysis of a database of membrane protein structures provides additional support for this idea. These observations raise new challenges for the structure prediction of membrane proteins and suggest that the classical two-stage model often used to describe membrane protein folding needs to be modified.     

One-dimensional Cu(I) camphor hydrazone polymers: Structure and catalytic properties

Complexes of general formula [{CuX}₂(YNC₁₀H₁₄O)] (X = Cl; Y = NHMe, NH₂ or X = Br; Y = NH₂) were synthesised from camphor hydrazone ligands (YNC₁₀H₁₄O) by reaction with the suitable copper(I) halide. Structural analysis by X-rays performed on a red crystal of [{CuCl}₂(Me₂NNC₁₀H₁₄O)] revealed that the complex is a one-dimensional copper polymer formed by two rather different copper units bridged by chloride. One of the copper units displays a tetrahedral geometry while the other is linear. Although the geometries and neighbourhoods of the two copper units are very different the oxidation state of the metal is the same, i.e. Cu(I) as corroborated by magnetic and electrochemical measurements.The ability of [{CuCl}₂(Me₂NNC₁₀H₁₄O)] to promote the activation of 4-pentyn-1-ol towards cyclization was studied under homogeneous or heterogeneous experimental conditions. The best results were obtained under homogeneous conditions at 40 °C.     

LmxMPK4, an essential mitogen-activated protein kinase of Leishmania mexicana is phosphorylated and activated by the STE7-like protein kinase LmxMKK5

The essential mitogen-activated protein kinase (MAP kinase), LmxMPK4, of Leishmania mexicana is minimally active when purified following recombinant expression in Escherichia coli and was therefore unsuitable for drug screening until now. Using an E. coli protein co-expression system we identified LmxMKK5, a STE7-like protein kinase from L. mexicana, which phosphorylates and activates recombinant LmxMPK4 in vitro. LmxMKK5 is comprised of 525 amino acids and has a calculated molecular mass of 55.9 kDa. The co-expressed, purified LmxMPK4 showed strong phosphotransferase activity in radiometric kinase assays and was confirmed by immunoblot and tandem mass spectrometry analyses to be phosphorylated on threonine 190 and tyrosine 192 of the typical TXY MAP kinase activation motif. The universal protein kinase inhibitor staurosporine reduced the phosphotransferase activity of co-expressed and activated LmxMPK4 in a dose-dependent manner. To our knowledge this is the first time that an in vitro activator of an essential Leishmania MAP kinase was identified and our findings form the basis for the development of drug screening assays to identify small molecule inhibitors of LmxMPK4 in the search for new therapeutic drugs against leishmaniasis.     

A bioreactor test system to mimic the biological and mechanical environment of oral soft tissues and to evaluate substitutes for connective tissue grafts

Gingival cells of the oral connective tissue are exposed to complex mechanical forces during mastication, speech, tooth movement and orthodontic treatments. Especially during wound healing following surgical procedures, internal and external forces may occur, creating pressure upon the newly formed tissue. This clinical situation has to be considered when developing biomaterials to augment soft tissue in the oral cavity. In order to pre‐evaluate a collagen sponge intended to serve as a substitute for autogenous connective tissue grafts (CTGs), a dynamic bioreactor system was developed. Pressure and shear forces can be applied in this bioreactor in addition to a constant medium perfusion to cell‐material constructs. Three‐dimensional volume changes and stiffness of the matrices were analyzed. In addition, cell responses such as cell vitality and extracellular matrix (ECM) production were investigated. The number of metabolic active cells constantly increased under fully dynamic culture conditions. The sponges remained elastic even after mechanical forces were applied for 14 days. Analysis of collagen type I and fibronectin revealed a statistically significant accumulation of these ECM molecules (P < 0.05–0.001) when compared to static cultures. An increased expression of tenascin‐c, indicating tissue remodeling processes, was observed under dynamic conditions only. The results indicate that the tested in vitro cell culture system was able to mimic both the biological and mechanical environments of the clinical situation in a healing wound. Biotechnol. Bioeng. 2010;107: 1029–1039. © 2010 Wiley Periodicals, Inc.     

Prolyl Hydroxylase Domain (PHD) 2 Affects Cell Migration and F-actin Formation via RhoA/Rho-associated Kinase-dependent Cofilin Phosphorylation

Cells are responding to hypoxia via prolyl-4-hydroxylase domain (PHD) enzymes, which are responsible for oxygen-dependent hydroxylation of the hypoxia-inducible factor (HIF)-1α subunit. To gain further insight into PHD function, we generated knockdown cell models for the PHD2 isoform, which is the main isoform regulating HIF-1α hydroxylation and thus stability in normoxia. Induction of a PHD2 knockdown in tetracycline-inducible HeLa PHD2 knockdown cells resulted in increased F-actin formation as detected by phalloidin staining. A similar effect could be observed in the stably transfected PHD2 knockdown cell clones 1B6 and 3B7. F-actin is at least in part responsible for shaping cell morphology as well as regulating cell migration. Cell migration was impaired significantly as a consequence of PHD2 knockdown in a scratch assay. Mechanistically, PHD2 knockdown resulted in activation of the RhoA (Ras homolog gene family member A)/Rho-associated kinase pathway with subsequent phosphorylation of cofilin. Because cofilin phosphorylation impairs its actin-severing function, this may explain the F-actin phenotype, thereby providing a functional link between PHD2-dependent signaling and cell motility.     

Calcitonin gene-related peptide activates different signaling pathways in mesenteric lymphatics of guinea pigs

The effects of calcitonin gene-related peptide (CGRP) on constriction frequency, smooth muscle membrane potential (V(m)), and endothelial V(m) of guinea pig mesenteric lymphatics were examined in vitro. CGRP (1-100 nM) caused an endothelium-dependent decrease in the constriction frequency of perfused lymphatic vessels. The endothelium-dependent CGRP response was abolished by the CGRP-1 receptor antagonist CGRP-(8-37) (1 microM) and pertussis toxin (100 ng/ml). This action of CGRP was also blocked by the nitric oxide (NO) synthase inhibitor N(G)-nitro-L-arginine (L-NNA; 10 microM), an action that was reversed by the addition of L-arginine (100 microM). cGMP, adenylate cyclase, cAMP-dependent protein kinase (PKA), and ATP-sensitive K+ (K+(ATP)) channels were all implicated in the endothelium-dependent CGRP response because it was abolished by methylene blue (20 microM), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (10 microM), dideoxyadenosine (10 microM), N-[2-(p-bromociannamylamino)-ethyl]-5-isoquinolinesulfonamide-dichloride (H89; 1 microM) and glibenclamide (10 microM). CGRP (100 nM), unlike acetylcholine, did not alter endothelial intracellular Ca2+ concentration or V(m). CGRP (100 nM) hyperpolarized the smooth muscle V(m), an effect inhibited by L-NNA, H89, or glibenclamide. CGRP (500 nM) also caused a decrease in constriction frequency. However, this was no longer blocked by CGRP-(8-37). CGRP (500 nM) also caused smooth muscle hyperpolarization, an action that was now not blocked by L-NNA (100 microM). It was most likely mediated by the activation of the cAMP/PKA pathway and the opening of K+(ATP) channels because it was abolished by H89 or glibenclamide. We conclude that CGRP, at low to moderate concentrations (i.e., 1-100 nM), decreases lymphatic constriction frequency primarily by the stimulation of CGRP-1 receptors coupled to pertussis toxin-sensitive G proteins and the release of NO from the endothelium or enhancement of the actions of endogenous NO. At high concentrations (i.e., 500 nM), CGRP also directly activates the smooth muscle independent of NO. Both mechanisms of activation ultimately cause the PKA-mediated opening of K+(ATP) channels and resultant hyperpolarization.     

The great escape of glutamate from the depth of presynaptic invaginations

The basal pole of a cone photoreceptor is in close contact with hundreds of bipolar cell dendrites. The function and properties of these unconventional junctions are a long-standing mystery. In this issue of Neuron, DeVries and colleagues provide compelling evidence that glutamate release from a single quanta can diffuse to distant AMPA/KA receptors on these basal junctions to generate slow mEPSCs.     

Deletion mutagenesis of p22phox subunit of flavocytochrome b558: identification of regions critical for gp91phox maturation and NADPH oxidase activity

The heterodimeric flavocytochrome b558, comprised of the two integral membrane proteins p22phox and gp91phox, mediates the transfer of electrons from NADPH to molecular oxygen in the phagocyte NADPH oxidase to generate the superoxide precursor of microbicidal oxidants. This study uses deletion mutagenesis to identify regions of p22phox required for maturation of gp91phox and for NADPH oxidase activity. N-terminal, C-terminal, or internal deletions of human p22phox were generated and expressed in Chinese hamster ovary cells with transgenes for gp91phox and two other NADPH oxidase subunits, p47phox, and p67phox. The results demonstrate that p22phox-dependent maturation of gp91phox carbohydrate, cell surface expression of gp91phox, and the enzymatic function of flavocytochrome b558 are closely correlated. Whereas the 5 N-terminal and 25 C-terminal amino acids are dispensable for these functions, the N-terminal 11 amino acids of p22phox are required, as is a hydrophilic region between amino acids 65 and 90. Upon deletion of 54 residues at the C terminus of p22phox (amino acids 142-195), maturation and cell surface expression of gp91phox was still preserved, although NADPH oxidase activity was absent, as expected, due to removal of a proline-rich domain between amino acids 151-160 that is required for recruitment of p47phox. Antibody binding studies indicate that the extreme N terminus of p22phox is inaccessible in the absence of cell permeabilization, supporting a model in which both the N- and C-terminal domains of p22phox extend into the cytoplasm, anchored by two membrane-embedded regions.