TNF-alpha inhibits macrophage clearance of apoptotic cells via cytosolic phospholipase A2 and oxidant-dependent mechanisms

Removal of apoptotic cells from inflammatory sites is an important step in the resolution of inflammation. Both murine and human macrophages stimulated with TNF-alpha or directly administered arachidonic acid showed an impaired ability to ingest apoptotic cells (efferocytosis). The inhibition was shown to be due to generation of reactive oxygen species, was blocked with a superoxide dismutase mimetic, MnTBAP, and was mimicked by direct addition of H2O2. To determine the mechanism of TNF-alpha-stimulated oxidant production, bone marrow-derived macrophages from gp91(phox)-deficient mice were examined but shown to still produce oxidants and exhibit defective apoptotic cell uptake. In contrast, a specific cytosolic phospholipase A2 inhibitor blocked the oxidant production and reversed the inhibited uptake. The suppressive effect of endogenous or exogenous oxidants on efferocytosis was mediated through activation of the GTPase, Rho. It was reversed in macrophages pretreated with C3 transferase to inactivate Rho or with an inhibitor of Rho kinase. During maturation of human monocyte-derived macrophages, only mature cells exhibited TNF-alpha-induced suppression of apoptotic cell clearance. The resistance of immature macrophages to such inhibition was shown to result not from defective generation of oxidants, but rather, from lack of response of these cells to the oxidants. Overall, the data suggest that macrophages in a TNF-alpha- and oxidant-rich inflammatory environment are less able to remove apoptotic cells and, thereby, may contribute to the local intensity of the inflammatory response.     

Discovery, characterization, and kinetic analysis of an alditol oxidase from Streptomyces coelicolor

A gene encoding an alditol oxidase was found in the genome of Streptomyces coelicolor A3(2). This newly identified oxidase, AldO, was expressed at extremely high levels in Escherichia coli when fused to maltose-binding protein. AldO is a soluble monomeric flavoprotein with subunits of 45.1 kDa, each containing a covalently bound FAD cofactor. From sequence alignments with other flavoprotein oxidases, it was found that AldO contains a conserved histidine (His(46)) that is typically involved in covalent FAD attachment. Covalent FAD binding is not observed in the H46A AldO mutant, confirming its role in covalent attachment of the flavin cofactor. Steady-state kinetic analyses revealed that wild-type AldO is active with several polyols. The alditols xylitol (K(m) = 0.32 mm, k(cat) = 13 s(-1)) and sorbitol (K(m) = 1.4 mm, k(cat) = 17 s(-1)) are the preferred substrates. From pre-steady-state kinetic analyses, using xylitol as substrate, it can be concluded that AldO mainly follows a ternary complex kinetic mechanism. Reduction of the flavin cofactor by xylitol occurs at a relatively high rate (99 s(-1)), after which a second kinetic event is observed, which is proposed to represent ring closure of the formed aldehyde product, yielding the hemiacetal of d-xylose. Reduced AldO readily reacts with molecular oxygen (1.7 x 10(5) m(-1) s(-1)), which confirms that the enzyme represents a true flavoprotein oxidase.     

Structure of compstatin in complex with complement component C3c reveals a new mechanism of complement inhibition

Undesired complement activation is a major cause of tissue injury in various pathological conditions and contributes to several immune complex diseases. Compstatin, a 13-residue peptide, is an effective inhibitor of the activation of complement component C3 and thus blocks a central and crucial step in the complement cascade. The precise binding site on C3, the structure in the bound form, and the exact mode of action of compstatin are unknown. Here we present the crystal structure of compstatin in complex with C3c, a major proteolytic fragment of C3. The structure reveals that the compstatin-binding site is formed by the macroglobulin (MG) domains 4 and 5. This binding site is part of the structurally stable MG-ring formed by domains MG 1-6 and is far away from any other known binding site on C3. Compstatin does not alter the conformation of C3c, whereas compstatin itself undergoes a large conformational change upon binding. We propose a model in which compstatin sterically hinders the access of the substrate C3 to the convertase complexes, thus blocking complement activation and amplification. These insights are instrumental for further development of compstatin as a potential therapeutic.     

A novel secondary acyl chain in the lipopolysaccharide of Bordetella pertussis required for efficient infection of human macrophages

Lipopolysaccharide is one of the major constituents of the Gram-negative bacterial outer membrane and is a potent stimulator of the host innate immune response. The biosynthesis of the lipid A moiety of lipopolysaccharide is a complex process in which multiple gene products are involved. Two late lipid A acyl transferases, LpxL and LpxM, were first identified in Escherichia coli and shown to be responsible for the addition of secondary acyl chains to the 2' and 3' positions of lipid A, respectively. Here, we describe the identification of two lpxL homologues in the genome of Bordetella pertussis. We show that one of them, LpxL2, is responsible for the addition of the secondary myristate group that is normally present at the 2' position of B. pertussis lipid A, whereas the other one, LpxL1, mediates the addition of a previously unrecognized secondary 2-hydroxy laurate at the 2 position. Increased expression of lpxL1 results in the appearance of a hexa-acylated lipopolysaccharide form with strongly increased endotoxic activity. In addition, we show that an lpxL1-deficient mutant of B. pertussis displays a defect in the infection of human macrophages.     

Discovery of a eugenol oxidase from Rhodococcus sp. strain RHA1

A gene encoding a eugenol oxidase was identified in the genome from Rhodococcus sp. strain RHA1. The bacterial FAD-containing oxidase shares 45% amino acid sequence identity with vanillyl alcohol oxidase from the fungus Penicillium simplicissimum. Eugenol oxidase could be expressed at high levels in Escherichia coli, which allowed purification of 160 mg of eugenol oxidase from 1 L of culture. Gel permeation experiments and macromolecular MS revealed that the enzyme forms homodimers. Eugenol oxidase is partly expressed in the apo form, but can be fully flavinylated by the addition of FAD. Cofactor incorporation involves the formation of a covalent protein-FAD linkage, which is formed autocatalytically. Modeling using the vanillyl alcohol oxidase structure indicates that the FAD cofactor is tethered to His390 in eugenol oxidase. The model also provides a structural explanation for the observation that eugenol oxidase is dimeric whereas vanillyl alcohol oxidase is octameric. The bacterial oxidase efficiently oxidizes eugenol into coniferyl alcohol (KM=1.0 microM, kcat=3.1 s-1). Vanillyl alcohol and 5-indanol are also readily accepted as substrates, whereas other phenolic compounds (vanillylamine, 4-ethylguaiacol) are converted with relatively poor catalytic efficiencies. The catalytic efficiencies with the identified substrates are strikingly different when compared with vanillyl alcohol oxidase. The ability to efficiently convert eugenol may facilitate biotechnological valorization of this natural aromatic compound.     

A Highlights from MBoC Selection: The rod domain is not essential for the function of plectin in maintaining tissue integrity

Epidermolysis bullosa simplex associated with late-onset muscular dystrophy (EBS-MD) is an autosomal recessive disorder resulting from mutations in the plectin gene. The majority of these mutations occur within the large exon 31 encoding the central rod domain and leave the production of a low-level rodless plectin splice variant unaffected. To investigate the function of the rod domain, we generated rodless plectin mice through conditional deletion of exon 31. Rodless plectin mice develop normally without signs of skin blistering or muscular dystrophy. Plectin localization and hemidesmosome organization are unaffected in rodless plectin mice. However, superresolution microscopy revealed a closer juxtaposition of the C-terminus of plectin to the integrin β4 subunit in rodless plectin keratinocytes. Wound healing occurred slightly faster in rodless plectin mice than in wild-type mice, and keratinocytes migration was increased in the absence of the rod domain. The faster migration of rodless plectin keratinocytes is not due to altered biochemical properties because, like full-length plectin, rodless plectin is a dimeric protein. Our data demonstrate that rodless plectin can functionally compensate for the loss of full-length plectin in mice. Thus the low expression level of plectin rather than the absence of the rod domain dictates the development of EBS-MD.     

A new research strategy for integrating studies of paleoclimate,paleoenvironment, and paleoanthropology

Paleoanthropologists (scientists studying human origins) universally recognize the evolutionary significance of ancient climates and environments for understanding human origins. 1 - 6 Even those scientists working in recent phases of human evolution, when modern humans evolved, agree that hunter‐gatherer adaptations are tied to the way that climate and environment shape the food and technological resource base. 7 - 10 The result is a long tradition of paleoanthropologists engaging with climate and environmental scientists in an effort to understand if and how hominin bio‐behavioral evolution responded to climate and environmental change. Despite this unusual consonance, the anticipated rewards of this synergy are unrealized and, in our opinion, will not reach potential until there are some fundamental changes in the way the research model is constructed. Discovering the relation between climate and environmental change to human origins must be grounded in a theoretical framework and a causal understanding of the connection between climate, environment, resource patterning, behavior, and morphology, then move beyond the strict correlative research that continues to dominate the field.     

A mRNA landscape of bovine embryos after standard and MAPK-inhibited culture conditions: a comparative analysis

Background

Genes and signalling pathways involved in pluripotency have been studied extensively in mouse and human pre-implantation embryos and embryonic stem (ES) cells. The unsuccessful attempts to generate ES cell lines from other species including cattle suggests that other genes and pathways are involved in maintaining pluripotency in these species. To investigate which genes are involved in bovine pluripotency, expression profiles were generated from morula, blastocyst, trophectoderm and inner cell mass (ICM) samples using microarray analysis. As MAPK inhibition can increase the NANOG/GATA6 ratio in the inner cell mass, additionally blastocysts were cultured in the presence of a MAPK inhibitor and changes in gene expression in the inner cell mass were analysed.

Results

Between morula and blastocyst 3,774 genes were differentially expressed and the largest differences were found in blastocyst up-regulated genes. Gene ontology (GO) analysis shows lipid metabolic process as the term most enriched with genes expressed at higher levels in blastocysts. Genes with higher expression levels in morulae were enriched in the RNA processing GO term. Of the 497 differentially expressed genes comparing ICM and TE, the expression of NANOG, SOX2 and POU5F1 was increased in the ICM confirming their evolutionary preserved role in pluripotency. Several genes implicated to be involved in differentiation or fate determination were also expressed at higher levels in the ICM. Genes expressed at higher levels in the ICM were enriched in the RNA splicing and regulation of gene expression GO term. Although NANOG expression was elevated upon MAPK inhibition, SOX2 and POU5F1 expression showed little increase. Expression of other genes in the MAPK pathway including DUSP4 and SPRY4, or influenced by MAPK inhibition such as IFNT, was down-regulated.

Conclusion

The data obtained from the microarray studies provide further insight in gene expression during bovine embryonic development. They show an expression profile in pluripotent cells that indicates a pluripotent, epiblast-like state. The inability to culture ICM cells as stem cells in the presence of an inhibitor of MAPK activity together with the reported data indicates that MAPK inhibition alone is not sufficient to maintain a pluripotent character in bovine cells.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1448-x) contains supplementary material, which is available to authorized users.     

Type 2 inhibitor leads of human tropomyosin receptor kinase (hTrkA)

In silico virtual screening using the ligand-based ROCS approach and the commercially purchasable compound collection from the ZINC database resulted in the identification of distinctly different and novel acetamide core frameworks with series representatives 1a and 2a exhibiting nanomolar affinity in the kinase domain only hTrkA HTRF biochemical assay. Additional experimental validation using the Caliper technology with either the active or inactive kinase conditions demonstrated the leads, 1a and 2a, to preferentially bind the kinase inactive state. X-ray structural analysis of the kinase domain of hTrkA…1a/2a complexes confirmed the kinase, bind the inhibitor leads in the inactive state and to exhibit a type 2 binding mode with the DFG-out and αC-helix out conformation. The leads also demonstrated sub-micromolar activity in the full length hTrkA cell-based assay and selectivity against the closely related hTrkB isoform. However, the poor microsomal stability and permeability of the leads is suggestive of a multiparametric lead optimization effort requirement for further progression.