Heterotrimeric G Proteins Directly Regulate MMP14/Membrane Type-1 Matrix Metalloprotease: A NOVEL MECHANISM FOR GPCR-EGFR TRANSACTIVATION*

Agonist stimulation of G protein-coupled receptors (GPCRs) can transactivate epidermal growth factor receptors (EGFRs), but the precise mechanisms for this transactivation have not been defined. Key to this process is the protease-mediated “shedding” of membrane-tethered ligands, which then activate EGFRs. The specific proteases and the events involved in GPCR-EGFR transactivation are not fully understood. We have tested the hypothesis that transactivation can occur by a membrane-delimited process: direct increase in the activity of membrane type-1 matrix metalloprotease (MMP14, MT1-MMP) by heterotrimeric G proteins, and in turn, the generation of heparin-binding epidermal growth factor (HB-EGF) and activation of EGFR. Using membranes prepared from adult rat cardiac myocytes and fibroblasts, we found that MMP14 activity is increased by angiotensin II, phenylephrine, GTP, and guanosine 5′-O-[γ-thio]triphosphate (GTPγS). MMP14 activation by GTPγS occurs in a concentration- and time-dependent manner, does not occur in response to GMP or adenosine 5′-[γ-thio]triphosphate (ATPγS), and is not blunted by inhibitors of Src, PKC, phospholipase C (PLC), PI3K, or soluble MMPs. This activation is specific to MMP14 as it is inhibited by a specific MMP14 peptide inhibitor and siRNA knockdown. MMP14 activation by GTPγS is pertussis toxin-sensitive. A role for heterotrimeric G protein βγ subunits was shown by using the Gβγ inhibitor gallein and the direct activation of recombinant MMP14 by purified βγ subunits. GTPγS-stimulated activation of MMP14 also results in membrane release of HB-EGF and the activation of EGFR. These results define a previously unrecognized, membrane-delimited mechanism for EGFR transactivation via direct G protein activation of MMP14 and identify MMP14 as a heterotrimeric G protein-regulated effector.     

Normalized cDNA libraries from a porcine model of orthopedic implant-associated infection

Staphylococcal infections that result from an alteration in a patient's immune response at the surgical site are a major problem in procedures that incorporate biomaterials in trauma surgery and joint replacement. Diagnosis of infection based on pathogen detection is difficult and exacerbated by increasing numbers of partially or totally resistant strains of nosocomial pathogens, particularly Staphylococcus aureus. Expression profiling of a host's cellular immune response could facilitate the identification of the pathways involved in pathogen recognition and eradication and could lead to more rational design of drugs and therapies. To this end, we constructed and characterized ten individually tagged and directionally cloned cDNA libraries from peripheral blood cells (PBC), spleen (Sp), thymus (Th), lymph node (LN), and bone marrow (BM) from immunologically naive and challenged pigs as part of an implant-associated orthopedic model of deep infection. Three of these libraries were normalized at C _{0 t} values 5, 10, 20, and 30. The libraries comprise more than 20 million primary transformants with an average insert length >1.4 kb. Cluster analysis of 7620 ESTs revealed 1029 clusters containing an average of 3.6 sequences and 3846 singletons. Gene discovery is estimated to be ∼64%. Searches of public databases resulted in 49.3% annotated porcine sequences, of which 22.2% had significant homologies to ESTs from a variety of species, and 28.5% were without a significant match in any public database. We also identified 9.1% ESTs as involved in host cell and organism defense and 11.5% related to cell signaling and communication. These sequences, together with the 28.5% appearing as novel, are of specific interest to the infectious disease process.     

Liquid chromatographic determination of the enantiomers of ibuprofen in plasma using a chiral AGP column

A reversed-phase high-performance liquid chromatographic method has been developed for the determination of the R- and S-enantiomers of ibuprofen. The enantiomers and the internal standard 4-pentylphenylacetic acid are extracted from plasma, separated and quantified on a Chiral-AGP column using ultraviolet detection. The simplicity, sensitivity and precision of the method makes it convenient for use in pharmacokinetic studies.     

Western Diet Triggers NLRP3-Dependent Innate Immune Reprogramming

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Purification, characterization and stability of barley grain peroxidase BP 1, a new type of plant peroxidase

The major peroxidase of barley grain (BP 1) has enzymatic and spectroscopic properties that are very differeant from those of other known plant peroxidases (EC 1.11.1.7) and can therefore contribute to the understanding of the many physiological functions ascribed to these enzymes. To study the structure-function relationships of this unique model peroxidase, large-scale and Jaboratory-scale purifications have been developed. The two batches of pure BP 1 obtained were identical in their enzymatic and spectral properties, and confirmed that BP 1 is different from the prototypical horseradish peroxidase isoenzyme C (HRP C). However, when measuring the specific activity of BP 1 at pH 4.0 in the presence of 1 m M C_aCl₂, the enzyme was as competent as HRP C at neutral pH towards a variety of substrates (m M mg⁻¹ min⁻¹): coniferyl alcohol (930±48), caffeic acid (795±53), ABTS (2,2'-azino-di-[3-ethyl-benzothiazoline-(6)-sulfonic acid]) (840±47), ferulic acid (415±20), p -coumaric acid (325±12), and guaiacol (58±3). The absorption spectrum of BP 1 is blue-shifted compared to that of HRP C with a Soret maximum of 399–402 nm, depending on pH. The prosthetic group was shown to be iron-protoporphyrin IX, which is characteristic of plant peroxidases. BP 1 is stable from pH 3 to 11, indicating that its unusual spectral characteristics do not result from enzyme instability. The thermostability is also normal with a melting temperature of 75°C at pH 6.6, and 67°C at pH 4.0 and 8.3. It is clear that the unusual properties of BP 1 are genuine, and reflect a novel regulation of plant peroxidase function.     

Ultrastructure of snail grazing damage to calcicolous lichens

The identification of damaged lichens is often difficult due to changes in the morphology of regenerating specimens. We examined the Ultrastructure of grazing damages to four species of calcicolous lichens ( Aspicilia calcarea, Physcia adscendens, Tephromela atra and Xanthoria parietina ) and free-living cyanobacteria (family Chroococcaceae) caused by individuals of four species of land snails ( Chondrina clienta, Balea perversa, Clausilia bidentata and Helicigona lapicida ). We also investigated the radular structure of the four lichen-feeding snails to examine whether differences in radular morphology result in species-specific grazing damages. Individuals of all four snail species removed the cyanobacteria layer covering the limestone or lichen surfaces. The four lichen species were grazed to a different extent by the different snail species. SEM-images showed that B. perversa left distinct depressions on the thalli of A. calcarea , whereas H. lapicida grazed off the thalli of this lichen rather evenly. Both snail species left visible radular traces on the lichen surface. In contrast, Ch. clienta left shallow depressions without radular traces on the thalli of A. calcarea. In Tephromela atra , grazing damages were observed on both thallus and ascocarp. Ascocarps of T. atra were partly grazed by B. perversa. Helicigona lapicida grazing on T. atra removed more or less evenly the entire lichen tissue including the ascocarps. In foliose lichens, grazing by Ch. clienta, B. perversa and Cl. bidentata resulted in depressions of different depths, while H. lapicida removed entire pieces of the thalli. In general, radular traces were less distinct in foliose lichens than in crustose lichens.     

A molecular phylogeny of the African widowbirds and bishops, Euplectes spp. (Aves: Passeridae: Ploceinae)

The elaborate male displays and plumage ornaments in the African widowbirds and bishops (Euplectes spp.) have inspired classic studies on mating systems and sexual selection. In order to study the extreme divergence in ornament design and expression in this group, we present and discuss a well-supported molecular phylogeny of the genus and its placement within the Ploceinae subfamily. Parsimony and Bayesian analyses were performed on 2557bp of mitochondrial DNA (ATP6, Cyt b, ND2 and ND3) and a nuclear intron (G3PDH). All 17 Euplectes species, and 31 of 51 suggested subspecies, were included, as well as eight Ploceinae outgroups from four genera (Amblyospiza, Ploceus, Quelea and Foudia). Our results show monophyly of Euplectes, but not of the intrageneric groupings of bishops and widowbirds. Most notably, the Red-collared Widowbird E. ardens belongs to a subclade of bishops, and not to the sister subclade of 'true' widowbirds. Furthermore, the two bishops E. afer and E. aureus represent lineages that branched off before this basal split, which also refutes the proposed superspecies of E. afer and E. diadematus. Also somewhat surprisingly, and despite the striking plumage similarities among the red bishops, E. franciscanus is not closely related to either E. nigroventris or E. orix (of which it until recently was considered a subspecies). Finally, the Mountain Marsh Widowbird E. psammocromius is likely closest to the Long-tailed Widowbird E. progne, and not, as previously thought, to the Marsh Widowbird E. hartlaubi.