Gingipains inactivate a cell surface ligand on Porphyromonas gingivalis that induces TLR2-and TLR4-independent signaling

Arginine-specific gingipain and lysine-specific gingipain are two major cysteine proteinases produced by Porphyromonas gingivalis. To clarify the role of gingipains in the interaction between P. gingivalis and the innate immune system, CHO reporter cells expressing TLR2 or TLR4 were stimulated with wildtype or gingipain-deficient P. gingivalis cells and activation of nuclear factor-kappaB in these cells was examined. While CHO/CD14 cells and 7.19 cells, an MD-2-defective mutant derived from CHO/CD14 cells, failed to respond to wild-type P. gingivalis, they responded to gingipain-deficient P. gingivalis. On the other hand, CHO/CD14/TLR2 cells responded to both wild-type and gingipain-deficient P. gingivalis. These results suggested that gingipains have no effects on TLR2-dependent signaling from P. gingivalis but have inhibitory effects on TLR2-and TLR4-independent signaling in CHO cells. Indeed, the activity of gingipain-deficient P. gingivalis to induce the activation of 7.19 cells was diminished after treatment of the bacterial cells with gingipains. We next partially purified bacterial cell components activating 7.19 cells from gingipain-deficient P. gingivalis. The activity of the partially purified components was diminished by treatment with heat or gingipains. It is also noteworthy that anti-CD14 mAb inhibited the activation of 7.19 cells induced by the partially purified components. These results indicated that the components of P. gingivalis that were able to induce TLR2-and TLR4-independent signaling were inactivated by gingipains before being recognized by CD14. The inactivation of the components would be helpful for P. gingivalis to escape from the innate immune system.     

Crystallographic studies of the Escherichia coli quinol-fumarate reductase with inhibitors bound to the quinol-binding site

The quinol-fumarate reductase (QFR) respiratory complex of Escherichia coli is a four-subunit integral-membrane complex that catalyzes the final step of anaerobic respiration when fumarate is the terminal electron acceptor. The membrane-soluble redox-active molecule menaquinol (MQH(2)) transfers electrons to QFR by binding directly to the membrane-spanning region. The crystal structure of QFR contains two quinone species, presumably MQH(2), bound to the transmembrane-spanning region. The binding sites for the two quinone molecules are termed Q(P) and Q(D), indicating their positions proximal (Q(P)) or distal (Q(D)) to the site of fumarate reduction in the hydrophilic flavoprotein and iron-sulfur protein subunits. It has not been established whether both of these sites are mechanistically significant. Co-crystallization studies of the E. coli QFR with the known quinol-binding site inhibitors 2-heptyl-4-hydroxyquinoline-N-oxide and 2-[1-(p-chlorophenyl)ethyl] 4,6-dinitrophenol establish that both inhibitors block the binding of MQH(2) at the Q(P) site. In the structures with the inhibitor bound at Q(P), no density is observed at Q(D), which suggests that the occupancy of this site can vary and argues against a structurally obligatory role for quinol binding to Q(D). A comparison of the Q(P) site of the E. coli enzyme with quinone-binding sites in other respiratory enzymes shows that an acidic residue is structurally conserved. This acidic residue, Glu-C29, in the E. coli enzyme may act as a proton shuttle from the quinol during enzyme turnover.     

A fluorescent GTP analog as a specific, high-precision label of microtubules

Fluorescent imaging of cytoskeletal structures permits studies of both organization within the cell and dynamic reorganization of the cytoskeleton itself. Traditional fluorescent labels of microtubules, part of the cytoskeleton, have been used to study microtubule localization, structure, and dynamics, both in vivo and in vitro. However, shortcomings of existing labels make imaging of microtubules with high precision light microscopy difficult. In this paper, we report a new fluorescent labeling technique for microtubules, which involves a GTP analog modified with a bright, organic fluorophore (TAMRA, Cy3, or Cy5). This fluorescent GTP binds to a specific site, the exchangeable site, on tubulin in solution with a dissociation constant of 1.0±0.4 μM. Furthermore, the label becomes permanently incorporated into the microtubule lattice once tubulin polymerizes. We show that this label is usable as a single molecule fluorescence probe with nanometer precision and expect it to be useful for modern subdiffraction optical microscopy of microtubules and the cytoskeleton.     

WNT5A signaling affects pituitary gland shape

Wnt signaling is important in organogenesis, and aberrant signaling in mature cells is associated with tumorigenesis. Several members of the Wnt family of signaling molecules are expressed in the developing pituitary gland. Wnt5a is expressed in the neuroectoderm that induces pituitary gland development and has been proposed to influence pituitary cell specification. We discovered that mice deficient in Wnt5a display abnormal morphology in the dorsal part of the developing pituitary. The expression of downstream effectors of the canonical Wnt pathway is not altered, and expression of genes in other signaling pathways such as Shh, Fgf8, Fgf10 and Fgfr2b is intact. Prop1 and Hesx1 are also important for normal shape of the pituitary primordium, but their expression is unaltered in the Wnt5a mutants. Specification of the hormone-producing cell types of the mature anterior pituitary gland occurs appropriately. This study suggests that the primary role of Wnt5a in the developing pituitary gland is in establishment of the shape of the gland.     

Biosynthesis of the beta-lactam antibiotic, thienamycin, by Streptomyces cattleya

Radioactive- and stable isotope-containing substrates were used to identify the biosynthetic precursors of the beta-lactam antibiotic, thienamycin, in Streptomyces cattleya. Acetate is utilized by the organism to form C(6) and C(7) of the beta-lactam ring. The two carbons of the hydroxyethyl group attached to C(6) are both derived from the methyl of methionine. The cysteaminyl side chain attached to C(2) is derived from cysteine. Selective inhibition of thienamycin and cephamycin C biosynthesis has been achieved either through the addition of metabolic inhibitors or through manipulation of the growth medium. These results suggest that the two beta-lactam antibiotics, thienamycin and cephamycin C, are formed by different biosynthetic pathways.     

Is the epithelium-derived inhibitory factor in guinea-pig trachea a prostanoid?

To examine further the possible prostanoid involvement in the influence of the epithelium on guinea-pig tracheal smooth muscle responsiveness, we have analyzed the effects of LTD₄, methacholine and histamine on the level of airway smooth muscle tone and on the amounts of PGE_2α and PGI₂ (determined by radioimmunoassay) in the presence and absence of the epithelium. Removal of the epithelium increased the sensitivity of guinea-pig trachea to the contractile effects of LTD₄, methacholine and histamine. LTD₄ (3–100 nM), methacoline (0.1–10 μM) or histamine (0.3–30 μM) did not increase prostanoid release above control values in either the presence or absence of the epithelium. The unstimulated release of PGE₂ and PGF_2α but not PGI₂, was decreased in tissues lacking epithelium. Indomethacin (1 μM) reduced the baseline tone to a smaller extent in the absence of epithelium. In the presence but not the absence of the epithelium, indomethacin increased the sensitivity of preparations to the contractile effect of methacholine. The results support the postulate of an epithelium-derived inhibitory factor modulating guinea-pig tracheal smooth muscle responsiveness. The identity of this factor is not known but is not PGI₂ and is unlikely to be PGF_2α or PGE₂. However, the possibility remains that the basal release of PGE₂ and/or PGF_2α derived from the epithelium may markedly affect the responsiveness of guinea-pig tracheal smooth muscle. Furthermore, the epithelium is a significant source of PGE₂ and PGF_2α which may be involved in the maintenance of baseline tone.     

Nuclear targeting of IGF-1 receptor in orbital fibroblasts from Graves' disease: apparent role of ADAM17

Insulin-like growth factor-1 receptor (IGF-1R) comprises two subunits, including a ligand binding domain on extra- cellular IGF-1Rα and a tyrosine phosphorylation site located on IGF-1Rβ. IGF-1R is over-expressed by orbital fibroblasts in the autoimmune syndrome, Graves' disease (GD). When activated by IGF-1 or GD-derived IgG (GD-IgG), these fibroblasts produce RANTES and IL-16, while those from healthy donors do not. We now report that IGF-1 and GD-IgG provoke IGF-1R accumulation in the cell nucleus of GD fibroblasts where it co-localizes with chromatin. Nuclear IGF-1R is detected with anti-IGF-1Rα-specific mAb and migrates to approximately 110 kDa, consistent with its identity as an IGF-1R fragment. Nuclear IGF-1R migrating as a 200 kDa protein and consistent with an intact receptor was undetectable when probed with either anti-IGF-1Rα or anti-IGF-1Rβ mAbs. Nuclear redistribution of IGF-1R is absent in control orbital fibroblasts. In GD fibroblasts, it can be abolished by an IGF-1R-blocking mAb, 1H7 and by physiological concentrations of glucocorticoids. When cell-surface IGF-1R is cross-linked with (125)I IGF-1, (125)I-IGF-1/IGF-1R complexes accumulate in the nuclei of GD fibroblasts. This requires active ADAM17, a membrane associated metalloproteinase, and the phosphorylation of IGF-1R. In contrast, virally encoded IGF-1Rα/GFP fusion protein localizes equivalently in nuclei in both control and GD fibroblasts. This result suggests that generation of IGF-1R fragments may limit the accumulation of nuclear IGF-1R. We thus identify a heretofore-unrecognized behavior of IGF-1R that appears limited to GD-derived fibroblasts. Nuclear IGF-1R may play a role in disease pathogenesis.     

Effects of local and landscape-scale habitat variables on abundance and reproductive success of wetland birds

Both local and landscape-scale habitat variables influence the abundance of wetland breeding birds. Few studies, however, simultaneously assess the effects of habitat variables at multiple spatial scales or consider effects on reproductive success. Therefore, we examined the effects of wetland and landscape-scale habitat variables on the abundance of nine breeding bird species and the effects of nest, wetland, or landscape-scale habitat variables on the nest success, clutch size, or number of fledglings of four species at 15 cattail (Typha sp.)-dominated wetlands in an agricultural region around Peterborough, Ontario, Canada. The abundance of Least Bittern (Ixobrychus exilis), Common Moorhen (Gallinula chloropus), and Marsh Wren (Cistothorus palustris) increased as wetland water depth increased; the abundance of Common Moorhen and Marsh Wren increased as wetland size increased; and the abundance of Marsh Wren increased as the amount of wetland in the surrounding landscape increased. Red-winged Blackbird (Agelaius phoeniceus) nest success decreased as nest cover increased. Clutch sizes were uninfluenced by the habitat variables that we considered. The number of Red-winged Blackbird fledglings per successful nest increased as wetland size increased and as the amount of wetland in the surrounding landscape increased. We speculate that food limitation in small wetlands may be responsible for the pattern in Red-winged Blackbird fledging success. The abundance and nest success of Virginia Rail (Rallus limicola) and Sora (Porzana carolina) were uninfluenced by the habitat variables we considered. Future research should consider mate attraction and productivity in relation to local and landscape-scale habitat variables for these and other secretive species. Our study suggests that wetland conservation will be most effective if it considers habitat variables at multiple spatial scales.