CD46 is phosphorylated at tyrosine 354 upon infection of epithelial cells by Neisseria gonorrhoeae

The Neisseria type IV pilus promotes bacterial adhesion to host cells. The pilus binds CD46, a complement-regulatory glycoprotein present on nucleated human cells (Källström et al., 1997). CD46 mutants with truncated cytoplasmic tails fail to support bacterial adhesion (Källström et al., 2001), suggesting that this region of the molecule also plays an important role in infection. Here, we report that infection of human epithelial cells by piliated Neisseria gonorrhoeae (GC) leads to rapid tyrosine phosphorylation of CD46. Studies with wild-type and mutant tail fusion constructs demonstrate that Src kinase phosphorylates tyrosine 354 in the Cyt2 isoform of the CD46 cytoplasmic tail. Consistent with these findings, infection studies show that PP2, a specific Src family kinase inhibitor, but not PP3, an inactive variant of this drug, reduces the ability of epithelial cells to support bacterial adhesion. Several lines of evidence point to the role of c-Yes, a member of the Src family of nonreceptor tyrosine kinases, in CD46 phosphorylation. GC infection causes c-Yes to aggregate in the host cell cortex beneath adherent bacteria, increases binding of c-Yes to CD46, and stimulates c-Yes kinase activity. Finally, c-Yes immunoprecipitated from epithelial cells is able to phosphorylate the wild-type Cyt2 tail but not the mutant derivative in which tyrosine 354 has been substituted with alanine. We conclude that GC infection leads to rapid tyrosine phosphorylation of the CD46 Cyt2 tail and that the Src kinase c-Yes is involved in this reaction. Together, the findings reported here and elsewhere strongly suggest that pilus binding to CD46 is not a simple static process. Rather, they support a model in which pilus interaction with CD46 promotes signaling cascades important for Neisseria infectivity.     

4-Hydroxybenzyl alcohol accumulates in flowers and developing fruits of carrot and inhibits seed formation

Somatic embryogenesis in carrot (Daucus carota) is autonomously inhibited by 4-hydroxybenzyl alcohol (4HBA), which is produced by embryogenic cells. Because somatic embryogenesis is used as a model of zygotic embryogenesis, we assayed for 4HBA in carrot seeds and analyzed the effect of 4HBA on seed formation to determine whether 4HBA is also produced during zygotic embryogenesis. HPLC analysis showed that 4HBA accumulated in flowers and immature and mature fruits, but not in vegetative tissues. The concentration of 4HBA was highest after flowering, when the zygote developed into the early globular-stage embryo. 4HBA accumulation then decreased with seed development. Exogenous application of 4HBA to immature carrot fruits inhibited seed formation. Many 4HBA-treated seeds did not include a mature embryo. These results indicate that the production and accumulation of 4HBA occurs during carrot seed development and that 4HBA has an inhibitory effect on carrot seed formation.     

neurotic,a novel maternal neurogenic gene,encodes an O-fucosyltransferase that is essential for Notch-Delta interactions

Notch signalling, which is highly conserved from nematodes to mammals, plays crucial roles in many developmental processes. In the Drosophila embryo, deficiency in Notch signalling results in neural hyperplasia, commonly referred to as the neurogenic phenotype. We identify a novel maternal neurogenic gene, neurotic, and show that it is essential for Notch signalling. neurotic encodes a Drosophila homolog of mammalian GDP-fucose protein O-fucosyltransferase, which adds fucose sugar to epidermal growth factor-like repeats and is known to play a crucial role in Notch signalling. neurotic functions in a cell-autonomous manner, and genetic epistasis tests reveal that Neurotic is required for the activity of the full-length but not an activated form of Notch. Further, we show that neurotic is required for Fringe activity, which encodes a fucose-specific beta1, 3 N-acetylglucosaminyltransferase, previously shown to modulate Notch receptor activity. Finally, Neurotic is essential for the physical interaction of Notch with its ligand Delta, and for the ability of Fringe to modulate this interaction in Drosophila cultured cells. We present an unprecedented example of an absolute requirement of a protein glycosylation event for a ligand-receptor interaction. Our results suggest that O-fucosylation catalysed by Neurotic is also involved in the Fringe-independent activities of Notch and may provide a novel on-off mechanism that regulates ligand-receptor interactions.     

Preparation of radioactive aldehyde-containing phosphatidylcholine

Short-chain, aldehyde-containing phosphatidylcholine (PC), formed during the oxidation of PC, is thought to be involved in cellular responses in atherosclerosis and inflammation. Here we report a convenient procedure for a small-scale preparation of aldehyde-containing PC. PC containing an unsaturated fatty acyl chain was treated with osmium tetroxide followed by sodium periodate at room temperature. The reaction product was purified by TLC. This preparation showed a single peak on reverse-phase HPLC, and its identity was confirmed by fast atom bombardment mass spectrometry. This procedure does not require special equipment and is easily applicable for preparation of radioactive materials.     

Suppression of polydactyly of the Gli3 mutant (extra toes) by deltaEF1 homozygous mutation

Digit patterning is established through multiple genetic interactions. Delta-crystallin enhancer/E2-box factor (deltaEF1) is a zinc finger and homeodomain containing repressor protein, and is expressed in the posterior half of the forelimb bud and in the entire hindlimb bud during the early stage of limb development. The 6EF1-deficient mutant mice display various skeletal abnormalities, among which inferior ossification and abnormal patterning of autopodial bones are similar to those observed in Hox and Gli gene mutants. Gli3 mutant mice, extra toes (Xt), exhibit pre-axial polydactyly losing the identity of digit I. It is demonstrated here that deltaEF1null(lacZ) homozygosity suppressed formation of the extra digit, uniquely of the hindlimb, in both Gli3XtJ heterozygous and homozygous mutants, but with no restoration of digit I identity. In Gli3XtJ mutants, the Hoxd13 expression domain was expanded more dramatically in homozygotes. In Gli3XtJ;deltaEF1null(lacZ) double homozygous mutants, Hoxd13 expression once expanded in Gli3XtJ homozygous mutant was reduced, more conspicuously in the hindlimbs, which may account for hindlimb-restricted suppression of formation of the extra digit. The data suggest the possibility that the extent of Hoxd13 expression along the distal margin of the limb bud is determinative in defining the digit number.     

Characterization of new conjugated metabolites in bile of rats administered 24,25-dihydroxyvitamin D(3) and 25-hydroxyvitamin D(3)

The characterization of new conjugated vitamin D metabolites in rat bile was performed using HPLC, liquid chromatography/tandem mass spectrometry combined derivatization, and GC-MS. After the administration of 24,25-dihydroxyvitamin D(3) to rats, 23, 25-dihydroxy-24-oxovitamin D(3) 23-glucuronide, 3-epi-24, 25-dihydroxyvitamin D(3) 24-glucuronide, and 24,25-dihydroxyvitamin D(3) 3-sulfate were obtained as new biliary metabolites together with 24,25-dihydroxyvitamin D(3) 3- and 24-glucuronides. The above metabolites, except 24,25-dihydroxyvitamin D(3) 3-glucuronide, were obtained from rats dosed with 25-hydroxyvitamin D(3). 23, 25-Dihydroxyvitamin D(3) 23-glucuronide was also obtained from the bile of rats administered 25-hydroxyvitamin D(3) in addition to its 3-glucuronide, 25-glucuronide, and 3-sulfate. Thus, it was found that 24,25-dihydroxyvitamin D(3) and 25-hydroxyvitamin D(3) were directly conjugated as glucuronide and sulfate, whereas at the C-23 position, they were hydroxylated and then conjugated. Furthermore, we found that the C-3 epimerization acts as one of the important pathways in vitamin D metabolism.     

Integration of the temperate phage phiU into the putative tRNA gene on the chromosome of its host Rhizobium leguminosarum biovar trifolii

The plasmid pCI6, carrying the attP site of the temperate phage phiU, integrates into the attB site on the chromosome of Rhizobium leguminosarum biovar trifolii strain 4S. The 4 kb EcoRI-HindIII region of pCI6 involved in site-specific integration was subcloned as the attP fragment of phage phiU and sequenced. The attL fragment, one of the new DNA junctions generated from the insertion of pCI6 into the chromosome of the host Rhizobium, was used as a hybridization probe for isolation of the attB fragment of strain 4S. The nucleotide sequence of the 2 kb PstI fragment of strain 4S, which hybridized with the attL fragment, was decided and compared with that of the attP fragment. A 53 bp common sequence was expected to be the core sequence of site-specific integration between phage phiU and strain 4S. One of the ORFs on the attP fragment, which was located adjacent to the core sequence, had structural homology to the integrase family. However, the attB fragment showed high homology with the tRNA genes of Agrobacterium tumefaciens and E. coli. A 47 bp sequence of the 53 bp core sequence overlapped with this tRNA-like sequence. This indicates that the target site of phage phiU integration is the putative tRNA gene on the chromosome of the Rhizobium host.     

Introduction of the cDNA for shape Arabidopsis glycerol-3-phosphate acyltransferase (GPAT) confers unsaturation of fatty acids and chilling tolerance of photosynthesis on rice

The chilling sensitivity of several plant species is closely correlated with the levels of unsaturation of fatty acids in the phosphatidylglycerol (PG) of chloroplast membranes. Plants with a high proportion of unsaturated fatty acids, such as Arabidopsis thaliana, are resistant to chilling, whereas species like squash with only a low proportion are rather sensitive to chilling. The glycerol-3-phosphate O-acyltransferase (GPAT) enzyme of chloroplasts plays an important role in determining the levels of PG fatty acid desaturation.A cDNA for oleate-selective GPAT of Arabidopsis under the control of a maize Ubiquitin promoter was introduced into rice (Oryza sativa L.) using the Agrobacterium-mediated gene transfer method. The levels of unsaturated fatty acids in the phosphatidylglycerol of transformed rice leaves were found to be 28% higher than that of untransformed controls. The net photosynthetic rate of leaves of transformed rice plants was 20% higher than that of the wild type at 17°C. Thus, introduction of cDNA for the Arabidopsis GPAT causes greater unsaturation of fatty acids and confers chilling tolerance of photosynthesis on rice.     

Possible involvement of PPARγ in the regulation of basal channel opening of P2X7 receptor in cultured mouse astrocytes

AimsRecently, we demonstrated that cultured mouse astrocytes exhibited basal channel opening of P2X7 receptor (P2X7R) in the absence of any exogenous ligand, but the regulatory mechanism involved was not elucidated. Since our preliminary experiments suggested possible involvement of peroxisome proliferator-activated receptor (PPAR) γ in the regulation, we examined whether PPARγ regulated P2X7R basal channel opening in mouse astrocytes.Main methodsP2X7R channel opening was assessed as to the uptake of a marker dye, YO-PRO-1^® (YP), in the presence or absence of agonists and antagonists for PPARγ under a fluorescence microscope. Expression of PPARγ was evaluated by Western blotting and immunocytochemistry.Key findingsNSAIDs such as flufenamic acid (FFA) and indomethacin, which are a cyclooxygenase inhibitor and a PPARγ agonist, showed enhancing and inhibiting effects on YP uptake at low and high concentrations, respectively, and the enhanced uptake was abolished by periodate-oxidized ATP (oxATP), a selective P2X7R antagonist. The PPARγ agonists 15-deoxy-Δ^12,14-prostaglandin J₂ and ciglitazone decreased the basal and FFA-enhanced YP uptake, while the antagonist GW9662 increased YP uptake, this effect being blocked by the agonists and also by oxATP. PPARγ was distributed in the nucleus and cytosolic/membrane fraction of cultured mouse astrocytes.SignificanceThese findings indicate that basal channel opening of P2X7R in mouse astrocytes is at least in part regulated by PPARγ.