Water relations of the parasite: host relationship between the mistletoe Amyema linophyllum (Fenzl) Tieghem and Casuarina obesa Miq

Summary Seasonal and diurnal gas exchange and water relations of Amyema linophyllum and its host Casuarina obesa were studied at Gingin Western Australia. As recorded elsewhere for other species of mistletoe, stomatal conductances and transpiration rates were consistently higher in parasite than host, but assimilation rates did not differ significantly between partners, and water use efficiency was accordingly substantially lower in the parasite. Parallel responses of the species to environmental conditions suggested closely coordinated stomatal behaviour. However, sunlit and artifically shaded clumps of Amyema maintained high leaf conductances even when foliage fell below turgor loss point, yet their tissue capacitance values indicated maintenance of greater tissue water reserves during stress than in the host. Pressure-volume relationships indicated that differences in tissue water relations were unlikely to contribute significantly to the observed gradient in leaf water potential between partners. An experiment measuring changes in water potential of freshly detached host: parasite systems cut with the host shoot end immersed in water indicated that the haustorial junction was the principal site of resistance to transpiration-driven water flow into the parasite. A parallel experiment on intact attached shoots with mistletoe clumps enclosed and darkened just before dawn, demonstrated that, once the host commenced rapid transpiration, the water potential gradient between partners became reversed.     

Electron-nuclear double resonance spectroscopy of 15N-enriched phthalate dioxygenase from Pseudomonas cepacia proves that two histidines are coordinated to the [2Fe-2S] Rieske-type clusters

We have performed ENDOR spectroscopy at microwave frequencies of 9 and 35 GHz at 2 K on the reduced Rieske-type [2Fe-2S] cluster of phthalate dioxygenase (PDO) from Pseudomonas cepacia. Four samples have been examined: (1) 14N (natural abundance); (2) uniformly 15N labeled; (3) [15N]histidine in a 14N background; (4) [14N]histidine in a 15N background. These studies establish unambiguously that two of the ligands to the Rieske [2Fe-2S] center are nitrogens from histidine residues. This contrasts with classical ferredoxin-type [2Fe-2S] centers in which all ligation is by sulfur of cysteine residues. Analysis of the polycrystalline ENDOR patterns has permitted us to determine for each nitrogen ligand the principal values of the hyperfine tensor and its orientation with respect to the g tensor, as well as the 14N quadrupole coupling tensor. The combination of these results with earlier M?ssbauer and resonance Raman studies supports a model for the reduced cluster with both histidyl ligands bound to the ferrous ion of the spin-coupled [Fe2+ (S = 2), Fe3+ (S = 5/2)] pair. The analyses of 15N hyperfine and 14N quadrupole coupling tensors indicate that the geometry of ligation at Fe2+ is approximately tetrahedral, with the (Fe)2(N)2 plane corresponding to the g1-g3 plane, and that the planes of the histidyl imidazoles lie near that plane, although they could not both lie in the plane. The bonding parameters of the coordinated nitrogens are fully consistent with those of an spn hybrid on a histidyl nitrogen coordinated to Fe. Differences in 14N ENDOR line width provide evidence for different mobilities of the two imidazoles when the protein is in fluid solution. We conclude that the structure deduced here for the PDO cluster is generally applicable to the full class of Rieske-type centers.     

Intravenously injected sialidase inactivates attachment sites for lymphocytes on high endothelial venules

Blood-borne lymphocytes initiate entry into secondary lymphoid organs, such as peripheral lymph nodes (PN) and gut-associated Peyer's patches (PP), by a highly specific adhesive interaction between the lymphocytes and the endothelium of specialized blood vessels known as a high endothelial venules (HEV). The selectivity with which functional subpopulations of lymphocytes migrate into particular lymphoid organs is believed to be regulated by the expression of cell adhesion receptors and complementary ligands on lymphocytes and HEV, respectively. The entry of lymphocytes into PN and PP has clearly been shown to involve distinct receptor-ligand pairs. Employing the Stamper-Woodruff in vitro adhesion assay, which measures lymphocyte attachment to HEV in cryostat-cut sections of lymphoid organs, we have previously shown that treatment of PN sections with two different sialidases inactivates HEV-adhesive ligands, whereas treatment of PP tissue sections has no effect on HEV-adhesive function. We now report that in vivo exposure of HEV to sialidase (after i.v. injection of the enzyme) also selectively prevents subsequent in vitro attachment of lymphocytes to PN HEV but not to PP HEV. Consistent with this organ-selective impairment of HEV-adhesive function by sialidase, i.v. injection of the enzyme is shown to prevent short term lymphocyte accumulation within peripheral lymph nodes while having no significant effect on accumulation in PP, blood, or nonlymphoid organs. Histologic examination with the sialic acid-specific lectin from Limax flavus verified that i.v. injected sialidase effectively removes stainable sialic acid moieties from HEV in both PN and PP. This study confirms that sialic acid is required for the adhesive function of PN HEV-ligands. A role for sialic acid as either a recognition determinant or as a regulatory molecule can be envisioned. In view of the fact that many pathogens release sialidase and cause substantially elevated serum levels of this enzyme, the present observations may have pathophysiologic significance. One mechanism by which such pathogens may avoid destruction is to inactivate susceptible HEV-ligands and disrupt the entry of lymphocytes into lymphoid organs where immune responses against the pathogens would normally be initiated.     

Molecular Cloning, Expression, and Pharmacological Characterization of humEAA1, a Human Kainate Receptor Subunit

Abstract: Kainate is a potent neuroexcitatory agent; its neurotoxicity is thought to be mediated by an ionotropic receptor with a nanomolar affinity for kainate. In this report, we describe the cloning of a cDNA encoding a human glutamate ionotropic receptor subunit protein from a human hippocampal library. This cDNA, termed humEAA1, is most closely related to rat and human cDNAs for kainate receptor proteins and, when expressed in COS or Chinese hamster ovary cells, is associated with high-affinity kainate receptor binding. We have successfully established cell lines stably expressing humEAA1. This is the first report of establishment of stable cell lines expressing a glutamate receptor subunit. The relative potency of compounds for displacing [³H] kainate binding of humEAA1 receptors expressed in these stable cell lines was kainate > quisqualate > domoate > L-glutamate > ( RS )-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid > dihydro-kainate > 6, 7-dinitroquinoxaline-2, 3-dione > 6-cyano-7-nitroquinoxaline-2, 3-dione. Homooligomeric expression of humEAA1 does not appear to elicit ligand-gated ion channel activity. Nevertheless, the molecular structure and pharmacological characterization of high-affinity kainate binding of the humEAA1 expressed in the stable cell line (ppEAA1–16) suggest that the humEAA1 is a subunit protein of a human kainate receptor complex.     

Low codon bias and high rates of synonymous substitution in Drosophila hydei and D. melanogaster histone genes

We have evaluated codon usage bias in Drosophila histone genes and have obtained the nucleotide sequence of a 5,161-bp D. hydei histone gene repeat unit. This repeat contains genes for all five histone proteins (H1, H2a, H2b, H3, and H4) and differs from the previously reported one by a second EcoRI site. These D. hydei repeats have been aligned to each other and to the 5.0-kb (i.e., long) and 4.8-kb (i.e., short) histone repeat types from D. melanogaster. In each species, base composition at synonymous sites is similar to the average genomic composition and approaches that in the small intergenic spacers of the histone gene repeats. Accumulation of synonymous changes at synonymous sites after the species diverged is quite high. Both of these features are consistent with the relatively low codon usage bias observed in these genes when compared with other Drosophila genes. Thus, the generalization that abundantly expressed genes in Drosophila have high codon bias and low rates of silent substitution does not hold for the histone genes.      

An EXAFS study of the interaction of substrate with the ferric active site of protocatechuate 3,4-dioxygenase

X-ray crystallographic studies of the intradiol cleaving protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa have shown that the enzyme has a trigonal bipyramidal ferric active site with two histidines, two tyrosines, and a solvent molecule as ligands [Ohlendorf, D.H., Lipscomb, J.D., & Weber, P.C. (1988) Nature 336, 403-405]. Fe K-edge EXAFS studies of the spectroscopically similar protocatechuate 3,4-dioxygenase from Brevibacterium fuscum are consistent with a pentacoordinate geometry of the iron active site with 3 O/N ligands at 1.90 A and 2 O/N ligands at 2.08 A. The 2.08-A bonds are assigned to the two histidines, while the 1.90-A bonds are associated with the two tyrosines and the coordinated solvent. The short Fe-O distance for the solvent suggests that it coordinates as hydroxide rather than water. When the inhibitor terephthalate is bound to the enzyme, the XANES data indicate that the ferric site becomes 6-coordinate and the EXAFS data show a beat pattern which can only be simulated with an additional Fe-O/N interaction at 2.46 A. Together, the data suggest that the oxygens of the carboxylate group in terephthalate displace the hydroxide and chelate to the ferric site but in an asymmetric fashion. In contrast, protocatechuate 3,4-dioxygenase remains 5-coordinate upon the addition of the slow substrate homoprotocatechuic acid (HPCA). Previous EPR data have indicated that HPCA forms an iron chelate via the two hydroxyl functions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dietary shift in corallivorous Drupella snails following a major bleaching event at Koh Tao, Gulf of Thailand

The island Koh Tao in the western Gulf of Thailand suffered severe coral bleaching in 2010. Its mushroom coral fauna of 20 species was surveyed during the bleaching in 2010 and after the bleaching in 2011. Multi-species assemblages of free-living mushroom corals occurred around the island, two of which were invaded by corallivorous Drupella snails after the bleaching. Previously these gastropods were known to mainly consume branching corals and hardly any mushroom corals. The snails were found preying on four fungiid species, three of which were susceptible to bleaching. The dietary shift became apparent after populations of preferred prey species (Acroporidae and Pocilloporidae) had died during the bleaching event. It seems that bleaching mortality reduced the availability of preferred prey, causing the corallivores to switch to less preferred species that occur in dense aggregations.     

Thrombin-induced p65 homodimer binding to downstream NF-kappa B site of the promoter mediates endothelial ICAM-1 expression and neutrophil adhesion

We investigated the mechanisms by which proinflammatory mediator, thrombin, released during intravascular coagulation and tissue injury, induces ICAM-1 (CD54) expression in endothelial cells. Stimulation of HUVEC with thrombin resulted in dose- and time-dependent increases in ICAM-1 mRNA and cell surface expression and in ICAM-1-dependent endothelial adhesivity toward polymorphonuclear leukocytes. Transient transfection of endothelial cells with ICAM-1 promoter luciferase reporter gene (ICAM-1LUC) constructs indicated that deletion of upstream NF-kappa B site (-533 bases from translation start site) had no effect on thrombin responsiveness, whereas mutation/deletion of downstream NF-kappa B site (-223 bases from the translation start site) prevented the activation of ICAM-1 promoter, indicating that the downstream NF-kappa B site is critical for thrombin inducibility. NF-kappa B-directed luciferase activity increased approximately 3-fold when cells transfected with the plasmid pNF-kappa BLUC containing five copies of consensus NF-kappa B site linked to a minimal adenovirus E1B promoter-luciferase gene were exposed to thrombin, indicating that activation of NF-kappa B was essential for thrombin response. Gel supershift assays demonstrated that thrombin induced binding of NF-kappa Bp65 (Rel A) to downstream NF-kappa B site of the ICAM-1 promoter. Thrombin receptor activation peptide, a 14-amino-acid peptide representing the new NH2 terminus of proteolytically activated receptor-1, mimicked thrombin's action in inducing ICAM-1 expression. These data indicate that thrombin activates endothelial ICAM-1 expression and polymorphonuclear leukocyte adhesion by NF-kappa Bp65 binding to the downstream NF-kappa B site of ICAM-1 promoter after proteolytically activated receptor-1 activation.