Neuronal process outgrowth of human sensory neurons on monolayers of cells transfected with cDNAs for five human N-CAM isoforms

Full length cDNAs for a variety of human N-CAM isoforms have been transfected into mouse L-cells and/or 3T3 cells. Three independent clones of each cell line that were shown to express human N-CAM were tested for their ability to support the morphological differentiation of sensory neurons. The cell surface expression of N-CAM isoforms, linked to the membrane directly by an integral transmembrane spanning domain or indirectly via covalent attachment to a glycosyl-phosphatidylinositol moiety, were consistently found to be associated with a significant increase in the morphological differentiation of both human and rat dorsal root ganglion neurons. Modification of the extracellular structure of both classes of N-CAM, consequent to the expression of a glycosylated 37-amino acid sequence normally found expressed exclusively in muscle N-CAM isoforms did not obviously affect the ability of transfected cells to support increased neuronal differentiation. 3T3 cells that were transfected with a full length cDNA encoding a secreted N-CAM isoform, and that have previously been shown to secrete N-CAM into the growth media rather than link it to the membrane did not significantly differ from control cells in their ability to support neuronal differentiation. These data provide direct evidence for both transmembrane and lipid-linked N-CAM isoforms being components of the regulatory machinery that determines neuronal morphology and process outgrowth.     

The effect of anti-intercellular adhesion molecule-1 on phorbol-ester-induced rabbit lung inflammation

The role of the CD18 complex of leukocyte glycoproteins in adhesion-dependent functions of human leukocytes in vitro has been well documented. A ligand, intercellular adhesion molecule-1 (ICAM-1), for at least one member of the CD18 complex has been identified. This molecule is inducible on many cell types including vascular endothelium and keratinocytes by inflammatory mediators such as IL-1, TNF, and IFN-gamma. ICAM-1 has been shown to mediate, in part, the in vitro adhesion of lymphocytes and neutrophils to endothelial cells expressing ICAM-1. In the present study we have shown that mAb's to the human CD18 complex and to human ICAM-1 cross react with rabbit cells and that both anti-CD18 and anti-CD11b but neither anti-CD11a nor anti-ICAM-1 mAb's inhibit neutrophil migration, an adhesion-dependent function, in vitro. Pretreatment of rabbits with anti-CD18 and anti-ICAM-1 but not anti-CD11a mAb inhibited by greater than 60% neutrophil migration into PMA-induced inflamed rabbit lungs. This effect of anti-ICAM-1 mAb on pulmonary neutrophil influx after PMA injection has important implications. Specifically, that ICAM-1 can function as a ligand for CD18 and can mediate, at least in part, the migration of neutrophils to inflammatory sites.     

Recognition of G-U mismatches by tris(4,7-diphenyl-1,10-phenanthroline)rhodium(III).

The coordination complex tris(4,7-diphenyl-1,10-phenanthroline)rhodium(III) [Rh(DIP)3(3+)], which promotes RNA cleavage upon photoactivation, has been shown to target specifically guanine-uracil (G-U) mismatches in double-helical regions of folded RNAs. Photoactivated cleavage by Rh(DIP)3(3+) has been examined on a series of RNAs that contain G-U mismatches, yeast tRNA(Phe) and yeast tRNA(Asp), as well as on 5S rRNAs from Xenopus oocytes and Escherichia coli. In addition, a "microhelix" was synthesized, which consists of seven base pairs of the acceptor stem of yeast tRNA(Phe) connected by a six-nucleotide loop and contains a mismatch involving residues G4 and U69. A U4.G69 variant of this sequence was also constructed, and cleavage by Rh(DIP)3(3+) was examined. In each of these cases, specific cleavage is observed at the residue which lies to the 3'-side of the wobble-paired U; some cleavage by the rhodium complex is also evident in several structured RNA loops. The remarkable site selectivity for G-U mismatches within double-helical regions is attributed to shape-selective binding by the rhodium complex. This binding furthermore depends upon the orientation of the G-U mismatch, which produces different stacking interactions between the G-U base pair with the Watson-Crick base pair following it on the 5'-side of U compared to the Watson-Crick pair preceding it on the 3'-side of U. Rh(DIP)3(3+) therefore serves as a unique probe of G-U mismatches and may be useful both as a model and in probing RNA-protein interactions as well as in identifying G-U mismatches within double-helical regions of folded RNAs.     

Delineation of structural domains in eukaryotic 5S rRNA with a rhodium probe.

The three-dimensional folding of Xenopus oocyte 5S rRNA has been examined using the coordination complex Rh(phen)2phi3+ (phen = phenanthroline; phi = phenanthrenequinone diimine) as a structural probe. Rh(phen)2phi3+ binds neither double-helical RNA nor unstructured single-stranded regions of RNA. Instead, the complex targets through photoactivated cleavage sites of tertiary interaction which are open in the major groove and accessible to stacking. The sites targeted by the rhodium complex have been mapped on the wild-type Xenopus oocyte RNA, on a truncated RNA representing the arm of the molecule comprised of helix IV-loop E-helix V, and on several single-nucleotide mutants of the 5S rRNA. On the wild-type 5S rRNA, strong cleavage is found at residues U73, A74, A101, and U102 in the E loop and U80 and G81 in helix IV; additional sites are evident at A22 and A56 in the B loop, C29 and A32 in helix III, and C34, C39, A42, and C44 in the C loop. Given the similarity observed in cleavage between the full 5S RNA and the truncated fragment as well as the absence of any long-range effects on cleavage in mutant RNAs, the results do not support models which involve long-range tertiary interactions. Cleavage results with Rh(phen)2phi3+ do, however, indicate that the apposition of several noncanonical bases as well as stem--loop junctions may result in intimately stacked structures with opened major grooves. In particular, on the basis of cleavage results on mutant RNAs, both loops C and E represent structures where the strands constituting each loop are not independent of one another but are intrinsically structured.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of dipyridophenazine complexes of ruthenium(II): the light switch effect as a function of nucleic acid sequence and conformation.

Spectroscopic parameters for two novel ruthenium complexes on binding to nucleic acids of varying sequences and conformations have been determined. These complexes, Ru(bpy)2dppz2+ and Ru(phen)2dppz2+ (bpy = 2,2'-bipyridine; phen = 1,10-phenanthroline; dppz = dipyrido[3,2:a-2',3':c]-phenazine) serve as "molecular light switches" for DNA, displaying no photoluminescence in aqueous solution but luminescing intensely in the presence of DNA. The luminescent enhancement observed upon binding is attributed to the sensitivity of the excited state to quenching by water; in DNA, the metal complex, upon intercalation into the helix, is protected from the aqueous solvent, thereby preserving the luminescence. Correlations between the extent of protection (depending upon the DNA conformation) and the luminescence parameters are observed. Indeed, the strongest luminescent enhancement is observed for intercalation into DNA conformations which afford the greatest amount of overlap with access from the major groove, such as in triple helices. Differences are observed in the luminescent parameters between the two complexes which also correlate with the level of water protection. In the presence of nucleic acids, both complexes exhibit biexponential decays in emission. Quenching studies are consistent with two intercalative binding modes for the dppz ligand from the major groove: one in which the metal-phenazine axis lies along the DNA dyad axis and another where the metal-phenazine axis lies almost perpendicular to the DNA dyad axis. Ru(bpy)2dppz2+ and Ru(phen)2dppz2+ are shown here to be unique reporters of nucleic acid structures and may become valuable in the design of new diagnostics for DNA.     

Helminth infracommunities in Litoria genimaculata (Amphibia: Anura) from Birthday Creek, and upland rainforest stream in Northern Queensland, Australia

The helminth fauna of Litoria genimaculata, a rainforest frog from northern Queensland, was quantified from 53 adult male frogs collected at monthly intervals between April 1990 and March 1991. The helminth fauna of this species was depauperate (6 species: Mesocoelium sp., Parapolystoma bulliense, Austraplectana sp., Onchocercidae gen. sp., Cosmocerca sp. and an unidentified nematode larva). The most commonly encountered species was P. bulliense, but the intestinal infracommanity was dominated by the digenean Mesocoelium sp. Fifty-five per cent of frogs were infected with only 1 helminth species and only 1 frog had more than 2 species, resulting in low diversity values. These results support previous studies which indicate that amphibians have depauperate helminth communities.     

Patterns of meiotic double-strand breakage on native and artificial yeast chromosomes

The preferred positions for meiotic double-strand breakage were mapped on Saccharomyces cerevisiae chromosomes I and VI, and on a number of yeast artificial chromosomes carrying human DNA inserts. Each chromosome had strong and weak double-strand break (DSB) sites. On average one DSB-prone region was detected by pulsed-field gel electrophoresis per 25 kb of DNA, but each chromosome had a unique distribution of DSB sites. There were no preferred meiotic DSB sites near the telomeres. DSB-prone regions were associated with all of the known ”hot spots” for meiotic recombination on chromosomes I, III and VI. Received: 19 March 1996; in revised form: 26 July 1996 / Accepted: 18 August 1996     

Effect of mutations in the PINHEAD gene of Arabidopsis on the formation of shoot apical meristems

The primary shoot apical meristem of angiosperm plants is formed during embryogenesis. Lateral shoot apical meristems arise postembryonically in the axils of leaves. Recessive mutations at the PINHEAD locus of Arabidopsis interfere with the ability of both the primary shoot apical meristem as well as lateral shoot apical meristems to form. However, adventitious shoot apical meristems can form in pinhead mutant seedlings from the axils of the cotyledons and also from cultred root explants. In this report, the phenotype of pinhead mutants is described, and a hypothesis for the role of the wild-type PINHEAD gene product in shoot meristem initiation is presented. © 1995 Wiley-Liss, Inc.