Characterization of O-glycosidically linked oligosaccharides of rat erythrocyte membrane sialoglycoproteins

The carbohydrate units of the rat erythrocyte membrane sialoglycoprotein rSGP-4 [Edge, A. S. B., & Weber, P. (1981) Arch. Biochem. Biophys. 209, 697-705] have been characterized. All of the carbohydrate of this Mr 19,000 glycoprotein occurs in O-glycosidic linkage to the peptide; following alkaline borohydride treatment and chromatography on Bio-Gel P-2, sialic acid containing oligosaccharides terminating in N-acetylgalactosaminitol were obtained. Their structures were determined by compositional analysis, exoglycosidase digestions, alkaline sulfite degradation, and periodate oxidation. The oligosaccharides were characterized for molecular weight and linkage by direct chemical ionization and gas-liquid chromatography/mass spectrometry, respectively. The structures are proposed to be NeuAc alpha 2----3Gal beta 1----3GalNAc-ol, Gal beta 1----3(NeuAc alpha 2----6)GalNAc-ol, NeuAc alpha 2----3Gal beta 1----3(NeuAc alpha 2----6)GalNAc-ol, and NeuAc alpha 2----3Gal beta 1----3(NeuAc alpha 2----3Gal beta 1----4GlcNAc beta 1----6)GalNAc-ol. Two of the N-acetylglucosamine-containing hexasaccharides were present per molecule of rSGP-4 along with two trisaccharides and seven tetrasaccharides.     

Presence of an O-glycosidically linked hexasaccharide in fetuin

Examination by gel filtration, thin layer and anion exchange chromatography of the O-linked carbohydrate units released from fetuin by alkaline borohydride treatment indicated the presence in this glycoprotein of an acidic glucosamine-containing hexasaccharide in addition to the previously described tetra- and trisaccharides. The structure of the hexasaccharide was determined to be NeuAc alpha 2----3Gal beta 1----3[NeuAc alpha 2----3Gal beta 1----4GlNAc beta 1----6]GalNAc, on the basis of exoglycosidase digestion, periodate oxidation, and methylation analysis as well as hydrazine-nitrous acid fragmentation. The latter procedure when carried out on the reduced asialohexasaccharide yielded Gal----2-deoxygalactitol and Gal----anhydromannose which were shown to be derived, respectively, from Gal----N-acetylgalactosaminitol and Gal----GlcNAc sequences. Reductive amination of the Gal----anhydromannose disaccharide with [14C] methylamine permitted identification of its linkage as 1----4. While Diplococcus pneumoniae endo-alpha-DN-acetylgalactosaminidase acting on asialofetuin released the sialic acid-free tetra- and trisaccharides (Gal beta 1----3GalNAc), this enzyme did not cleave the peptide attachment of the asialohexasaccharide (Gal beta 1----3 [Gal beta 1----4GlcNAc beta 1----6] GalNAc). The number of O-linked hexa-, tetra-, and trisaccharides per fetuin molecule was determined to be 0.2, 0.7, and 2.1, respectively, on the basis of galactosaminitol analyses. The absence of O-linked N-acetylglucosamine-containing tetra- or pentasaccharides in fetuin suggest that the attachment of this sugar is a rate-limiting step; furthermore, the limited occurrence of the hexasaccharide may indicate that the addition of sialic acid to Gal beta 1----3GalNAc to form the NeuAc alpha 2----3Gal linkage precludes action of the GlcNAc transferase to form the branch point on the GalNAc residue.     

Isoforms of apolipoprotein A-II in human plasma and thoracic duct lymph. Identification of proapolipoprotein A-II and sialic acid-containing isoforms

Proapolipoprotein (apo-) A-II and several isoforms of apo-A-II including sialylated isoforms were identified in human plasma and thoracic duct lymph. Proapo-A-II secreted by HepG2 cells was identified by a combination of immunoblots and [14C]arginine incorporation. Proapo-A-II which contains 2 arginine residues could be readily differentiated from mature apo-A-II which contains no arginine. The pI of proapo-A-II is 6.79, whereas the pI of the major apo-A-II isoform in plasma and lymph is 4.90. Minor apo-A-II isoforms have pI values of 5.17, 4.68, 4.42, and 4.20, respectively. Sialoisoforms of apo-A-II were identified, which had a higher apparent molecular weight on sodium dodecyl sulfate-gel electrophoresis than the major isoform and disappeared following neuraminidase treatment. The relative quantity of proapo-A-II was relatively constant in lymph very low density lipoproteins, lymph high density lipoproteins, and plasma high density lipoproteins, whereas the sialoforms and the other minor isoforms of apo-A-II were greater in lymph very low density lipoproteins and the lowest in plasma high density lipoproteins.     

Human serum amyloid P component. cDNA isolation, complete sequence of pre-serum amyloid P component, and localization of the gene to chromosome 1

Complementary DNA clones corresponding to the human serum amyloid P component (SAP) were isolated, and the complete nucleotide and derived amino acid sequence of preSAP was determined. PreSAP biosynthesis is directed by a 1.1-kilobase mRNA. Synthesis and postsynthetic processing of preSAP in Xenopus oocytes result in secretion of a protein with mobility similar to native purified SAP when analyzed by sodium dodecyl sulfate gel electrophoresis. The human SAP gene is on chromosome 1, probably closely linked to the gene for C-reactive protein which encodes the related acute phase reactant found in human plasma.     

MAJOR CLADES OF THE ANGIOSPERMS

Abstract— Our knowledge of fundamental angiosperm interrelationships is still very incomplete. The absence of a narrowly circumscribed gymnosperm outgroup, ideally the sister group, makes character evaluation, necessary for a cladistic analysis, difficult. According to current views the superorder Magnoliiflorae with a number of other groups, for example the monocotyledons, may represent a complex of families near the base of the angiosperms. Interrelationships of groups within the monocotyledons are much better understood than those between groups within the dicotyledons. A cladogram of monocotyledon orders based on earlier work by R. Dahlgren, H. T. Clifford, and F. N. Rasmussen is presented. A data matrix for a sample of the angiosperms with 61 characters for 49 taxa, mostly magnoliifloran and related families, is presented. The characters are polarized mainly according to the current view that the primitive angiosperm morphotype is a woody dicotyledon with strobiloid flowers. As an alternative the matrix is adjusted following W. C. Burger's conjecture that the primitive angiosperm was a herbaceous monocotyledon with trimerous flowers. Both matrices were run in a computerized parsimony analysis, resulting in numerous equally parsimonious solutions. This result is illustrative of the great homoplasy in the available character information, and also of how little actually is known about fundamental angiosperm interrelationships or phylogeny.     

Isolation of sulfited oligosaccharides from glycoproteins treated with alkaline sulfite

The oligosaccharide products resulting from treatment of mucin-type glycoproteins with alkali in the presence of the sulfite anion have been investigated. Treatment of fetuin and of tryptic glycopeptides from the human erythrocyte with this reagent resulted in the release of sulfited oligosaccharides identified as N-acetylsulfohexosamine (HexNAcSO3), alpha-NeuAc-(2----6)-HexNAcSO3, and alpha-NeuAc-(2----3)-Gal-(1----3 or 4)-[GlcNAc-(1----6)]-HexNAcSO3. In addition, 2.7 moles of sialic acid were released per mole of alpha-NeuAc-(2----6)-HexNAcSO3 from fetuin. The sulfohexosamine moiety is formed via unsaturated intermediates from a 3-O-substituted 2-acetamido-2-deoxy-D-galactosyl residue at the carbohydrate-peptide linkage site when this residue is not substituted at O-4 by another sugar residue. A reaction mechanism accounting for the release of the sulfited oligosaccharides from a 3-O- and 6-O-substituted hexosamine is proposed in which the oligosaccharide branch attached to O-6 is obtained as a specific fragment terminating in sulfohexosamine.     

Assignment of orthologous relationships among mammalian alpha-globin genes by examining flanking regions reveals a rapid rate of evolution

In order to study the relationships among mammalian alpha-globin genes, we have determined the sequence of the 3' flanking region of the human alpha 1 globin gene and have made pairwise comparisons between sequenced alpha-globin genes. The flanking regions were examined in detail because sequence matches in these regions could be interpreted with the least complication from the gene duplications and conversions that have occurred frequently in mammalian alpha-like globin gene clusters. We found good matches between the flanking regions of human alpha 1 and rabbit alpha 1, human psi alpha 1 and goat I alpha, human alpha 2 and goat II alpha, and horse alpha 1 and goat II alpha. These matches were used to align the alpha-globin genes in gene clusters from different mammals. This alignment shows that genes at equivalent positions in the gene clusters of different mammals can be functional or nonfunctional, depending on whether they corrected against a functional alpha-globin gene in recent evolutionary history. The number of alpha-globin genes (including pseudogenes) appears to differ among species, although highly divergent pseudogenes may not have been detected in all species examined. Although matching sequences could be found in interspecies comparisons of the flanking regions of alpha- globin genes, these matches are not as extensive as those found in the flanking regions of mammalian beta-like globin genes. This observation suggests that the noncoding sequences in the mammalian alpha-globin gene clusters are evolving at a faster rate than those in the beta-like globin gene clusters. The proposed faster rate of evolution fits with the poor conservation of the genetic linkage map around alpha-globin gene clusters when compared to that of the beta-like globin gene clusters. Analysis of the 3' flanking regions of alpha-globin genes has revealed a conserved sequence approximately 100-150 bp 3' to the polyadenylation site; this sequence may be involved in the expression or regulation of alpha-globin genes.      

P48-Triggered transmembrane signaling transduction of human monocytes:mobilization of calcium ion and activation of protein kinase C(PKC)

INTRODUCTI0NThedifferentiati0nofcelIsalongthemonocyte-macr0phagepathwayandthesig-nalsinvo1vedinthesecel1sacquiringtheabilitytokilltum0rcellsarenotfllllyundersto0d.Wehavebeenstudingamoleculewhichappearst0beanimportantmemberofthecytokinenetworkinvo1vedintheregulati0nmonocyteactivation.ThiscytokinetermedP48wasisolatedfr0mthehllmannullcellleukemiacell1ineReh.IthasbeenpurifiedtohomogeneityandfOundtobedistinctfrominterferongamma,col0nystimulatingfactors(CSFs)andTNFalphaalldbeta[1,2].Func-ti…     

Clofentezine and hexythiazox resistance in Tetranychus urticae Koch in Australia

Clofentezine resistance in T. urticae was first confirmed in Australia in 1987 after Queensland glasshouse roses had been exposed to 40 applications of clofentezine over a 10 month period. Clofentezine resistance in this strain was extremely high (>2.500x) and conferred high level cross-resistance to the chemically unrelated compound hexythiazox. Clofentezine resistance in T. urticae was detected in pome fruit orchards in the Goulburn Valley, Victoria in 1988 and caused field control failure after 5–6 sprays. Resistance was subsequently detected in Adelaide during 1988 and the Bathurst Orange region, NSW in 1989. Clofentezine and hexythiazox resistance appears particularly stable making it difficult to manage.This work contributes in part for the fulfilment of the requirements of the degree of PhD. at the University of Sydney.