Biomedical subjects
Publications and source records attributed to H Paulsen.
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The steady state level of mRNA for the light-harvesting chlorophyll a/b binding protein (LHCP) varies considerably during a day-night light cycle in expanding leaves of tobacco (Nicotiana tabacum, cv Wisconsin 38). The maximum (day) level is at least one hundred-fold higher than the minimum (night) level. The oscillation in the LHCP mRNA level continues for several days even under constant illumination. However, no such circadian rhythm can be detected in constant darkness. Plants that have been maintained in constant darkness for a prolonged period reinitiate the circadian rhythm upon illumination: the LHCP mRNA level resumes its rhythmic behavior with a period of roughly 24 hours. The same circadian fluctuation of LHCP mRNA is observed upon illuminating etiolated tobacco seedlings, demonstrating that the circadian rhythm is not dependent on the presence of mature chloroplasts. In vitro transcription experiments with nuclei isolated from green plants during various times of the day indicate that the diurnal as well as the circadian changes of the level of LHCP RNA are due, at least in part, to changes in the rate of LHCP mRNA synthesis.
The tetrasaccharides O-alpha-D-mannopyranosyl-(1----3)-O-[alpha-D- mannopyranosyl-(1----6)]-O-(4-deoxy-beta-D-lyxo-hexopyranosyl)-(1- ---4)-2- acetamido-2-deoxy-alpha, beta-D-glycopyranose (22) and O-alpha-D-mannopyranosyl-(1----3)-O-[alpha-D-mannopyranosyl-(1----6)]-O- beta-D-talopyranosyl-(1----4)-2-acetamido-2-deoxy-alpha, beta-D- glucopyranose (37), closely related to the tetrasaccharide core structure of N-glycoproteins, were synthesized. Starting with 1,6-anhydro-2,3-di-O-isopropylidene-beta-D-mannopyranose, the glycosyl donors 3,6-di-O-acetyl-2-O-benzyl-2,4-dideoxy-alpha-D-lyxo- hexopyranosyl bromide (10) and 3,6-di-O-acetyl-2,4-di-O-benzyl-alpha-D-talopyranosyl bromide (30), were obtained in good yield. Coupling of 10 or 30 with 1,6-anhydro-2-azido-3-O-benzyl-beta-D-glucopyranose to give, respectively, the disaccharides 1,6-anhydro-2-azido-3-O-benzyl-2-deoxy-4-O-(3,6-di-O-acetyl-2-O-benzyl-4 -deoxy- beta-D-lyxo-hexopyranosyl)-beta-D-glucopyranose and 1,6-anhydro-2-azido-3-O-benzyl-2-deoxy-4-O-(3,6-di-O-acetyl-2,4-di-O-ben zyl- beta-D-talopyranosyl)-beta-D-glucopyranose was achieved with good selectivity by catalysis with silver silicate. Simultaneous glycosylation of OH-3' and OH-6' of the respective disaccharides with 2-O-acetyl-3,4,6-tri-O-benzyl-alpha-D-mannopyranosyl chloride yielded tetrasaccharide derivatives, which were deblocked into the desired tetrasaccharides 22 and 37.
The conformational analysis of the recently synthesized tetrasaccharides alpha-D-Manp (1----3)-[alpha-D-Manp-(1----6)]-4-deoxy-beta-D-lyx-hexp+ ++-(1----4)-D-GlcNAc (2) and alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-beta-D-Talp -(1----4)-D-GlcNAc (3) will be described. The preferred solution conformation of 2 and 3 is a gt-conformation, which is nearly identical with the preferred conformation of the naturally occurring tetrasaccharide alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-beta-D-Manp -(1----4)-D-GlcNAc (1). The main structural feature is the backfolding of the alpha-(1----6)-linked D-Man to the reducing D-GlcNAc unit. Conformational analysis of the tetrasaccharides alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-beta-D-Manp -(1----4)-1,6- anhydro-beta-D-GlcNAc (4), alpha-D-Manp-(1----3)-alpha-D-Manp-(1----6)]-4-deoxy-beta-D- lyx-hexp-(1----4)- 1,6-anhydro-beta-D-GlcNAc (5), and alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-beta-D-Talp -(1----4)- 1,6-anhydro-beta-D-GlcNAc (6) gave additional proof for this backfolding. The substitution of the reducing unit leads to a smaller amount of gt- and a greater amount of gg-conformers. The method used for conformational analysis of 2-6 is a combination of n.m.r.-experiments and HSEA-calculations with the program GESA. Concerning the application of new 2D-techniques, the COLOC-experiment turned out to be extremely useful in sequencing oligosaccharides.
Mouse monoclonal antibodies were raised against heat-killed bacteria of the Re mutant R595 of Salmonella minnesota and characterized by the passive hemolysis and passive hemolysis inhibition tests and by double immunodiffusion experiments using lipopolysaccharide (LPS) from different rough mutants of S. minnesota and synthetic antigens. The latter were copolymerization products of acrylamide with the alpha- and beta-allylglycosides of 3-deoxy-D-manno-octulosonic acid (KDO) and the alpha-2,4-linked KDO disaccharide [poly-alpha-KDO, poly-beta-KDO, and poly-(alpha-KDO)2, respectively], and sodium (3-deoxy-D-manno-octulopyranosyl)onate-(2----6)-(2-deoxy-2-[ (R)-3- hydroxytetradecanoylamino]- beta-D-glucopyranosyl)-(1----6)-(2-deoxy-2-[(R)-3-hydroxytetradecanoy lam ino]-D-glucose) [alpha-KDO-(GlcNhm)2], representing a part structure of Re LPS. One antibody (clone 20, immunoglobulin M) was found to recognize a terminal alpha-linked KDO residue, since it reacted in the passive hemolysis assay with alpha-KDO-(GlcNhm)2 and all LPS tested, it was inhibited by all synthetic antigens containing alpha-linked KDO residues, and it gave a reaction of identity with poly-alpha-KDO and poly-(alpha-KDO)2 in double immunodiffusion experiments. A second antibody (clone 25, immunoglobulin G3) was identified as specific for an alpha-2,4-linked KDO disaccharide, since it reacted in immunodiffusion exclusively with synthetic poly-(alpha-KDO)2 and not with the monosaccharide derivatives in either anomeric configuration, and it was inhibited only with poly-(alpha-KDO)2 and with LPS from S. minnesota R595 (Re) and R345 (Rb2). The reaction of this antibody with R345 LPS is attributed to the quantitative substitution with KDO disaccharide present as a side chain, which is not present in stoichiometric amounts in the other LPS.
The two partial reactions of elongation factor G dependent translocation, the release of deacylated tRNA from the P site and the displacement of peptidyl tRNA from the A to the P site, have been studied with the stopped-flow technique. The experiments were performed with poly(U)-programmed ribosomes from Escherichia coli carrying deacylated tRNAPhe in the P site and N-AcPhe-tRNAPhe in the A site in the presence of GTP. The kinetics of the reaction were followed by monitoring either the intensity or the polarization of the fluorescence of both wybutine and proflavine located in the anticodon loop or of proflavine located in the D loop of yeast tRNAPhe or N-AcPhe-tRNAPhe. Both displacement and release fluorescence changes could be described by three exponentials, exhibiting apparent first-order rate-constants (20 degrees C) of 2 to 5 s-1 (15 s-1, 35 degrees C), 0.1 to 0.3 s-1, and 0.01 to 0.02 s-1, measured with a saturating concentration of elongation factor G (1 microM). The activation energy for the fast process of both reactions was found to be 70 kJ/mol (17 kcal/mol), while the intermediate process exhibits an activation energy of 30 kJ/mol (7 kcal/mol). The fast step is assigned to the displacement of the N-AcPhe-tRNAPhe from the A to the P site, and to the release of the tRNAPhe from the P site. The reactions take place simultaneously to form an intermediate post-translocation complex. The latter, in the intermediate step, rearranges to form a post-translocation complex carrying the deacylated tRNAPhe in an exit site and N-AcPhe-tRNAPhe in the P site, both in their equilibrium states. In parallel, or subsequently, the deacylated tRNAPhe spontaneously dissociates from the ribosome, thus completing the translocation process. The slow process has not been assigned.
In the conformational analysis of the pentasaccharide alpha-D-Manp-(1----3)-[beta-D-GlcpNAc-(1----4)]-[alpha-D- Manp-(1----6)]-beta-D-Manp-(1----4)-D-GlcNAc, which represents the essential core structure within the bisected type of N-glycoproteins, n.m.r. experiments and GESA calculations indicated that a gg conformation is preferred for the (1----6)-glycosidic linkage. This is in contrast to the results obtained for the tetrasaccharide alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-beta-D- Manp-(1----4)- D-GlcNAc, which indicated that a gt conformation with the (1----6)-linked alpha-D-mannose group folded back is preferred. The conformations of the remaining glycosidic linkages in the tetra- and penta-saccharide are similar.
The synthesis is described of alpha-beta-D-xylopyranosyl-L-serylglycyl-L-isoleucyl-glycine (7), a glycopeptide containing the amino acid sequence of the protein core-structure of beef-skin proteodermatan sulfate; coupling of N-protected O-XylpSer with protected GlyIleuGly followed by deprotection afforded 7.
In the presence of silver silicate, the reaction of 3,4,6-tri-O-acetyl-2-azido-2-deoxy-alpha-D-mannopyranosyl bromide with 1,6-anhydro-2-azido-3-O-benzyl-2-deoxy-beta-D-glucopyranose gave beta-glycosidically linked 1,6-anhydro-2-azido-3-O-benzyl-2-deoxy-4-O-(3,4,6- tri-O-acetyl-2-azido-2-deoxy-beta-D-mannopyranosyl)-beta-D- glucopyranose. After deacetylation and catalytic oxidation with oxygen and platinum, 1,6-anhydro-2-azido-4-O-[(2-azido-2-deoxy-beta-D-mannopyranosyl) uronic acid]-3-O-benzyl-2-deoxy-beta-D-glucopyranose was obtained. A series of intermediate steps led to the glycosyl donor 6-O-acetyl-2-azido-3-O-benzyl-4-O-[benzyl (3,4-di-O-acetyl-2-azido-2-deoxy-beta-D-mannopyranosyl)uronate]-2-deoxy- alpha, beta-glucopyranosyl chloride which was coupled with 8-methoxycarbonyloctyl 4-azido-2-O-benzyl-4,6-dideoxy-alpha-D-galactopyranoside to give 8-methoxycarbonyloctyl O-[benzyl (3,4-di-O-acetyl-2-azido-2-deoxy-beta-D-mannopyranosyl)uronate]- (1----4)-O-(6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-alpha-D-glucopyranosyl )-(1----3)-4-azido-2-O-benzyl-4,6-dideoxy-alpha-D-galactopyranoside. Deblocking gave the spacer-linked repeating unit of the enterobacterial common antigen, beta-D-ManpNAcA-(1----4)-alpha-D-GlcpNAc-(1----3)-alpha- D-Fucp4NACO(CH2)8CO2CH3.
The distances between the anticodon loops of fluorescent tRNAPhe bound to the E site and to either the A or the P site of poly(U)-programmed Escherichia coli ribosomes were measured by fluorescence energy transfer. Donor and acceptor molecules were wybutine and proflavin, respectively, both located 3' to the anticodon of tRNAPhe. The anticodon loops were found to be separated by 42 +/- 10 A (A to E site) and 34 +/- 8 A (P to E site). The latter distance is much larger than the one measured between the anticodon loops of A and P site bound tRNAs [24 +/- 4 A; Paulsen, H., Robertson, J. M., & Wintermeyer, W. (1983) J. Mol. Biol. 167, 411-426], rendering unlikely simultaneous codon-anticodon interaction in the P and E sites. In kinetic stopped-flow measurements, the energy transfer between the anticodon loops of the tRNA molecules was followed during translocation. The transfer efficiency decreases in three steps with apparent rate constants on the order of 1, 0.1, and 0.01 s-1. The fast step is ascribed to the simultaneous displacement of the deacylated tRNAPhe out of the P site and of the N-AcPhe-tRNAPhe from the A site to the P site. The distance between the anticodon loops does not change appreciably during this reaction. A significant separation of the two tRNAs occurs during the intermediate and the slow steps. The latter most likely represents a rearrangement of the posttranslocation complex containing both tRNA molecules.(ABSTRACT TRUNCATED AT 250 WORDS)
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We have tested various carbohydrate structures and neoglycoproteins (carbohydrate haptens attached to BSA) as inhibitors of the invasion of human red blood cells by Plasmodium falciparum merozoites (strain FCB) in synchronous in vitro cultures, using 3H-hypoxanthine incorporation into intraerythrocytic parasites as analytical tool, and have got the following results: The inhibition rate of N-acetyl-D-glucosamine glycosides is increased with increasing lipophilicity of the aglycon or spacer: CH3 less than (CH2)2NHCO(CH2)2COOCH3 less than (CH2)8COOCH3; p-Nitrophenyl glycosides proved to exhibit a toxic effect; The inhibition rate of synthetic disaccharide glycosides increased in the order beta DGal(1----4) beta DGlcNAcOCH3 less than beta DGlcNAc(1----4) beta DGlcNAcOCH3 less than beta DGlcNAc(1----4) alpha DManOCH3 less than beta DGlcNAc(1----4) beta DManO(CH2)8COOCH3; The O-linked tetrasaccharide alpha NeuAc(2----3) beta DGal(1----3) [alpha NeuAc(2----6)] alpha DGalNAcol, isolated from glycophorin A, was the best carbohydrate inhibitor tested so far; The inhibition rate of carbohydrates attached to BSA by an aliphatic spacer [-(CH2)8COOCH3] was not enhanced compared to the haptens; [DNP]33-BSA proved to be an extraordinary inhibitor of invasion which, however, most likely has to be attributed to a toxic effect; Observed toxicities appear to be attributable to hydrophobic interactions between the inhibitors and the RBC and merozoite membranes, which hampers both, intraerythrocytic growth of the parasite and its capability of RBC invasion.
For the synthesis of the threefold-branched pentasaccharide, O-alpha-D-mannopyranosyl-(1----3)-O-[(2-acetamido-2-deoxy-beta-D- glucopyranosyl)-(1----4)]-O-[alpha-D-mannopyranosyl-(1----6)]-O-beta-D- mannopyranosyl-(1----4)-2-acetamido-2-deoxy-D-glucopyranose (20), which is a part of the structure of the N-glycoproteins, the disaccharide 4-O-(4-O-acetyl-3,6-di-O-allyl-2-O-benzyl-beta-D-mannopyranosyl) -1,6-anhydro-2-azido-3-O-benzyl-2-deoxy-beta-D-glucopyranose was synthesized as a key compound by use of the silver silicate-catalyst procedure. After elimination of the 4-O-acetyl group, a 2-acetamido-2-deoxy-beta-D-glucopyranosyl group was attached according to the phthalimido method. Further elimination of the allyl groups allowed the linkage of two alpha-D-mannopyranosyl groups in the presence of mercury salt. A deblocking sequence consisting of four steps gave 20.
Antibodies formed in rabbits to synthetic glycoproteins with the disaccharide structure beta-D-Gal-(1-3)-D-GalNAc, the carbohydrate moiety of desialylated glycophorin A, were investigated for their carbohydrate specificity by hemagglutination techniques and radioimmunoassay. The synthetic disaccharide was coupled to human serum albumin as carrier protein using different spacer groups and different configurations of the disaccharide spacer linkage. Radioimmunological inhibition experiments using a number of different derivatives of the carbohydrate group as inhibitors revealed a significant specificity of the antibodies for the oligosaccharide structure. However, the nature of the conjugation of the disaccharide to the carrier protein was found to be important for defining the specificity of the antibodies. Furthermore the arrangement of the hapten groups in the synthetic antigen had an important influence on the antigen-antibody reaction. No cross-reactions were observed between asialo-glycophorin A and the different synthetic antigens. Synthetic antigens provide a valuable tool for generating carbohydrate-specific antibodies. However, if the synthetic hapten molecule is small compared to the combining site of antibodies, the specificity of the immunological reaction is significantly influenced by the synthetic spacer arm or even the carrier protein.
The panagglutination phenomenon described by Thomsen and Friedenreich (TF) is due to the reaction of naturally occurring TF-antibodies with the carbohydrate group beta-D-Gal-(1-3)-D-GalNAc of desialylated glycophorin A, the major glycoprotein component of the erythrocyte membrane. The specificity of human TF-antibodies reacting with this disaccharide was investigated by hemagglutination inhibition assay and radioimmunoassay using various synthetic oligosaccharides and neoglycoproteins as well as asialoglycophorin A. The results indicate that TF-antibodies represent a heterogeneous mixture of carbohydrate-specific antibodies. The disaccharide beta-D-Gal-(1-3)-D-GalNAc is the common structure recognized by all TF-antibodies. However, the conjugation mode of the carbohydrate to the carrier protein is important for defining the specificity of different subpopulations of TF-antibodies. The immunological reaction depends on the configuration of the glycosidical linkage as well as on the chemical nature of the aglycon, which is coupled to the disaccharide. These findings suggest that the heterogeneity of natural TF-antigens is due to the wide distribution of the carbohydrate structure beta-D-Gal-(1-3)-D-GalNAc. The characterization of TF- or TF-like antibodies directed to particular natural TF-antigens (e.g. asialoglycophorin A, tumor TF-antigens, glycolipids, bacterial antigens) requires TF-analogues, which contain the additional molecular regions together with the TF-disaccharide. These structures, apart from the TF-hapten, are obviously important for defining the immunodeterminant group of TF-antigens of different origin.
The crystal structure of 3-O-(6-O-acetyl-2,4-diazido-3-O-benzyl-2,4-dideoxy-alpha-D- glucopyranosyl)-1,6-anhydro- 2,4-diazido-2,4-dideoxy-beta-D-glucopyranose, C21H24N12O7, mol. wt. 556.5, was investigated by X-ray analysis. The disaccharide crystallizes in the triclinic space group P1, with a = 889.3(5), b = 869.6(5), and c = 999.5(6) pm, and alpha = 105.83(4) degrees, beta = 116.22(4) degrees, gamma = 88.42(4) degrees, Z = 1, and rho = 1.394 g.cm-3. Phase determination failed with direct methods, but, as the 1,6-anhydride component of the molecule was already known from a previous structure analysis, the vector-search method could be applied in solving the structure. Diffractometer data were refined to an R value of 0.063 (Rw = 0.080) for 2102 observed reflections. The anhydro-bridged system has a distorted 1C4(D) conformation, in agreement with that of other anhydropyranoses so far investigated. A comparison shows that, for the specific kind of distortion, mainly the anti-reflex effect is responsible, whereas 1,3-diaxial interactions have a minor influence. The nonbridged ring adopts an almost perfect 4C1(D) conformation. The anomeric effect is observed in both of the sugar-ring systems in terms of bond-length shortening. The disaccharide has an alpha-D-Glc-4C1-(1a----3e)-D-Glc-1C4 glycosidic linkage. No previous X-ray investigation of a compound of this type is known. The pyranoid rings are almost perpendicular to each other. The phi, psi angles of the glycosidic linkage are +78.1(5) and -86.0(4) degrees.(ABSTRACT TRUNCATED AT 250 WORDS)
The 3'-terminal A-C-C-A sequence of yeast tRNAPhe has been modified by replacing either adenosine 76 or 73 with the fluorescent analogues 1,N6-ethenoadenosine (epsilon A) or 2-aza-1,N6-ethenoadenosine (aza-epsilon A). T4 RNA ligase was used to join the nucleoside 3',5'-bisphosphates to the 3' end of the tRNA which was shortened by one [tRNAPhe(-A)] or four [tRNAPhe(-ACCA)] nucleotides. It was found that the base-paired 3'-terminal cytidine 72 in tRNAPhe(-ACCA) is a more efficient acceptor in the ligation reaction than the unpaired cytidine 75 at the A-C-C terminus of tRNAPhe(-A). This finding indicates that the mobility of the accepting nucleoside substantially influences the ligation reaction, the efficiency being higher the lower the mobility. This conclusion is corroborated by the observation that the ligation reaction with the double-stranded substrate exhibits a positive temperature dependence rather than a negative one as found for single-stranded acceptors. The replacement of the 3'-terminal adenosine 76 with epsilon A and aza-epsilon A leads to moderately fluorescent tRNAPhe derivatives, which are inactive in the aminoacylation reaction. A number of other tRNAs (Met, Ser, Glu, Lys and Leu-specific tRNAs both from yeast and Escherichia coli) are also inactivated by epsilon A incorporation. Replacement of adenosine 73 followed by repair of the C-C-A end using nucleotidyl transferase leads to tRNAPhe derivatives which are fully active in the aminoacylation reaction and in polyphenylalanine synthesis. The fluorescence of epsilon A and aza-epsilon A at position 73 is virtually completely quenched, suggesting a stacked arrangement of bases around this position. There is no fluorescence increase when the epsilon A-labeled tRNAPhe is complexed with phenylalanyl-tRNA synthetase, elongation factor Tu, or ribosomes. These observations indicate that the stacked conformation of the 3' terminus is not changed appreciably in these complexes.