Conformations in solution of angiotensin II, and its 1-7 and 1-6 fragments.

High-resolution proton spectra at 620 MHz of human angiotensin II (1-8), angiotensin II (1-7), and angiotensin II (1-6) have been obtained in aqueous solution at acidic pH, and in dimethylsulfoxide solution. Complete chemical shift assignments for all three angiotensin peptides were made based on two-dimensional (2D) correlated spectroscopy and 2D-CA-MELSPIN spectra. Based on the measured values of 3JHNCH, the pattern of observed transverse Overhauser effects, and side-chain coupling constants, it is concluded that all three analogues exist in H2O or DMSO-d6 as a mixture of conformers that is largely extended, with negligible content of folded structures, such as beta-turns, gamma-turns, or helix content. The results fit well with those of Nikiforovich et al.     

Gerbich blood group deficiency of the Ge:-1,-2,-3 and Ge:-1,-2,3 types. Immunochemical study and genomic analysis with cDNA probes

Human erythrocytes carry several transmembrane glycoproteins, among which the two minor species associated with the blood group Gerbich (Ge) antigens, GP C and GP D, play pivotal role since they interact with the membrane cytoskeleton and contribute to maintain the normal red cell shape. On the red cells from two categories of homozygous donors lacking the Ge determinants (Ge:-1,-2,-3 and Ge:-1,-2,3), GP C and GP D are missing but instead there is a new glycoprotein, easily detected by SDS/polyacrylamide gel electrophoresis, which exhibits some properties shared by GP C and GP D. This was shown by immunochemical analyses with a murine monoclonal antibody, extraction of the glycoproteins by organic solvents and binding studies with the 125I-labelled Lens culinaris lectin. The red cells from obligate heterozygotes for the Ge:-1,-2,-3 condition also carry this new glycoprotein component but in a much lesser amount than expected on the basis of one gene dose response. Using a cDNA probe containing the coding sequence of human GP C and the entire 3' untranslated region of its mRNA, we have demonstrated by Southern analyses that the Ge:-1,-2,-3 and the Ge:-1,-2,3 conditions are associated with a constant 3-kbp deletion within the GP C gene. Similar studies indicated that this gene is present as a unique copy per haploid genome of Ge-positive control donors (Ge:1,2,3). To account for these data and for the glycoprotein profile of Ge-negative erythrocytes, it is proposed that a unique Gerbich gene encodes for GP C and GP D, either by alternative RNA splicing or by different post-translational events, and that, following a 3-kbp deletion within this gene, a new glycoprotein having properties common to GP C and GP D can be produced.     

Purification of human haptoglobin 1-1, 2-1, and 2-2 using monoclonal antibody affinity chromatography

Similar to blood type, human plasma haptoglobin (Hp) is classified as 3 phenotypes: Hp 1-1, 2-1, or 2-2. The structural and functional relationship between the phenotypes, however, has not been studied in detail due to the complicated and difficult isolation procedures. This report provides a simple protocol that can be used to purify each Hp phenotype. Plasma was first passed through an affinity column coupled with a high affinity Hp monoclonal antibody. The bound material was washed with a buffer containing 0.2M NaCl and 0.02 M phosphate, pH 7.4, eluted at pH 11, and collected in tubes containing 1M Tris-HCl, pH 6.8. The crude Hp fraction was then chromatographed on a HPLC Superose 12 column in 0.05 M ammonium bicarbonate at a flow rate of 0.5 ml/min. The homogeneity of purified Hp 1-1, 2-1, or 2-2 was greater than 95% as judged by SDS-polyacrylamide gel electrophoresis. Essentially, each Hp isolated was not contaminated with hemoglobin and apolipoprotein A-I as that reported from the other methods, and was able to bind hemoglobin. Neuraminidase treatment demonstrated that the purified Hp possessed a carbohydrate moiety, while Western blot analysis confirmed alpha and beta chains corresponding to each Hp 1-1, 2-1, and 2-2 phenotype. The procedures described here represent a significant improvement in current purification methods for the isolation of Hp phenotypes. Circular dichroic spectra showed that the alpha-helical content of Hp 1-1 (29%) was higher than that of Hp 2-1 (22%), and 2-2 (21%). The structural difference with respect to its clinical relevance is discussed.     

Solution conformation of asparagine-linked oligosaccharides: alpha(1-2)-, alpha(1-3)-, beta(1-2)-, and beta(1-4)-linked units

The solution conformation is presented for representatives of each of the major classes of asparaginyl oligosaccharides. In this report the conformation of alpha(1-3)-, alpha(1-2)-, beta(1-2)-, and beta(1-4)-linked units is described. The conformational properties of these glycopeptides were determined by high-resolution 1H nuclear magnetic resonance in conjunction with potential energy calculations. The NMR parameters that were used in this analysis were chemical shifts and nuclear Overhauser enhancements. Potential energy calculations were used to evaluate the preferred conformers available for the different linkages in glycopeptides and to draw conclusions about the behavior in solution of these molecules. It was found that the linkage conformation of the Man alpha 1-3 residues was not affected by substitution either at the 2-position by alpha Man or beta GlcNAc or at the 4-position by beta GlcNAc or by the presence of a bisecting GlcNAc on the adjacent beta Man residue.     

Kaempferol-3-O-glucosyl(1-2)rhamnoside from Ginkgo biloba and a reappraisal of other gluco(1-2, 1-3 and 1-4)rhamnoside structures.

A kaempferol-3-O-glucorhamnoside from Ginkgo biloba is defined as the 3-O-alpha-L-[ beta-D-glucopyranosyl(1-2)rhamnopyranoside] on the basis of 2D NMR evidence. Complete assignments of the 1H and 13C NMR spectra of this compound and of its known p-coumaroyl derivative are presented for the first time. The NMR distinctions of 1-2, 1-3 and 1-4 linked glucopyranosylrhamnopyranosides are discussed and indicate (i) that the 13C NMR assignments for one published gluco(1-3)rhamnoside are in need of modification, (ii) that the published structure of hordenine-O-[6-O-t-cinnamoyl-beta-glucosyl(1-4)-alpha-rhamnoside] from Selaginella doederleinii is not distinguished from the 1-3 linked glucorhamnoside structure, and (iii) that the 8-prenylkaempferol-3-O-[glucosyl(1-4)rhamnoside]-7-O-glucoside and the equivalent 4'-O-methylated xylosyl(1-4)rhamnoside from Epimedium pubescens and E. washanense, respectively, are (1-2)-linked.     

Expression of matrilin-1, -2 and -3 in developing mouse limbs and heart.

The expression of matrilin-1, -2 and -3 was studied in the heart and limb during mouse development. Matrilin-1 is transiently expressed in the heart between days 9.5 and 14.5 p.c. Matrilin-2 expression was detected in the heart from day 10.5 p.c. onwards. In the developing limb bud, both matrilin-1 and -3 were observed first at day 12.5 p.c. Throughout development matrilin-3 expression was strictly limited to cartilage, while matrilin-1 was also found in some other forms of connective tissue. Matrilin-2, albeit present around hypertrophic chondrocytes in the growth plate, was mainly expressed in non-skeletal structures. The complementary, but in part overlapping, expression of matrilins indicates the possibility for both redundant and unique functions among the members of this novel family of extracellular matrix proteins.     

Base Modified Oligodeoxynucleotides. II. Increase of Stability to Nucleases by 5-Alkyl-, 5-(1-Alkenyl)-, and 5-(1-Alkynyl)-pyrimidines

Abstract

The effect of pyrimidine base substitution on the sensitivity of oligonucleotides to nucleases has been studied with two series of self complementary deoxyoligonucleotides containing n-alkyl, n-(1-alkenyl) or n-(1-alkynyl) groups at C5 of pyrimidines, (dA-r⁵dU)₁₀ and (dG-r^sdC)₆. The rate of hydrolysis by snake venom phosphodiesterase and in human serum decreased with increasing length and unsaturation of the substituent.