Utilization of thermal neutron induced in-situ chain reactions and the (n,p) reaction with fast neutrons for compositional characterization of lithium titanate.

A methodology has been developed for the complete compositional characterization of lithium titanate (LTO) using neutron activation, which is quite challenging and no literature report is available so far. The concept of thermal neutron induced in-situ chain reactions 6Li(n,α)3H and 16O(3H,n)18F has been used for the determination of Li and O through the measurement of 18F activity. The method is capable of analyzing Li and O in percentage level as reported in the present analysis of two types of lithium titanate samples. Spectroscopic interference of the elements which can directly or indirectly affect the outcome, were evaluated meticulously. Determination of Ti was carried out using fast neutron activation through the product isotopes like 47Sc, 48Sc, generated via (n,p) nuclear reactions. Fast neutron activation methodology seems to be advantageous for Ti determination over thermal neutron activation, as it offers self validation through different isotopes and multiple gamma lines.

Unusual uniformity of the N-linked oligosaccharides of HLA-A, -B, and -C glycoproteins.

MHC class I glycoproteins possess an invariant site for N-linked oligosaccharide addition at position 86 of the heavy chain. For human HLA-A, -B, and -C class I glycoproteins, position 86 is the only site of N-linked glycosylation. Comparison of the size and relative abundance of oligosaccharides associated with nine HLA-A, -B, or -C allotypes isolated from EBV-transformed B cell lines and mixtures of HLA-A, -B, and -C allotypes isolated from pooled PBLs revealed a very restricted set of structures. Allotypes encoded by the HLA-A and -B loci have two predominant glycan structures that were almost exclusively di-sialylated. In contrast, HLA-C allotypes have four glycan structures, comprising those associated with HLA-A and -B and two additional glycans. Identical oligosaccharides were present on different allotypes of a class I HLA locus, and in particular, HLA-C allotypes defining two inhibitory specificities for NK cells were shown to possess the same set of oligosaccharides. The uniformity of oligosaccharide structure associated with different HLA-A, -B, and -C products and the relative lack of heterogeneity for any given allotype are unusual features for a mammalian glycoprotein. Particularly striking is that such conserved oligosaccharide structures juxtapose the major regions of amino acid sequence variation within the Ag recognition site, including the polymorphisms of the alpha 1 helix that determine the inhibitory ligands for human NK cells.

Nonselective and efficient fluorescent labeling of glycans using 2-amino benzamide and anthranilic acid.

Reaction conditions for conjugation of two fluorescent ortho-substituted aniline derivatives, 2-amino benzamide (2-AB) and 2-anthranilic acid (2-AA), to N- and O-glycans have been investigated. Conjugation conditions for attaching 2-AB and 2-AA to core-fucosylated and non-fucosylated glycans were developed using complex N-glycans radiolabeled at the nonreducing terminus with [3H]C6-galactose. Optimal conditions for each of the following reaction parameters were experimentally defined: [glycans], [2-AB] or [2-AA], solvent and acid composition, temperature and time of Schiff's base formation, nature of reductant, and temperature and time of reduction. Using the optimized reaction conditions it has been shown with several standard glycans and glycoprotein-derived glycan libraries that (i) molar labeling efficiencies are high and essentially independent of the amount of glycans; (ii) negligible (< 2 mol%) desialylation occurs during conjugation; (iii) glycan labeling is nonselective, i.e., independent of glycan structure; and (iv) insignificant fluorescent or chemical "blank" is recovered during the glycan-labeling and purification protocol. Labeling glycan pools with 2-AB or 2-AA therefore allows representative glycan profiles to be obtained and also allows relative molar quantitation of individual glycans in a pool. The 2-AB label is compatible with several chromatographic means for separation of carbohydrates including Bio Gel P4 gel permeation, high-performance anion-exchange chromatography with fluorescence detection, and a variety of HPLC procedures, as well as with mass spectrometric methods including matrix-assisted laser desorption-mass spectrometry and electrospray-mass spectrometry. The 2-AA label is particularly well-suited for electrophoretic separations by polyacrylamide gel electrophoresis. These fluorophores show high intrinsic sensitivity and thus facilitate very sensitive analysis of protein glycosylation.

Identification of endogenous protein-associated carbohydrate ligands for E-selectin.

A comparative analysis of carbohydrate libraries derived from cell lines binding E-selectin was used to identify endogenous protein-associated carbohydrate ligands for E-selectin. Three structures, which together constitute less than 1% of the total cell surface protein-associated carbohydrate, were unique to cell lines capable of binding E-selectin, including neutrophils and the monocytic cell line U937. All are tetra-antennary N-linked structures, with a sialic acid alpha 2 --> 3 galactose beta 1 --> 4 (fucose alpha 1 --> 3) N-acetyl glucosamine beta 1 --> 3 galactose beta 1 --> 4 (fucose alpha 1 --> 3) N-acetyl glucosamine lactosaminoglycan extension (sialyl-di-Lewis X [S-diLe(x)]) on the arm linked through the C4 residue on the mannose. While all contained the expected 3-SLe(x) sub-structure, these native structures have an additional fucosylated lactosamine unit. Direct evidence that these S-di-Lex-containing structures are high-affinity ligands for E-selectin came from the use of recombinant soluble E-selectin-agarose affinity chromatography. These three carbohydrate structures bound specifically to the E-selectin column, while 3-SLe(x) itself does not bind under identical conditions.

Isolation and characterization of natural protein-associated carbohydrate ligands for E-selectin.

A comparative analysis of carbohydrate 'libraries' derived from cell lines binding E-selectin with differing avidity identified endogenous protein-associated carbohydrate ligand candidates for E-selectin. Three unusual structures, which constitute less than 3% of cell surface protein-associated carbohydrate, were unique to the E-selectin-binding cells, including neutrophils and the monocytic cell line U937. All are tetraantennary N-linked structures with a NeuAc alpha 2-->3Gal beta 1-->4(Fuc alpha 1-->3)GlcNAc beta 1-->3Gal beta 1-->4- (Fuc alpha 1-->3)GlcNAc lactosaminoglycan extension (diSLex) on the arm linked through the C4 residue on the mannose. While all contained the expected SLex [NeuAc alpha 2-->3Gal beta 1-->4(Fuc alpha 1-->3)GlcNAc] moiety, these structures have an additional fucosylated lactosamine unit. Direct evidence that these diSLex-containing structures are, indeed, high-affinity ligands for E-selectin came from the use of recombinant soluble E-selectin-agarose affinity chromatography. We found that these three carbohydrate structures bound specifically to the E-selectin column. SLex itself does not bind under identical conditions. In summary, these related structures: (1) all possess an unusual 3-sialyl di-Lewis x extension on one arm of an N-linked tetraantennary glycan; (2) of the cells tested, are present only on E-selectin-binding leukocytes and leukocytic cell lines; (3) bind to E-selectin with a relatively high affinity (Kd < microM) and one greater than that of 3-sialyl Lewis x or 3-sialyl Lewis a; and (4) represent a very small percentage of the protein-associated carbohydrate. These carbohydrate structures appear to be present on only a very small number of cell surface proteins and may alone be responsible for the specificity of E-selectin-dependent adhesion.

Different culture methods lead to differences in glycosylation of a murine IgG monoclonal antibody.

A monoclonal IgG-1 was produced by culture of a murine hybridoma (3.8.6) by three different methods, namely culture in ascites, in serum-free media and in serum-supplemented media. IgG-1 was purified to homogeneity (as judged by SDS/PAGE under reducing conditions) from each medium by ion-exchange chromatography and h.p.l.c. Protein A chromatography. Oligosaccharides were released from each IgG-1 preparation by hydrazinolysis and radiolabelled by reduction with alkaline sodium borotritide, and 'profile' analysis of the radiolabelled oligosaccharide alditols was performed by a combination of paper electrophoresis and gel-filtration chromatography. This analysis indicated clear and reproducible differences in the glycosylation patterns of the three IgG-1 preparations. Sequential exoglycosidase analysis of individual oligosaccharides derived from each IgG-1 preparation was used to define these differences. Ascites-derived material differed from serum-free-culture-derived material only with respect to the content of sialic acid. IgG-1 derived from culture in serum-containing media had an intermediate sialic acid content and a lower incidence of outer-arm galactosylation than the other two preparations. These differences in glycosylation could not be induced in any IgG-1 preparation by incubating purified IgG-1 with ascites or culture medium. It is concluded that the glycosylation pattern of a secreted monoclonal IgG is dependent on the culture method employed to obtain it.

Comparing the glycosylation patterns of recombinant glycoproteins.

The glycosylation profile of a recombinant glycoprotein can not only significantly affect its therapeutic profile, but is also extremely sensitive to cell-culture and purification conditions. To define glycosylation patterns and to ensure consistency of recombinant glycoproteins among different preparations requires highly sensitive and reproducible analytical methods that can be used routinely. New strategies and instrumentation are being developed which should allow such analysis to be largely automated.

Synthesis and biophysical studies of short oligodeoxynucleotides with novel modifications: a possible approach to the problem of mixed base oligodeoxynucleotide synthesis.

The syntheses of 1,2-dideoxy-D-ribofuranose and 1,2-dideoxy-1-phenyl-beta-D-ribofuranose are described. Oligodeoxynucleotides containing these analogues have been synthesised and hybridized to their complementary strands. Hypochromicity studies have shown that these duplices are less stable than either the totally complementary duplex or those containing A.C and G.T mismatches.

A rapid deprotection procedure for phosphotriester DNA synthesis.

An equimolar solution of aldoxime and tetramethylguanidine at 70 degrees C removes both the base and phosphate protection from oligonucleotides prepared by solid phase phosphate triester technology. The rate of cleavage from the succinyl linkage commonly used for solid phase synthesis is also increased. The method is simpler, faster and more easily automated than existing methods.

The influence of messenger RNA secondary structure on expression of an immunoglobulin heavy chain in Escherichia coli.

A gene for murine mu heavy chain immunoglobulin has been inserted into a bacterial expression plasmid containing the Escherichia coli trp promoter and ribosome binding site. A low level expression of mu protein was detected. Secondary structure analysis showed the presence of a hairpin loop burying the mu initiation codon. Alteration of secondary structure at this site by oligonucleotide replacement mutagenesis revealed a correlation between mu expression levels and accessibility of the ribosome binding site. Abolition of secondary structure increased mu protein expression over ninety-fold, to a level approximately equal to that of a trpE -mu fusion protein using the native trpE ribosome binding site.

Improvements to solid phase phosphotriester synthesis of deoxyoligonucleotides.

A solution of benzenesulphonic acid (3%, w/v) in a dimethylformamide and dichloromethane mixture (9:1, v/v) is shown to be a very effective reagent for the detritylation of deoxyoligonucleotides attached to a solid support. The levels of depurination with this reagent were lower than those observed with other reagents such as trichloroacetic acid. Coupling reactions are described using above ambient temperatures with no detectable increase in side products. Both procedures have been successfully incorporated into an automated system, which can compete with the rate of synthesis by the phosphite approach.

Molecular cloning and nucleotide sequence of cDNA coding for calf preprochymosin.

DNA complementary to calf stomach mRNA has been synthesised and inserted into the Pst1 site of pAT153 by G-C tailing. Clones containing sequences coding for prochymosin were recognised by colony hybridisation with cDNA extended from a chemically synthesised oligodeoxynucleotide primer, the sequence of which was predicted from the published amino acid sequence of calf prochymosin. Two clones were identified which together contained a complete copy of prochymosin mRNA. The nucleotide sequence is in substantial agreement with the reported amino acid sequence of prochymosin and shows that this protein has a mol.wt. of 40431 and chymosin a mol.wt. of 35612. The sequence also indicates that prochymosin is synthesised as a precursor molecule, preprochymosin, having a 16 amino acid hydrophobic leader sequence analogous to that reported for other secreted proteins.

The complete amino acid sequence of human fibroblast interferon as deduced using synthetic oligodeoxyribonucleotide primers of reverse transcriptase.

Using synthetic oligodeoxyribonucleotides to prime the transcription of interferon mRNA and cDNA, we recently determined the mRNA sequence coding for the 47 amino-terminal amino acids of mature human fibroblast interferon (1). From this sequence, we have now synthesised an oligodeoxyribonucleotide that is homologous with the mRNA sequence coding for amino acids 42-45 and used it as a primer to selectively transcribe an interferon cDNA template. The sequence of the newly synthesised DNA predicted the sequence of amino acids 48-109 in the interferon polypeptide. By repeating this process with one more primer, we have determined the complete amino acid sequence of mature human fibroblast interferon, a polypeptide of 166 amino acids.

The amino-terminal sequence of human fibroblast interferon as deduced from reverse transcripts obtained using synthetic oligonucleotide primers.

From recently published data on the amino-terminal structures of human and mouse interferons, we have predicted and synthesised an oligonucleotide capable of priming specifically the reverse transcription of human fibroblast interferon mRNA present within a total mRNA population. From these transcripts we determined the sequence of the 5'-terminus of the mRNA and identified a putative pre-peptide signal sequence. This enabled us to predict the sequence of another primer capable of directing the synthesis of interferon double-stranded cDNA corresponding to the entire coding region of the mRNA. Further sequencing studies also enabled us to establish the identity of 47 consecutive amino acids beginning with the methionine residue at the amino-terminus of the mature protein.