The elimination ofO-linked glycans from glycoproteins under non-reducing conditions

A simple procedure is described for the elimination ofO-linked glycans from bovine submaxillary mucin under non-reducing conditions, using triethylamine in aqueous hydrazine. The glycans were isolated as the hydrazones, which were converted to the reducing glycans by exchange with acetone in neutral aqueous solution. The glycan alditols obtained after reduction corresponded to those obtained by the reductive -elimination ofO-glycans.     

The apoptotic pathway contributing to the deletion of naive CD8 T cells during the induction of peripheral tolerance to a cross-presented self-antigen

The maintenance of T cell tolerance in the periphery proceeds through several mechanisms, including anergy, immuno-regulation, and deletion via apoptosis. We examined the mechanism underlying the induction of CD8 T cell peripheral tolerance to a self-Ag expressed on pancreatic islet beta-cells. Following adoptive transfer, Ag-specific clone 4 T cells underwent deletion independently of extrinsic death receptors, including Fas, TNFR1, or TNFR2. Additional experiments revealed that the induction of clone 4 T cell apoptosis during peripheral tolerance occurred via an intrinsic death pathway that could be inhibited by overexpression of Bcl-2 or targeted deletion of the proapoptotic molecule, Bim, thereby resulting in accumulation of activated clone 4 T cells. Over-expression of Bcl-2 in clone 4 T cells promoted the development of effector function and insulitis whereas Bim-/- clone 4 cells were not autoaggressive. Examination of the upstream molecular mechanisms contributing to clone 4 T cell apoptosis revealed that it proceeded in a p53, E2F1, and E2F2-independent manner. Taken together, these data reveal that initiation of clone 4 T cell apoptosis during the induction of peripheral tolerance to a cross-presented self-Ag occurs through a Bcl-2-sensitive and at least partially Bim-dependent mechanism.     

8-Modified-2'-deoxyadenosine analogues induce delayed polymerization arrest during HIV-1 reverse transcription

The occurrence of resistant viruses to any of the anti-HIV-1 compounds used in the current therapies against AIDS underlies the urge for the development of new drug targets and/or new drugs acting through novel mechanisms. While all anti-HIV-1 nucleoside analogues in clinical use and in clinical trials rely on ribose modifications for activity, we designed nucleosides with a natural deoxyribose moiety and modifications of position 8 of the adenine base. Such modifications might induce a steric clash with helix αH in the thumb domain of the p66 subunit of HIV-1 RT at a distance from the catalytic site, causing delayed chain termination. Eleven new 2'-deoxyadenosine analogues modified on position 8 of the purine base were synthesized and tested in vitro and in cell-based assays. In this paper we demonstrate for the first time that chemical modifications on position 8 of 2'-deoxyadenosine induce delayed chain termination in vitro, and also inhibit DNA synthesis when incorporated in a DNA template strand. Furthermore, one of them had moderate anti-HIV-1 activity in cell-culture. Our results constitute a proof of concept indicating that modification on the base moiety of nucleosides can induce delayed polymerization arrest and inhibit HIV-1 replication.     

Binding of carbohydrates to solid supports Part 3: Reaction of sugar hydrazones with polystyrene substituted with aromatic aldehydes

The hydrazones of glucose and N-acetylglucosamine, as models for the residues at the reducing termini of glycans, were covalently and reversibly bound in good yield to hydroxybenzaldehydo ligands attached to a polymer support. The binding, by a sugar azine linkage, occurred within two hours at room temperature at neutral pH, and efficient recoveries of sugars from the beads were achieved by displacement with aqueous hydrazine hydrate, ethanolic benzaldehyde, or aqueous acetone. Enzyme modification of glycans was demonstrated by separation of the products of hydrolysis of lactose hydrazone with -galactosidase, using hydroxybenzaldehyde-derivatized polystyrene beads. Addition of a spacer arm to aminopolystyrene beads, for binding of reducing sugars as Amadori compounds to the aromatic amine function, was also investigated.     

Interphotoreceptor retinoid-binding protein. Gene characterization, protein repeat structure, and its evolution

The gene for bovine interphotoreceptor retinoid-binding protein (IRBP) has been cloned, and its nucleotide sequence has been determined. The IRBP gene is about 11.6 kilobase pairs (kb) and contains four exons and three introns. It transcribed into a large mRNA of approximately 6.4 kb and translated into a large protein of 145,000 daltons. To prove the identity of the genomic clone, we determined the protein sequence of several tryptic and cyanogen bromide fragments of purified bovine IRBP protein and localized them in the protein predicted from its nucleotide sequence. There is a 4-fold repeat structure in the protein sequence with 30-40% sequence identity and many conservative substitutions between any two of the four protein repeats. The third and fourth repeats are the most similar pair. All three of the introns in the IRBP gene fall in the fourth protein repeat. Two of the exons, the first and the fourth, are large, 3173 and 2447 bases, respectively. The introns are each about 1.5-2.2 kb long. The human IRBP gene has a sequence that is similar to one of the introns from the bovine gene. The unexpected gene structure and protein repeat structure in the bovine gene lead us to propose a model for the evolution of the IRBP gene.     

Deep sequencing reveals clonal evolution patterns and mutation events associated with relapse in B-cell lymphomas

Background

Molecular mechanisms associated with frequent relapse of diffuse large B-cell lymphoma (DLBCL) are poorly defined. It is especially unclear how primary tumor clonal heterogeneity contributes to relapse. Here, we explore unique features of B-cell lymphomas - VDJ recombination and somatic hypermutation - to address this question.

Results

We performed high-throughput sequencing of rearranged VDJ junctions in 14 pairs of matched diagnosis-relapse tumors, among which 7 pairs were further characterized by exome sequencing. We identify two distinctive modes of clonal evolution of DLBCL relapse: an early-divergent mode in which clonally related diagnosis and relapse tumors diverged early and developed in parallel; and a late-divergent mode in which relapse tumors developed directly from diagnosis tumors with minor divergence. By examining mutation patterns in the context of phylogenetic information provided by VDJ junctions, we identified mutations in epigenetic modifiers such as KMT2D as potential early driving events in lymphomagenesis and immune escape alterations as relapse-associated events.

Conclusions

Altogether, our study for the first time provides important evidence that DLBCL relapse may result from multiple, distinct tumor evolutionary mechanisms, providing rationale for therapies for each mechanism. Moreover, this study highlights the urgent need to understand the driving roles of epigenetic modifier mutations in lymphomagenesis, and immune surveillance factor genetic lesions in relapse.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0432-0) contains supplementary material, which is available to authorized users.