Structural and immunochemical identification of Leb glycolipids in the plasma of a group O Le(a-b-) secretor

Total non-acid glycosphingolipids were isolated from the plasma of a healthy red blood cell group O Le(a-b-) salivary ABH secretor individual. Glycolipids were fractionated by HPLC and combined into eight fractions based on chromatographic and immunoreactive properties. These glycolipid fractions were analysed by thin-layer chromatography and tested for Lewis activity with antibodies reactive to the type 1 precursor (Le^c), H type 1 (Le^d), Le^a and Le^b epitopes. Fractions were structurally characterized by mass spectrometry (EI-MS and LSIMS) and proton NMR spectroscopy. Expected blood group glycolipids, such as H type 1, (Fuc1-2Gal1-3GlcNAc1-3Gal1-4Glc1-1Cer) were immunochemically and structurally identified. Inconsistent with the red cell phenotype and for the first time, small quantities of Le^b blood group glycolipids (Fuc1-2Gal1-3(Fuc1-4)GlcNAc1-3Gal1-4Glc1-1Cer) were immunochemically and structurally identified in the plasma of a Lewis-negative individual. These findings confirm recent immunological evidence suggesting the production of small amounts of Lewis antigens by Lewis negative individuals. Abbreviations: HPLC, high performance liquid chromatography; TLC, (high performance) thin layer chromatography; EI-MS, electron impact ionisation mass spectrometry; LSIMS, liquid secondary ion mass spectrometry; NMR, nuclear magnetic resonance spectroscopy. The sugar types are abbreviated to Hex for hexose, HexNAc forN-acetylhexosamine and dHex for deoxyhexose (fucose). The ceramide types are abbreviated to d for dihydroxy and t for trihydroxy base, n for non-hydroxy and h for hydroxy fatty acids; LCB, long chain base.     

Default biosynthesis pathway for blood group-related glycolipids in human small intestine as defined by structural identification of linear and branched glycosylceramides in a group O Le(a-b-) nonsecretor

Glycoconjugates of the GI tract are important for microbial interactions. The expression of histo-blood group glycosyltransferases governs both the expression of blood group determinants and in part the structure and size of the glycoconjugates. Using neutral glycolipids isolated from the small intestine of a rare blood group O Le(a-b-) ABH secretor-negative (nonsecretor) individual we were able to map the "default" pathway of the individual lacking ABO, Lewis, and secretor glycosyltransferases. Structures were deduced with combined analysis of mass spectrometry (MALDI-TOF and ESI-MS/MS), and 1H NMR (500 and 600 MHz). All structures present at a level >5% were structurally resolved and included two extended structures: Galbeta4(Fucalpha3)GlcNAcbeta3(Galbeta4[Fucalpha3]GlcNAcbeta6)Galbeta4GlcNAcbeta3Galbeta4Glcbeta1Cer and Galbeta3GlcNAcbeta3(Galbeta4[Fucalpha3]GlcNAcbeta6)Galbeta3GlcNAcbeta3Galbeta4Glcbeta1Cer. The first, a novel component, is based on a type 2 chain and bears the Lex glycotopes on both its branches. The second, a major component, is based on a type 1 chain, which bears a 3-linked type 1 precursor (Lec) glycotope and a 6-linked Lex glycotope on its branches. This latter structure is identical to that previously isolated from plasma and characterized by MS and GC-MS but not by NMR. Structural resolution of these structures was supported by reanalysis of the blood group H-active decaosylceramides previously isolated from rat small intestine. Other minor linear monofucosylated penta-, hepta-, and difucosylated octaosylceramides, some bearing blood group determinants, were also identified. The cumulative data were used to define a default biosynthesis pathway where it can be seen that carbohydrate chain extension, in the absence of blood group glycosyltransferases, is controlled and regulated by non-blood group fucosylation and branching with type 2 Galbeta4GlcNAc branches.     

Beta-hairpin formation in aqueous solution and in the presence of trifluoroethanol: a (1)H and (13)C nuclear magnetic resonance conformational study of designed peptides

In order to check our current knowledge on the principles involved in beta-hairpin formation, we have modified the sequence of a 3:5 beta-hairpin forming peptide with two different purposes, first to increase the stability of the formed 3:5 beta-hairpin, and second to convert the 3:5 beta-hairpin into a 2:2 beta-hairpin. The conformational behavior of the designed peptides was investigated in aqueous solution and in 30% trifluoroethanol (TFE) by analysis of the following nuclear magnetic resonance (NMR) parameters: nuclear Overhauser effect (NOE) data, and C(alpha)H, (13)C(alpha), and (13)C(beta) conformational shifts. From the differences in the ability to adopt beta-hairpin structures in these peptides, we have arrived to the following conclusions: (i) beta-Hairpin population increases with the statistical propensity of residues to occupy each turn position. (ii) The loop length, and in turn, the beta-hairpin type, can be modified as a function of the type of turn favored by the loop sequence. These two conclusions reinforce previous results about the importance of beta-turn sequence in beta-hairpin folding. (iii) Side-chain packing on each face of the beta-sheet may play a major role in beta-hairpin stability; hence simplified analysis in terms of isolated pair interactions and intrinsic beta-sheet propensities is insufficient. (iv) Contributions to beta-hairpin stability of turn and strand sequences are not completely independent. (v) The burial of hydrophobic surface upon beta-hairpin formation that, in turn, depends on side-chain packing also contributes to beta-hairpin stability. (vi) As previously observed, TFE stabilizes beta-hairpin structures, but the extent of the contribution of different factors to beta-hairpin formation is sometimes different in aqueous solution and in 30% TFE.     

Structural and serological characterization of the O-chain polysaccharide of Aeromonas salmonicida strains A449, 80204 and 80204-1

The O-chain polysaccharide (O-PS) of Aeromonas salmonicida was studied by a combination of compositional, methylation, CE-ESMS and one- and two-dimensional NMR analyses. It was found to be a branched polymer of trisaccharide-repeating units composed of L-rhamnose (Rha), D-glucose (Glc), 2-acetamido-2-deoxy-D-mannose (ManNAc) and O-acetyl group (OAc) and having the following structure: CE-ESMS analysis of A. salmonicida cells from strains A449, 80204 and 80204-1 grown under different conditions confirmed that the O-PS structure was conserved. ELISA-based serological study with native LPS-specific antisera performed on the native O-PS and its O-deacetylated and periodate-oxidized derivatives confirmed the importance of the O-PS backbone structure as an immunodominant determinant.     

Chemical synthesis of the 3-sulfooxy-7-N-acetylglucosaminyl-24-amidated conjugates of 3beta,7beta-dihydroxy-5-cholen-24-oic acid, and related compounds: unusual, major metabolites of bile acid in a patient with Niemann-Pick disease type C1

The chemical synthesis of 3beta,7beta-dihydroxy-5-cholen-24-oic acid, triply conjugated by sulfuric acid at C-3, by N-acetylglucosamine (GlcNAc) at C-7, and by glycine or taurine at C-24, is described. These are unusual, major metabolites of bile acid found to be excreted in the urine of a patient with Niemann-Pick disease type C1. Analogous double-conjugates of 3beta-hydroxy-7-oxo-5-cholen-24-oic acid were also prepared. The principal reactions involved were: (1) beta-d-N-acetylglucosaminidation at C-7 of methyl 3beta-tert-butyldimethylsilyloxy (TBDMSi)-7beta-hydroxy-5-cholen-24-oate with 2-acetamido-1alpha-chloro-1,2-dideoxy-3,4,6-tri-O-acetyl-d-glucopyranose in the presence of CdCO(3) in boiling toluene; (2) sulfation at C-3 of the resulting 3beta-TBDMSi-7beta-GlcNAc with sulfur trioxide-trimethylamine complex in pyridine; and (3) direct amidation at C-24 of the 3beta-sulfooxy-7beta-GlcNAc conjugate with glycine methyl ester hydrochloride (or taurine) using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride as a coupling agent in DMF. The structures of the multi-conjugated bile acids were characterized by liquid chromatography-mass spectrometry with an electrospray ionization probe under the positive and negative ionization modes.     

The mechanism of alkylation of DNA by 5-(3-methyl-1-triazeno)imidazole-4-carboxamide (MIC), a metabolite of DIC (NSC-45388). Non-involvement of diazomethane

Comparison of the nuclear magnetic resonance spectra of chemically synthesized methyl-d₁-methanol with the methanol produced in the solvolytic decompostion of 5-(3-methyl-1-triazeno)imidazole-4-carboxamide (MIC) in D₂O under acidic, basic or neutral conditions indicated that no deuterium was exchanged for the hydrogens on the methyl group. Diazomethane can therefore be ruled out as an intermediate in this reaction.The methyl-d₃-guanine isolated after incubation of methyl-d₃-MIC with calfthymus DNA in vitro displayed, on chemical ionization mass spectrometry, a quasimolecular ion (MH⁺) at m/e 169, which was 3 mass units higher than the quasimolecular ion for an undeuterated 7-methylguanine standard. The major fragment ions for 7-methyl-d₃-guanine on electron impact mass spectrometry likewise were situated at positions 3 mass units higher than the fragment ions for 7-methylguanine itself.These data indicate that the methylation of biological macromolecules by MIC must involve the transfer of an intact methyl group.     

Structural flexibility and the positive charges are the key factors in bacterial cell selectivity and membrane penetration of peptoid-substituted analog of Piscidin 1

Piscidin 1 (Pis-1) is a novel cytotoxic peptide with a cationic α-helical structure isolated from the mast cells of hybrid striped bass. In our previous study, we showed that Pis-1[PG] with a substitution of Pro⁸ for Gly⁸ in Pis-1 had higher bacterial cell selectivity than Pis-1. We designed peptoid residue-substituted peptide, Pis-1[NkG], in which Gly⁸ of Pis-1 was replaced with Nlys (Lys peptoid residue). Pis-1[NkG] had higher antibacterial activity and lower cytotoxicity against mammalian cells than Pis-1 and Pis-1[PG]. We determined the tertiary structure of Pis-1[PG] and Pis-1[NkG] in the presence of DPC micelles by NMR spectroscopy. Both peptides had a three-turn helix in the C-terminal region and a bent structure in the center. Pis-1[PG] has a rigid bent structure at Pro⁸ whereas Pis-1[NkG] existed as a dynamic equilibrium of two conformers with a flexible hinge structure at Nlys⁸. Depolarization of the membrane potential of Staphylococcus aureus and confocal laser-scanning microscopy study revealed that Pis-1[NkG] effectively penetrated the bacterial cell membrane and accumulated in the cytoplasm, whereas Pis-1[PG] did not penetrate the membrane but remained outside or on the cell surface. Introduction of a lysine peptoid at position 8 of Pis-1 provided conformational flexibility and increased the positive charge at the hinge region; both factors facilitated penetration of the bacterial cell membrane and conferred bacterial cell selectivity on Pis-1[NkG].     

Design,synthesis, in vitro and in vivo evaluation,and structure-activity relationship (SAR) discussion of novel dipeptidyl boronic acid proteasome inhibitors as orally available anti-cancer agents for the treatment of multiple myeloma and mechanism studies

A series of novel dipeptidyl boronic acid inhibitors of 20S proteasome were designed and synthesized. Aliphatic groups at R¹ position were designed for the first time to fully understand the SAR (structure–activity relationship). Among the screened compounds, novel inhibitor 5c inhibited the CT-L (chymotrypsin-like) activity with IC₅₀ of 8.21?nM and the MM (multiple myeloma) cells RPMI8226, U266B and ARH77 proliferations with the IC₅₀ of 8.99, 6.75 and 9.10?nM, respectively, which showed similar in vitro activities compared with the compound MLN2238 (biologically active form of marketed MLN9708). To investigate the oral availability, compound 5c was esterified to its prodrug 6a with the enzymatic IC₅₀ of 6.74?nM and RPMI8226, U266B and ARH77 cell proliferations IC₅₀ of 2.59, 4.32 and 3.68?nM, respectively. Furthermore, prodrug 6a exhibited good pharmacokinetic properties with oral bioavailability of 24.9%, similar with MLN9708 (27.8%). Moreover, compound 6a showed good microsomal stabilities and displayed stronger in vivo anticancer efficacy than MLN9708 in the human ARH77 xenograft mouse model. Finally, cell cycle results showed that compound 6a had a significant inhibitory effect on CT-L and inhibited cell cycle progression at the G2M stage.     

Novel chromosomal translocation t(11;9)(p15;p23) involving deletion and duplication of 9p in a girl associated with autism and mental retardation

We describe a 5-year-old girl presented with autism and mental retardation features. Conventional karyotyping revealed a novel unidirectional translocation t(11;9)(p15;p23). HumanCytoSNP-12 Chip analysis identified a 13 Mb deletion from 9p24.3 to 9p23 and a 12.5Mb duplication from 9p23 to 9p21.2. The karyotype was described as 45,XX,psu dic(11; 9)(p15;p23), which was reported for the first time here. The deleted region, extending from 9p24.3 to 9p23, overlaps with the candidate region for monosomy 9p syndrome and contains a potential autism spectrum disorders (ASD) locus. The duplication region extending from 9p23 to 9p21.2 was previously identified as a critical region for the 9p duplication syndrome. These results suggested that the apparently balanced de novo translocations could produce cryptic deletions or duplications, and the precise mapping of the abnormal area may improve clinical management.     

Natural products as modulators of the nuclear receptors and metabolic sensors LXR,FXR and RXR

Nuclear receptors (NRs) represent attractive targets for the treatment of metabolic syndrome-related diseases. In addition, natural products are an interesting pool of potential ligands since they have been refined under evolutionary pressure to interact with proteins or other biological targets.This review aims to briefly summarize current basic knowledge regarding the liver X (LXR) and farnesoid X receptors (FXR) that form permissive heterodimers with retinoid X receptors (RXR). Natural product-based ligands for these receptors are summarized and the potential of LXR, FXR and RXR as targets in precision medicine is discussed.