Urinary excretion ofin vivo13C-labelled milk oligosaccharides in breastfed infants

Recent observations indicate that human milk oligosaccharides (HMO) are involved in a variety of physiological processes in infants. Their metabolic fate, however, is virtually unknown. We investigated metabolic aspects in infants after endogenous13C-labelling of HMO. An oral bolus of natural and13C-labelled galactose (Gal; 23 g Gal+4 g13C-Gal) was given to ten lactating women. Aliquots of milk at each nursing as well as breath samples from the mothers and urine from their infants were collected over 36 h. The13C-enrichment of HMO and their renal excretion was determined by isotope ratio-MS; characterisation was achieved by fast atom bombardment-MS. After the Gal bolus was given, an immediate13C-enrichment in milk and in infants' urine was observed which lasted 36 h. Mass spectrometric analysis of13C-enriched urinary fractions confirmed the excretion of a variety of neutral and acidic HMO without metabolic modification of their structures. Components with glucose split off at the reducing end were also detectable. Quantitative data regarding the infants' intake of lacto-N-tetraose and its monofucosylated derivative lacto-N-fucopentaose II ranged from 50 to 160 mg with each suckling, respectively; renal excretion of both components varied between 1 and 3 mg/d. Since the intake of individual HMO by the infants was in the range of several hundred mg per suckling, i.e. several g/d, and some of these components were excreted in mg amounts as intact HMO with the infants' urine, not only local but also systemic effects might be expected.

Structural elucidation of novel phosphocholine-containing glycosylinositol-phosphoceramides in filamentous fungi and their induction of cell death of cultured rice cells

Novel ZGLs (zwitterionic glycosphingolipids) have been found in and extracted from the mycelia of filamentous fungi (Acremonium sp.) isolated from soil. Five ZGLs (ZGL1–ZGL5) were structurally elucidated by sugar compositional analysis, methylation analysis, periodate oxidation, matrix-assisted laser-desorption ionization–time-of-flight MS, 1H-NMR spectroscopy and fast-atom bombardment MS. Their chemical structures were as follows: GlcN(α1-2)Ins1-P-1Cer (ZGL1), Man(α1-6)GlcN(α1-2)Ins1-P-1Cer (ZGL2), Man(α1-6)Man(α1-6)GlcN(α1-2)Ins1-P-1Cer (ZGL3), PC→6Man(α1-6)GlcN(α1-2)Ins1-P-1Cer (ZGL4), and PC→6Man(α1-6)Man(α1-6)GlcN(α1-2)Ins1-P-1Cer (ZGL5) (where Cer is ceramide and PC is phosphocholine). In addition, one acidic glycosphingolipid, which was the precursor of ZGLs, was also characterized as inositol-phosphoceramide. The core structure of the ZGLs, GlcN(α1-2)Ins1-P, is rather different from those found in other fungi, such as Man(α1-2)Ins1-P and Man(α1-6)Ins1-P. Interestingly, the terminal mannose residue of ZGL4 and ZGL5 was modified further with a PC group. The presence of PC-containing glycosylinositol-phosphoceramides has not been reported previously in any organism. The ceramide constituents of both ZGLs and acidic glycosphingolipid were essentially the same, and consisted of a 4-hydroxyoctadecasphinganine (phytosphingosine) as the sole sphingoid base and 2-hydroxytetracosanoic acid (>90%) as the major fatty acid. ZGLs were found to cause cell death in suspensions of cultured rice cells. The cell death-inducing activity of ZGLs is probably due to the characteristic glycan moiety of Man(α1-6)GlcN, and PC-containing ZGLs had high activity. This study is the first to demonstrate that fungal glycosylinositol-phosphoceramides induce cell death in cultured rice cells.

Characterization of a putative α-mannosyltransferase involved in phosphatidylinositol trimannoside biosynthesis in Mycobacterium tuberculosis

Phosphatidyl-myo-inositol mannosides (PIMs), lipomannan (LM) and lipoarabinomannan (LAM) are an important class of bacterial factors termed modulins that are found in tuberculosis and leprosy. Although their structures are well established, little is known with respect to the molecular aspects of the biosynthetic machinery involved in the synthesis of these glycolipids. On the basis of sequence similarity to other glycosyltransferases and our previous studies defining an α-mannosyltransferase from Mycobacterium tuberculosis, named PimB [Schaeffer, Khoo, Besra, Chatterjee, Brennan, Belisle and Inamine (1999) J. Biol. Chem. 274, 31625–31631], which catalysed the formation of triacyl (Ac3)-PIM2 (i.e. the dimannoside), we have identified a related gene from M. tuberculosis CDC1551, now designated pimC. The use of a cell-free assay containing GDP-[14C]mannose, amphomycin and membranes from Myobacterium smegmatis overexpressing PimC led to the synthesis of a new alkali-labile PIM product. Fast-atom-bombardment MS established the identity of the new enzymically synthesized product as Ac3PIM3 (i.e. the trimannoside). The results indicate that pimC encodes an α-mannosyltransferase involved in Ac3PIM3 biosynthesis. However, inactivation of pimC in Myobacterium bovis Bacille Calmette—Guérin (BCG) did not affect the production of higher PIMs, LM and LAM when compared with wild-type M. bovis BCG, suggesting the existence of redundant gene(s) or an alternate pathway that may compensate for this PimC deficiency. Further analyses, which compared the distribution of pimC in a panel of M. tuberculosis strains, revealed that pimC was present in only 22% of the clinical isolates examined.

Structural determination of a 5-acetamido-3,5,7,9-tetradeoxy-7-(3-hydroxybutyramido)-L-glycero-L-manno-nonulosonic acid-containing homopolysaccharide isolated from Sinorhizobium fredii HH103

The structure of a polysaccharide from Sinorhizobium fredii HH103 has been determined. This polysaccharide was isolated by following the protocol for lipopolysaccharide extraction. On the basis of monosaccharide analysis, methylation analysis, fast atom bombardment MS, matrix-assisted laser desorption ionization MS, electron-impact high-resolution MS, one-dimensional 1H-NMR and 13C-NMR and two-dimensional NMR experiments, the structure was shown to consist of a homopolymer of a 3:1 mixture of 5-acetamido-3,5,7,9-tetradeoxy-7-[(R)- and (S)-3-hydroxybutyramido]-L-glycero-L-manno-nonulosonic acid. The sugar residues are attached via a glycosidic linkage to the OH group of the 3-hydroxybutyramido substituent and thus the monomers are linked via both glycosidic and amidic linkages. In contrast with the Sinorhizobium K-antigens previously reported, which are composed of a disaccharide repeating unit, the K-antigen polysacharide of S. fredii HH103 is a homopolysaccharide.

Structural determination of a 5-O-methyl-deaminated neuraminic acid (Kdn)-containing polysaccharide isolated from Sinorhizobium fredii

The structure of a polysaccharide from Sinorhizobium frediiSVQ293, a thiamine auxotrophic mutant of S. fredii HH103, has been determined. This polysaccharide was isolated following the protocol for lipopolysaccharide extraction. On the basis of monosaccharide analysis, methylation analysis, fast atom bombardment MS, collision-induced dissociation tandem MS, one-dimensional 1H and 13C NMR and two-dimensional NMR experiments, the structure was shown to consist of the following trisaccharide repeating unit → 2)-α-d-Galp-(1 → 2)-β-d-Ribf-(1 → 9)-α-5-O-Me-Kdnp-(2 →, in which Kdn stands for deaminated neuraminic acid; 25% of the Kdn residues are not methylated. The structure of this polysaccharide is novel and this is the first report of the presence of Kdn in a rhizobial polysaccharide, as well as being the first structure described containing 5-O-Me-Kdn. This Kdn-containing polysaccharide is not present in the wild-type strain HH103, which produces a 3-deoxy-d-manno-2-octulosonic acid (Kdo)-rich polysaccharide. We conclude that it is likely that the appearance of this new Kdn-containing polysaccharide is a consequence of the mutation.

Glycoinositol phospholipids from Endotrypanum species express epitopes in common with saccharide side chains of the lipophosphoglycan from Leishmania major

We have characterized glycoinositol phospholipids (GIPLs) from three strains of the trypanosomatid parasites Endotrypanum schaudinni and Endotrypanum monterogeii. Methanolysis of the intact GIPLs liberated methyl esters of tetracosanoic acid, docosanoic acid, octadecanoic acid and hexadecanoic acid and C20 and C21 phytosphingosines. Phosphoinositol oligosaccharides were released from the GIPLs by mild base treatment, and their structures were determined by compositional analysis, fast-atom-bombardment MS and NMR spectroscopy. Similar compounds were detected in all three strains, although their relative proportions varied. The predominant components in E. schaudinni strain LV59 and E. monterogeii LV88 were Galpβ1-3Galpβ1-3Manα1-3Manα1-4GlcNα1-6Ins-1-P and Arapβ1-2Galpβ1-3Galpβ1-3Manα1-3Manα1-4GlcNα1-6Ins-1-P, and the major phosphoinositol oligosaccharide in E. schaudinni LV58 was the hybrid-type GIPL Manα1-2(EtNP-6)Manα1-6(Galpβ1-3Manα1-3)Manα1-4GlcNα1-6Ins-1-P (where EtNP is ethanolamine phosphate). Several minor oligosaccharides containing additional galactose and/or arabinose residues were also detected.

Galactosamine in walls of slow-growing mycobacteria

Galactosamine was found consistently as a minor component of the envelope of five species of slow-growing mycobacteria, including all the major human pathogens, but not three rapid-growing species. The amino sugar was a component of the arabinogalactan of the cell wall skeleton, and occurred at the level of about one residue per arabinogalactan chain. Its amino group was in the free, un-N-acetylated state. Examination of oligosaccharides released by partial acid hydrolysis of arabinogalactan by fast atom bombardment-MS and gas chromatography-MS identified a series of oligoarabinans, each possessing one GalN unit, linked to position 2 of arabinose. It is proposed that the GalN residues occur as stub branches of 1 → 5-linked arabinose chains in the arabinogalactan. Possible functions of GalN are discussed.

Structural elucidation of XR586, a peptaibol-like antibiotic from Acremonium persicinum

A novel peptide, XR586, has been isolated from fermentations of Acremonium persicinum (Xenova culture collection number X21488). The structure of XR586 has been elucidated by means of NMR spectroscopy, electrospray and fast-atom bombardment MS, derivatization and enzymic digestion. It has been shown to be helical by CD measurements. XR586 shows many structural and conformational features in common with peptaibols, particularly the zervamicins. Peptaibol antibiotics are peptides, typically of 15–20 residues, containing a large proportion of α-aminoisobutyric acid (Aib) residues. These peptides adopt a helical conformation in solution and display anti-bacterial and toxic properties due to their ability to form pores in membranes. However, while XR586 contains several Aib residues, it lacks a terminal phenylalaninol and terminates in the sequence Phe-Gly. The lack of reduction of the penultimate residue at the C-terminus may indicate that this step is normally at the end of the biosynthetic pathway of peptaibols and occurs with cleavage of Gly. The 1H chemical shift assignments of XR586 are reported in Supplementary Publication SUP 50179 (3 pages), which has been deposited at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1996) 313, 9 (‘Deposition of data’).

Photoaffinity labelling of cyanomethaemoglobin with derivatives of tryptophan and 5-bromotryptophan

Tryptophan and 5-bromotryptophan (5-BrTrp) are relatively potent inhibitors of sickle-haemoglobin polymerization. The binding sites of these compounds to normal and sickle haemoglobin (HBA and HBS) have been suggested, but not firmly established, through the use of spin-labelled derivatives and/or computer modeling. In the present study we approached the problem by utilizing the technique of photoaffinity labelling. The cyanomet forms of HBA and HBS were subjected to photoaffinity labelling with N alpha-(4-azidotetrafluorobenzoyl)tryptophan and N alpha-(1-ethyl-2-diazomalonyl)-5-bromotryptophan respectively. Both irradiated samples of HBA and HBS were denatured, digested with trypsin, and then separated by reversed-phase HPLC. A labelled tryptic peptide was isolated from the photolabelling of HBS with N alpha-(1-ethyl-2-diazomalonyl)-5-bromotryptophan. The peptide was identified to be Val1(alpha)-Lys7(alpha), with the label attached to Val1(alpha), by virtue of amino acid analysis and sequencing, in conjunction with fast-atom-bombardment MS. The binding mode of N alpha-(1-ethyl-2-diazomalonyl)-5-bromotryptophan is proposed and its relevance to the potency of the 5-BrTrp-based anti-sickling agents is discussed.

Labeled Fumonisins: Production and Use of Fumonisin B1 Containing Stable Isotopes

Fumonisin B1 (FB1) labeled on the branch methyl groups with deuterium was produced in liquid cultures, and methyl-D3-labeled methionine was added. The isolated FB1 had 90% incorporation of 6 deuterium atoms and 9% incorporation of 3 deuterium atoms. The labeled FB1 was used as an internal standard for 2 analytical methods to measure FB1 in extracts of corn, corn products, and cultures. One method was hydrolysis followed by gas chromatography/mass spectrometry (GC/MS) of the derivatized backbone, and the other was analysis by fast atom bombardment MS (FAB/MS). Incorporation of labeled FB1 into samples resulted in a GC/MS method with improved precision and accuracy and allowed for a quantitative FAB/MS method.