Lekh Raj Juneja

Occurrence of a sialylglycopeptide and free sialylglycans in hen's egg yolk

Free sialylglycans (FSGs) and a sialylglycopeptide (SGP) as components of hens egg yolk were found and their chemical structures were determined. SGP and FSGs were isolated from fresh egg yolk by treatment with phenol, gel filtration and successive chromatographies on columns of anion- and cation-exchangers. They were localized in the yolk plasma. The glycan moiety of SGP, which was liberated by PNGase digestion, was studied for the chemical structure by HPLC mapping with p-aminobenzoic ethylester-derivatization, sugar composition analysis, 1H nuclear magnetic resonance and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and the glycomoiety was found to be an N-linked disialyl-biantennary glycan. The amino acid sequence of the peptide moiety of SGP was determined to consist of Lys-Val-Ala-Asn-Lys-Thr, the Asn of which is modified with the disialylglycan moiety. FSGs were determined to be two free disialyl-biantennary glycans whose reducing end was either Man beta1-4GlcNAc (FSG-I) or Man beta1-4GlcNAc beta1-4GlcNAc (FSG-II). Since the molar value of SGP present in one egg yolk (2.8 micromol) is comparable to those of well-known major yolk proteins, low density lipoprotein, lipovitellins and phosvitin, it can be considered that SGP is one of the major components in hens egg yolk.

A micronised, dispersible ferric pyrophosphate with high relative bioavailability in man.

Ferric pyrophosphate is a water-insoluble Fe compound used to fortify infant cereals and chocolate-drink powders as it causes no organoleptic changes to the food vehicle. However, it is only of low absorption in man. Recently, an innovative ferric pyrophosphate has been developed (Sunactive Fe trade mark ) based on small-particle-size ferric pyrophosphate (average size 0.3 microm) mixed with emulsifiers, so that it remains in suspension in liquid products. The aim of the present studies was to compare Fe absorption of micronised, dispersible ferric pyrophosphate (Sunactive Fe trade mark ) with that of ferrous sulfate in an infant cereal and a yoghurt drink. Two separate Fe absorption studies were made in adult women (ten women/study). Fe absorption was based on the erythrocyte incorporation of stable isotopes ((57)Fe and (58)Fe) 14 d after the intake of labelled test meals of infant cereal (study 1) or yoghurt drink (study 2). Each test meal was fortified with 5 mg Fe as ferrous sulfate or micronised, dispersible ferric pyrophosphate. Results are presented as geometric means. There was no statistically significant difference between Fe absorption from micronised, dispersible ferric pyrophosphate- and ferrous sulfate-fortified infant cereal (3.4 and 4.1 % respectively; P=0.24) and yoghurt drink (3.9 and 4.2 % respectively; P=0.72). The results of the present studies show that micronised, dispersible ferric pyrophosphate is as well absorbed as ferrous sulfate in adults. The high relative Fe bioavailability of micronised, dispersible ferric pyrophosphate indicates the potential usefulness of this compound for food fortification.

Conversion of Complex Sialooligosaccharides into Polymeric Conjugates and their Anti-Influenza Virus Inhibitory Potency

ABSTRACT To investigate the specificity of various influenza virus strains we have prepared polyacrylic type conjugates of undecasaccharide (Neu5Acα2-6Galβ1-4GlcNAcβ1-2Manα1)2-3,6Manβ1-4GlcNAcβ1-4GlcNAc (YDS), and trisaccharides 6‵-sialyl-N-acetyllactosamine (6‵SLN), 6‵-sialyllactose (6‵SL), and 3‵-sialyllactose (3‵SL). Free oligosaccharides were transformed to glycosylamine-1-N-glycyl derivatives by sequential action of NH4HCO3, chloroacetic anhydride, and aqueous NH3. The known derivatization protocol has been optimized for these sialooligosaccharides. Coupling of obtained amino-spacered derivatives with poly(4-nitrophenyl acrylate) gave rise to two types of conjugates, namely with polyacrylic acid and polyacrylamide backbones; the conversion proceeded quantitatively and without destruction of the oligosaccharides. The content of oligosaccharides in the conjugates was 10, 20, and 30% mol for 3‵SL, 6‵SL, 6‵SLN, and 2, 5 and 10% mol for YDS. Free oligosaccharides and the glycoconjugates were tested as inhibitors of influenza virus adhesion, and also as blockers of virus infectivity in MDCK cell culture. Biantennary YDS demonstrated similar activity to trisaccharide 6‵SLN both as the free form and neoglycoconjugate.

Enzymatic synthesis of a sialyl Lewis X dimer from egg yolk as an inhibitor of E-selectin

A dimeric sialyl Lewis X (SLex) glycopeptide was synthesized enzymatically in three steps from an N-linked oligosaccharide prepared from egg yolk. Treatment of delipidated hen egg yolk with the protease Orientase and neuraminidase gave a dimeric N-acetyllactosamine-containing oligosaccharide linked to asparagine. Addition of sialic acid and fucose catalyzed by alpha-2,3-sialyltransferase and alpha-1,3-fucosyltransferase provided the dimeric SLex, which was shown to be as active as monomeric SLex as an inhibitor of E-selectin with IC50 0.75 mM. The synthetic dimeric SLex of the mucin type (i.e. SLex linked to the 3- and 6-OH groups of Gal) is, however, about five times as active as the monomer. It is suggested that dimeric SLex glycopeptides of the mucin type would be effective ligands for E-selectin.

Preparation of N-acetylneuraminic acid from delipidated egg yolk

Egg yolk, a large proportion of the egg, was studied for the preparation ofN-acetylneuraminic acid (Neu5Ac). The delipidated hen egg yolk (DEY; 500 kg containing 0.2% w/w, Neu5Ac) was hydrolysed with HCl (pH 1.4) at 80 °C and neutralized with NaOH (pH 6.0). The mixture was filtered and electrodialysed until the conductivity was 240 µS cm−1. The filtrate was applied on a column of Dowex HCR-W2 (20–50 mesh), followed by a column of Dowex 1-X8 (200–400 mesh). The latter column was washed with water, and then eluted with a linear gradient of HCO2H (0–2m). The eluates containing Neu5Ac were concentrated using a reverse osmosis membrane and, finally, rotary evaporated at 40 °C. The residue was then lyophilized to yield 500 g Neu5Ac. The purity of Neu5Ac was >98% (TBA method). HPLC, NMR spectroscopy and TLC chromatography of the product obtained from the DEY showed that Neu5Ac was the sole derivative present in egg yolk. The DEY, a byproduct from egg processing plants, was found to be an excellent source for the large-scale preparation of Neu5Ac.

Comparison of egg-yolk protein hydrolysate and soyabean protein hydrolysate in terms of nitrogen utilization

Egg-yolk protein hydrolysate (YPp) is an alternative protein source in formulas for infants with intolerance to cows milk or soyabean protein, or for patients with intestinal disorders. However, the nutritional value of YPp has never been investigated. YPp was prepared by enzymic hydrolysis of delipidated yolk protein, which led to an average peptide length of 2.6 residues. Three experiments were performed. In Expt 1, we compared the intestinal absorption rate of YPp and soyabean protein hydrolysate (SPp) in rats. YPp and SPp solutions were injected into the duodenum of anaesthetized rats and blood samples were taken from the portal vein at 7, 15, 30, 60, and 120 min. A higher amino acid concentration in the serum of the YPp group demonstrated that YPp was absorbed faster than SPp. In Expt 2, the effects of dietary YPp and SPp on body-weight gain, protein efficiency ratio (PER) and feed efficiency ratio (FER) were determined. At the end of the experiment, body weight had increased in both groups, while PER and FER were significantly higher in rats fed on YPp. In Expt 3, to investigate the effects of dietary YPp and SPp on N metabolism, we determined the biological value and net protein utilization. Yolk protein was the reference protein. Biological value and net protein utilization values were very similar between animals fed on yolk protein and YPp diets, and significantly higher than in rats fed on the SPp diet. The present findings demonstrate that there is no adverse effect of hydrolysis of yolk protein on N utilization, and that the nutritive value of YPp is similar to that of yolk protein and superior to that of SPp.

An Efficient Preparation and Structural Characterization of Sialylglycopeptides from Protease Treated Egg Yolk

Abstract Delipidated egg yolk (DEY) was digested with protease. The digest was ultrafiltered using a molecular weight cut-off of 10,000, and the UF permeate was treated with a reverse osmosis membrane. The resulting sialylglycopeptides-rich fraction was purified by the combination of anion exchange and gel filtration chromatography. Two major sialylglycopeptides, A-I and A-II, were characterized as biantennary complex type sialylglycopeptides having one or two N-acetylneuraminic acid and 1 - 3.5 amino acids including one asparagine residue by composition analyses and 1H NMR spectroscopy.

SYNTHETIC SIALYLPHOSPHATIDYLETHANOLAMINE DERIVATIVES BIND TO HUMAN INFLUENZA A VIRUSES AND INHIBIT VIRAL INFECTION

We synthesized the sialylphosphatidylethanolamine (sialyl PE) derivatives Neu5Ac-PE, (Neu5Ac)2-PE, Neu5Ac-PE (amide) and Neu5Ac-PE (methyl). We examined the anti-viral effects of the derivatives on human influenza A virus infection by ELISA/virus-binding, hemagglutination inhibition, hemolysis inhibition and neutralization assays. The sialyl PE derivatives that we examined bound to A/Aichi/2/68, A/Singapore/1/57 and A/Memphis/1/71 strains of H3N2 subtype, but not to A/PR/8/34 strain of H1N1 subtype. The derivatives inhibited viral hemagglutination and hemolysis of human erythrocytes with A/Aichi/2/68 and A/Singapore/1/57 (H3N2), but not with A/PR/8/34 (H1N1). The inhibitory activity of the (Neu5Ac)2-PE derivative was the strongest of all sialyl PE derivatives (IC50, 35 μM to 40 μM). Sialyl PE derivatives also inhibited the infection of A/Aichi/2/68 in MDCK cells. Complete inhibition was observed at a concentration between 0.3 to 1.3 mM. IC50 of (Neu5Ac)2-PE was 15 μM in A/Aichi/2/68 strain. Taken together, the synthetic sialyl PE derivatives may be effective reagents against infection of some types of influenza A viruses.

S15.9 Characterization and antiviral function of sialyloligosaccharides from egg yolk

Influenza virus attachment to host cells is mediated by specific interaction of viral hemagglutinin with the cell-surface sialooligosaccharides. In effort to produce potent synthetic inhibitors of viral attachment a number of polyacrylates and dextrans bearing a-benzylsialoside residue (Neu5Aca2OCH2C6H4NHCOCH2NH-) were prepared and tested as inhibitors of virus binding activity and multiplication in tissue culture. 20 various strains of influenza A and B virus were tested. The greatest affinity for inhibitors was shown mostly by H3 strains emerged after 1977, while HI , H2 and type B strains were of lower affinity. Polyacrylic acid containing 12 mole % of sialoside (P~2) was found to be the best inhibitor, those containing 5 and 20 mole % were significantly less effective. Elongation of spacer by aminocaproyl led only to 2 4 fold higher inhibition activity for HI , H2 and type B viruses and had no effect on H3 high affinity strains. The influence of carrier on inhibitory activity was demonstrated by comparison of polyacrylates. Polyacrylic acid as carrier was the best for H1 strains, some H3 and type B viruses have higher affinity for polyacrylamide and -ethanolamide type polymers, in case of H3 (after 1977) strains all three polymers had equal binding constants. Additional substituents (alkyls, Gal, aminoacids) of carrier did not increase the binding affinity of polysialoside. Hydrophobic (n-cetyl) or strongly ionized (sulfonic acid) substituents lowered inhibition ability. The polymer-virus binding was amplified with the increase of polymer MW. For A/Phil ippines/2/82 the 1000 kDa polymer affinity was 500-fold higher comparing to 20 kDa. Further increase of MW resulted in decrease of affinity. Activity of dextran based inhibitors increased tenfold when going from D70 to D500.