N-acetyl-neuraminic acid (NANA) serum level in the diagnostics of preleukemic states, acute micromyeloblastic leukemias and pancytopenias.

The levels of N-acetyl-neuraminic acid were determined in patients with preleukemic states, acute micromyeloblastic leukemias and pancytopenias. A statistically significant increase of NANA was found in patients with micromyeloblastic leukemia in comparison with preleukemic states and pancytopenias. A significant rise in the NANA level was observed in preleukemic states in comparison with pancytopenia of other origins. The assay of the NANA level may be employed as a sensitive biochemical test for differential diagnostics of these diseases.     

Novel biological function of sialic acid (N-acetylneuraminic acid) as a hydrogen peroxide scavenger

We have found that N-acetylneuraminic acid (NANA) consumes toxic hydrogen peroxide (H(2)O(2)) under physiological conditions. Close investigation of this finding revealed that NANA was oxidized by an equimolar amount of H(2)O(2) to provide its decarboxylated product, 4-(acetylamino)-2,4-dideoxy-D-glycero-D-galacto-octonic acid (ADOA). To date, there have been little data on this reaction, and its physiological significance has not been discussed. Examining the detoxification of H(2)O(2) in cultured cells with NANA, we were able to confirm that the cell death caused by H(2)O(2) was suppressed by NANA in a dose-dependent manner. These results revealed a novel role for NANA as a reactive oxygen scavenger. It is known that terminal NANA residues are removed by neuraminidase and that free NANA molecules are recycled or degraded by enzymes. We propose that released monomeric NANA is the potent defense molecule against oxidative damage.     

Identification and characterization of N-acetyl-2,3-didehydro-2-deoxyneuraminic acid as a metabolite in mammalian brain

We have identified N-acetyl-2,3-didehydro-2-deoxyneuraminic acid (NADNA) in bovine and in rat brain. Identification was made by mass spectrometric and gas-liquid chromatographic analysis of the per(trimethylsilyl) derivative of the purified brain compound. Central nervous system NADNA hitherto has escaped detection; it behaves chromogenically and chromatographically during purification on ion-exchange chromatography as free N-acetylneuraminic acid (NANA) that also occurs in brain. Although NADNA is a dehydro analogue of NANA, we have ascertained that brain NANA does not give rise to NADNA as an artifact during its purification from brain. Three hours after intracranial injection of [14C]-N-acetylmannosamine [( 14C]ManNAc), we detected [14C]NANA but no [14C]NADNA in rat brain. ManNAc is a brain NANA precursor, and at this time, formation of cytidine 5'-phosphate (CMP)-[14C]NANA from [14C]ManNAc is at a maximum. This finding precludes decomposition of CMP-NANA as a source of brain NADNA. Upon intracranial injection of [14C]ManNAc, [14C]NADNA became detectable at 19 h and reached a maximum level around 40 h later; this maximum of labeling of NADNA coincides with the maximum label in brain sialo conjugate-NANA. These findings clearly demonstrate the occurrence of NADNA in mammalian brain. From the evidence, NADNA may derive enzymatically from brain sialo conjugates.     

Determination of neuraminidase-susceptible and total N-acetylneuraminic acid in cells by selected ion monitoring.

N-acetylneuraminic acid (NANA) is released from glycoproteins by neuraminidase or acid hydrolysis and quantified by monitoring the protonated molecular ions of fully silylated NANA ($\text{m}\text{e}\text{= 814}$) and neuraminic acid β-methylglycoside (internal standard, $\text{m}\text{e}\text{= 714}$) in a gas chromatograph—mass spectrometer system using isobutane ionization. Detection limit is 200 pg (0.6 pmol, underivatized weight) of NANA injected. In biological samples 5 ng (15 pmol) of NANA can be detected in 50 μl of hydrolysate. Only 1 to 50 μl of hydrolysate is needed, sample preparation is simple, NANA is positively identified in every analysis, 2-deoxy carbohydrates and other sialic acids do not interfere, only free NANA is determined, and the internal standard increases reliability. The NANA content of neuraminidase-treated human leukemic cells was on the order of $\text{0.3μ}\text{mol}\text{10}{}^9$ cells. NANA was quantified using as few as 5 × 10⁴ cells, in contrast to the conventional colorimetric (thiobarbituric) technique which requires 2.5 × 10⁷ cells.     

Relationship between the structure and activity of a histamine-blocking serum glycoprotein]

A plasmatic glycoprotein is submitted to a mild periodate oxydation and its pharmacological activity is studied. This glycoprotein contains much N acetyl Neuraminic Acid (NANA = 15 p. cent), and it reduces the biological activity of histamine on smooth muscle such as guinea pig ileum. See article. We also identify the 8 NANA and 7 NANA derivaties. Th only 8 carbon derivative is obtained when about one mole of m-periodate is consumed for one mole of NANA. The 7 carbon derivative appears as soon as the consumption of a second mole leads ta a second cleavage. These results prove that the oxydation islimited to the sole N acetyl neuraminic acid and more precisely to the lateral polyhydroxylic chain. Under these conditions, pharmacological activity gradually decreases, it disappears as soon as the lateral polyhydroxylic chain is completely cut off.     

Binding of an influenza A virus to a neomembrane measured by surface plasmon resonance

Neomembranes composed of either bovine brain lipid that contains sialoglycolipids or egg yolk lecithin that does not, were formed on an HPA sensor chip and used to study the binding of influenza A virus in real time by surface plasmon resonance. Virus bound only to the bovine brain lipid membrane. This was confirmed by an 84% reduction in virus binding after treatment of the neomembrane with neuraminidase. Binding was temperature dependent, being highest at 30-35 degrees C and lower at 10 degrees C. Surprisingly, the rate of complex formation was enhanced, rather than inhibited, by the presence of 1.34-25.2 x 10(6) molecules of free NANA per virus binding site and the rate of dissociation was lower suggesting that the complex was more stable. The free energy of association to form the transition complex was increased by 3 kJ mol(-1) and there was an almost 10-fold increase in the enthalpy of complex formation in the presence of free NANA. These results show the value of surface plasmon resonance for measuring complex molecular interactions in real time, and provide a model that can be used to study the effectiveness of inhibitors of attachment of influenza virus to its receptor molecules.     

Binding ability of sialic acid towards biological and toxic metal ions. NMR, potentiometric and spectroscopic study.

The binary complexes of 5-amino-3,5-dideoxy-D-glycero-D-galactononulosic acid (NANA), commonly called N-acetyl neuraminic acid, formed with biological metal ions such as Co(II) and Cu(II) and toxic metal ions such as Cd(II) and Pb(II) were investigated in aqueous solution by means of potentiometry, UV and NMR spectroscopy. The corresponding ternary systems with 2,2'-bipyridine were studied in aqueous solution by potentiometry and UV spectroscopy. NANA co-ordinates all metal ions, in both binary and ternary systems through the carboxylic group (protonated or deprotonated according to pH), pyranosidic ring oxygen and glycerol chain alcoholic hydroxy groups. The prevailing species in the pH range 2-7 are of [M(NANA)(2)] type, and their stability constants are greater than those of simple carboxylate complexes. Above pH 7, the species [M(NANA)(2)OH](-) are also formed, but they do not prevent the precipitation of metal hydroxides. This work provides information on the solution state chemistry of NANA in the presence of bivalent metal ions; its great affinity for the toxic metals Cd(II) and Pb(II), near physiological conditions, and the relatively high stability of the complex species found may also account for the mechanism of toxicity.     

Cloning and constitutive expression of the N-acetylneuraminate lyase gene of Escherichia coli

The N-acetylneuraminate (NANA) lyase (EC 4.1.3.3) gene from Escherichia coli was self-cloned in E. coli. Transformants were selected by complementation of a NANA lyase-deficient E. coli strain. One clone was found to produce NANA lyase, and it contained a recombinant plasmid, pNAL1, with a 9.0-kilobase HindIII insert. The cloning of the NANA lyase gene resulted in the change from inducible to constitutive production of the enzyme. The level of expression of the NANA lyase gene in E. coli(pNAL1) clones was two- to three-fold higher than that in the fully induced wild-type strains.     

Theoretical and experimental study on the competition of NANA-aldolase and cytidine-5'-monophosphosialate-synthase for their common substrate N-acetylneuraminic acid

The competition of NANA-Aldolase and Cytidine-5'-monophosphosialate-Synthase for their common substrate NANA has been studied, with, (i) the two enzymes in solution, (ii) NANA-Aldolase in solution and Cytidine-5'-monophosphosialate-Synthase immobilized in an artificial membrane (diffusion coefficient: 1.2 X 10(-3) cm2 h-1). The relation of the reaction rates for both enzyme was found 1:1 in case (i) and 2:1 in case (ii), in favor of NANA-Aldolase. These results are in agreement with the results obtained by computer simulation, where the Michaelian assumption and the diffusion effects had been considered. It was also calculated that the regulation of this branch point for the metabolic pathway of NANA is dependent on the input of NANA produced by the previous steps of the pathway and not on the concentration of CTP (second substrate of Cytidine-5'-monophosphosialate-Synthase) or the parameters controlling the diffusion of NANA. Computer simulations were performed by numerical analysis.     

Luminescent solid phase for sialic acid determination: a promising sensor for milk‐adulterated samples

This article presents the synthesis, characterization and spectroscopic study of silica modified with thenoyltrifluoroacetonate (SilTTA) and coordinated to an europium (III) ion, for the determination of sialic acid (NANA). Elemental analysis and infrared spectroscopy suggest silica functionalization, as well as coordination of beta‐diketone to the lanthanide ion. The emission spectra of compound‐free and coordinated Eu–SilTTA to NANA showed significant changes with respect to the maximum emission and spectral profile, suggesting that the NANA ion is coordinated to the Eu(III). The values of the phenomenological intensity parameters show an increase in polarizability around the Eu(III) in the case of Eu–SilTTA coordinated to NANA, as expected, since water molecules are less polarizable than sialic acid. The results of the batch assay showed that luminescent silica can be used for sialic acid determination in milk‐adulterated samples, with a correlation coefficient of 0.9992; and a detection limit of 0.4 mg/L; relative standard deviation (RSD%) = 0.0028. Copyright © 2014 John Wiley & Sons, Ltd.     

Cloning and constitutive expression of the N-acetylneuraminate lyase gene of Escherichia coli.

The N-acetylneuraminate (NANA) lyase (EC 4.1.3.3) gene from Escherichia coli was self-cloned in E. coli. Transformants were selected by complementation of a NANA lyase-deficient E. coli strain. One clone was found to produce NANA lyase, and it contained a recombinant plasmid, pNAL1, with a 9.0-kilobase HindIII insert. The cloning of the NANA lyase gene resulted in the change from inducible to constitutive production of the enzyme. The level of expression of the NANA lyase gene in E. coli(pNAL1) clones was two- to three-fold higher than that in the fully induced wild-type strains.     

Transferrin variants in Japan and New Zealand. Report of an unusually sialyzed TF variant

The genetic polymorphism of transferrin (TF) was studied in 2,167 Japanese individuals and in 448 New Zealanders. The three TF C subtypes were identified, but TF C3 was absent from Japanese populations. In addition, three TF B and six TF D variants were observed, each of which occurred either in Japanese or in New Zealanders. Stepwise removal of N-acetyl-neuraminic acid (NANA) with neuraminidase revealed that the most cathodal variant TF DShinnanyo found in a Japanese family was characterized by having only two NANA residues.     

The structure of NSD1 reveals an autoregulatory mechanism underlying histone H3K36 methylation

The Sotos syndrome gene product, NSD1, is a SET domain histone methyltransferase that primarily dimethylates nucleosomal histone H3 lysine 36 (H3K36). To date, the intrinsic properties of NSD1 that determine its nucleosomal substrate selectivity and dimethyl H3K36 product specificity remain unknown. The 1.7 Å structure of the catalytic domain of NSD1 presented here shows that a regulatory loop adopts a conformation that prevents free access of H3K36 to the bound S-adenosyl-l-methionine. Molecular dynamics simulation and computational docking revealed that this normally inhibitory loop can adopt an active conformation, allowing H3K36 access to the active site, and that the nucleosome may stabilize the active conformation of the regulatory loop. Hence, our study reveals an autoregulatory mechanism of NSD1 and provides insight into the molecular mechanism of the nucleosomal substrate selectivity of this disease-related H3K36 methyltransferase.     

Unusual sialilation of three different rare genetic variants of serum DBP: Gc 1A17, Gc 1A16, and Gc 1A11

Summary The proteins of three anodal Gc1 variants, Gc 1A16, 1A11, and 1A17, are characterized by the most acidic isoelectric points observed so far among the different Gc mutants. Stepwise removal of N-acetylneuraminic acid (NANA) by treatment with neuraminidase was performed to estimate the degree of sialilation of these Gc variants. The results indicate that both proteins, the anodal and the cathodal component of these Gc 1 mutants, carry sialic acid residues. This observation is remarkable in so far as usually only the anodal component of the Gc 1 protein contains NANA and only a single residue. From the experiments carried out it can be deduced that Gc 1A16 has two NANA residues in the anodal and one NANA residue in the cathodal component. Gc 1A16 was found in four members of three generations in a Danish family; the variant segregated as a Mendelian trait. More difficult to interprete are the results obtained with the variants Gc 1A11 and Gc 1A17. Gc 1A11 probably has three NANA residues in the anodal and two NANA residues in the cathodal component. Gc 1A11 has been observed in two mother-child pairs and is presumably also a simple genetic trait. Gc 1A17 has also several NANA residues in both Gc proteins; it is suggested that the anodal component has either three or four NANA residues and the cathodal component either two or three NANA residues. Family information on this variant is not yet available.     

Fractionation and Characterization of 9 Subcomponents of Bovine κ-Casein A

κ-casein A was fractionated into 9 subcomponents, all of which were identified as κ-casein from immunological analyses. The microheterogeneity of the subcomponents was explained by stepwise increase of their carbohydrate contents (0~4mol/mol of GalNAc, and 0~8mol/mol of NANA). The micelle-stabilizing ability of κ-casein subcomponents increased with the increase of their carbohydrate contents: the carbohydrate rich subcomponent 7 possessed twice the stabilizing ability of the carbohydrate free subcomponent 1. The sensitivity of synthetic casein micelle composed of κ-casein subcomponents and α_sl-casein to the wheat germ lectin-induced aggregation also increased with the increase of their NANA contents.     

Sialyl-Tn-KLH, glycoconjugate analysis and stability by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD)

The quantitation of sialyl-Tn (STn) conjugated to keyhole limpethaemocyanin (KLH) can be determined by quantitating the amountof ^N-acetylneuraminic acid (NANA) released by acid or enzymaticdigestion. An optimal 0.1 N H₂SO₄ acid hydrolysis at 80°Cresults in quantitative release of NANA with minimal loss. Arapid isocratic method for the quantitation and separation ofNANA is described using high-pH anion-exchange chromatographyand pulsed amperometric detection (PAD). Multiple injectionof NANA standard and/or samples containing protein led to adecrease in the PAD response which was corrected by additionof internal standard, -2-keto-3-deoxyoctonate (KDO). The ratioof NANA/KDO peak area or peak height gives a linear responsewith increasing amount of NANA in the range 2.5–20 µg/ml(r² = 0.99). The limit of quantitation (LOQ) for NANA usingthis isocratic method is 1.9 µg/ml ({small tilde}160 pmol/25µl injection). Based on the multiple determination theglycoconjugate, STn-KLH, showed a NANA content of 2.9% (w/w).Acid hydrolysis and the sialidase treatment of STn-KLH bothyielded a similar NANA content. The carrier protein, KLH, showedthe absence of NANA. The stability of glycoconjugate STn-KLHwas monitored by a gradient method which separated possibledegradation products STn-crotyl, NANA and GalNAc. Subjectingthe glycoconjugate STn-KLH to various stress conditions of temperature,pH and oxidation does not result in any release of sialic acid,GalNac and STn-crotyl group. high-pH anion-exchange chromatography mono-saccharide analysis pulsed amperometric detection sialyl-Tn stability of glycoconjugate     

Physiology of sialic acid capsular polysaccharide synthesis in serogroup B Neisseria meningitidis

The pathway for biosynthesis of sialic acid capsular polysaccharide was examined in Neisseria meningitidis serogroup B strain M986 and in strain PRM102, an isogenic mutant defective in polysaccharide production. Strain PRM102 was found to possess only 25% of the level of sialyltransferase activity that was found in strain M986, but it had wild-type levels of both the N-acetylneuraminic acid (NANA) condensing enzyme and the CMP-NANA synthetase. A new meningococcal enzyme, a CMP-NANA hydrolase, was found in both meningococcal strains. This enzyme generated CMP and NANA from CMP-NANA, had a Km of 0.88 microM, had a Vmax of 10.75 nmol of NANA produced per h per mg of protein, and was completely inhibited by 45.3 microM CMP. The sialyltransferase, which also had CMP-NANA as substrate, was insensitive to CMP addition. Subcellular fractionation and purification of cytoplasmic and outer membranes on sucrose density gradients revealed that both the sialyltransferase and the CMP-NANA hydrolase were cytoplasmic membrane associated. The NANA condensing enzyme and the CMP-NANA synthetase were found to be cytosolic. A working hypothesis for the regulation of sialic acid polysaccharide synthesis was developed. The CMP-NANA hydrolase with its high affinity for CMP-NANA regulates polysaccharide formation by the sialyltransferase, whereas CMP, a product of both the sialyltransferase and the CMP-NANA hydrolase, modulates the activity of the hydrolase on the cytoplasmic membrane.     

NSD1 mutations are the major cause of Sotos syndrome and occur in some cases of Weaver syndrome but are rare in other overgrowth phenotypes

Sotos syndrome is a childhood overgrowth syndrome characterized by a distinctive facial appearance, height and head circumference >97th percentile, advanced bone age, and developmental delay. Weaver syndrome is characterized by the same criteria but has its own distinctive facial gestalt. Recently, a 2.2-Mb chromosome 5q35 microdeletion, encompassing NSD1, was reported as the major cause of Sotos syndrome, with intragenic NSD1 mutations identified in a minority of cases. We evaluated 75 patients with childhood overgrowth, for intragenic mutations and large deletions of NSD1. The series was phenotypically scored into four groups, prior to the molecular analyses: the phenotype in group 1 (n=37) was typical of Sotos syndrome; the phenotype in group 2 (n=13) was Sotos-like but with some atypical features; patients in group 3 (n=7) had Weaver syndrome, and patients in group 4 (n=18) had an overgrowth condition that was neither Sotos nor Weaver syndrome. We detected three deletions and 32 mutations (13 frameshift, 8 nonsense, 2 splice-site, and 9 missense) that are likely to impair NSD1 functions. The truncating mutations were spread throughout NSD1, but there was evidence of clustering of missense mutations in highly conserved functional domains between exons 13 and 23. There was a strong correlation between presence of an NSD1 alteration and clinical phenotype, in that 28 of 37 (76%) patients in group 1 had NSD1 mutations or deletions, whereas none of the patients in group 4 had abnormalities of NSD1. Three patients with Weaver syndrome had NSD1 mutations, all between amino acids 2142 and 2184. We conclude that intragenic mutations of NSD1 are the major cause of Sotos syndrome and account for some Weaver syndrome cases but rarely occur in other childhood overgrowth phenotypes.     

Gangliosides of the peripheral nervous system of the rat

Rats sciatic nerves gangliosides have been studied. We found about 120 nmoles NANA/g dry weight. We studied the relative distribution of the gangliosides, after TLC and densitometry. G4 (G_M1) accounts for about 31 % of total NANA. G5 is absent from our samples ; we found a very low level of G7. Our distribution is different from the data of Svennerholm's group for the gangliosides of the human femoral nerve, and from Yates and Wherrett for the gangliosides of the rabbit sciatic nerves.     

Thrombocytes and aging. Protein and neuraminic acid content of thrombocytes of male donors of various age groups

Thrombocytes of male donors of different age groups were investigated in terms of their content of N-acetylneuraminic acid (NANA) and protein. For this purpose NANA was split up hydrolytically as well as enzymatically by neuraminidase. Furthermore, the relation of NANA to protein was determined in the lysed thrombocytes of these donors. A significant increase in the protein content of platelets could be observed between group I and III (young and old donors). The relation of NANA to protein (acid hydrolysis) existing between these groups decreased significantly with growing age. Appropriate results could also be attained after having made a separate analysis in lysed thrombocytes. The results achieved allow the conclusion to be drawn that there is a changed thrombocytopoiesis in men of old age.