Sialic Acid

PH-20 is a glycoprotein located on the surface of the sperm plasma membrane and on the inner acrosomal membrane. The best understood function of sperm surface PH-20 is its hyaluronidase activity, which results in hydrolysis of the hyaluronic acid-rich cumulus matrix during sperm penetration of this extracellular oocyte investment. In this study, we investigated whether alterations in the secondary and tertiary structures of sperm surface PH-20 would affect its enzyme activity. Proteins were isolated from the sperm plasma membrane by treatment of living cells with phosphatidylinositol-specific phospholipase C (PI-PLC). PH-20 was purified from the PI-PLC released proteins by immunoaffinity chromatography. Two-dimensional electrophoresis of purified PH-20 revealed 6 isoforms with isoelectric points ranging from 5.1 to 6.0. Removal of the N-linked glycans from PH-20 with N-glycosidase F shifted the molecular weight from 64 kd to approximately 54 kd, its deduced molecular weight based on sequence analysis, suggesting that most if not all, of the potential N-glycosylation sites are linked to oligosaccharides. The lectins Con A and PSA recognized purified sperm surface PH-20 after Western blotting, suggesting that mannose is a major sugar within or at the terminal end of the linked glycan. The lectins UEA and LPA did not recognize PH-20 Western blot, suggesting that fucose and sialic acid are not terminal sugars of sperm surface PH-20. Deglycosylation of sperm surface PH-20 resulted in a complete loss of its hyaluronidase activity. The reduction of disulfide bonds with ␤-mercaptoethanol or dithiothreitol also resulted in loss of enzyme activity. We conclude that the hyaluronidase activity of sperm surface PH-20 is dependent on structural features established by sulfhydryl linkages, as well as glycosylation.

Conversions of the C-5 acetamide group in sialic acid into two kinds of C@C double bond substituents were accomplished under Shotten-Baumann conditions. The polymerizable glycomonomers also contain a hydrophobic chain or hydroxyl group at the anomeric position. Radical polymerizations of the fully protected glycomonomers were carried out with acryl amide in the presence of ammonium persulfate (APS) and N,N,N 0 ,N 0 -tetramethylethylenediamine (TEMED), followed by deprotection to furnish water-soluble glycopolymers. The activities of the deprotected glycopolymers and glycomonomers against human influenza viruses (H1N1 and H3N2) and avian influenza virus (H5N3) were evaluated. Biological evaluations showed that the glycomonomers containing a long hydrophobic chain at the anomeric position had both hemagglutination and neuraminidase inhibitory activities.

Actinomyces naeslundii genospecies 1 and 2 express type-2 fimbriae (FimA subunit polymers) with variant Galbeta binding specificities and Actinomyces odontolyticus a sialic acid specificity to colonize different oral surfaces. However, the fimbrial nature of the sialic acid binding property and sequence information about FimA proteins from multiple strains are lacking. Here we have sequenced fimA genes from strains of A.naeslundii genospecies 1 (n = 4) and genospecies 2 (n = 4), both of which harboured variant Galbeta-dependent hemagglutination (HA) types, and from A.odontolyticus PK984 with a sialic acid-dependent HA pattern. Three unique subtypes of FimA proteins with 63.8-66.4% sequence identity were present in strains of A. naeslundii genospecies 1 and 2 and A. odontolyticus. The generally high FimA sequence identity (> 97.2%) within a genospecies revealed species specific sequences or segments that coincided with binding specificity. All three FimA protein variants contained...

Sialidases (EC 3.2.1.18) are a group of glycohydrolytic enzymes, widely distributed in nature, that cleave sialic acid residues from the oligosaccharide components of glycoconjugates. All of the sialidase enzymes thus far characterized share an Asp block, repeated three to five times in the primary structure, and an F/YRIP sequence motif that is part of the active site. Using a sequence homology-based approach, we have identified a novel human gene, named NEU2, mapping to chromosome 2q37. The nucleotide sequence analysis of the gene has shown that it contains only one intron of about 1.25 kb, and the longest open reading frame encodes a protein of 380 amino acids, with a two-Asp block consensus, and the YRIP sequence. In the putative promoter sequence there are a classical TATAA box and four E boxes, which are consensus binding sites for muscle-specific transcription factors. Northern blot analysis revealed expression of the NEU2 transcript only in skeletal muscle.

Sialic acid-containing glycoconjugates are generally considered to be unique to the deuterostomes, a lineage of the animal kingdom which includes animals from the echinoderms up to the vertebrates. There are, however, two isolated reports of sialic acid occurring in the insect species Drosophila melanogaster and Galleria mellonella. Since insects are classified as protostomes, these findings call previous assumption on the phylogenetic distribution and thus on the evolution of sialic acids into question. Here, we report the occurrence of N-acetylneuraminic acid (Neu5Ac) in larvae of the cicada Philaenus spumarius. Cytochemical analysis of larval sections with lectins from Sambucus nigra and Limax flavus suggested the presence of sialic acids in the concrement vacuoles of the Malpighian tubules. The monoclonal antibody MAb 735, which is specific for polysialic acid, labelled the same structures. A chemical analysis performed by HPLC of fluorescent derivatives of sialic acids and by GLC-MS provided sound evidence for the presence of Neu5Ac in the Philaenus spumarius larvae. These data suggest that in this cicada Neu5Ac occurs in α2,8-linked polysialic acid structures and in α2,6-linkages. The results provide further evidence for the existence of sialic acids in insects and in linkages known to occur in glycoconjugates of deuterostomate origin.

The amino acid composition of CHO cell culture media for manufacturing an antibody fusion protein (B1) was optimized using data obtained through metabolic flux analysis. The metabolic flux analysis was performed using the metabolic rates derived from two semi-steady states of continuous culture. The amino acid composition was the same for media used in both the fed-batch and continuous cultures, which made the modification of amino acid composition suggested by the metabolic flux analysis applicable to the fed-batch culture. The flux analysis indicated that methionine, tryptophan, asparagine and serine were limiting, while alanine, arginine, glutamine and glycine were in excess at the semi-steady states. Based on this result, the limiting amino acids were increased while those in excess were reduced to obtain balanced amino acid composition in both the batch and feed media. The fed-batch cell cultures using the modified media generated less lactate and ammonia, and more significantly, increased peak cell densities by 55% and B1 protein titer by 27% without any impact on final sialic acid content, a quality attribute of the B1 protein.

Background. This study aimed to assess the therapeutic efficacy of oral vitamin E supplementation on the biochemical and kinetic properties of Tamm-Horsfall glycoprotein (THP) in hypertensive and hyperoxaluric patients. Methods. Newly detected hypertensives (n ¼ 200) and stone formers (n ¼ 200) were each subdivided into two groups. One group (n ¼ 100) was administered the antioxidant vitamin E at 400 mg/day given as an oral supplement along with standard therapeutic drugs for hypertension and hyperoxaluria and the patients were followed for a period of 9 months. The other group (n ¼ 100) did not receive vitamin E (placebo controls). Age and sex-matched controls (n ¼ 100) were monitored simultaneously. THP was isolated from 24 h urine samples before and at the end of every third month during a period of 9 months from the vitamin Etreated hypertensive and hyperoxaluric groups. THP samples were also collected from control subjects, and at the end of the ninth month from placebo controls. The isolated protein was assessed for purity by SDS-PAGE. The purity-checked proteins were subjected to spectrophotometric crystallization assay, calcium oxalate (CaOx) crystal interaction studies, and biochemical analysis of sialic acid, thiol and carbonyl content. Plasma superoxide, hydroxyl radical, hydrogen peroxide and vitamin E levels as well as superoxide dismutase and catalase activities were also monitored. Results. The THP from the hypertensive and hyperoxaluric subjects exhibited a significant promoting effect on the nucleation and aggregation phases and

A periodate-resorcinol microassay for the quantitation of total sialic acid (TSA) has been developed. A 96-well microtiter plate format was used as a single reagent mixing well. The principle of this newly-developed assay is the periodate-resorcinol procedure. Conditions for the quantitation of sialic acid in serum, such as the concentration of periodate, resorcinol, incubation time and human serum volume, were optimized. It was found that the optimal concentration of periodic acid and resorcinol reagent were 1.3 mM and 0.6%, respectively. An incubation time of 60 minutes for the reaction of periodic acid and resorcinol with samples was found to give the highest absorbance. These new procedures were used for the quantitation of sialic acid in human serum. It was found that only 5 µL samples were needed to give a varied coefficient of the intra- and inter-assay of 0.79% and 4.68%, respectively. Furthermore, this method demonstrated the recovery percentage of 94.25% by the addition of...

This study was undertaken to determine the occurrence of uronic acid in chicken eggshell membranes and to compare chemical compositions among the inner and outer eggshell membranes and the organic matter of eggshell. We report here for the first time the occurrence of uronic acid in chicken eggshell membranes. Uronic acid concentrations were similar (P > 0.05) between the inner shell membrane and outer shell membrane but approximately fivefold higher (P < 0.05) in the organic matter of eggshell. Sialic acid concentrations were (

Avian influenza viruses preferentially use a2,3-linked sialic acid as a receptor for binding and entry into target cells. The sialic acid is the terminal residue of various types of glycan. There are two major types of a2,3-linked sialic acid differing in the penultimate bond: Neu5Aca2-3Galb1-3GalNAc and Neu5Aca2-3Galb1-4GlcNAc. In the human airway, while Neu5Aca2-3Galb1-3GalNAc is present only in alveolar epithelial cells, the Neu5Aca2-3Galb1-4GlcNAc is expressed in both the upper and lower airway. Previous data showed preferential binding of hemagglutinin from H5N1 highly pathogenic influenza virus to Neu5Aca2-3Galb1-4GlcNAc. We further show here that suppression of this sialic acid by siRNA against a sialyltransferase, ST3GAL4, can inhibit H5N1 avian influenza virus infection and that this gene is abundantly expressed in human pharynx, trachea and bronchus. These data suggest that the ST3GAL4 gene is responsible for biosynthesis of the viral receptor and may play a crucial role in infection of H5N1 avian influenza virus in humans.

CD33 also known as Siglec-3 is endogenously expressed in stem cells and is a marker for the myeloid lineage
of cells. Increased expression of CD33 thus allows it to bind to any Sialic Acids (SIAs). These acids are binding
sites for pathogens and toxins. By binding to these acids, CD33 can prevent invasion of hosts by these pathogens.
Down-regulation of CD33, increase the release of the pro-inflammatory cytokine TNF-α by monocytes that increases
reactive oxygen species that are involved in diseases like diabetes mellitus, Alzheimer’s, cardiovascular diseases
asthma, and in various cancers.
The up-regulation of CD33 using Metadichol® was studied using Wharton’s Jelly Mesenchymal Stem Cells
(MSCs) isolated from human umbilical cord and were grown in p-35 dishes until confluent and treatment was
carried out with different concentrations. One dish was untreated and considered as control. The treated and
untreated cells were analyzed using Flow Cytometry. The cells treated at 100 pg of Metadichol® has shown the
highest increase (>400 fold) in CD33++ expression (48.77%) compared to untreated control (0.11%).

Background: The Cronobacter genus is composed of seven species, and can cause infections in all age groups. Of particular concern is C. sakazakii, as this species is strongly associated with severe and often fatal cases of necrotizing enterocolitis and meningitis in neonates and infants. Whole genome sequencing has revealed that the nanAKT gene cluster required for the utilisation of exogenous sialic acid is unique to the C. sakazakii species (ESA_03609-13). Sialic acid is found in breast milk, infant formula, intestinal mucin, and gangliosides in the brain, hence its metabolism by C. sakazakii is of particular interest. Therefore its metabolism could be an important virulence factor. To date, no laboratory studies demonstrating the growth of C. sakazakii on sialic acid have been published nor have there been reports of sialidase activity. The phylogenetic analysis of the nan genes is of interest to determine whether the genes have been acquired by horizontal gene transfer.

The use of CNS cultures for detection and quantification of neuronotrophic activity in the CNS has been analyzed. In particular the development, i.e., neurotransmitter uptake characteristics, and survival of dopaminergic and GABAergic neurons in fetal mouse (E13)-dissociated mesencephalic cells cultured in serum-free, hormone-supplemented medium have been assessed as a function of culture time and cell density. At all times, more than 98% of the cells were classified as neurons on the basis of immunocytochemical criteria. Results indicate that the increase of cell density in vitro significantly enhances specific high-affinity dopamine uptake per dopaminergic cell and cell survival. This effect is not limited to the dopaminergic cells and suggests that the development of neurotransmitter-related traits and cell survival are influenced by cell density-derived trophic signals. The above-mentioned cultures and parameters have also been used to detect neuronotrophic activity in adult mam...

Background: The problems of adherence to energy restriction in humans are well known. Objective: To compare the feasibility and effectiveness of intermittent continuous energy (IER) with continuous energy restriction (CER) for weight loss, insulin sensitivity and other metabolic disease risk markers. Design: Randomized comparison of a 25% energy restriction as IER (B2710 kJ/day for 2 days/week) or CER (B6276 kJ/day for 7 days/week) in 107 overweight or obese (mean ( ± s.d.) body mass index 30.6 ( ± 5.1) kg m À2 ) premenopausal women observed over a period of 6 months. Weight, anthropometry, biomarkers for breast cancer, diabetes, cardiovascular disease and dementia risk; insulin resistance (HOMA), oxidative stress markers, leptin, adiponectin, insulin-like growth factor (IGF)-1 and IGF binding proteins 1 and 2, androgens, prolactin, inflammatory markers (high sensitivity C-reactive protein and sialic acid), lipids, blood pressure and brain-derived neurotrophic factor were assessed at baseline and after 1, 3 and 6 months. Results: Last observation carried forward analysis showed that IER and CER are equally effective for weight loss: mean (95% confidence interval ) weight change for IER was À6.4 (À7.9 to À4.8) kg vs À5.6 (À6.9 to À4.4) kg for CER (P-value for difference between groups ¼ 0.4). Both groups experienced comparable reductions in leptin, free androgen index, high-sensitivity C-reactive protein, total and LDL cholesterol, triglycerides, blood pressure and increases in sex hormone binding globulin, IGF binding proteins 1 and 2. Reductions in fasting insulin and insulin resistance were modest in both groups, but greater with IER than with CER; difference between groups for fasting insulin was À1.2 (À1.4 to À1.0) mU ml À1 and for insulin resistance was À1.2 (À1.5 to À1.0) mU mmol À1 l À1 (both P ¼ 0.04). Conclusion: IER is as effective as CER with regard to weight loss, insulin sensitivity and other health biomarkers, and may be offered as an alternative equivalent to CER for weight loss and reducing disease risk.

Objective: To investigate the effects of cadmium (Cd) and copper (Cu) on sialic acid levels of brain and blood tissues of Cyprinus carpio. Methods: Adult carps were exposed to 0.1, 0.5 mg/L Cu, 0.1, 0.5 and 1.0 mg/L Cd and 0.1 mg/ L Cu+0.1 mg/L Cd under static experiment conditions for 1 week. At the end of exposure period, heavy metal accumulations and sialic acid levels in blood and brain tissues of the test animals were analyzed. Results: Cu and Cd accumulated in tissues in a dramatically increasing dose-dependent manner. Sialic acids level of the fish exposed to 0.1, 0.5 and 1.0 mg/L Cu and Cd and control grups for 1 week were 0.834, 1.427, 0.672, 0.934, 2.968, 4.714 mg/mL respectively. The results also showed that Cu has an antagonistic effect on tissue sialic acid level. Conclusions: We propose that Cd and Cu make a complex with sialic acids of membranes in the tissues researched. This complex between metal ions and sialic acid migth account for the cellular toxicity based on Cu and Cd.

Enamel matrix consists of amelogenin and non-amelogenins. Though amelogenin is not involved in nucleation of minerals, the enamel mineralization is impaired when amelogenin or other matrix protein (ameloblastin/enamelin) genes are mutated. We hypothesize that amelogenin may promote enamel mineralization by interacting with the calcium-binding matrix proteins. Specific binding of amelogenin to N-acetylglucosamine (GlcNAc), GlcNAc-mimicking peptides (GMps), and their carrier proteins and the identification of amelogenin-trityrosyl-motif-peptide (ATMP) as a GlcNAc/GMp-binding domain in amelogenin favor the hypothesis. This study tested the interaction of amelogenin with ameloblastin, a carrier of GMp sequence at intermittent sites. Neither GlcNAc nor sialic acids were identified in the recombinant-ameloblastin. Amelogenin bound to recombinant-ameloblastin in both Western blots and in ELISA. More specifically, [ 3 H]ATMP bound to both recombinant and native ameloblastins. Dosimetry and Scatchard analyses showed the specific interaction between ATMP and ameloblastin, suggesting that amelogenin may interact with ameloblastin to form a heteromolecular assembly.

In the present study, a thrombin-like enzyme named BpSP-I was isolated from Bothrops pauloensis snake venom and its biochemical, enzymatic and pharmacological characteristics were determined. BpSP-I is a glycoprotein that contains both N-linked carbohydrates and sialic acid in its structure, with M r ¼ 34,000 under reducing conditions and pI w 6.4. The N-terminal sequence of the enzyme (VIGGDECDINEHPFL) showed high similarity with other thrombin-like enzymes from snake venoms. BpSP-I showed high clotting activity upon bovine and human plasma and was inhibited by PMSF, benzamidine and leupeptin. Moreover, this enzyme showed stability when examined at different temperatures (À70 to 37 C), pH values (3-9) or in the presence of divalent metal ions (Ca 2þ , Mg 2þ , Zn 2þ and Mn 2þ ). BpSP-I showed high catalytic activity upon substrates, such as fibrinogen, TAME, S-2238 and S-2288. It also showed kallikrein-like activity, but was unable to act upon factor Xa and plasmin substrates. Indeed, the enzyme did not induce hemorrhage, myotoxicity or edema. Taken together, our data showed that BpSP-I is in fact a thrombin-like enzyme isoform isolated from Bothrops pauloensis snake venom.

Paramyxoviruses are the leading cause of respiratory disease in children. Several paramyxoviruses possess a surface glycoprotein, the hemagglutinin-neuraminidase (HN), that is involved in attachment to sialic acid receptors, promotion of fusion, and removal of sialic acid from infected cells and progeny virions. Previously we showed that Newcastle disease virus (NDV) HN contained a pliable sialic acid recognition site that could take two states, a binding state and a catalytic state. Here we present evidence for a second sialic acid binding site at the dimer interface of HN and present a model for its involvement in cell fusion. Three different crystal forms of NDV HN now reveal identical tetrameric arrangements of HN monomers, perhaps indicative of the tetramer association found on the viral surface.

S. α s" -, α s# -, βand κ-caseins from Somali camels (Camelus dromedarius) were purified by acid precipitation at pH 4n4, crudely separated into an α-CN and a β-CN fraction and further purified by reversed-phase HPLC. Fragments of tryptic digests were sequenced. Amino acid patterns obtained were used to screen a cDNA library constructed from mRNA from lactating udder tissue. Full length clones corresponding to the four caseins were sequenced. The numbers of residues in the sequences deduced were α s" -CN 207, α s# -CN 178, β-CN 217, κ-CN 162. Percentage similarity to bovine proteins was α s" -CN A 39, α s# -CN 56, β-CN 64, κ-CN 56. Acidprecipitated casein of pooled milk was separated by reversed-phase HPLC and monitored at 220 nm, and its composition, estimated from peak integration, was (g\kg total casein) α s" -CN 220, α s# -CN 95, β-CN 650, κ-CN 35. Degrees of phosphorylation and glycosylation were determined by laser ionization mass spectrometry and sequence pattern analysis. Molecular masses determined were (kDa) α s" -CN A, 24n755 and 24n668 ; α s" -CN B, 25n293 ; α s# -CN 21n993 ; β-CN, 24n900 ; κ-CN 22n294-22n987. The pH values of the most probable isoelectric points were : α s" -CN A 6P 4n41, α s" -CN B 6P 4n40, α s# -CN 9P 4n58, β-CN 4P 4n66, κ-CN 1P, with ten sialic acid residues bound, 4n10.

Drying is one of the essential processing steps for dried edible bird’s nest; however, sialic acid and
antioxidant can be highly thermosensitive and unstable. Therefore, aim of this study was to
determine the degradation kinetics of sialic acid and antioxidants during low-temperature drying at
25–40°C as compared to conventional hot air-drying at 70°C. These compounds’ degradation
exhibited first-order kinetics. Sialic acid and antioxidant retentions were 83.9 and 96.6%,
respectively, at 25°C, and 78.7 and 91.5% at 40°C, respectively, by low-temperature drying; while,
42.5 and 38.7%, respectively, at 70°C by conventional hot air-drying. Finally, empirical models were
significantly fitted to predict sialic acid and antioxidant retention as edible bird’s nest reached a
certain level of drying, which may be useful from the processing standpoint and validate the usage
of low-temperature drying as a process tool for retention of sialic acid and antioxidant in edible
bird’s nest.

Sphingolipids were discovered more than a century ago in the brain. Cerebrosides and sphingomyelins were named so because they were first isolated from neural tissue. Although glycosphingolipids and especially those containing sialic acid in their oligosaccharide moiety are particularly abundant in the brain, sphingolipids are ubiquitous cellular membrane components. They form cell-and species-specific profiles at the cell surfaces that characteristically change in development, differentiation, and oncogenic transformation, indicating the significance of these lipid molecules for cell-cell and cell-matrix interactions as well as for cell adhesion, modulation of membrane receptors and signal transduction. This review summarizes sphingolipid metabolism with emphasis on aspects particularly relevant in neural cell types, including neurons, oligodendrocytes and neuroblastoma cells. In addition, the reader is briefly introduced into the methodology of lipid evaluation techniques and also into the putative physiological functions of glycosphingolipids and their metabolites in neural tissue.

The remarkable similarity among the genomes of humans and the African great apes could warrant their classification together as a single genus. However, whereas there are many similarities in the biology, life history, and behavior of humans and great apes, there are also many striking differences that need to be explained. The complete sequencing of the human genome creates an opportunity to ask which genes are involved in those differences. A logical approach would be to use the chimpanzee genome for comparison and the other great ape genomes for confirmation. Until such a great ape genome project can become reality, the next best approach must be educated guesses of where the genetic differences may lie and a careful analysis of differences that we do know about. Our group recently discovered a human-specific inactivating mutation in the CMP-sialic acid hydroxylase gene, which results in the loss of expression of a common mammalian cell-surface sugar throughout all cells in the human body. We are currently investigating the implications of this difference for a variety of issues relevant to humans, ranging from pathogen susceptibility to brain development. Evaluating the uniqueness of this finding has also led us to explore the existing literature on the broader issue of genetic differences between humans and great apes. The aim of this brief review is to consider a listing of currently known genetic differences between humans and great apes and to suggest avenues for future research. The differences reported between human and great ape genomes include cytogenetic differences, differences in the type and number of repetitive genomic DNA and transposable elements, abundance and distribution of endogenous retroviruses, the presence and extent of allelic polymorphisms, specific gene inactivation events, gene sequence differences, gene duplications, single nucleotide polymorphisms, gene expression differences, and messenger RNA splicing variations. Evaluation of the reported findings in all these categories indicates that the CMP-sialic hydroxylase mutation is the only one that has so far been shown to result in a global biochemical and structural difference between humans and great apes. Several of the other known genetic dissimilarities deserve more exploration at the functional level. Among the areas of focus for the future should be genes affecting development, mental maturation, reproductive biology, and other aspects of life history. The approaches taken should include both going from the genome up to the adaptive potential of the organisms and going from novel adaptive regimes down to the relevant repercussions in the genome. Also, as much as we desire a simple genetic explanation for the human phenomenon, it is much more probable that our evolution occurred in multiple genetic steps, many of which must have left detectable footprints in our genomes. Ultimately, we need to know the exact number of genetic steps, the order in which they occurred, and the temporal, spatial, environmental, and cultural contexts that determined their impact on human evolution.

β-amyloid (Aβ) is the primary protein component of senile plaques in Alzheimer's disease and is believed to be associated with neurotoxicity in the disease. We and others have shown that Aβ binds with relatively high affinity to clustered sialic acid residues on cell surfaces and that removal of cell surface sialic acids attenuate Aβ toxicity. We have also shown that sialic acid functionalized dendrimeric polymers can act as mimics of cell surface sialic acid clusters and attenuate Aβ induced neurotoxicity. In the current study, we prepared sialic acid conjugated dendrimers using a physiologically relevant attachment of the sialic acid to the dendrimeric termini, and evaluated the Aβ toxicity attenuation properties of the dendrimers. We compared performance of sialic acid conjugated dendrimeric polymers in which the sialic acid moeties were attached to dendrimeric termini via the anomeric hydroxyl group of the sialic acid, a physiological attachment, to polymers in which the attachment was made via the carboxylic acid group on the sialic acid, a non-physiological attachment. This work enhances our understanding of Aβ-cell surface binding and is a step towards the development of new classes of sequestering agents as therapeutics for the prevention of Aβ toxicity in AD.

Electronegative LDL (LDL(-)) constitutes a plasma subfraction of LDL with proinflammatory properties. Its proportion is increased in familial hypercholesterolemia (FH); however, the characteristics of LDL(-) isolated from FH subjects have not been previously studied. In this work, the composition, oxidative status, and inflammatory capacity on endothelial cells of LDL(-) from FH and normolipemic (NL) subjects were evaluated. LDL(-) from FH was relatively enriched in esterified and free cholesterol and triglyceride, and had lower apoB and phospholipid content compared with the non-electronegative fraction (LDL(+)). LDL(-) also contained increased amounts of apoE, apoC-III, sialic acid, and non-esterified fatty acids (NEFAs). The same was observed in NL subjects, except that esterified cholesterol and phospholipid were similar in LDL(-) and LDL(+). No difference was observed between the two fractions concerning malondialdehyde, fatty acid hydroxides, and antioxidants, thereby indicating the absence of increased oxidation of LDL(-) compared with LDL(+). When LDL(-) (100 mg/l) from NL and FH subjects was incubated for 24 h with human umbilical vein endothelial cells (HUVECs), interleukin 8 (IL-8) and monocyte chemotactic protein 1 (MCP-1) increased twofold in the culture medium compared with LDL(+). Vascular cell adhesion molecule 1 (VCAM-1) expression was not increased by LDL(-). Our data indicate that LDL(-) from FH or NL subjects shows no evidence of increased oxidative modification compared to LDL(+); however, LDL(-) induces twofold the release of chemokines by endothelial cells. This effect, which may contribute to leukocyte recruitment and promote atherogenesis, may be greater in FH subjects in which LDL(-) can be up to eightfold higher than in NL subjects.

Homozygous cattle transferrin has been fractionated into six main peaks by DEAE-Sephadex chromatography. These correspond to the six transferrin components seen in starch gel electrophoresis of normal serum. In addition, six more minor components were isolated from DEAE-Sephadex, making 12 in all, and these could be divided into six pairs. Treatment of whole transferrin with neuraminidase yielded only two bands. Treatment of the individual fractionated bands showed that the slower band of each pair had the same mobility as the slow band from treated whole transferrin, while the faster band from each pair corresponded with the fast band of treated whole transferrin. These observations, and the results of sialie acid assays, showed that the difference between the pairs of bands was caused by differing numbers of sialic acid residues (0-5)per molecule of protein, but that sialic acid was not responsible for the difference between the bands within a pair. The mode of genetic control is discussed and probably involves three loci. Other physicoehemical properties of cattle transferrins, namely, molecular weight, effect of iron addition, and behavior in isoelectrie focusing, were also studied.

Humans face a constant threat from pathogens like influenza varieties H1N1, H5N1, and others and there is a need to prevent these from epidemics. The pathogens depend on successful colonization of the host in order to reproduce and multiply. Sialidases are known as neuraminidases are a group of enzymes, the most abundant of these being the exo-sialidases that can catalyze the cleavage of sialic acids from carbohydrates, glycoproteins or glycolipids. Sialidases have been thoroughly studied since their discovery 75 years ago and their occurrence in bacteria and viruses is widespread. They are found in diverse virus families and bacteria and other microbes. Moreover, sialic acids serve as a receptor for various pathogens. This allows bacteria like H1N1 or other influenza viruses, to enter the host cell. There is a need to block sialidases as they release sialic acid that serves as nutrition for the microbes and as well allows them to bind and invade the host cell where they can proliferate. This makes sialidases an interesting target to control pathogenic activity. Metadichol ® is nanoemulsion of long-chain lipid alcohols derived from food ingredients. In rats, it has an LD50 of 5000 mg/kilo and its ingredients are present in many foods we consume on a daily basis. It has antiviral and antibacterial and anti-parasitic properties. We studied inhibition of Sialidases by inducing it with Lipopolysaccharide (LPS) using THP1 cells. Metadichol showed inhibition at 1 picogram per ml to 1 nanogram per/ml. Compared to Prednisone. It is 100 times more active. Previous studies on Metadichol ® showed that it is toxic to cancer cells at higher concentrations. Since it is safer, it has the potential of being directly tested on humans without side effects and could have a potential role in mitigating the pathogens that a burden on the Public health system.

The present study investigated aspects of lipid and carbohydrate metabolism in Sparus aurata semen and tested the effect of lipids, carbohydrates and related metabolites on sperm viability using in vitro incubation experiments. Sparus aurata semen contained enzyme systems to metabolize sugars and lipids. Also key enzymes of the tricarboxylic acid cycle and enzymes involved in ATP metabolism were detected. When spermatozoa were incubated in sperm motility inhibiting saline solution for 48 h phospholipid levels decreased constantly and triglycerides levels during the first 24 h of incubation indicating that spermatozoa utilize lipids as energy resources. After 24 h triglycerides levels started to re-increase indicating a change in sperm metabolism, in particular the onset of triglycerides synthesis by the fatty acid synthase complex. In the incubation period from 0 to 24 h glucose levels were constant, and decreased thereafter. Glycogen levels did not change at all. Semen contained also considerable amounts of sialic acid, glucuronic acid and hexosamines, components of mucopolysaccharides. To find out whether lipids, carbohydrates, and related metabolites had a positive effect on sperm functionality semen was incubated together with the described compounds in sperm motility inhibiting saline solution and motility when activated was determined. In the control 37.2 ± 10.1% of the spermatozoa were locally motile and 38.3 ± 13.3% motile after 24 h, 36.4 ± 5.2% were locally motile and 9.6 ± 4.5% were motile after 48 h. The swimming velocity was 89.0 ± 13.1 µm/s after 24 h and 61.3 ± 12.6% after 48 h. Different types of lipids (arachidic acid, linoleic acid, and glycerol trimyristate) and metabolites acting as fuel for the tricarboxylic acid cycle (hydroxybutyrate, ketoglutarate, and pyruvate) had a positive effect on the sperm viability. Tested carbohydrates (fucose, galactose, glucosamine, glucose, glucoheptose, glycogen, and sialic acid) had no effect. Also lactate and fructose-6phosphate had no effect on sperm viability while glucose-6-phosphate, oxalacetate, and phosphoglycerate had negative effects.

Our results show that if the 2-thiobarbituric acid concentration is decreased, its co-precipitation with the chromophore is diminished. Subsequent running of this reaction mixture by highperformance liquid chromatography still allows measurement of Neu-5-Ac in the picomole order, with a substantial time and reactive saving, as compared with the original assay. © 1997 Elsevier Science B. V.

Complex biomolecular machinery carrying diverse glycan chains are involved in a wide range of physiological activities including blood group determination, cancer recognition protein stabilization and sperm-egg interaction. Diversity of glycan chains, linked to lipids and proteins is due to isomeric and conformational modifications of various sugar residues, giving rise to unique carbohydrate structures with a wide range of anomeric linkages. This unique and significant structural diversity of naturally occurring oligosaccharide structures make them the best recognition markers for countless physiological activities. This is a challenging task to explore the relationship between biological processes and stereochemical behavior of sugar residues. Current review article

Sialic acid, oral glucose and plasma lipids were determined in 70 control subjects age range 20-60 and 150 individuals of age range 20-60 who were offspring of one type 2 diabetic parent. The number of first degree relatives who had impaired glucose tolerance were 50. The total sialic acid concentration was significantly higher (p&lt;0.05) in the first degree relatives when compared to the control subjects. However the total sialic acid concentration was significantly higher in the offspring with normal glucose tolerance than those with impaired glucose tolerance. There was no significant difference in the lipids between the control subjects and the offspring with normal glucose tolerance. But the total cholesterol and triglycerides were significantly higher in the offspring with impaired glucose tolerance when compared to the control subjects and the offspring with normal glucose tolerance. Our study shows that the desialylation of the vascular endothelium is an early event that pr...

Purpose: Many biochemical processes are closely related to ion exchange, adsorption, and catalysis. Zeolites reversibly bind small molecules such as oxygen or nitric oxide; they possess size and shape selectivity, the possibility of metalloenzyme mimicry, and immunomodulatory activity. These properties make them interesting for pharmaceutical industry and medicine. Methods: The experiments were performed on mice. Dierent biochemical and molecular methods were used. Results: Micronized zeolite (MZ) administered by gastric intubation to mice injected with melanoma cells signi®cantly reduced the number of melanoma metastases. In mice fed MZ for 28 days, concentration of lipid-bound sialic acid (LSA) in serum increased, but lipid peroxidation in liver decreased. The lymphocytes from lymph nodes of these mice provoked a signi®cantly higher alogeneic graft-versus-host (GVH) reaction than cells of control mice. After i.p. application of MZ, the number of peritoneal macrophages, as well as their production of superoxide anion, increased. However, NO generation was totally abolished. At the same time, translocation of p65 (NFjB subunit) to the nucleus of splenic cells was observed. Conclusion: Here we report antimetastatic and immunostimulatory eect of MZ and we propose a possible mechanism of its action.

Sialidases are glycohydrolytic enzymes present from virus to mammals that remove sialic acid from oligosaccharide chains. Four different sialidase forms are known in vertebrates: the lysosomal NEU1, the cytosolic NEU2 and the membrane-associated NEU3 and NEU4. These enzymes modulate the cell sialic acid content and are involved in several cellular processes and pathological conditions. Molecular defects in NEU1 are responsible for sialidosis, an inherited disease characterized by lysosomal storage disorder and neurodegeneration. The studies on the biology of sialic acids and sialyltransferases, the anabolic counterparts of sialidases, have revealed a complex picture with more than 50 sialic acid variants selectively present in the different branches of the tree of life. The gain/loss of specific sialoconjugates have been proposed as key events in the evolution of deuterostomes and Homo sapiens, as well as in the host-pathogen interactions. To date, less attention has been paid to the evolution of sialidases. Thus we have conducted a survey on the state of the sialidase family in metazoan. Using an in silico approach, we identified and characterized sialidase orthologs from 21 different organisms distributed among the evolutionary tree: Metazoa relative (Monosiga brevicollis), early Deuterostomia, precursor of Chordata and Vertebrata (teleost fishes, amphibians, reptiles, avians and early and recent mammals). We were able to reconstruct the evolution of the sialidase protein family from the ancestral sialidase NEU1 and identify a new form of the enzyme, NEU5, representing an intermediate step in the evolution leading to the modern NEU3, NEU4 and NEU2. Our study provides new insights on the mechanisms that shaped the substrate specificity and other peculiar properties of the modern mammalian sialidases. Moreover, we further confirm findings on the catalytic residues and identified enzyme loop portions that behave as rapidly diverging regions and may be involved in the evolution of specific properties of sialidases.

The clostridial neurotoxins include the closely related tetanus (TeNT) and botulinum (BoNT) toxins. Botulinum toxin is used to treat severe muscle disorders and as a cosmetic wrinkle reducer. Large quantities of botulinum toxin have also been produced by terrorists for use as a biological weapon. Because there are no known antidotes for these toxins, they thus pose a potential threat to human health whether by an accidental overdose or by a hostile deployment. Thus, the discovery of high specificity and affinity compounds that can inhibit their binding to neural cells can be used as antidotes or in the design of chemical detectors. Using the crystal structure of the C fragment of the tetanus toxin (TetC), which is the cell recognition and cell surface binding domain, and the computational program DOCK, sets of small molecules have been predicted to bind to two different sites located on the surface of this protein. While Site-1 is common to the TeNT and BoNTs, Site-2 is unique to TeNT. Pairs of these molecules from each site can then be linked together synthetically to thereby increase the specificity and affinity for this toxin. Electrospray ionization mass spectroscopy was used to experimentally screen each compound for binding. Mixtures containing binders were further screened for activity under biologically relevant conditions using nuclear magnetic resonance (NMR) methods. The screening of mixtures of compounds offers increased efficiency and throughput as compared to testing single compounds and can also evaluate how possible structural changes induced by the binding of one ligand can influence the binding of the second ligand. In addition, competitive binding experiments with mixtures containing ligands predicted to bind the same site could identify the best binder for that site. NMR transfer nuclear Overhauser effect (trNOE) confirm that TetC binds doxorubicin but that this molecule is displaced by N-acetylneuraminic acid (sialic acid) in a mixture that also contains 3-sialyllactose (another predicted site 1 binder) and bisbenzimide 33342 (nonbinder). A series of five predicted Site-2 binders were then screened sequentially in the presence of the Site-1 binder doxorubicin. These experiments showed that the compounds lavendustin A and naphthofluorescein-di-(P-D-galactopyranoside) binds along with doxorubicin to TetC. Further experiments indicate that doxorubicin and lavendustin are potential candidates to use in preparing a bidendate inhibitor specific for TetC. The simultaneous binding of two different predicted Site-2 ligands to TetC suggests that they may bind multiple sites. Another possibility is that the conformations of the binding sites are dynamic and can bind multiple diverse ligands at a single site depending on the pre-existing conformation of the protein, especially when doxorubicin is already bound.

Updated information and services can be found at: These include: REFERENCES http://iai.asm.org/content/68/7/3894#ref-list-1 at: This article cites 34 articles, 17 of which can be accessed free CONTENT ALERTS more» articles cite this article), Receive: RSS Feeds, eTOCs, free email alerts (when new http://journals.asm.org/site/misc/reprints.xhtml Information about commercial reprint orders: http://journals.asm.org/site/subscriptions/ To subscribe to to another ASM Journal go to: on February 26, 2014 by guest http://iai.asm.org/ Downloaded from on February 26, 2014 by guest

Background: Sialic acid has been shown to be a major virulence determinant in the pathogenesis of otitis media caused by the bacterium Haemophilus influenzae. This study aimed to characterise the expression of genes required for the metabolism of sialic acid and to investigate the role of these genes in virulence. Results: Using qRT-PCR, we observed decreased transcriptional activity of genes within a cluster that are required for uptake and catabolism of 5-acetyl neuraminic acid (Neu5Ac), when bacteria were cultured in the presence of the sugar. We show that these uptake and catabolic genes, including a sialic acid regulatory gene (siaR), are highly conserved in the H. influenzae natural population. Mutant strains were constructed for seven of the nine genes and their influence upon LPS sialylation and resistance of the bacteria to the killing effect of normal human serum were assessed. Mutations in the Neu5Ac uptake (TRAP transporter) genes decreased virulence in the chinchilla model of otitis media, but the attenuation was strain dependent. In contrast, mutations in catabolism genes and genes regulating sialic acid metabolism (siaR and crp) did not attenuate virulence. Conclusion: The commensal and pathogenic behaviour of H. influenzae involves LPS sialylation that can be influenced by a complex regulatory interplay of sialometabolism genes.

OBJETIVO: Caracterizar la hemorragina aislada de B. brazili y determinar si el suero antibotrópico polivalente es capaz de neutralizarlo. MATERIAL Y MÉTODOS: Se ha purificado una hemorragina del veneno de Bothrops brazili a través de dos pasos cromatográficos en Sephadex G-100 y CM Sephadex C-50, usando buffer acetato de amonio 0,05M pH 7. En este último sistema, la proteína fue eluida con NaCl 0,3M y el análisis electroforético en PAGE-SDS mostró una sola banda proteica. Usando caseína como substrato se determinó una purificación de 8,4 veces, habiéndose calculado la dosis hemorrágica mínima (DHM) en 6,61 ug usando ratones albinos. RESULTADOS: El análisis de carbohidratos asociados a la hemorragina reveló un contenido de 8,05% de hexosas, 11,62% de hexosamina y 0,69% de ácido siálico, en tanto que su resistencia térmica fue registrada hasta 50°C por 10 minutos, ya que temperaturas mayores la inactivaron. La hemorragina fue muy inestable a pH 5,0 mientras que el tratamiento con EDTA, ácido glutámico y glutatión 5 mM también produjeron una severa inhibición, tanto en su acción hemorrágica como en su actividad caseinolítica. En cambio, por adición de iones calcio (10mM) se incrementó la intensidad del área hemorrágica. CONCLUSIONES: Los ensayos de inmunodifusión e inmunoelectroforesis demostraron que el suero antibotrópico polivalente (Instituto Nacional de Salud [INS] -Lima) reconoció a la fracción hemorrágica y las pruebas de neutralización in vivo dieron como resultado la inhibición del veneno crudo, pero no de la hemorragina purificada. Palabras clave: Botropos; veneno de crotálidos; viperidae; inmunodifusión; inmunoelectrofóresis.

by human antibodies. antigen 175 polypeptides and their recognition Plasmodium falciparum erythrocyte binding Baculovirus-mediated expression of http://iai.asm.org/content/65/9/3631 Updated information and services can be found at: These include: CONTENT ALERTS more» cite this article), Receive: RSS Feeds, eTOCs, free email alerts (when new articles http://journals.asm.org/site/misc/reprints.xhtml Information about commercial reprint orders:

Benzoic acid and pyridine derivatives inhibit recombinant trans-sialidase from Trypanosoma cruzi with I 50 values between 0.4 and 1 mM. The best compounds, 4-acetylamino-3-hydroxymethylbenzoic acid and 5-acetylamino-6-aminopyridine-2-carboxylic acid, provide new leads to inhibitors not containing the synthetically complex sialic acid structure. The weak inhibition by such compounds contrasts with their much stronger inhibition of neuraminidase from Influenza virus.

Gangliosides from livers of weanling rats were analyzed after 15% partial hepatectomy (PH) and different pre-and post-operative hyberbaric oxygenation (pre-and postHBO). Neu5Ac was the predominant ganglioside-derived sialic acid (>85%) compared to Neu5Gc. Almost identical low total sialic acid content (Neu5Ac + Neu5Gc) of the control and operated nonHBO animals opposed a 6.4-to 7.6-fold increase in pre-and postHBO animals (69.26 and 81.64 pmol/mg wet weight, respectively). NanoESI-QTOF mass spectrometry combined with HPTLC immunostaining revealed GM3(Neu5Ac) and GM3(Neu5Gc) as major gangliosides, correlating with the respective sialic acid concentrations. Minor neolacto-series gangliosides were enhanced in preHBO and postHBO, but GM1-core gangliosides only in preHBO rats. GM2 and GalNAc-GM1b were clearly detectable in oxygenated rats compared to traces in the control and nonHBO animals. These results point at a functional role of gangliosides in liver growth regulation and reconstitution after PH combined with pre-and post-operative HBO treatment.

Avian influenza A viruses exhibit a strong preference for using a2,3-linked sialic acid as a receptor. Until recently, the presumed lack of this receptor in human airways was believed to constitute an efficient barrier to avian influenza A virus infection of humans. Recent zoonotic outbreaks of avian influenza A virus have triggered researchers to analyse tissue distribution of sialic acid in further detail. Here, we review and extend the current knowledge about sialic acid distribution in human tissues, and discuss viruses with ocular tropism and their preference for a2,3linked sialic acid.

Trypanosoma cruzi, the agent of Chagas disease, expresses a unique enzyme, the trans-sialidase (TcTS) involved in the transfer of sialic acid from host glycoconjugates to mucins of the parasite. The enzyme is shed to the medium and may affect the immune system of the host. We have previously described that lactose derivatives effectively inhibited the transfer of sialic acid to Nacetyllactosamine. Lactitol also prevented the apoptosis caused by the TcTS, although it is rapidly eliminated from the circulatory system. In this paper we report covalent conjugation of polyethylene glycol (PEG) with lactose, lactobionolactone and benzyl β-D-galactopyranosyl-(1→6)-2-amino-2-deoxy-α-D-glucopyranoside (1) with the hope to improve the bioavailability, though retaining their inhibitory properties. Different conjugation methods have been used and the behavior of the PEGylated products in the TcTS reaction was studied.