Sialic Acid Sugar Research Articles

Total sialic acid, total protein and total sugar levels in serum and saliva of oral squamous cell carcinoma patients: A case control study.

Background:Detection of cancer at an early stage is of utmost importance to decrease the morbidity and mortality of the disease. Apart from the conventional biopsy, non-invasive methods like analysis of serum and saliva may provide cost-effective approach for screening a large population. Tumor markers are a major part of secondary prevention and thus, the detection of malignancies. The aim of this study was to evaluate total sialic acid (TSA), total protein and total sugar (TS) in serum and saliva of oral squamous cell carcinoma (OSCC) and controls to assess their role as a diagnostic marker.Materials and Methods:Unstimulated whole saliva and sera were collected from 40 squamous cell carcinoma patients and 20 controls. Serum and salivary TSA, total protein and TS estimation was carried out. This was correlated with clinical stages and histopathological grades of OSCC. The data obtained was analyzed statistically using Chi-square test, ANOVA and Student's t-test with SPSS statistical software.Results:A highly significant rise in the salivary sialic acid, serum sialic acid and serum protein was noted in OSCC subjects compared to controls. Salivary protein, serum and salivary sugar did not show any significance. Furthermore, serum and salivary sialic acid levels were found to be significantly increased with increasing level of histopathological grading.Conclusion:The present study showed a significant increase in serum sialic acid, salivary sialic acid and serum protein from control to OSCC and suggests that these markers may be reliable in diagnosis and predicting prognosis.

Fine-tuning the specificity of boronate affinity monoliths toward glycoproteins through pH manipulation

Boronate affinity functionalized materials have recently drawn increasing attention due to their capability to selectively isolate and enrich glycoproteins and glycopeptides. As cheaper and more stable competitors to lectins, boronic acids are generally believed to yield a relatively wider spectrum specificity to glycoproteins. For better understanding and effective utilization of boronate affinity, it is necessary to establish if boronic acids exhibit lectin-like narrow specificity towards individual or a sub-class of glycoproteins. Here we report a pH manipulation strategy for fine-tuning the specificity of boronate affinity monoliths towards two sub-classes of glycoproteins, sialylated and nonsialylated glycoproteins. When the binding pH > the pK(a) of the boronic acid by one pH unit or more, the boronate affinity monolith preferentially binds to glycoproteins containing neutral sugars and excludes sialic acid containing glycoproteins due to electrostatic repulsion. When the binding pH < the pK(a) by one pH unit or more, the boronate affinity monolith binds to sialylated glycoproteins due to the exceptional binding affinity of the boronic acid towards sialic acid residues. The alternative specificity towards sialic acid and neutral sugar was first verified using an off-line combination of boronate affinity extraction with nano-ESI-Orbitrap MS/MS detection. The alternative specificity towards sialylated and nonsialylated glycoproteins was then demonstrated by means of off-line combination of boronate affinity extraction with MALDI-TOF MS. Finally, the developed approach was applied to the alternative extraction of intact sialylated and nonsialylated glycoproteins spiked in human serum.

Jatropha curcas hemagglutinin is similar to a 2S albumin allergen from the same source and has unique sugar affinities

We have previously reported the purification and preliminary X-ray characterization of a hemagglutinin from the seeds of Jatropha curcas and, with the detailed sequencing information available now, we find that it is similar to a 2S albumin allergen isolated from the same source. Through a search of Jatropha genome database (http://www.kazusa.or.jp/jatropha/), we map it to the sequence id JcCA0234191 (now referred to as Jcr4S00619.70 in the new version, release 4.5) which has a conserved alpha amylase inhibitor/seed storage protein domain found in the 2S albumin allergens. The putative sequence of the small and large chains of the protein is assigned and the total mass of the two subunits matches with the intact mass 10 kDa determined through MALDI. The protein retains hemagglutination activity between pH 6-9 and up to 60 °C on heat treatment and its hemagglutination activity is inhibited by sialic acid and fetuin. Bioinformatics studies show that the isolated protein sequence clusters in close association with a 2S albumin from Ricinus communis in phylogeny analysis and has a conservation of the characteristic four disulfide linkage pattern. Hemagglutinins and lectins are known to have allergenic effects through their interaction with immunoglobulin E and histamine release and earlier studies have shown that this interaction can be inhibited by lectin-specific sugars. We hope this report bridges the plant allergens and hemagglutinins further for exploring possible mediation of allergenic activity through sialic acid and complex sugar interactions and generates further interest in the area.

Inhibition of Aspergillus fumigatus conidia binding to extracellular matrix proteins by sialic acids: a pH effect?

Infection by Aspergillus fumigatus, which causes the life-threatening disease invasive aspergillosis, begins with the inhalation of conidia that adhere to and germinate in the lung. Previous studies have shown that A. fumigatus conidia express high levels of the negatively charged 9-carbon sugar sialic acid, and that sialic acid appears to mediate the binding of A. fumigatus conidia to basal lamina proteins. However, despite the ability of sialic acid to inhibit adherence of A. fumigatus conidia, the exact mechanism by which this binding occurs remains unresolved. Utilizing various free sialic acids and other carbohydrates, sialic acid derivatives, sialoglycoconjugates, glycoproteins, alpha-keto acid related compounds and amino acids we have found that the binding of A. fumigatus conidia to type IV collagen and fibrinogen was inhibited by (i) glycoproteins (in a sialic acid-independent manner), and (ii) free sialic acids, glucuronic acid and alpha-keto acid related compounds. However, inhibition by the latter was found to be the result of a shift in pH from neutral (pH 7.4) to acidic (less than pH 4.6) induced by the relatively high concentrations of free sialic acids, glucuronic acid and alpha-keto acid related compounds used in the binding assays. This suggests that previous reports describing inhibition of A. fumigatus conidia binding by free sialic acid may actually be due to a pH shift similar to that shown here. As previously reported, we found that A. fumigatus conidia express only N-acetylneuraminic acid, the most common sialic acid found in nature. However, A. fumigatus appears to do so by an alternative mechanism to that seen in other organisms. We report here that A. fumigatus (i) does not incorporate sialic acid obtained from the environment, (ii) does not synthesize and incorporate sialic acid from exogenous N-acetylmannosamine, and (iii) lacks homologues of known sialic acid biosynthesizing enzymes.

Portrait of an Enzyme, a Complete Structural Analysis of a Multimodular β-N-Acetylglucosaminidase from Clostridium perfringens

Common features of the extracellular carbohydrate-active virulence factors involved in host-pathogen interactions are their large sizes and modular complexities. This has made them recalcitrant to structural analysis, and therefore our understanding of the significance of modularity in these important proteins is lagging. Clostridium perfringens is a prevalent human pathogen that harbors a wide array of large, extracellular carbohydrate-active enzymes and is an excellent and relevant model system to approach this problem. Here we describe the complete structure of C. perfringens GH84C (NagJ), a 1001-amino acid multimodular homolog of the C. perfringens micro-toxin, which was determined using a combination of small angle x-ray scattering and x-ray crystallography. The resulting structure reveals unprecedented insight into how catalysis, carbohydrate-specific adherence, and the formation of molecular complexes with other enzymes via an ultra-tight protein-protein interaction are spatially coordinated in an enzyme involved in a host-pathogen interaction.

Lectin-based proteomic profiling of aged skeletal muscle: Decreased pyruvate kinase isozyme M1 exhibits drastically increased levels of N-glycosylation

Since various neuromuscular diseases are associated with abnormal glycosylation, it was of interest to determine whether this key post-translational modification is also altered in aged skeletal muscle. Lectins represent highly versatile carbohydrate-binding proteins that are routinely employed for the characterization of glycoproteins. Here, we used the lectin wheat germ agglutinin (WGA) for the proteomic profiling of senescent fibers. WGA labeling of the soluble proteome from 3-month- versus 30-month-old rat gastrocnemius muscle, following two-dimensional gel electrophoretic separation, resulted in the identification of 13 distinct protein species. Analysis of WGA binding levels, in conjunction with mass spectrometric fingerprinting, revealed that one isoform of a major metabolic muscle protein exhibited a drastic alteration in the content of sialic acid and N-acetylglucosaminyl sugar residues. Pyruvate kinase isoform M1 with protein accession number gi|16757994|, exhibiting a pI of 6.6 and an apparent molecular mass of 57.8 kDa, showed a six fold increase in N-glycosylation and a three fold decrease in protein expression. In contrast to comparable levels of N-glycosylated proteins in young adult versus senescent muscle, as judged by fluorescein-conjugated WGA labeling of transverse muscle cryosections, staining with antibodies to the M1 isoform of pyruvate kinase showed reduced expression of this cytosolic element. Furthermore, activity assays demonstrated a reduced activity of this glycolytic enzyme in senescent muscle. This agrees with the idea that abnormal post-translational modifications in key metabolic enzymes may be involved in the conversion of aged muscle to slower twitch patterns and a drastic shift to more aerobic-oxidative metabolism.

SUGAR LINK CALMS INFLAMMATION

RESEARCHERS HAVE IDENTIFIED a specific sugar linkage on an antibody that is responsible for the antibody’s anti-inflammatory activity, a discovery that may lead to improved treatments for autoimmune diseases ( Science 2008, 320 , 373). Physicians sometimes soothe inflammation in patients with lupus or rheumatoid arthritis with an antibody treatment called intravenous immunoglobulin G. IVIG is actually a mixture of antibodies modified with different branched sugar chains, and patients need high doses to relieve inflammation. Immunologist Jeffrey V. Ravetch from Rockefeller University and colleagues from the University of New Hampshire and the Scripps Research Institute have now shown that anti-inflammatory activity depends on how terminal sialic acid sugars in IVIG are linked to the next sugar in the chain. The scientists used that information to generate a synthetic antibody that relieves inflammation in arthritic mice at a dose 30 times lower than IVIG. In previous work, Ravetch’s team showed that only a...

Evaluation of salivary sialic acid, total protein, and total sugar in oral cancer: A preliminary report

Detection of cancer at the early stage is of utmost importance to decrease the morbidity and mortality of the disease. Apart from the conventional biopsy, noninvasive methods like analysis of saliva may provide a cost-effective approach for screening a large population. Thus, this study aimed to estimate salivary levels of sialic acid, total protein, and total sugar in the oral cancer patients and in healthy control group to evaluate their role in diagnosis and prognosis of oral cancer. Unstimulated whole saliva samples were collected from 30 healthy controls (Group I) and 30 squamous cell carcinoma patients (group II). Estimations of salivary levels of sialic acid, total protein, and total sugar were performed. This was correlated histopathologically with the grades of carcinoma. The Student's ' t ' test and multivariate regression analysis were performed. The results showed that salivary levels of total protein, total sugar, protein-bound sialic acid, and free sialic acid were significantly higher in oral cancer patients compared to those of normal healthy controls ( P values in all the results were less than 0.001). The salivary free sialic acid levels were found to be significantly higher in well-differentiated squamous cell carcinoma than in moderately differentiated carcinoma ( P < 0.001). However, protein-bound sialic acid, total proteins, and total sugars did not show any statistical significance between well and moderately differentiated carcinomas. Biochemical analysis of saliva can be used in early detection of cancer and is best correlated with histopathological degree of squamous cell carcinoma.

Mucous domains: microchemical heterogeneity in the mucociliary complex of the olfactory epithelium.

Access to and clearance of odorants from binding sites on olfactory cilia are regulated by a complex interplay of molecular, physical and cellular factors. These perireceptor events occur primarily in the mucociliary complex. The use of gold-labelled lectinoprobes, one from Limax flavus (LFA) which is specific for terminal sialic acid residues, and one from Datura stramonium (DSA) specific for N-acetylglucosamine residues, demonstrated intricate patterns of binding in mucous domains of the olfactory mucus and ectodomains of the glycocalyx of olfactory cilia. In electron micrographs of Lowicryl-embedded salamander olfactory mucosa, the mucus consisted of an electron-dense domain that lay superficial to an electron-lucent domain; the interface between the two was irregular. A significantly higher density of binding sites for both lectins was present in the superficial than in the deeper domain. The two domains were not homogeneous: there were small electron-lucent domains (hsL) within the superficial electron-dense domain (hsD) that bound a 4.8-fold lower density of gold-labelled DSA than the surrounding matrix, and the olfactory cilia, which project into hsD, were surrounded by an electron-lucent sheath that appeared to be continuous with the deeper domain. Ectodomains of the glycocalyx associated with olfactory cilia exhibited a higher density of binding sites for both LFA and DSA than did either microvilli of sustentacular cells or respiratory cilia. Specificity of the lectinoprobes was confirmed by inhibition of binding with specific sugars or enzymic removal of specific sugar residues. These results demonstrated microchemical heterogeneity of the non-homogeneous mucous domains in olfactory mucus and in the attendant glycocalyx of olfactory cilia based on the differential localization of sialic acid and N-acetylglucosamine sugar residues.

The rainbow trout CMP-sialic acid synthetase utilises a nuclear localization signal different from that identified in the mouse enzyme

The terminal sugar sialic acid (Sia) plays a pivotal role in cell-cell interaction and recognition. A prerequisite for the biosynthesis of sialoglycoconjugates is the activation of Sia to cytidine monophosphate-Sia (CMP-Sia), by CMP-Sia synthetases (CMP-Sia-syn). CMP-Sia-syn are conserved from bacteria to man, and have been found to reside in the nucleus of all vertebrate species analysed to date. We previously cloned the CMP-Sia-syn from rainbow trout (rt) and identified three clusters of basic amino acids (BC) that might act as nuclear localization signals (NLS). Here, we utilised chimeric proteins and rt CMP-Sia-syn mutants in which putative NLS sequences were deleted, to identify the nuclear transport signal. Divergent from the mouse enzyme, where the crucial NLS is part of the enzyme's active site, in the rt CMP-Sia-syn the NLS and active site are disparate. The crucial NLS in the fish enzyme is bipartite and the functionality depends on a free N-terminus. Comparative analysis of all putative rt NLS in mouse and fish cells identified a second inferior motif (rtBC5-6), which was functional only in fish cells suggesting some differences in transport mechanism or folding variabilities in fish. Moreover, based on computational analyses of putative CMP-Sia-syn from distant deuterostomian organisms it was concluded that CMP-Sia-syn nuclear localization is a relatively recent invention, originating in echinoderms. In summary, our data describing structural differences in the NLS of vertebrate CMP-Sia-syn, and the independence of Sia activation from the subcellular localization of the enzyme, provide supporting evidence that nuclear localization is linked to a second yet unknown function.

Fluorescent assay for studying the substrate specificity of neuraminidase

Toyopearl 650 M was purchased from Tosoh (Japan). The 10-~1 pipette tips with filter (cat. no. FI08-96R-I0) and thin-well 0.2-ml PCR microtubes (cat. no. 430-01XNBZ-Q) with caps were obtained from Porex. Black polystyrene 96-well microtiter plates were purchased from Nunc (Denmark, cat. no. 437111). Vibrio cholerae neuraminidase (3.2.1.18) was purchased from Sigma (USA). 3'SiaLac and 6'SiaLac were purchased from Lectinity (Russia). ro-Aminospacered 3'SiaLac, 3'SiaLacNAc (Neu5Ac Cl2-3GaI131-4GlcNAc), and 6'SiaLacNAc (Neu5Ac Cl2-6GaI131-4GlcNAc) were synthesized previously [5-7]. ro-Aminospacered trisaccharide (5 ~ol), followed by triethylamine (5 ~l), was added to BODIPY FLSE (5.5 ~ol) in dimethylformamide (0.5ml). The reaction mixture was stirred at room temperature for 2-3 h and was subjected to gel filtration on a Sephadex LH-20 column equilibrated with acetonitrile/water (1:1, v/v). The yield of BODIPYlabeled trisaccharides was 80-90%. (Complete lH nuclear magnetic resonance data (500 MHz, DzO) are available from www.carbohydrate.ru/docs/nmrdata_OOl.rtf.) Virus NBulgaria/120/82 (HINl) and virus A!FPV/ Rostock/34 (H7Nl) were obtained from the virus repository of the Ivanovsky Institute of Virology in Moscow, Russia, and were propagated in 10-day-old embryonated chick~n eggs. A Madin-Darby canine kidney (MDCK)derived variant of human influenza virus A/Hongkong/ 1143/99 (H3N2) was obtained from the Institute of Applied Microbiology in Vienna, Austria. Primary isolation, propagation of human influenza virus, and purification of all viruses were done as described previously [8]. A titer of viruses was determined in a hemagglutination reaction with human erythrocytes Neuraminidase, or sialidase, is an exoglycosidase that hydrolyzes tX-ketosidic linkage between sialic acid and adjacent sugar residue. There are several methods of neuraminidase activity determination, usually based on fluorescent or chemiluminescent detection of an aglycon formed due to neuraminic acid cleavage [1-3]. Substrate specificity of neuraminidase is usually defined as the enzyme ability to distinguish the type of bond between neuraminic acid and galactose residues. As a rule, 3'SiaLac (Neu5ActX2-3Galf31-4Glc)! and 6'SiaLac (Neu5Ac tX2-6Galf31-4Glc) are used as substrates, and the amount of released neuraminic acid is measured [4]. In this article, we propose a simple and sensitive fluorescent method for determining the neuraminidase specificity using BODIPY -labeled trisaccharides as substrates. The method is based on the quantitative separation of neutral disaccharide-BODIPY from negatively charged uncleaved substrate using an anion exchange microcartridge, with the reaction product being registered by its fluorescence (Scheme 1). BODIPY FLSE (4,4-difluoro-5,7-dimethyl-4-bora3a,4a-diaza-s-indacene-3-propionic acid) was purchased from Molecular Probes (The Netherlands). All other chemicals used were obtained from Fluka (Switzerland). All chemicals used were of analytical grade. DEAE-

Structure and function of vertebrate CMP-sialic acid synthetases.

Activation of sugars into nucleotide sugars is critical for their entry into biosynthetic pathways. In eukaryotic cells, the activation of the acidic nine-carbon sugar sialic acid to CMP-sialic acid takes place in the cell nucleus, whereas all other nucleotide sugars are made in the cytoplasm. Molecular cloning of vertebrate CMP-sialic acid synthetases confirmed the nuclear localization and introduced new molecular tools for directly exploring the functional mechanisms of the enzymes, as well as the physiological relevance of their nuclear transport. Although major advances have been made in understanding structure-function relationships and defining elements involved in the nuclear transport, the riddle surrounding the physiological relevance of nuclear localization awaits resolution.

Sialic acids are absent from the dermatophytes Trichophyton mentagrophytes and Trichophyton rubrum.

Experiments were performed to determine whether sialic acids are expressed in two dermatophytes: Trichophyton rubrum and T. mentagrophytes, similarly to other fungal pathogens. Chemical extraction of mycelia and microconidia followed by high-performance thin-layer chromatography and colorimetric assays were all negative for sialic acid. Incubation of dermatophytes in the presence of Limax flavus agglutinin, specific for sialic acid, was negative in a fluorescence staining test. We have also used other lectins that bind to sialic acid and/or other sugar residues, and these ligands coupled to fluorescein strongly stained these fungi. Such fluorescence staining was not abolished or reduced when fungi were pretreated with sialidase. Different strains of influenza virus which recognize sialic acid residues were also used, but no agglutination of the dermatophytes was observed. Based on these methods, which successfully revealed the presence of sialic acids in other fungal pathogens, we show that these monosaccharides do not occur in both dermatophyte species. Thus, sialic acids do not seem to play a role in the pathogenicity of these fungi.

Immunocytohistochemical characterization of pituitary cells of the bluefin tuna, Thunnus thynnus L.

In this paper we report the first complete mapping of the pituitary in a tuna species. The various different adenohypophysis cell types of the bluefin tuna, Thunnus thynnus L. have been identified and located using different antisera against mammalian and piscine hormones and various histochemical techniques: PAS, Alcian Blue pH 2.5 and lectins -ConA and WGA(Neutral and Acidic Glycoproteins); Bromophenol Blue (Proteins) and Tioglycollate-Ferric-Ferricianide-FeIII (-S-S- groups). Prolactin (PRL) and adrenocorticotrophic (ACTH) cells were located in the rostral pars distalis (RPD) of the pituitary, while the proximal pars distalis (PPD) displayed gonadotrophic (GTH), thyrotrophic (TSH), somatotrophic (GH) and also a few PRL cells. Moreover, somatolactin (SL) and melanotrophic (MSH) cells were identified inside the pars intermedia (PI). Interestingly, some SL-immunoreactive fibers were also detected in the neurohypophysis. Some GTH cells were also located on the exterior surface of the PI. Glycoproteins containing mannose (Man) and/or glucose (Glc); N-acetyl-glucosamine (GlcNAc) and/or sialic acid sugar residues, as well as -S-S- groups, were observed in GTH, TSH and SL cells. The Bromophenol Blue technique stained amphiphilic SL, acidophilic GH cells and weakly ACTH cells. GH and ACTH cells were unreactive to PAS, Alcian Blue, Tioglycollate-Ferric-Ferricianide-FeIII and lectin (Con A and WGA) techniques. Finally, PAS reaction was positive in amphiphilic SL cells, which were PbH unreactive, while MSH and ACTH cells were stained with PbH technique.

An assessment of the in vitro toxicology of Clostridium perfringens type D epsilon-toxin in human and animal cells.

The epithelial Madin Darby canine kidney (MDCK) cell line and 17 human cell lines were examined for sensitivity to Clostridium perfringens type D epsilon-toxin. MDCK cells were confirmed as being sensitive to the toxin. In addition, the Caucasian renal leiomyoblastoma (G-402) human cell line was identified as being epsilon-toxin sensitive. Using the MTS/PMS assay system the concentration of toxin reducing cell culture viability by 50% (LC50) was found to be 2 microg/ml in MDCK cells. The LC50 for G-402 cells was 280 microg/ml. Epsilon-Toxin was found to be rapid acting in MDCK cells exposed to a maximum lethal dose of the toxin (40% loss of viability after a 0.5 h exposure), but slower acting in G-402 cells (40% loss of viability after 1.7 h exposure). Photomicrography of toxin exposed cultures indicated necrotic cell death on exposure to epsilon-toxin. Investigations using an antibody probe indicated that epsilon-toxin could bind to many cell surface proteins in both MDCK, G-402 and a toxin insensitive human cell line (CAKI-2). It has previously been found that the toxin may bind to the cell surface via glycosylated moieties. However, exposing MDCK and G-402 cells to epsilon-toxin in the presence of sialic acid and several different sugars did not reduce the lethal effects of the toxin.

Studies of the chemical structure of gangliosides in deer antler, Cervus nippon.

The biological activity of deer antler has been considered to originate in the gangliosides, although the structures of gangliosides have not been well elucidated. The quality of deer antler as an Asian folk medicine has often been evaluated by the amount of gangliosides contained in the crude drug. We have completed the structural determination of five gangliosides isolated from deer antler in the present study. Five ganglioside fractions were isolated and purified from deer antler, Cervus nippon, by the Folch-Suzuki partition method, DEAE-Sephadex A-25, and further by silica gel column chromatography. High field proton nuclear magnetic resonance spectroscopy, gas chromatography/mass spectrometry, and fast atom bombardment-mass spectrometry studies characterized the isolated ganglioside fractions. GM3 and GD3 were present in the isolated ganglioside fractions. Samples were hydrolyzed in trifluoroacetic acid for direct compositional analysis and analyzed for sialic acid and neutral sugar without prior derivatization. Separation of the monosaccharides was achieved by HPLC on a Dionex CarboPac column eluted at a high pH. The resolved monosaccharides were identified using standard monosaccharides by pulsed amperometric detection. N-Acetyl GM3 (Neu5Ac), N-glycolyl GM3 (Neu5Gc), and N-acetyl GD3 (Neu5Ac) were present in the antler. The major ceramide moiety was composed of C16:0 or C22:0 fatty acids along with either C18 sphingosine or C20 eicosasphingosine.

Histochemical study of skin and gills of Senegal sole, Solea senegalensis larvae and adults.

A battery of horseradish peroxidase-conjugated lectins (Con A, WGA and DBA), as well as conventional histochemical techniques (PAS, saponification, Alcian Blue pH 0.1, 1, 2.5, chlorhydric hydrolisis, neuraminidase, Bromophenol blue, Tioglycollate reduction and Ferric-ferricyanide-FeIII) were used to study the content and distribution of carbohydrates, proteins and glycoconjugate sugar residues on the skin and gills of Senegal sole, Solea senegalensis larvae and adults: During larval development of Solea senegalensis (from hatching until day 45 posthatching), epidermal sacciform, as well as branchial and epidermal chloride cells were unreactive with all cytochemical tests performed in this paper. Mucous or goblet cells of the corporal skin and gills containing strongly sulphated acid glycoproteins were evident on days 15-20 of larval development, as well as in epidermal and branchial mucous cells of adult specimens, which also contained GlcNAc and/or sialic acid. In adult specimen, the proteic content was higher in branchial mucous cells than in epidermal cells. In larvae, variable amounts of glycoproteins containing sialic acid, GlcNAc, GalNAc, Man and/or Glc residues were observed in epithelial cells and/or cuticle. GlcNAc and/or sialic acid sugar residues were only weakly detected in glycoproteins of some epidermal and branchial mucous cells of larvae by day 45, because from hatching until metamorphosis, lectin reactions (WGA, Con A and DBA) were negative in mucous cells.

Effect of sugar chain structure on in vivo biological activity of a recombinant human glycoprotein expressed in SP2/0-AG14 cells

We have reported previously that recombinant human erythropoietin (rhEPO) produced from Sp2/0-Ag14 transformant had a low biological activity in vivo as compared with other human EPOs. rhEPO from SP2/0 has now been found to have a lower amount of sialic acid and different sugar chains from other hEPOs. This recombinant molecule contains disialobranches as major and complex sugar chains which showed broad peaks on gel-filtration chromatography. These differences may be responsible for its low in vivo bioactivity.

An immunocytochemical study of pituitary cells of the Senegalese sole, Solea senegalensis (Kaup 1858).

Different antisera directed against mammalian and piscine pituitary hormones, as well as a battery of various conventional histochemical techniques (PAS, Alcian Blue pH 2.5, Bromophenol Blue) and lectins, were used to identify the different hormonal cell types in the pituitary of the Senegalese sole, Solea senegalensis. Prolactin and adrenocorticotrophic cells were located in the rostral pars distalis of the pituitary. Gonadotrophic, thyrotrophic and growth hormone cells were distributed in the proximal pars distalis, but gonadotrophic cells appear also at the border of the pars intermedia. Somatolactin cells, as well as alpha-melanotrophic cells were located in the pars intermedia of the Solea senegalensis pituitary. The PAS reaction was positive in somatolactin cells, which were unreactive with the lead-Haematoxylin technique, whereas melanotrophic cells were positive. Glycoproteins containing mannose and/or glucose, as well as N-acetyl-glucosamine and sialic acid sugar residues, are synthesized and secreted by gonadotrophic, thyrotrophic and somatolactin cells. Adrenocorticotrophic cells and, especially, the amphiphilic somatolactin and acidophilic growth hormone cells were stained with the Bromophenol Blue technique that identifies proteins in general, but adrenocorticotrophic and growth hormone cells were unreactive towards PAS, Alcian Blue pH 2.5 and lectins (Con A and WGA).

Analysis of different glycosylation states in IgG subclasses

Altered IgG glycosylation affects certain immunological activities of human IgG. Enzyme-linked lectin assays (ELLA) were developed for detecting the glycosylation on IgG and its individual subclasses in sera from healthy controls. Biotinylated Sambucus nigra, Ricinus communis agglutinin I and Bandeiraea simplicifolia II were used to detect the terminal sialic acid (SA), galactose (Gal) and N-acetylglucosamine (GlcNAc) sugar residues, respectively on the captured IgG. A mild oxidation step of the anti-IgG-coated plates obviated background reaction with the lectins. Terminal glycosylation varied significantly with age. The old age group (>65 years) had less SA in IgG1, and IgG2, when compared to young (0–19 years) and adult (20–39 years) groups, respectively. Also the old age group had less Ga1 in IgG2, IgG3 and IgG4 subclasses compared to the adult group, and to the young group in the case of IgG3. This ELLA system may be a valuable tool in the detection of glycosylation disorders in patients' sera.