Sialic Acids

PTA lectin causes haemagglutination by acting on sialic acid ligand on the surface of RBC's. In malarial infection, the parasite causes surface changes in RBC leading to blockage of sialic acid ligand. Resulting in to the decrease in haemaggulinations by PTA lectin methods. Purified lectin prepared from pedilanthus tithymaloides is used to determine haemagglutination in normal and malarial parasite RBC's. The titer of lectine causing haemagglutination is observed. haemagglutination by PTA lectin in parasitized RBC's is significantly low as compared to haemaglutination in normal RBC's. Changes in surface ligands on RBC's due to malarial infection changes the haemagglutination property of RBC's by PTA lectin, with resultant decrease in haemagglitunation can have application in the diagnosis and treatment of malarial infection.

Gonorrhea has become resistant to most conventional antimicrobials used in clinical practice. The global spread of multidrug-resistant isolates of Neisseria gonorrhoeae could lead to an era of untreatable gonorrhea. New therapeutic modalities with novel mechanisms of action that do not lend themselves to the development of resistance are urgently needed. Gonococcal lipooligosaccharide (LOS) sialylation is critical for complement resistance and for establishing infection in humans and in experimental mouse models. Here we describe two immunotherapeutic approaches that target LOS sialic acid: i) a fusion protein that comprises the region in the complement inhibitor factor H (FH) that binds to sialylated gonococci and IgG Fc (FH/Fc fusion protein) and ii) analogs of sialic acid that are incorporated into LOS but fail to protect the bacterium against killing. Both molecules showed efficacy in the mouse vaginal colonization model of gonorrhea and may represent promising immunotherapeutic...

-specific Abs are able to enhance or suppress immune responses depending on the receptors that they bind on immune cells. Recent studies have shown that pro-or antiinflammatory effector functions of IgG Abs are also regulated through their Fc N-linked glycosylation patterns. IgG Abs that are agalactosylated (nongalactosylated) and asialylated are proinflammatory and induced by the combination of T cell-dependent (TD) protein antigens and proinflammatory costimulation. Sialylated IgG Abs, which are immunosuppressive, and Tregs are produced in the presence of TD antigens under tolerance conditions. T cell-independent (TI) B cell activation via B cell receptor (BCR) crosslinking through polysaccharides or via BCR and TLR costimulation also induces IgG Abs, but the Fc glycosylation state of these Abs is unknown. We found in mouse experiments that TI immune responses induced suppressive sialylated IgGs, in contrast to TD proinflammatory Th1 and Th17 immune responses, which induced agalactosylated and asialylated IgGs. Transfer of low amounts of antigen-specific sialylated IgG Abs was sufficient to inhibit B cell activation and pathogenic immune reactions. These findings suggest an immune regulatory function for TI immune responses through the generation of immunosuppressive sialylated IgGs and may provide insight on the role of TI immune responses during infection, vaccination, and autoimmunity.

We have characterized the transport of GSH and the mechanism for impaired GSH transport in mutant Eisai hyperbilirubinemic rats (EHBR) using isolated canalicular membrane-enriched vesicles (cLPM). In control animals, the transport of GSH is an electrogenic process and is trans-stimulated by preloading the vesicles with GSH and is not enhanced in the presence of ATP. GSH transport in cLPM is saturable with a single component having a K , of -16 mM and a Vma, of 6.7 nmol/mg/l5 s. EHBR is a Sprague-Dawley rat with hyperbilirubinemia due to impaired bile secretion of organic anions by the ATP-dependent organic anion/ GSH-conjugate transporter. In cLPM from EHBR we confirmed the defective stimulation by ATP of the transport of LTC., and GSSG. In the mutant cLPM, the characteristics and kinetics of GSH transport were the same as in the controls. 2,4-(dinitropheny1)-glutathione (DNP-GSH), which is a substrate for the ATPdependent canalicular organic anion carrier, in the absence of ATP, cis-inhibited the transport of GSH into cLPM vesicles; however, when the vesicles were preloaded with DNP-GSH, there was a dose-dependent trans-stimulation of GSH transport. In contrast, in the presence of ATP, DNP-GSH enhanced GSH transport in cLPM vesicles; at 0.25 mM DNP-GSH, a concentration which did not cis-inhibit GSH, addition of ATP resulted in accelerated GSH transport; at 1.0 mM DNP-GSH, cis-inhibition was completely reversed by the addition of ATP despite a negligible fall in the medium DNP-GSH. Interestingly, sulfobromophthalein-glutathione (BSP-GSH) neither cis-inhibited nor transstimulated GSH transport in cLPM. This contrasts with bLPM where BSP-GSH interacts with the GSH carrier. Therefore, GSH is transported into bile by a multispecific low affinity electrogenic carrier which is distinct from the multispecific high affinity ATPdriven organic anion transporter. Although both carriers have overlapping specificities, BSP-GSH and GSH are uniquely specific for only one of the carriers. The near absence of GSH in the bile of mutant rats can be best explained as a secondary defect due to cisinhibition from retained substrates for the defective *

Dendritic cells (DCs) play a pivotal role in the regulation of the immune response. DC development and activation is finely orchestrated through transcriptional programs. GATA1 transcription factor is required for murine DC development, and data suggest that it might be involved in the fine-tuning of the life span and function of activated DCs. We generated DC-specific Gata1 knockout mice (Gata1-KO(DC)), which presented a 20% reduction of splenic DCs, partially explained by enhanced apoptosis. RNA sequencing analysis revealed a number of deregulated genes involved in cell survival, migration, and function. DC migration toward peripheral lymph nodes was impaired in Gata1-KO(DC) mice. Migration assays performed in vitro showed that this defect was selective for CCL21, but not CCL19. Interestingly, we show that Gata1-KO(DC) DCs have reduced polysialic acid levels on their surface, which is a known determinant for the proper migration of DCs toward CCL21.

Introduction: Routine surveillance samples disclosed seropositivity to influenza A virus (IAV) in a Norwegian turkey breeder flock. Simultaneous reports of influenza-like symptoms in farm workers and a laboratory confirmed influenza A(H1N1)pdm09 (H1N1pdm09) infection in one person led to the suspicion of a H1N1pdm09 infection in the turkeys. Animals and methods: H1N1pdm09 infection was confirmed by a positive haemaggutinin inhibition test using H1N1pdm09 antigens, and detection of H1N1pdm09 nucleic acid in reproductive organs of turkey hens. The flock showed no clinical signs except for a temporary drop in egg production. Previous reports of H1N1pdm09 infection in turkeys suggested human-to-turkey transmission (anthroponosis) during artificial insemination. Results and discussion: The flock remained seropositive to IAV and the homologous H1N1pdm09 antigen throughout the following 106 days, with decreasing seroprevalence over time. IAV was not detected in fertilised eggs or in turkey...

Adding to the Antiviral Arsenal The envelope of influenza virus contains two immunodominant glycoproteins: hemagglutinin and neuraminidase (NA). Existing antivirals like zanamivir (Relenza) and oseltamivir (Tamiflu) target NA; however, the development of drug resistance is a problem. Kim et al. (p. 71 , published online 21 February) now report a different class of NA inhibitors. NA catalyzes the removal of sialic acids from the surface of host cells to initiate entry. Discovery of a NA–sialic acid intermediate led to the production of sialic acid analogs that bound covalently to NA and inhibited its enzymatic activity. These compounds showed activity against a wide variety of influenza strains, inhibited viral replication in cell culture, and were able to protect mice against influenza infection. Protection of mice was equivalent to protection seen from zanamivir. Moreover, the compounds showed activity against drug-resistant strains in vitro. These compounds represent a potentially...

Letters to the Editor high concentrations of spleen type alone. Of the patients with high serum ferritin concentrations (473-762 ,ug/l), two had increases in both heart and spleen type fer- ritin and two of spleen type ferritin alone. Patients with idiopathic sideroblastic anaemia have considerably increased intracytoplasmic ferritin concentrations in addition to mitochondrial iron deposi- tion.34 This study shows that although high concentrations of erythrocyte femtin are common in patients with myelodysplastic syndromes, these are often associated with transfusional iron overload and in such cases it is difficult to determine whether the increase in ferritin is due solely to an intracorpuscular abnormality. Among the non-transfused patients, however, high erythrocyte ferritin concentrations were found in all four patients with idiopathic macrocytosis, seven of nine patients with sideroblastic anaemia, one of seven patients with refractory anaemia, and one of three patients with chronic myelomonocytic leukaemia. The reason for this increase in red cell ferritin content is not clear. It does, how- ever, indicate that an abnormality of ery- throid iron metabolism is common in this condition and is not confined to patients with sideroblastic anaemia, thus supporting the suggestion that sideroblastic anaemia is not a discrete entity but merely signifies a more prominent abnormality in some patients with myelodysplastic syndromes.25

We previously reported that pericontusional extracellular chondroitin sulfate proteoglycans (CSPGs) are profoundly reduced for 3 weeks after experimental traumatic brain injury, indicating a potential growthpermissive window for plasticity. Here, we investigate the extracellular environment of sprouting neurons after controlled cortical impact injury in adult rats to determine the spatial and temporal arrangement of inhibitory and growth-promoting molecules in relation to growthassociated protein 43Ypositive (GAP43+) neurons. Spontaneous cortical sprouting was maximal in pericontused regions at 7 and 14 days after injury but absent by 28 days. Perineuronal nets containing CSPGs were reduced at 7 days after injury in the pericontused region (p G 0.05), which was commensurate with a reduction in extracellular CSPGs. Sprouting was restricted to the perineuronal nets and CSPGdeficient regions at 7 days, indicating that the pericontused region is temporarily and spatially permissive to new growth. At this time point, GAP43+ neurons were associated with brain regions containing cells positive for polysialic acid neural cell adhesion molecule but not with fibronectin-positive cells. Brain-derived neurotrophic factor was reduced in the immediate pericontused region at 7 days. Along with prior Western blot evidence, these data suggest that a lowered intrinsic growth stimulus, together with a later return of growthinhibitory CSPGs, may contribute to the ultimate disappearance of sprouting neurons after traumatic brain injury.

We previously reported that pericontusional extracellular chondroitin sulfate proteoglycans (CSPGs) are profoundly reduced for 3 weeks after experimental traumatic brain injury, indicating a potential growthpermissive window for plasticity. Here, we investigate the extracellular environment of sprouting neurons after controlled cortical impact injury in adult rats to determine the spatial and temporal arrangement of inhibitory and growth-promoting molecules in relation to growthassociated protein 43Ypositive (GAP43+) neurons. Spontaneous cortical sprouting was maximal in pericontused regions at 7 and 14 days after injury but absent by 28 days. Perineuronal nets containing CSPGs were reduced at 7 days after injury in the pericontused region (p G 0.05), which was commensurate with a reduction in extracellular CSPGs. Sprouting was restricted to the perineuronal nets and CSPGdeficient regions at 7 days, indicating that the pericontused region is temporarily and spatially permissive to new growth. At this time point, GAP43+ neurons were associated with brain regions containing cells positive for polysialic acid neural cell adhesion molecule but not with fibronectin-positive cells. Brain-derived neurotrophic factor was reduced in the immediate pericontused region at 7 days. Along with prior Western blot evidence, these data suggest that a lowered intrinsic growth stimulus, together with a later return of growthinhibitory CSPGs, may contribute to the ultimate disappearance of sprouting neurons after traumatic brain injury.

One of the female causes of infertility is anovulation which is treatable with gonadotropin hormones. These hormones affect the molecular organization of the uterus such as glycoconjugates that are the first site of contact between the blastocyst and the uterus. The objective of this project was to study the alteration of glycoconjugates on the uterine apical, Golgi zone, and basement membrane of epithelial cells and the uterine gland after hyperstimulation with pregnant mare serum gonadotropin (PMSG) (4, 8, 16, 24, and 40 IU), during the implantation period. Injection of PMSG (in experimental groups) and injection of distilled water (in the control group) were followed by HCG administration (10 IU), mating, isolation of positive vaginal plug rats, and killing at 5.5 days of pregnancy. Histochemistry was done on the pregnant uterine horns with the use of WGA, DBA, PNA, ConA, SBA, and UEA lectins. The intensity of the immunohistochemical staining was scored, and quantitative data wer...

Desialation of cell surfaces has been associated with the initiation or modification of diverse cellular functions. In these studies we have examined the subcellular distribution of sialidase (SE) in human neutrophils as well as the mobilization of this enzyme following neutrophil activation. Separation of subcellu- lar fractions by density gradient centrifugation showed that SE is present not only in neutrophil primary and secondary granule populations, like lysozyme, but also in plasma membrane fractions. Neutrophil activation was associated with a redistribu- tion of SE from secondary granule-enriched fractions to the plasma membrane. Furthermore, SE activity detected on the surface of intact neutrophils with a fluorescent SE substrate increased rapidly after activation with kinetics that matched both the loss of total cell-associated sialic acid and release of free sialic acid from the cells. These activation-dependent events were in each case blocked by incubation of neutrophils with the SE inhibitor, 2-deoxy-N-acetyl-neuraminic acid. Ag- gregation responses of neutrophils as well as adhesion re- sponses to nylon and plastic surfaces were also inhibited by 2-deoxyNANA. Our findings indicate that the activation-de- pendent desialation of the neutrophil surface is associated with mobilization of an endogenous SE to the plasma membrane and has a role in stimulated adhesion responses of these cells. (J.

Desialation of cell surfaces has been associated with the initiation or modification of diverse cellular functions. In these studies we have examined the subcellular distribution of sialidase (SE) in human neutrophils as well as the mobilization of this enzyme following neutrophil activation. Separation of subcellu- lar fractions by density gradient centrifugation showed that SE is present not only in neutrophil primary and secondary granule populations, like lysozyme, but also in plasma membrane fractions. Neutrophil activation was associated with a redistribu- tion of SE from secondary granule-enriched fractions to the plasma membrane. Furthermore, SE activity detected on the surface of intact neutrophils with a fluorescent SE substrate increased rapidly after activation with kinetics that matched both the loss of total cell-associated sialic acid and release of free sialic acid from the cells. These activation-dependent events were in each case blocked by incubation of neutrophils with the SE inhibitor, 2-deoxy-N-acetyl-neuraminic acid. Ag- gregation responses of neutrophils as well as adhesion re- sponses to nylon and plastic surfaces were also inhibited by 2-deoxyNANA. Our findings indicate that the activation-de- pendent desialation of the neutrophil surface is associated with mobilization of an endogenous SE to the plasma membrane and has a role in stimulated adhesion responses of these cells. (J.

The isoelectdc point (pl) of the human red cell glucose transporter (Glut 1) was determim~ lacemisteut values of 6.0, 6.4-6.5 and 8 have been reported earlier, lnteIwal membrane lmJteius from Itmmm red cells wet~ altaknt~ by two-dimensional electrophore~:~ with isoelectric focusing and sodium dede~l sulfate 8el el~ (2D-PAGE). A zone of mnomerie Glut 1 ,?as found at pH 8.7, but most of the Glut I focused at pil 6-7 telgtheg ~ the transporter and other compenents. PurUk~ Glut ! focused ~ at pH 8.$ + ll2 (S.D., • -12) mul Im~ Glut I only at pH 8.4 + 0.1 (n ~ 5), as shown by 2D-PAGE. The absmce ~g Glut I bekm lull 8 t. the latter cases was confirmed by immunoblotting with a monodonal antibody. Furtherme~ Glut I was pimteummi~labeged with PH]cytocludasin B and sub~.cted to isoelectric focusins in one dimembL Tile pl of the IdMled Glut I was &6 ± 0.3 (n : 11). A pl of 9.1 was calculated for the Glut I polypeptJde mt the basis of amJuo add eomimeitJ~ mid pK, values for amino acid side groups. The sialic acid content of the 8tY~ transputer frem fresh red edls was determined at approlimately 2.1 sfallc acid residues per transporter, wbklt em'gmlmm~ to u cakulated pl of 8.8. The pl values of other human Iluease transporter polypeptides of the fa¢iililati~ ~ type (Glut 2, 3, 4 and 5) were calculated at 8,4, 7A, 7.1 and 6.2, respectively.

The isoelectric points (pl) of native proteins are important in several separation techniques. For estimating p l values the net charge of several proteins was calculated versus pH by use of the Henderson-Hasselbalch equation. Amino acid composition, pK, values for amino acid side chains and for the N-and C-terminal groups, and the presence of other charged groups were taken into account. A set of pK, values was chosen for amino acid residues with ionizable side chains. Each particular type of ionizable group was assumed to have pK, values distributed around the chosen value, thereby simulating the situation in proteins and polypeptides. The calculated p l values showed reasonably good agreement with experimental ones for most of 16 native proteins over a wide pH range (3.4-11) when charge contributions of heme groups, sialic acid residues, etc., were taken into account. The calculated pZ for the human red cell glucose transporter (Glutl) with one sialic acid residue was decreased from 8.8 to 8.5 by introducing pK, value spreading and became consistent with the experimental plvalue of 8.4 f 0.05 at 15°C determined in the presence of 6 M urea. The p l of the native Glutl was lower, 8.0 f 0.1, at 22°C. In general, the p l values for native proteins are affected by the three-dimensional structure of the proteins, which causes greater differences between calculated and experimental plvalues than in the case of polypeptides for which plvalues are determined in the presence of urea.

The isoelectdc point (pl) of the human red cell glucose transporter (Glut 1) was determim~ lacemisteut values of 6.0, 6.4-6.5 and 8 have been reported earlier, lnteIwal membrane lmJteius from Itmmm red cells wet~ altaknt~ by two-dimensional electrophore~:~ with isoelectric focusing and sodium dede~l sulfate 8el el~ (2D-PAGE). A zone of mnomerie Glut 1 ,?as found at pH 8.7, but most of the Glut I focused at pil 6-7 telgtheg ~ the transporter and other compenents. PurUk~ Glut ! focused ~ at pH 8.$ + ll2 (S.D., • -12) mul Im~ Glut I only at pH 8.4 + 0.1 (n ~ 5), as shown by 2D-PAGE. The absmce ~g Glut I bekm lull 8 t. the latter cases was confirmed by immunoblotting with a monodonal antibody. Furtherme~ Glut I was pimteummi~labeged with PH]cytocludasin B and sub~.cted to isoelectric focusins in one dimembL Tile pl of the IdMled Glut I was &6 ± 0.3 (n : 11). A pl of 9.1 was calculated for the Glut I polypeptJde mt the basis of amJuo add eomimeitJ~ mid pK, values for amino acid side groups. The sialic acid content of the 8tY~ transputer frem fresh red edls was determined at approlimately 2.1 sfallc acid residues per transporter, wbklt em'gmlmm~ to u cakulated pl of 8.8. The pl values of other human Iluease transporter polypeptides of the fa¢iililati~ ~ type (Glut 2, 3, 4 and 5) were calculated at 8,4, 7A, 7.1 and 6.2, respectively.

Purpose: Cellular response to cigarette smoke (CS) involves activation of recognition receptors resulting in changes in immune status, oxidative stress and cell turnover. We investigated the effects of CS on sialic acid-binding immunoglobulin type lectins (Siglecs) expression and their sialylated ligands in human immune and non-immune cells. Methods: Human monocytes (THP-1) and epithelial cells (A549) were cultured in CS-conditioned medium (CSM). Expression of Siglec-8 and Siglec-5/Siglec-14 was analysed in THP-1 cells using flow cytometry. The effects of CS on immune activity was evaluated flow cytometrically in these cells by assessment of phagocytosis and intracellular expression IL-1β and IL-10. Detection and differentiation of sialic acids was analyzed by dot blot, western blot and flow cytometry using plant lectins and antibodies. Results: Exposure to CS significantly increased expression of Siglec-8 and Siglec-5/Siglec-14 in THP-1 cells. These changes were accompanied by enhanced intracellular level of IL-1β and IL-10 but reduced phagocytic activity. In THP-1 and A549 cells, the level of α2,3-sialic acids, but not α2,6-sialic acid, was significantly increased when compared to naïve cells. The level of α2,8-sialic acids increased significantly in A549 cells, but not in THP-1 cells, after exposure to CS. Conclusion: These results show that cellular response to CS involves changes in expression of Siglec receptors and sialylated ligands functionally associated with immunity.

Abbreviations: (BCA) bicinchoninic acid, (CA) colominic acid, (CV) coefficient of variation, (EU) endotoxin unit, (N) Newton, (PEG) polyethylene glycol, (PSA) polysialic acid, (SDS-PAGE) Sodium dodecyl sulfate polyacrylamide gel electrophoresis, (SE-HPLC) size-exclusion high-performance liquid chromatography, (WB) Western blotting

Rotavirus is one of the common causes of gastroenteritis in children; in fact nearly every child in the world will have at least one rotavirus-induced gastroenteritis (marked by watery diarrhea) before age 5. Despite the introduction of vaccines, over 200,000 children die annually from rotavirus induced severe dehydrating gastroenteritis, most of the children being in the poor countries of the world. Rotaviruses belong to the non-enveloped double-stranded RNA (dsRNA) virus group. The RNA segment encodes six structural viral proteins (VPs); among them attachment of rotavirus to host cells is mediated through the VP8* domain of the outer capsid protein VP4. Picrasma javanica Blume belongs to the Simaroubaceae family and can be found in Bangladesh, India and other tropical countries. Since the plant has indigenous uses as an antiviral, it was of interest to evaluate through molecular docking studies whether phytochemicals reported from the plant can bind to VP8* domain of the outer cap...

Astrocytes show large morphological and functional heterogeneity and are involved in many aspects of neural function. Progress in defining astrocyte subpopulations has been hampered by the lack of a suitable antibody for their direct detection and isolation. Here, we describe a new monoclonal antibody, ACSA‐1, which was generated by immunization of GLAST1 knockout mice. The antibody specifically detects an extracellular epitope of the astrocyte‐specific L‐glutamate/L‐aspartate transporter GLAST (EAAT1, Slc1a3). As shown by immunohistochemistry, immunocytochemistry, and flow cytometry, ACSA‐1 was cross‐reactive for mouse, human, and rat. It labeled virtually all astrocytes positive for GFAP, GS, BLBP, RC2, and Nestin, including protoplastic, fibrous, and reactive astrocytes as well as Bergmann glia, Müller glia, and radial glia. Oligodendrocytes, microglia, neurons, and neuronal progenitors were negative for ACSA‐1. Using an immunomagnetic approach, we established a method for the is...

Recently we have described the molecular cloning of the genetic determinant coding for the S-fimbrial adhesin (Sfa), a sialic acid-recognizing pilus frequently found among extraintestinal Escherichia coli isolates. Fimbriae from the resulting Sfa+ E. coli K-12 clone were isolated, and an Sfa-specific antiserum was prepared. Western blots indicate that S fimbriae isolated from different uropathogenic and meningitis-associated E. coli strains, including O83:K1 isolates, were serologically related. The Sfa-specific antibodies did not cross-react with P fimbriae, but did cross-react with F1C fimbriae. Furthermore the sfa+ recombinant DNAs and some cloned sfa-flanking regions were used as probes in Southern experiments. Chromosomal DNAs isolated from O18:K1 and O83:K1 meningitis strains with and without S fimbriae and from uropathogenic O6:K+ strains were hybridized against these sfa-specific probes. Only one copy of the sfa determinant was identified on the chromosome of these strains. ...

Chagas' disease, caused by Trypanosoma cruzi, affects about 18 million people in Latin America, and no effective treatment is available to date. To acquire sialic acid from the host glycoconjugates, T. cruzi expresses an unusual surface sialidase with trans-sialidase activity (TcTS) that transfers the sugar to parasite mucins. Surface sialic acid was shown to have relevant functions in protection of the parasite against the lysis by complement and in mammalian host cell invasion. The recently determined 3D structure of TcTS allowed a detailed analysis of its catalytic site and showed the presence of a lactose-binding site where the b-linked galactose accepting the sialic acid is placed. In this article, the acceptor substrate specificity of lactose derivatives was studied by high pH anion-exchange chromatography with pulse amperometric detection. The lactose open chain derivatives lactitol and lactobionic acid, as well as other derivatives, were found to be good acceptors of sialic acid. Lactitol, which was the best of the ones tested, effectively inhibited the transfer of sialic acid to N-acetyllactosamine. Furthermore, lactitol inhibited parasite mucins re-sialylation when incubated with live trypanosomes and TcTS. Lactitol also diminished the T. cruzi infection in cultured Vero cells by 20±27%. These results indicate that compounds directed to the lactose binding site might be good inhibitors of TcTS.

Sialic acid sugars are overexpressed by cancer cells and contribute to the metastatic cascade at multiple levels. Therapeutic interference of sialic acids, however, has been difficult to pursue because of the absence of dedicated tools. Here we show that a rationally designed sialic acidblocking glycomimetic (P-3F ax -Neu5Ac) successfully prevents cancer metastasis. Formulation of P-3F ax -Neu5Ac into poly(lactic-co-glycolic acid) nanoparticles coated with anti-tyrosinaserelated protein-1 antibodies allowed targeted delivery of P-3F ax -Neu5Ac into melanoma cells, slow release, and long-term sialic acid blockade. Most importantly, intravenous injections of melanoma-targeting P-3F ax -Neu5Ac nanoparticles prevented metastasis formation in a murine lung metastasis model. These findings stress the importance of sialoglycans in cancer metastasis and advocate that sialic acid blockade using rationally designed glycomimetics targeted to cancer cells can effectively prevent cancer metastases. This targeting strategy to interfere with sialic acid-dependent processes is broadly applicable not only for different types of cancer but also in infection and inflammation. Evidence is accumulating that aberrant glycosylation and in particular overexpression of sialic acid sugars by cancer cells are involved in, and possibly drive distinct processes during cancer metastasis. Dissemination of cancer cells from primary tumors and metastases formation in distant organs is the major cause of cancer-associated mortality. Therefore, preventing dissemination of tumor cells and their growth at secondary sites are key to effective cancer

Staining with Congo red according to is the most commonly used method for the demonstration of amyloid, but structures other than amyloid can give false-positive results. To overcome this problem, Romha ´nyi (1971) introduced an aqueous Congo red staining with gum arabic as the mounting medium, which we have used in this and previous publications. Most histochemical studies on amyloid deposits to date have concentrated on conventional methods including staining with thioflavine, sirius red, alcian blue, methyl and crystal violet. In this study, we used topo-optical reactions with thiazine dyes on both the light and polarization microscopic level to establish the structure, distribution and location of carbohydrate components that occur within amyloid deposits, especially in the respiratory tract. Topo-optical staining reactions for the qualitative analysis of carbohydrate components in amyloid deposits included (1) reactions that identify the carbohydrate residues, (2) reactions that detect sialic acids and, (3) methods that visualize glycosaminoglycans. In conclusion, a comparison of consecutive serial sections stained with Congo red, aldehyde bisulfite toluidine blue reaction, sialic acid-specific topo-optical reaction, toluidine blue topo-optical reaction and chemically intensified basophilic reaction showed correlative staining patterns and anisotropic effects, corresponding to a close pathomorphological relationship between amyloid fibrils, periodate reactive carbohydrates, including sialic acids, and glycosaminoglycans.

Multiple myeloma (MM) is a neoplastic disorder characterized by proliferation of a single clone of plasma cells derived from B cells, which proliferates in the bone marrow and frequently invades the adjacent bone, producing skeletal destruction that results in bone pain and fractures. Patients with MM can furthermore present with anemia, hypercalcemia and renal failure. Non-secretory multiple myeloma (NSMM) is characterized by the absence of a monoclonal (M) protein in both the serum and urine. The reported incidence is 1-5% of all multiple myeloma cases. Development of amyloid tumors in NSMM has been described in the literature only occasionally. The clinical features of a 49-year-old female patient with NSMM and amyloid tumors in the breast, lung and rib are presented in this report. Conventional histology, Congo red staining with and without potassium permanganate pretreatment, aldehyde bisulfite-toluidine blue (ABT) reaction, sialic acid specific topooptical reaction, toluidine blue topo-optical reaction as well as immunohistochemistry were performed. An attempt is made to explain the lack of monoclonal immunoglobulins in the serum and urine, although extensive organ amyloidosis of AL type (k-light chains) has been found. It is assumed that the plasmocytic plasma cells possess an excretory mechanism, which allows the pathologic immunoglobulins to be secreted either as amyloid proteins polymerizing into amyloid fibrils, or as immunoglobulin fragments that are subject to degradation as soon as they are excreted out of the tumor cell. In this paper, we review the occurrence of amyloid tumors in non-secretory multiple myeloma and, in a single case report, we confirm the existence of carbohydrate residues, including sialic acids and sulfated GAGs, in amyloid deposits.

The glycosylation of tissue plasminogen activator (t-PA) obtained from the Bowes melanoma cell line was re-examined using methods of serial lectin affinity chromatography coupled with Bio-Gel P-4 gel filtration chromatography and exoglycosidase sequencing. This study clarified an earlier discrepancy in the literature and confirmed that the major complex N-linked glycans on Bowes t-PA that carry sialic acid as their sole charged group are bi-antennary, core fucosylated, with terminal N-acetylgalactosamine residues. We also report the characterization of a series of related and previously unidentified sialylated glycans. Further we show that Bowes t-PA expresses glucuronic acid/sulphate containing N-linked glycans and is recognized by anti-carbohydrate L2/HNK-1 monoclonal antibodies. The presence on Bowes t-PA of glycans associated primarily with the nervous system is consistent with its expression in a cell line of neuroectodermal origin.

Precursor cells have been shown to be affected by oxidative stress, in vivo and vitro, but little is known about the expression of antioxidant mechanisms in neuronal/glial differentiation. We have characterized the expression of Cu/Zn superoxide dismutase (Cu/Zn SOD), one of the main antioxidant proteins involved in the breakdown of superoxide, in the immature rat dorsolateral subventricular zone (SVZ), rostral migratory stream (RMS) and hippocampal subgranular zone (SGZ). Progenitor cells were identified immunohistochemically on cryostat sections by 5 Bromodeoxyuridine (BrdU) incorporation and expressing cells were further characterized using double labeling for progenitor markers. In the SVZ, only a subpopulation of BrdU+ cells, mostly found in the medial SVZ, expressed Cu/Zn SOD. These cells were mostly nestin+ and some were also vimentin+. In contrast, in the lateral SVZ few Cu/Zn SOD+/BrdU+ cells were found. These were primarily nestin+, vimentin-, showed some PSA-NCAM expression, but only a few were NG2+. In the RMS and SGZ virtually all BrdU+ progenitors were Cu/Zn SOD+ and expressed nestin and vimentin. Some RMS cells were also PSA-NCAM+. These findings show a heterogeneous expression of Cu/Zn SOD in restricted cell types in the germinative zones and suggest a role for antioxidant Cu/Zn SOD in progenitor cells of the immature rat brain.

The recent discovery that human noroviruses (huNoVs) recognize sialic acids (SAs) in addition to histo-blood group antigens (HBGAs) pointed to a new direction in studying virus-host interactions during calicivirus infection. HuNoVs remain difficult to study due to the lack of an effective cell culture model. In this study, we demonstrated that Tulane virus (TV), a cultivable primate calicivirus, also recognizes SAs in addition to the previously known TV-HBGA interactions. Evidence supporting this discovery includes that TV virions bound synthetic sialoglycoconjugates (SGCs) and that treatment of TV permissive LLC-MK2 cells with either neuraminidases or SA-binding lectins inhibited TV infectivity. In addition, we found that Maackia amurensis leukoagglutinin (MAL), a lectin that recognizes the α-2,3 linked SAs, bound LLC-MK2 cells, as well as TV, by which MAL promoted TV infectivity in cell culture. Our findings further highlight TV as a valuable surrogate for huNoVs, particularly in ...

have been involved in one or more previous or ongoing trials related to the use of uterotonics for the prevention of postpartum haemorrhage that were considered for inclusion in this review. Ferring Pharmaceuticals (Saint-Prex, Switzerland) and Novartis Pharmaceuticals UK Ltd (Surrey, UK) have supplied carbetocin and oxytocin to these studies. Ioannis Gallos, Metin Gülmezoglu, Justus Hofmeyr and Arri Coomarasamy have not participated in decisions regarding inclusion of these trials in this review or any tasks related to them such as data extraction or quality assessment. Arri Coomarasamy is involved in a World Health Organization-sponsored randomised controlled trial of carbetocin versus oxytocin, supported by Merck for Mothers (Merck & Co., Inc., Kenilworth, NJ, USA). Metin Gülmezoglu was involved in a large multicentre trial included in the review as part of the central co-ordination unit. As part of the central co-ordination unit, he is also involved in an ongoing World Health Organization-sponsored randomised controlled trial of carbetocin versus oxytocin supported by Merck for Mothers. Abi Merriel is part-funded by Ammalife (a UK-registered charity 1120236) and the Birmingham Women's NHS Foundation Trust. Harry Gee and Arri Coomarasamy are trustees of Ammalife. Jonathan Deeks is a member of the Health Technology Assessment (HTA) Commissioning Board and the HTA Efficient Study and Designs Board.

The identification of sialylated Thomsen–Friedenreich antigens in proteins poses much interest in the context of cancer research. MALDI‐TOF‐MS is a powerful technique for this purpose; still it shows considerable low sensitivity for sialylated molecules due to in‐source and metastable decomposition. Herein, we report a target‐driven strategy to identify these antigens in minute amounts of glycoproteins isolated in polyacrylamide gels. The glycans were recovered from gel spots by reductive β‐elimination, permethylated and analyzed by nano‐LC‐MALDI‐TOF‐MS. A computational algorithm was developed to filter spectral noise and enhance/isolate the signals of interest. Sialylated antigens were identified in minute amounts of fetuin (0.1 μg) and plasminogen (1.0 μg) by this approach.MS assignments were further validated by enzymatic methods. This methodology allowed a fivefold decrease in the current LOD of fetuin sialylated O‐glycans by MALDI‐TOF‐MS.

with tritium incorporated into its sialic acid residues has been prepared by the method of Van Lenten and Ashwell ((1970) J. Biol. Chem. 246, 1889-1894). The resulting radiolabeled protein ([siafyl-3H]prothrombin) was electrophoretically indistinguishable from the untreated protein. The thrombin activity generated by factor Xa activation of [siatyZ-3H]prothrombin was about 90% of that obtained from nonlabeled prothrombin. The sole products of the reaction of thrombin with [sialyZ-3H]prothrombin are intermediates 1 and 3. The products of the activation of [siaZyZ-3H]prothrombin with factor Xa are intermediates 1, 2, 3, and 4. Analysis of the carbohydrate distribution during activation suggests that about two-thirds of the sialic acid is present in intermediate 3, and about onethird of the sialic acid is attached to intermediate 1. Upon further activation, the carbohydrate which was attached to intermediate 1 is found in intermediate 2, and subsequently in cu-thrombin. Intermediate 4 contains no sialic acid. Activation of [sin2y2-3H]prothrombin in serum-thromboplastin produced intermediates identical in apparent molecular weight with those produced both upon activation in purified systems (HELDEBRANT, C.

ABSTRACTSkeletal muscle, at 30 to 40% of body mass, is the most abundant soft tissue in the body. Besides its primary function in movement and posture, skeletal muscle is a significant innate immune organ with the capacity to produce cytokines and chemokines and respond to proinflammatory cytokines. Little is known about the role of skeletal muscle during systemic influenza A virus infection in any host and particularly avian species. Here we used primary chicken and duck multinucleated myotubes to examine their susceptibility and innate immune response to influenza virus infections. Both chicken and duck myotubes expressed avian and human sialic acid receptors and were readily susceptible to low-pathogenicity (H2N3 A/mallard duck/England/7277/06) and high-pathogenicity (H5N1 A/turkey/England/50-92/91 and H5N1 A/turkey/Turkey/1/05) avian and human H1N1 (A/USSR/77) influenza viruses. Both avian host species produced comparable levels of progeny H5N1 A/turkey/Turkey/1/05 virus. Notabl...

A serum-susceptible, gmnea-ptg chamber-passaged, laboratory strata (BS4 (agar)) of Netssena gonorrhoeae was converted to serum resistance by incubauon vath cyudine 5 -monophospho-N-acetyl neuramimc acid (CMP-NANA) and exarmned by electron nucroscopy after staining with ruthemumored. In contrast to serum susceptible gonecocci incubated without CMP-NANA, the majority (60-70%) of the serum resistant organisms showed a surface accumulatmn of polysacchande. Tlus surface polysacchande was enhanced on all the resistent gonococci after mcubauon wRh fresh human serum. Control susceptible gonococci were devoid of the polysacchande after incubation wRh heated human serum. Indenucal results were ohtamed with a fresh gonococcal isolate which had lost serum resistance on subculture but which, in common with 3 other isolates, was restored to serum resistance by incubation wRh CMP-NANA

Rickettsia prowazeki will adsorb to ghosts derived from sheep erythrocytes by hypotonic lysis. Adsorption to ghosts, as to intact erythrocytes, is dependent on the metabolism of the rickettsiae. KCN and 0 C inhibit adsorption. Fluoride, while inhibiting hemolysis, has no effect on adsorption to ghosts or intact erythrocytes. The adsorption of rickettsiae to ghosts does not lead to lysis, in that fluorescent albumin can be retained by the ghost after adsorption. Both inside-out and right-side-out vesicles formed from the ghost are equally capable of adsorption, indicating that the receptor is stable during the process of vesiculation and is not localized to one aspect of the cell membrane. Adsorption to ghosts is slower and displays less affinity than to the intact erythrocyte. The ghost system will be useful in characterizing the membrane receptor for rickettsia and in establishing assays for adsorption to other cells.

The current investigation was designed to determine the total serum sialic acid (TSA), lipid associated sialic acid (LASA), total protein (TP), and TSA/TP values for 16 patients with active form of Behçet's disease, 12 patients with other uveitis types (pathologic controls) and 22 normal individuals. Data analysis indicated significant differences in mean TSA, LASA and TSA/TP values in patients with Behçet's disease (95.6 ± 15.3 mg/dl, 29.8 ± 7.4 mg/dl and 12.9 ± 2.1 mg/g, respectively) and in pathologic controls (75.8 ± 8.1 mg/dl, 26.6 ± 0.3 mg/dl and 10.0 ± 0.9 mg/g, respectively) when compared to normal controls (59.5 ± 7.5 mg/dl, 19.2 ± 3.2 mg/dl and 8.2 ± 0.9 mg/g, respectively) (p<0.01). No significant difference was observed in mean LASA values in patients with Behçet's disease when compared to pathologic controls (p>0.05). No significant difference was observed in mean TP values in patients with Behçet's disease (7.4 ± 0.7 g/dl), normal controls (7.3 ± ...

Proton NMR spectroscopy allows the detection in plasma of resonances arising from N-acetyl-glucosamine ~NAG) and N-acetyi-neuraminic acid (NANA) which have been shown to be borne by acute phase glycoproteins. These resonances can be identified using 2 different protocols of spectrum acquisition detecting different physical states in the global pool of glycoproteins. ie rnobile and less mobile moieties of glycosylated chains, in this study we demonstrate that NMR spectroscopy allows a precise monitoring of the variations of glycosylated residues in cancers, inflammatory processes and bone marrow transplantation. The most important lindings are that: i), the distribution of glycosylated residues varies with the origin of the cancerous tissue: ii). the level of these residues is a function of tumor development; iii). the concentrations in NAG and NANA are well correlated with the standard biological parameters of acute phase and leucocyte activation. Proton NMR spectroscopy of glycosylated residues in plasma may offer a new means of monitoring sialic acid in cancer and other pathological conditions. proton NMR spectroscopy / plasma / glycoproteins / glycolipids / cancer / inflammation

A characteristic feature of neurogenic sites in the postnatal brain is the expression of the polysialylated forms of the neural cell adhesion molecule (PSA-NCAM). To investigate the role of PSA-NCAM in generation of neuronal populations, we developed an in vitro model where neurogenesis occurs in primary cortical cultures following serum withdrawal. We show that removal or inactivation of the PSA tail of NCAM in these cultures leads to a significant decrease in the number of newly generated neurons. Similarly, cultures prepared from NCAM knock-out mice exhibit a significantly reduced neurogenesis. Pulse-chase experiments using the proliferation marker BrdU reveal that the lack of PSA does not affect the mitotic rate of neural progenitors but rather, it reduces the early survival of newly generated neurons. These results suggest that, in addition to its role in the migration of neuronal progenitors, PSA-NCAM is required for the adequate survival of these cells.

The cx-2,8-linked sialic acid polymer (PSA) on the neural cell adhesion molecule (NCAM) is an important regulator of cell surface interactions. We have examined the translocation of PSA-NCAM to the surface of cultured cortical neurons and insulin secreting ,3 cells under different conditions of cell activity. Endoneuraminidase N, an enzyme that specifically cleaves PSA chains, was used to remove pre-existing PSA from the plasma membrane and the re-expression of the molecule was monitored by immunocytochemistry. Punctate PSA immunostaining was restored on the surface of 68% of neurons within 1 h. This recovery was almost completely prevented by tetrodo- toxin, suggesting that spontaneous electrical activity is required. K+ depolarization (50 mM) allowed recovery of PSA surface staining in the presence of tetrodotoxin and this effect required the presence of extracellular Ca2+. Rapid redistribution of PSA-NCAM to the surface of , cells was observed under conditions that stimulate insulin secretion. Ca2+ channel inhibition decreased both PSA-NCAM expression and insulin secretion to control, non-stimulated levels. Finally, subcellular fractionation of an insulin-secreting cell line showed that the secretory vesicle fraction is highly enriched in PSA-NCAM. These results suggest that PSA-NCAM can be translocated to the cell surface via regulated exocytosis. Taken together, our results provide unprecedented evidence linking cell activity and PSA-NCAM expression, and suggest a mechanism for rapid modulation of cell surface interactions.

The capacity for long-distance migration of the oligodendrocyte precursor cell, oligodendrocyte-type 2 astrocyte (O-2A), is essential for myelin formation. To study the molecular mechanisms that control this process, we used an in vitro migration assay that uses neurohypophysial explants. We provide evidence that O-2A cells in these preparations express functional N-methyl-D-aspartate (NMDA) receptors, most likely as homomeric complexes of the NR1 subunit. We show that NMDA evokes an increase in cytosolic Ca2+ that can be blocked by the NMDA receptor antagonist AP-5 and by Mg2+. Blocking the activity of these receptors dramatically diminished O-2A cell migration from explants. We also show that NMDA receptor activity is necessary for the expression by O-2A cells of the highly sialylated polysialic acid-neural cell adhesion molecule (PSA-NCAM) that is required for their migration. Thus, glutamate or glutamate receptor ligands may regulate O-2A cell migration by modulating expression ...

A characteristic feature of neurogenic sites in the postnatal brain is the expression of the polysialylated forms of the neural cell adhesion molecule (PSA-NCAM). To investigate the role of PSA-NCAM in generation of neuronal populations, we developed an in vitro model where neurogenesis occurs in primary cortical cultures following serum withdrawal. We show that removal or inactivation of the PSA tail of NCAM in these cultures leads to a significant decrease in the number of newly generated neurons. Similarly, cultures prepared from NCAM knock-out mice exhibit a significantly reduced neurogenesis. Pulse-chase experiments using the proliferation marker BrdU reveal that the lack of PSA does not affect the mitotic rate of neural progenitors but rather, it reduces the early survival of newly generated neurons. These results suggest that, in addition to its role in the migration of neuronal progenitors, PSA-NCAM is required for the adequate survival of these cells.