Isolation, fractionation, and preliminary characterization of a novel class of sulfated glycans from the tunic of Styela plicata (Chordata Tunicata)

The sulfated glycans in the tunic of Styela plicata differ from the glycosaminoglycans of animal tissues and also from the sulfated polysaccharides isolated from marine algae. The ascidian glycans occur primarily as three fractions that differ markedly in molecular weight and chemical composition. The high molecular weight fraction encompasses a broad range of molecular weights but is chemically homogeneous and contains an unusual amount of galactose. The 20,000 molecular weight polysaccharide is rich in galactose and glucose while the 8,000 molecular weight fraction is rich in amino sugars and contains the neutral hexoses galactose, glucose, and mannose. All fractions contain large amounts of sulfate esters. The ascidians polysaccharides can be extracted from the tissue by proteolytic enzyme or by guanidine hydrochloride solutions. The high molecular weight fraction is preferentially extracted by papain while guanidine hydrochloride removes mainly the low molecular weight polysaccharides. We speculate that these sulfated glycans are essential for maintaining the structural integrity of the tunic, in analogy with the glycosaminoglycans of vertebrate connective tissues.     

N-myc down regulates neural cell adhesion molecule expression in rat neuroblastoma.

In human neuroblastoma, amplification of the N-myc oncogene is correlated with increased metastatic ability. We recently showed that transfection of the rat neuroblastoma cell line B104 with an N-myc expression vector resulted in an increase in metastatic ability and a significant reduction in the expression of major histocompatibility complex class I antigens. We examined whether N-myc causes additional phenotypic changes in these cells. We showed that expression of N-myc leads to a dramatic reduction in the levels of neural cell adhesion molecule (NCAM) polypeptides and mRNAs. Spontaneous revertants of the high N-myc phenotype were found to have regained significant levels of NCAM expression, indicating that the continued expression of N-myc is required to maintain the low NCAM phenotype. NCAM was not reduced in B104 cells transfected with the neomycin resistance vector alone, and other neuronal markers were not specifically reduced in N-myc-transfected B104 cells. As NCAM functions in cell-cell adhesion, decreased NCAM expression could contribute significantly to the increased metastatic potential of N-myc-amplified neuroblastomas.     

The biosynthesis, processing, and secretion of laminin by human choriocarcinoma cells

Laminin, a glycoprotein component of basal laminae, is synthesized and secreted in culture by a human malignant cell line (JAR) derived from gestational choriocarcinoma. Biosynthetically labeled human laminin subunits A (Mr approximately 400,000) and B (Mr = 200,000 doublet) are glycoslyated with asparagine-linked high mannose oligosaccharides that are processed to complex oligosaccharides before the laminin molecule is externalized by the cell. The rate-limiting step in the processing of the asparagine-linked glycans of laminin is at the point of action of alpha-mannosidase I since the principal laminin forms that accumulate in JAR cells contain Man9GlcNAc2 and Man8GlcNAc2 oligosaccharide units. The combination of subunits to form the disulfide-linked laminin molecule (Mr approximately 950,000) occurs rapidly within the cell at a time when the subunits contain these high mannose oligosaccharides. The production of laminin is limited by the availability of the A subunit such that excess B subunit forms accumulate intracellularly as uncombined B and a disulfide-linked B dimer. Pulse-chase kinetic studies establish these B forms as intermediates in the assembly of the laminin molecule. The fully assembled laminin undergoes further oligosaccharide processing and translocation to the cell surface, but uncombined B and B dimer are neither processed nor secreted to any significant extent. Therefore, laminin subunit combination appears to be a prerequisite for intracellular translocation, processing, and secretion. The mature laminin that contains complex oligosaccharides does not accumulate intracellularly but is rapidly externalized upon completion, either secreted into the culture medium (25%) or associated with the cell surface (75%) as determined by susceptibility to degradation by trypsin. About one-third of the laminin molecules secreted or shed by JAR cells into the chase medium contain a smaller A subunit form that appears to have been modified by limited proteolytic cleavage. The putative proteolytic event is closely timed to the release of the laminin into the culture medium.     

Balance of cell proliferation and death among dynamic populations: a mathematical model

Developmental changes in cell numbers represent the dynamic balance between cell proliferation and death. One obstacle to assessing this balance is an inability to quantify the total amount of cell death, i.e., with a positive indicator such as terminal dUTP nick end labeling (TUNEL) or caspase activity. A novel mathematical model is described wherein data on daily cell growth (the change in cell number) and cell cycle kinetics can be used to determine the total amount of cell death. Two sets of data from previously published studies were tested in this model; primary cultured cortical neurons and B104 neuroblastoma cells. These two preparations have contrasting features: neuronal cultures are heterogeneous and have relatively few cells that are actively cycling (i.e., the growth fraction for these cells is low), whereas B104 cells are relatively homogeneous cultures in which the growth fraction is high. In primary cortical cultures, there was a balance in cell production and death. Treatment with a potent anti-mitogen, ethanol (400 mg/dl), affected this balance principally by reducing cell production, although the rate of cell death was also increased. In untreated B104 cells, there was eight-fold more cell production than cell death. Growth factors such as platelet-derived growth factor BB doubled cell production. Ethanol reduced cell production by >60%, and it eliminated growth factor-mediated cell production. All of these changes occurred in the absence of an effect on the amount of cell death. Thus, the model is ideal for predicting the effects of an epigenetic factor (e.g., a growth factor, toxin, or pharmacological agent) on cell development and can be useful in determining the consequences of a genetic manipulation as well.     

The outer mantle epithelium of Haliotis tuberculata (Gastropoda Haliotidae): an ultrastructural and histochemical study using lectins

The mantle of molluscs has been the subject of many studies as it is the organ that forms the shell. Microscopic studies in particular focus on the outer mantle epithelium, but few studies address this epithelium in a histochemical way. In this study, the outer mantle epithelium in adult specimens of Haliotis tuberculata is studied, that is, in specimens involved in maintaining and repairing the shell rather than in generating it. The epithelial cells are studied by scanning (SEM) and transmission electron microscopy (TEM), and by histochemical techniques, including the use of lectins for their biochemical characterization. The epithelium is composed of pigmented epidermal cells with small microvilli and junctional complexes. It furthermore contains a few ciliated cells, as well as two types of secretory cells which differ in the ultrastructural appearance of their secretory granules and their glycoconjugate content. Histochemical study shows secretory cells containing sulphated glycoconjugates such as glycosaminoglycans or mucins rich in N‐acetylgalactosamine and N‐glycoproteins rich in fucose. Furthermore, the apical regions of the epidermal cells are positive for lectins that label fucose, mannose and N‐acetylglucosamine. The role of epithelial cells in the synthesis of structural components of the shell is discussed.     

Amphotericin B-induced carbohydrate changes on the Trypanosoma cruzi surface membrane.

Changes in the cell surface carbohydrates of Trypanosoma cruzi epimastigotes induced by Amphotericin B (AmB) were assessed by chemical methods and by agglutination assay employing a panel of highly purified lectins of various sugar specificities. Escherichia coli K12 with mannose-sensitive fimbriae was also used as an agglutination probe. Amphotericin B caused a decrease in the total carbohydrate content of all glycoconjugate fractions isolated. Exposure to AmB strongly affected the mannose/galactose ratio (1:5) in the CHCl3/methanol/H2O soluble fraction. These sugars in 1.4:1 ratio were the major hexose components of control cells. The decrease in the mannose content (48 to 15%) after AmB treatment agrees with the marked decrease in the T. cruzi cell surface receptors for fimbriated E. coli K12. Also, an increase in the galactose content (74%) as compared with control cells (34%) is in agreement with the peanut agglutinin and Euonymus europaeus lectins agglutination results. Differences in the cell surface carbohydrates induced by AmB could be associated with alterations in the membrane structure and organization.     

Amphotericin B-Induced Carbohydrate Changes on the Trypanosoma cruzi Surface Membrane

ABSTRACT Changes in the cell surface carbohydrates of Trypanosoma cruzi epimastigotes induced by Amphotericin B (AmB) were assessed by chemical methods and by agglutination assay employing a panel of highly purified lectins of various sugar specificities, Escherichia coli K12 with mannose-sensitive fimbriae was also used as an agglutination probe. Amphotericin B caused a decrease in the total carbohydrate content of all glycoconjugate fractions isolated. Exposure to AmB strongly affected the mannose/galactose ratio (1:5) in the CHCI3/methanol/H2O soluble fraction. These sugars in 1.4:1 ratio were the major hexose components of control cells. The decrease in the mannose content (48 to 15%) after AmB treatment agrees with the marked decrease in the T. cruzi cell surface receptors for fimbriated E. coli K12. Also, an increase in the galactose content (74%) as compared with control cells (34%) is in agreement with the peanut agglutinin and Euonymus europaeus lectins agglutination results. Differences in the cell surface carbohydrates induced by AmB could be associated with alterations in the membrane structure and organization.     

The fine structure of Caenorhabditis elegans N-glycans

We report the fine structure of a nearly contiguous series of N-glycans from the soil nematode Caenorhabditis elegans. Five major classes are revealed including high mannose, mammalian-type complex, hybrid, fuco-pausimannosidic (five mannose residues or fewer substituted with fucose), and phosphocholine oligosaccharides. The high mannose, complex, and hybrid N-glycan series show a high degree of conservation with the mammalian biosynthetic pathways. The fuco-pausimannosidic glycans contain a novel terminal fucose substitution of mannose. The phosphocholine oligosaccharides are high mannose type and are multiply substituted with phosphocholine. Although phosphocholine oligosaccharides are known immunomodulators in human nematode and trematode infections, C. elegans is unique as a non-parasitic nematode containing phosphocholine N-glycans. Therefore, studies in C. elegans should aid in the elucidation of the biosynthetic pathway(s) of this class of biomedically relevant compounds. Results presented here show that C. elegans has a functional orthologue for nearly every known enzyme found to be deficient in congenital disorders of glycosylation types I and II. This nematode is well characterized genetically and developmentally. Therefore, elucidation of its N-glycome, as shown in this report, may place it among the useful systems used to investigate human disorders of glycoconjugate synthesis such as the congenital disorders of glycosylation syndromes.     

Expression of an unusual acidic glycoconjugate in Leishmania donovani

An acidic glycoconjugate containing mannose, galactose and phosphate in approximately equimolar amounts was extracted from Leishmania donovani promastigotes and partially characterized. The glycoconjugate could be metabolically labeled with either [3H]mannose or [3H]galactose and was extractable from a delipidated residue fraction with water/ethanol/diethyl ether/pyridine/concentrated NH4OH (15:15:5:1:0.017) at 25 degrees C. The radioactively labeled glycoconjugate was found to possess the following characteristics: 1) comprised 45-60% of the total [3H]mannose label incorporated into macromolecules; 2) was soluble in alkaline solvents and 0.5% Triton X-100; 3) migrated as a broad band upon electrophoresis on sodium dodecyl sulfate-polyacrylamide gels with an approximate molecular weight of 15,000-30,000; 4) bound to DE52 cellulose and was eluted with a salt gradient of 0-0.1 M NaCl; 5) was insensitive to Pronase, hyaluronidase, chondroitinase, endo-beta-N-acetylglucosaminidase H, and endo-beta-galactosidase; and 6) possessed hydrophobic properties. An unusual feature of the glycoconjugate was its lability to mild acid hydrolysis (0.02 N HCl, 15 min, 60 degrees C). As determined by alkaline phosphatase and glycosidase digestion and paper chromatographic analysis, the major fragment generated by mild acid hydrolysis was found to be a phosphorylated galactosyl-beta-mannose disaccharide. All of these characteristics suggest that the glycoconjugate may be a polysaccharide and, possibly, may be important in parasite-host cell interactions.     

The processing of asparagine-linked oligosaccharides in HT-29 cells is a function of their state of enterocytic differentiation. An accumulation of Man9,8-GlcNAc2-Asn species is indicative of an impaired N-glycan trimming in undifferentiated cells

Studies on the regulation of the enterocytic differentiation of the human colon cancer cell line HT-29, which is differentiated in the absence (Glc-) but not in the presence of glucose (Glc+), have recently shown that the post-translational processing of sucrase-isomaltase and particularly its glycosylation vary as a function of cell differentiation (Trugnan G., Rousset, M., Chantret, I., Barbat, A., and Zweibaum, A. (1987) J. Cell Biol. 104, 1199-1205). Other studies indicate that in undifferentiated HT-29 Glc+ cells there is an accumulation of UDP-N-acetylhexosamine, which is involved in the glycosylation process (Wice, B. M., Trugnan, G., Pinto, M., Rousset, M., Chevalier, G., Dussaulx, E., Lacroix, B., and Zweibaum, A. (1985) J. Biol. Chem. 260, 139-146). The purpose of the present work is to investigate whether an overall alteration of protein glycosylation is associated with the inability of HT-29 cells to differentiate. At least three alterations are detected: (i) after a 10-min pulse, the incorporation of D-[2-3H]mannose in undifferentiated cells is severely reduced, compared to differentiated cells. (ii) After a 24-h period of labeling with D-[2-3H]mannose, undifferentiated cells accumulate more than 60% of the radioactivity in the high mannose glycopeptides, whereas differentiated HT-29 Glc- cells accumulate only 38%. (iii) The analysis of the high mannose oligosaccharides transferred "en bloc" from the lipid precursor shows that Man9,8-GlcNAc2 species accumulate in undifferentiated cells, whereas no such accumulation can be detected in differentiated cells. This glycosylation pattern is consistent with an impairment of the trimming of high mannose into complex glycans. It is concluded that N-glycan processing is correlated with the state of enterocytic differentiation of HT-29 cells.     

Granule-mediated killing by granzyme B and perforin requires a mannose 6-phosphate receptor and is augmented by cell surface heparan sulfate

During granule-mediated killing by cytotoxic T lymphocytes or natural killer cells, the serine protease granzyme B enters the target cell by endocytosis and induces apoptosis. Previous studies suggested a role for the mannose 6-phosphate receptor, but further experiments with purified granzyme B indicated this was not essential. Additionally, it is now clear that grB is exocytosed from killer cells in a high-molecular-weight complex with the proteoglycan serglycin. Here granzyme B was delivered as a purified monomer, or in complex with either glycosaminoglycans or serglycin, and killing was evaluated. When granzyme B was a monomer, soluble mannose 6-phosphate had a limited impact, whereas apoptosis induced by the complexed grB was effectively inhibited by mannose 6-phosphate. Most importantly, when granzyme B and perforin were delivered together from granules, inhibition by mannose 6-phosphate was also observed. In pulldown assays mediated by the cation-independent mannose 6-phosphate receptor, granzyme B bound to the receptor more intensely in the presence of immobilized heparan sulfate. We therefore propose the model that under physiological conditions serglycin-bound granzyme B is critically endocytosed by a mannose 6-phosphate receptor, and receptor binding is enhanced by cell surface heparan sulfate.     

Human helper T cell factor(s). IV. Demonstration of a human late-acting B cell differentiation factor acting on Staphylococcus aureus Cowan I-stimulated B cells

At least two distinct B cell stimulatory factors (BSF) were found to be involved in the differentiation of Staphylococcus aureus Cowan I (SAC)-stimulated human B cells to IgG-producing cells. A factor tentatively called B cell differentiation factor I (BCDF I) was found in one fraction, and a second factor, BCDF II was found in another fraction. The BCDF I fraction alone induces IgG-production in SAC-stimulated B cells, but the BCDF II fraction does not. The BCDF II fraction enhances IgG production in SAC-stimulated B cells in the presence of the BCDF I fraction. Studies concerning the time-course of the action of the BCDF II fraction revealed that it contains a late-acting differentiation factor that acts on B cells most effectively when it is added to the SAC-stimulated B cell culture after the addition of BCDF I fraction; it induces IgG plaque-forming cells within 1 day. The pI value of a late-acting BCDF was in the range of 5 to 6; this pI range is different from that of BCDF I but similar to that of BCDF II, which was shown in our previous studies to be able to induce IgG production in Epstein Barr Virus-transformed B lymphoblastoid cell lines. In addition, the m.w. of a late-acting BCDF were about 35,000 and 20,000, which are the same as those of BCDF II, and thus its identity with BCDF II was suggested.     

The Candida albicans phospholipomannan is a family of glycolipids presenting phosphoinositolmannosides with long linear chains of beta-1,2-linked mannose residues.

In a series of studies, we have shown that Candida albicans synthesizes a glycolipid, phospholipomannan (PLM), which reacted with antibodies specific for beta-1,2-oligomannosides and was biosynthetically labeled by [(3)H]mannose, [(3)H]palmitic acid, and [(32)P]phosphorus. PLM has also been shown to be released from the C. albicans cell wall and to bind to and stimulate macrophage cells. In this study, we show by thin layer chromatography scanning of metabolically radiolabeled extracts that the C. albicans PLM corresponds to a family of mannose and inositol co-labeled glycolipids. We describe the purification process of the molecule and the release of its glycan fraction through alkaline hydrolysis. Analysis of this glycan fraction by radiolabeling and methylation-methanolysis confirmed the presence of inositol and of 1, 2-linked mannose units. NMR studies evidenced linear chains of beta-1,2-oligomannose as the major PLM components. Mass spectrometry analysis revealed that these chains were present in phosphoinositolmannosides with degrees of polymerization varying from 8 to 18 sugar residues. The PLM appears as a new type of eukaryotic inositol-tagged glycolipid in relationship to both the absence of glucosamine and the organization of its glycan chains. This first structural evidence for the presence of beta-1, 2-oligomannosides in a glycoconjugate other than the C. albicans phosphopeptidomannan may have some pathophysiological relevance to the adhesive, protective epitope, and signaling properties thus far established for these residues.     

Cell surface sialylation and tumor metastasis. Metastatic potential of B16 melanoma variants correlates with their relative numbers of specific penultimate oligosaccharide structures

Numerous investigations suggest that cell surface glycoconjugates, and in particular sialic acids, are directly involved in determining the metastatic phenotype. To further evaluate this hypothesis, we have used a variety of techniques to probe the cell surfaces of several metastatic variants of the murine B16 melanoma that were selected for experimental lung-colonizing ability (Fidler, I. (1973) Nature 242, 148-149) or for their ability to spontaneously metastasize from the site of a subcutaneous injection (Stackpole, C. W., Alterman, A. L., and Fornabaio, D. M. (1985) Invasion & Metastasis 5, 125-142). Using a highly sensitive high performance liquid chromatography sialic acid assay in conjunction with Vibrio cholerae sialidase, we find that none of these metastatic variants differ significantly in their overall levels of cell surface sialic acid. Using highly purified, linkage-specific sialyltransferases, in conjunction with specific glycosidases, to probe the cell surface saccharide topography of specific penultimate oligosaccharides, we also find no significant differences between the efficient lung-colonizing variant, B16-F10 and the poorly-colonizing B16-F1 or B16-Flr variants. In contrast, the spontaneously metastatic variants examined contain substantially different levels of specific penultimate sialylation sites. The tumorigenic but nonmetastatic B16-LM3/G3.26 variant contains 4-fold more penultimate Gal beta 1-3GalNAc sialylation sites than the tumorigenic and highly metastatic B16-LM3/G3.12 variant when CMP[3H]NeuAc and the alpha 2-3Gal beta 1-3GalNAc sialyltransferase are used to probe the melanoma cell surfaces. Several prominent glycoconjugates of apparent Mr 43,000, 40,000, and 30,000 are especially evident upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the nonmetastatic cells. The nonmetastatic variant also contains 2-fold more Gal beta 1-4GlcNAc sialylation sites than the metastatic variant when the alpha 2-6Gal beta 1-4GlcNAc sialyltransferase is used as a cell surface probe. In this case, glycoconjugates of apparent Mr 74,000, 45,000, and 43,000 are more prominently observed on the cell surfaces of the nonmetastatic variant. These data indicate that the differences in lung-colonizing abilities of B16 melanoma metastatic variants do not correlate with the numbers or sialylation states of specific penultimate oligosaccharide structures on their surfaces. However, the relative levels of specific penultimate saccharide structures do correlate with the ability of the cells to undergo spontaneous metastasis from a subcutaneous tumor.     

Adhesive responses of fibroblast and neuroblastoma cells to substrata coated with polyvalent or monoclonal antibody to fibronectin

Both polyvalent and hybridoma-produced antibodies to fibronectin (Fn) were used to ‘map’ the immunoaccessible subsets of cell surface fibronectin on virus-transformed murine fibroblast SVT2 and rat neuroblastoma B104 cells. As one approach to this end, attachment and spreading responses of cells were measured on tissue culture substrata coated with antibody or with plasma fibronectin to compare their adhesive responses. Both SVT2 and B104 cells adhere poorly to polyvalent anti-Fn-coated substrata over short time intervals, but within several hours changes occur which permit cells to attach and spread as well on anti-Fn as on Fn (post-adsorption of the anti-Fn with Fn also generates a maximal response). This adhesive response could be completely prevented by predigesting the cells with Flavobacterium heparanase, but not with chondroitinase ABC, indicating that the cell surface Fn responsible for antibody-mediated adhesion is associated with heparan sulfate proteoglycans on the cell surface. The compositions of the substratum-attached material (left bound after EGTA-mediated detachment of cells) from cells attaching to anti-Fn or Fn were analysed by SDS-PAGE and found to be identical within the same cell type for the two different substrata. Three hybridoma-produced antibodies, which recognize different determinants on Fn, generated different adhesive responses for SVT2 or B104 cells when adsorbed to the substratum. SVT2 cells adhered well to antibody no. 32-coated substrata but poorly to antibodies 92 or 136; on the other hand, B104 cells responded similarly to all three antibodies over short times of attachment but much better to no. 32 after a several hour incubation. These experiments indicate that (1) much of the cell surface fibronectin is complexed with heparan sulfate proteoglycan and is initially inaccessible to bind to polyvalent antibody on the substratum to promote adhesion; (2) the surface of neuroblastoma cells contains a fibronectin-like molecule which is important in their substratum adhesion; and (3) monoclonal antibodies are valuable tools in ‘mapping’ the orientation of cell surface molecules like fibronectin by measuring adhesive responses to antibody-coated substrata.     

Characterization of a unique glycoprotein antigen expressed on the surface of human neuroblastoma cells

In order to develop a molecular probe to delineate chemical and biological characteristics of human neuroblastoma cells, a murine monoclonal antibody (Mab 5G3) was produced that is directed to a glycoprotein, preferentially expressed on the surface of such cells. This antibody is of IgG2a isotype, has an association constant of 8 X 10(9) M-1, and reacts preferentially with human neuroblastoma cell lines and fresh frozen tissue sections in enzyme-linked immunosorbent assay and immunoperoxidase assays, respectively. Minimal reactivity is observed with a variety of lymphoblastoid cell lines and normal fetal and adult tissues. Mab 5G3 specifically recognizes a neuroblastoma target glycoprotein antigen of 215 kDa that is derived from a 200-kDa precursor, as evident from pulse-chase biosynthetic studies. Treatment with tunicamycin revealed that both molecules contain N-asparagine-linked oligosaccharides; however, only the 215-kDa species is resistant to treatment with endo-beta-N-acetylglucosaminidase H and sensitive to neuraminidase, indicating that it contains trimmed and terminally sialylated oligosaccharides of the "complex" type. In contrast, the 200-kDa precursor is sensitive to endo-beta-N-acetylglucosaminidase H and resistant to neuraminidase treatment indicating that it contains high-mannose non-processed oligosaccharides. The 215-kDa molecule is sulfated, phosphorylated at serine residues, and expressed on the cell surface. A molecule of 200 kDa is detected by Mab 5G3 in spent culture medium of human neuroblastoma cells which is neither sulfated nor phosphorylated.     

Newly synthesized glycoconjugates from two cell lines derived from rat osteogenic sarcoma: effects of Matrigenin activity from bone

An activity isolated from bovine bone was previously shown to stimulate proteoglycan synthesis by several connective tissue cell lines from normal tissues (Matrigenin activity). The effect of this activity on glycoconjugate synthesis by two osteoblastic cell lines, ROS 17/2 and UMR-106, derived from rat osteogenic sarcoma, was examined after labelling of the cells with [3H]glucosamine and [35S]sulfate. The glycoconjugates from the cell layers and the media were separated by DEAE-Sephacel chromatography and the anionic glycoconjugates of the media were further analyzed by chromatography on Sepharose CL-2B and enzymatic digestion of the papain-released glycosaminoglycans. The ROS 17/2 cells secreted at least two distinct species of proteoglycan (one heparan sulfate rich and the other chondroitin sulfate rich), whereas the UMR-106 secreted primarily an anionic glycoprotein. The addition of Matrigenin activity to the ROS 17/2 cells resulted in stimulation of incorporation of radioactivity into the proteoglycan and hyaluronic acid, but in UMR-106 cultures it resulted in decreased incorporation into the anionic glycoprotein. The decrease in incorporation into the anionic glycoprotein from the medium was shown, by alkaline beta-elimination, to have occurred mainly in the oligosaccharide fraction, relative to control cultures.     

Resveratrol Inhibits Proliferation and Promotes Apoptosis of Neuroblastoma Cells: Role of Sirtuin 1

Resveratrol prolongs lifespan and prevent cancer formation; however, the mechanisms are not understood. Here we evaluated the cell-cycle inhibition and apoptosis of resveratrol in B65 neuroblastoma cells, and we also studied the effects of resveratrol on the mammalian silent information regulator 2 (SIRT1). Results show that resveratrol reduces cell viability and causes apoptosis at 24 h of treatment. Resveratrol partially blocked cell proliferation, and significantly increased the fraction of cells arrested in the S phase. The role of SIRT1 in cell-cycle effects mediated by resveratrol was studied through changes in the expression of SIRT1 using western blot. Exposure to resveratrol decreased SIRT1 content, concomitant with an increase in the acetylated form of sirtuin substrates p53 and NFκ-β. Treatment of B65 neuroblastoma cells with resveratrol also reduced the content of the phosphorylated form of AKT. Exposure to the SIRT1 inhibitors nicotinamide and sirtinol altered neither cell viability nor the fraction of apoptotic cells. Furthermore, when cells were exposed simultaneously to resveratrol and nicotinamide or sirtinol, no changes were observed in the fraction of apoptotic cells. Our results show that a decrease in SIRT1 content, caused by exposure to resveratrol, does not appear to be involved in cell-cycle arrest or activation of apoptosis.     

Biosynthesis of glycosaminoglycans by epithelial and lymphocytic components of murine thymus

Interactions between the epithelial and lymphocytic components of the thymus are required for T cell maturation, yet the molecular bases for these interactions remain elusive. In the development and function of other endodermally derived organs, glycosaminoglycan-containing proteins are known to play a critical role. In contrast, virtually nothing is known about the macromolecules that are major constituents of thymic interstitial spaces. For these reasons, we undertook metabolic labeling studies in vitro with D-(6-3H)glucosamine and 35SO4(-2) to begin to characterize systematically the relative amounts and types of glycosaminoglycans made by enriched subpopulations of cells within the thymus. Hydrocortisone, which depletes the thymus of 90% of its lymphocytes, was used both to enrich for epithelium-derived glycoconjugates and to determine if significant alterations in glycoconjugate metabolism accompany drug-induced premature thymic involution. Results indicate: 1) glycosaminoglycans account for a substantial proportion of the total glycoconjugates synthesized by both thymocytes and epithelium; 2) Glycosaminoglycans show a tissue-specific distribution. Hyaluronic acid is the major glycosaminoglycan synthesized by thymic epithelium, whereas it accounts for less than 15% of the total glycosaminoglycans made by thymocytes; 3) Similar proportions of sulfated glycosaminoglycans are made by thymic epithelium and thymocytes. Chondroitin sulfates predominate (75 to 90%) over heparan sulfates (10 to 25%). Chondroitin sulfates from both nonstimulated thymocytes and epithelium are nearly exclusively sulfated at the 4-position of their N-acetylgalactosamine residues; 4) The major high m.w. glycoconjugate of thymocytes, however, is nonsulfated and is resistant to pronase, hyaluronidase, chondroitinase ABC, nitrous acid, keratanase, and neuraminidase; 5) Although hydrocortisone treatment causes a dramatic inhibitory effect on the incorporation of radioactivity into smaller oligosaccharide side-chains by "cortisone-resistant" thymocytes, the drug exerts negligible effects on the metabolism of glycoconjugates by epithelium. These data, which quantify and categorize the complex arrays of glycoconjugates synthesized by the major cell types of the thymus, establish the necessary foundations for further investigations into the functional roles of these glycoconjugates in thymic epithelium-induced maturation of T lymphocytes.     

Identification and analysis of a lipopolysaccharide in cell walls of the blue-green alga Anacystis nidulans

Summary A lipopolysaccharide was isolated from cell walls of Anacystis nidulans by extraction with 45% aqueous phenol at 65°, and further purified by repeated high speed centrifugation. It contains 30–40% of lipid and about 60% of carbohydrate components. The carbohydrate moiety contains predominantly mannose and smaller amounts of galactose, glucose, fucose, rhamnose, 2-keto-3-deoxyoctonate, glucosamine and a second aminosugar. The latter was identified as a 2-amino-2-deoxyheptose with the gluco-configuration from C3 to C7. Thelipid moiety contains glucosamine and fatty acids (C22:0, C18:2, C16:0, C12:0 and C14:OH). The lipopolysaccharide has a very low phosphate content and does not contain heptose. It shows low pyrogenicity in rabbits and it is not toxic in mice.Abbreviations KDO 2-Keto-3-deoxy-octonate - LPS Lipopolysaccharide