High Molecular Weight Glycoconjugates Research Articles

Signaling Pathways Used by the Specialized Pro-Resolving Mediator Maresin 2 Regulate Goblet Cell Function: Comparison with Maresin 1.

Specialized pro-resolving mediators (SPMs), including Maresins (MaR)-1 and 2, contribute to tear film homeostasis and resolve conjunctival inflammation. We investigated MaR2′s signaling pathways in goblet cells (GC) from rat conjunctiva. Agonist-induced [Ca2+]i and high-molecular weight glycoconjugate secretion were measured. MaR2 increased [Ca2+]i and stimulated secretion. MaR2 and MaR1 stimulate conjunctival goblet cell function, especially secretion, by activating different but overlapping GPCR and signaling pathways, and furthermore counter-regulate histamine stimulated increase in [Ca2+]i. Thus, MaR2 and MaR1 play a role in maintaining the ocular surface and tear film homeostasis in health and disease. As MaR2 and MaR1 modulate conjunctival goblet cell function, they each may have potential as novel, but differing, options for the treatment of ocular surface inflammatory diseases including allergic conjunctivitis and dry eye disease. We conclude that in conjunctival GC MaR2 and MaR1, both increase the [Ca2+]i and stimulate secretion to maintain homeostasis by using one set of different, but overlapping, signaling pathways to increase [Ca2+]i and another set to stimulate secretion. MaR2 also resolves ocular allergy.

Indirect Evidence of Circulating Parasite Hapten Immune Complexes in Visceral Leishmaniasis.

Hypergammaglobulinemia is present in visceral leishmaniasis (VL), inducing the formation of immune complexes (ICs), which interferes in conventional serology. Parasitic haptens block antibodies, making it difficult to identify and detect them. ICs could be determined indirectly by acid dissociative ELISA (DE) seroconversion in natural and experimental VL. We determined the frequency of samples that seroconverted in DE or presented a 10% increase in DE (ΔDE) in 3 types of VLs-hamster, canine, and human samples-with larger antigen determination by direct antigen capture in experimental samples. The ΔDE frequency is increased in all VL models: human (34%), canine (27%), and hamster (25%) samples. Seroconversion was present in hamster (14%), dog (1%), and human (6%) samples. During experimental infection, higher frequencies (28%) of circulating antigens were observed at the 30th day, associated with higher ΔDE (47%) and seroconversion (22%), with lower frequencies in other periods. The frequency of ΔED and seroconversion samples found in natural and experimental infection suggests that specific antibodies can be blocked by low molecular weight antigens that interfere qualitatively (seroconversion) or quantitatively (ΔDE) in serology. Several antigen types may be involved, as high molecular weight proteins and low molecular weight glycoconjugates. The higher frequency of those indirect demonstrations of antibody-blocking antigen or haptens that could be acid-removed in VL has implications for the development of assays for detection of circulating or antibody-bound 1- to 3-kDa antigens, which could interfere in diagnosis and also in the immune response of the host.

Context-Dependent Regulation of Conjunctival Goblet Cell Function by Allergic Mediators

In the eye, goblet cells responsible for secreting mucins are found in the conjunctiva. When mucin production is not tightly regulated several ocular surface disorders may occur. In this study, the effect of the T helper (Th) 2-type cytokines IL4, IL5, and IL13 on conjunctival goblet cell function was explored. Goblet cells from rat conjunctiva were cultured and characterized. The presence of cytokine receptors was confirmed by Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Changes in intracellular [Ca2+], high molecular weight glycoconjugate secretion, and proliferation were measured after stimulation with Th2 cytokines with or without the allergic mediator histamine. We found that IL4 and IL13 enhance cell proliferation and, along with histamine, stimulate goblet cell secretion. We conclude that the high levels of IL4, IL5, and IL13 that characterize allergic conjunctivitis could be the reason for higher numbers of goblet cells and mucin overproduction found in this condition.

An engineered human conjunctival-like tissue to study ocular surface inflammatory diseases.

The aim of this study was to develop a three-dimensional model of the human conjunctiva that can be used to perform physiology and pathophysiology experiments. Fibrin-based matrices (derived from human plasma or plasma cryoprecipitate) were used as scaffolds, and primary cells were obtained from conjunctival tissue. Conjunctival constructs were analyzed by immunofluorescent staining and scanning electron microscopy and cell proliferation was measured with alamarBlue® assay. After characterizing the constructs, four different experimental conditions were analyzed in cryoprecipitate matrices: controls, air-lifted cultures (to increase cell stratification), partially desiccated cultures (to mimic dry eye disease), and IL-13-treated cultures (to mimic allergy). Constructs were stained with hematoxylin/eosin to observe changes in morphology. High molecular weight glycoconjugates were identified by HPA staining. MUC5AC and IL-6 secretion was evaluated by ELISA. The fibrin-based matrices supported conjunctival cell growth. Epithelial cells grew on the surface of the scaffolds and underwent stratification that increased over time. These cells had microvilli, which suggests cell polarization and functionality. Fibroblasts were integrated in the scaffold and showed elongated shape. Compared to controls, air-lifted construct had increased epithelial stratification and upregulated MUC5AC secretion. Increased MUC5AC secretion also occurred in partially desiccated and IL-13-treated cultures. The inflammatory status of cells was evaluated by IL-6 levels which were increased in air-lifted and partially desiccated cultures, but not in IL-13-treated ones. In conclusion, we have developed a new three-dimensional model of human conjunctiva that can be used to study ocular surface inflammatory diseases.

Interaction of IFN-γ with cholinergic agonists to modulate rat and human goblet cell function.

Goblet cells populate wet-surfaced mucosa including the conjunctiva of the eye, intestine, and nose, among others. These cells function as part of the innate immune system by secreting high molecular weight mucins that interact with environmental constituents including pathogens, allergens, and particulate pollutants. Herein we determined whether IFN-γ, a Th1 cytokine increased in dry eye, alters goblet cell function. Goblet cells from rat and human conjunctiva were cultured. Changes in intracellular [Ca2+] ([Ca2+]i), high molecular weight glycoconjugate secretion, and proliferation were measured after stimulation with IFN-γ with or without the cholinergic agonist carbachol. IFN-γ itself increased [Ca2+]i in rat and human goblet cells and prevented the increase in [Ca2+]i caused by carbachol. Carbachol prevented IFN-γ-mediated increase in [Ca2+]i. This cross-talk between IFN-γ and muscarinic receptors may be partially due to use of the same Ca2+i reservoirs, but also from interaction of signaling pathways proximal to the increase in [Ca2+]i. IFN-γ blocked carbachol-induced high molecular weight glycoconjugate secretion and reduced goblet cell proliferation. We conclude that increased levels of IFN-γ in dry eye disease could explain the lack of goblet cells and mucin deficiency typically found in this pathology. IFN-γ could also function similarly in respiratory and gastrointestinal tracts.

Effect of VIP on Intracellular [Ca2+], Extracellular Regulated Kinase 1/2, and Secretion in Cultured Rat Conjunctival Goblet Cells

To determine the intracellular signaling pathways that vasoactive intestinal peptide (VIP) uses to stimulate high molecular weight glycoconjugate secretion from cultured rat conjunctival goblet cells. Goblet cells from rat bulbar and forniceal conjunctiva were grown in organ culture. Presence and localization of VIP receptors (VPAC1 and 2) were determined by RT-PCR, immunofluorescence microscopy and Western blot analysis. Intracellular [Ca(2+)] ([Ca(2+)]i) was measured using fura-2. Extracellular signal-regulated kinase (ERK)-1/2 activity was determined by Western blot analysis. High molecular weight glycoconjugate secretion was measured with an enzyme-linked lectin assay on cultured goblet cells that were serum-starved for 2 hours before stimulation with VIP, VPAC1-, or VPAC2-specific agonists. Inhibitors were added 30 minutes prior to VIP. Activation of epidermal growth factor receptor (EGFR) was measured by immunoprecipitation using an antibody against pTyr followed by Western blot analysis with an antibody against EGFR. Both VIP receptors were present in rat conjunctiva and cultured goblet cells. VIP- and VPAC-specific agonists increased [Ca(2+)]i and secretion in a concentration-dependent manner. VIP also increased ERK1/2 activity, VIP-stimulated increase in [Ca(2+)]i. Secretion, but not ERK1/2 activity, was inhibited by the protein kinase A inhibitor, H89. VIP-stimulated secretion was inhibited by siRNA for ERK2 but not by siRNA for EGFR. VIP did not increase the phosphorylation of the EGFR. In conclusion, in cultured rat conjunctival goblet cells, VPAC1 and 2 receptors are functional. VIP stimulates a cAMP-dependent increase in [Ca(2+)]i and glycoconjugate secretion, but not ERK1/2 activation. VIP does not activate with EGFR.

Effect of Histamine on Ca2+-Dependent Signaling Pathways in Rat Conjunctival Goblet Cells

The purpose of this study was to determine the Ca(2+)-dependent cellular signaling pathways used by histamine to stimulate conjunctival goblet cell secretion. Cultured rat goblet cells were grown in RPMI 1640. Goblet cell secretion of high molecular weight glycoconjugates was measured by an enzyme-linked lectin assay. Intracellular [Ca(2+)] ([Ca(2+)](i)) was measured by loading cultured cells with the Ca(2+) sensitive dye fura-2. The level of [Ca(2+)](i) was measured using fluorescence microscopy. Extracellular regulated kinase (ERK) 2 was depleted using small interfering RNA (siRNA). Histamine-stimulated conjunctival goblet cell secretion of high molecular weight glycoproteins was blocked by removal of extracellular Ca(2+) and depletion of ERK2 by siRNA. Histamine increase in [Ca(2+)](i) was desensitized by repeated addition of agonist and blocked by a phospholipase C antagonist. Histamine at higher doses increased [Ca(2+)](i) by stimulating influx of extracellular Ca(2+), but at a lower dose released Ca(2+) from intracellular stores. Activation of each histamine receptor subtype (H(1)-H(4)) increased [Ca(2+)](i) and histamine stimulation was blocked by antagonists of each receptor subtype. The H(2) receptor subtype increase in [Ca(2+)](i) was cAMP dependent. We conclude that histamine activates phospholipase C to release intracellular Ca(2+) that induces the influx of extracellular Ca(2+) and activates ERK1/2 to stimulate conjunctival goblet cell mucous secretion, and that activation of all four histamine receptor subtypes can increase [Ca(2+)](i).

Role of Histamine and Its Receptor Subtypes in Stimulation of Conjunctival Goblet Cell Secretion

The purpose of this study was to determine the effect of histamine and its receptors on goblet cell secretion. Cultured rat and human goblet cells were grown in RPMI 1640. Goblet cell secretion of high molecular weight glycoconjugate was measured by an enzyme-linked lectin assay. Cultured rat goblet cells were homogenized and either RNA was isolated for RT-PCR or proteins were isolated for Western blot analysis for presence of histamine receptors subtypes H₁ through H₄. The localization of these receptors was determined in rat and human goblet cells by immunofluorescence microscopy. Histamine stimulated goblet cell secretion in a concentration- and time-dependent manner. All four histamine receptors were present in cultured rat and human goblet cells. Use of agonists specific to individual histamine receptor subtypes indicated that the rank order of agonist stimulation was H₁ = H₃ > H₄ > H₂. Using antagonists specific to individual histamine receptor subtypes determined that H₂ and H₃, but not the H₁ and H₄, antagonists, inhibited histamine-stimulated conjunctival goblet cell secretion. Rat and human conjunctival goblet cells are a direct target of histamine, which induces secretion. All four histamine receptors are present in rat and human conjunctiva and are active in rat conjunctival goblet cells. These findings suggest that all four histamine receptor subtypes are important for conjunctival goblet cell secretion. Blockage of histamine receptor subtypes could prevent the excess mucus production associated with ocular allergy.

Glycan Profiling of Gel Forming Mucus Layer from the Scleractinian Symbiotic Coral Oculina arbuscula

The gel forming mucus layer surrounding scleractinian corals play fundamental functions in the maintenance of a favorable microenvironment required for the survival of these organisms. In particular, it harbors a rich partially species-specific symbiotic community through yet poorly understood molecular interactions. However, removal or contamination of this community by exogenous bacteria is closely linked to the worldwide bleaching events that are presently devastating coral colonies. The present study investigates the structure of major high molecular weight glycoconjugates that are responsible for both rheological properties of mucus and sugar-protein interactions with microbial communities. We demonstrated that it is composed by two distinct types of sulfated macromolecules: mucin type glycoproteins densely substituted by short unusual O-linked glycans and repetitive polysaccharides.

Effect of OPC-12759 on EGF receptor activation, p44/p42 MAPK activity, and secretion in conjunctival goblet cells

The purpose of the study was to determine if OPC-12759 stimulates secretion from conjunctival goblet cells in culture and if it activates the EGF receptor (EGFR) and p44/p42 mitogen-activated protein kinase (MAPK) to cause mucin secretion. Conjunctival goblet cells were cultured from pieces of male rat conjunctiva. OPC-12759 was added at increasing concentrations and for varying times to the cultured cells. The cholinergic agonist carbachol was used as a positive control. In selected experiments an inhibitor of the EGFR, AG1478, or an inhibitor of the kinase that activates MAPK, U0126, were added before OPC-12759. Goblet cell secretion of high molecular weight glycoconjugates was measured by an enzyme-linked lectin assay using the lectin UEA-1. Activation of the EGFR and MAPK were determined with Western blotting analysis using antibodies specific to the phosphorylated and the total amounts of these proteins. We found that OPC-12759 induced goblet cell secretion in a time- and concentration-dependent manner. Inhibition of the EGFR with AG1478 blocked secretion stimulated by OPC-12759. Inhibition of MAPK with U0126 also blocked secretion stimulated by OPC-12759. OPC-12759 increased the phosphorylation of the EGFR and MAPK in a time-dependent manner. We concluded that OPC-12759 stimulates secretion from cultured conjunctival goblet cells by activating the EGFR, which then induces MAPK activity.

GPI-anchored Proteins and Free GPI Glycolipids of Procyclic FormTrypanosoma bruceiAre Nonessential for Growth, Are Required for Colonization of the Tsetse Fly, and Are Not the Only Components of the Surface Coat

The procyclic form of Trypanosoma brucei exists in the midgut of the tsetse fly. The current model of its surface glycocalyx is an array of rod-like procyclin glycoproteins with glycosylphosphatidylinositol (GPI) anchors carrying sialylated poly-N-acetyllactosamine side chains interspersed with smaller sialylated poly-N-acetyllactosamine-containing free GPI glycolipids. Mutants for TbGPI12, deficient in the second step of GPI biosynthesis, were devoid of cell surface procyclins and poly-N-acetyllactosamine-containing free GPI glycolipids. This major disruption to their surface architecture severely impaired their ability to colonize tsetse fly midguts but, surprisingly, had no effect on their morphology and growth characteristics in vitro. Transmission electron microscopy showed that the mutants retained a cell surface glycocalyx. This structure, and the viability of the mutants in vitro, prompted us to look for non-GPI-anchored parasite molecules and/or the adsorption of serum components. Neither were apparent from cell surface biotinylation experiments but [3H]glucosamine biosynthetic labeling revealed a group of previously unidentified high apparent molecular weight glycoconjugates that might contribute to the surface coat. While characterizing GlcNAc-PI that accumulates in the TbGPI12 mutant, we observed inositolphosphoceramides for the first time in this organism.

Partial characterisation of high-molecular weight glycoconjugates in the trail mucus of the freshwater pond snail Lymnaea stagnalis

We have studied the glycoconjugates in trail mucus of the pond snail Lymnaea stagnalis. The mucus was dissolved with 6 M guanidinium hydrochloride (GuHCl) and the major component was comprised of very high- M r glycoconjugates that were eluted in the void volume of a Sepharose CL-4B gel-filtration column. This high- M r material was pooled and thereafter subjected to density gradient centrifugation first in 4 M GuHCl/CsCl and subsequently 0.2 M GuHCl/CsCl to further remove non-glycosylated proteins and DNA. The harvested glycoconjugate pool chromatographed in the void volume of Sepharose CL-2B. However, reduction of disulfide bonds lowered the molecular size of approximately 80% of the void material yielding a major fragment and some minor smaller fragments in gel chromatography. The reduced glycoconjugates were digested with papain and yielded high molecular weight, proteinase-resistant glycopeptides. This fragmentation pattern is similar to that found for oligomeric gel-forming mucins in mammals and the amino acid composition (60% Ser/Thr) and sugar analysis of the glycopeptides is consistent with mucin-like molecules, there being no significant amounts of xylose or uronic acids. The residual 20% of the preparation, which apparently resisted reduction and protease digestion, had a similar amino acid composition to the bulk, but was somewhat different in sugar composition, containing some xylose and a significant amount of glucuronic acid. The two groups of molecules had very different morphologies in the electron microscope. Taken together, these data suggest that trail mucus is a complex mixture of at least two families of protein-glycoconjugate molecules based upon the gel-forming mucin and proteoglycan families, though we cannot rule out that polysaccharides may also be present.

Purification and chemical characterization of the high molecular weight glycoconjugate fraction of the bovine tear film and comparison to mucins from other sources.

The tear film is the layer of liquid that covers the surface of the eye, providing protection from invasion and a smooth surface for correct vision.1 It consists of three components: lipid at the air interface, aqueous in the interior and mucous at the epithelial surface.2 The mucous layer may be responsible for stabilizing the tear film.3 Mucins are high molecular weight glycoproteins that could be responsible for the gel-like properties of mucous.4 To date, 13 mucin genes have been reported in humans (MUC 1–9 including 5AC and 5B) of which five, MUC-1, -2, -4, -5AC and -7, have been identified in the eye.5–7 Analysis of the mucous layer of the tear film has been predominantly by monoclonal antibody staining and in situ hybridization techniques. The few chemical analyses that have been performed on the tear film do not, however, correspond to the reported values for the mucins that have been identified.8,9

Glycoconjugate Metabolism in a Cystic Fibrosis Knockout Mouse Model

Cystic fibrosis knockout mice (cftr−/−) die prematurely of obstruction of the intestine which may result from accumulation of dehydrated glycoconjugate-containing mucus. We noted an increase in the specific activity of [14C]glucosamine-labeled high-molecular weight glycoconjugates, probably mucin, in the lumen of the intestine of cftr−/− (homozygous) mice compared to cftr+/+ (wild-type) and cftr+/− (heterozygous) mice and a decrease in the turnover of glycoconjugates of several organs of the cftr−/− mice. No difference in the anionic composition of secreted intestinal glycoconjugates was detected and no difference in the amount of mucin 1 (Muc1) was found in the small intestine, colon, pancreas, and lungs of the different genotypes. In addition, the spleen of the cftr−/− mice was significantly smaller than that of control mice and the small intestine and colon were, respectively, longer and shorter compared to control mice. These results indicate modified glycoconjugate metabolism in cystic fibrosis knockout mice and morphologic changes to the spleen and intestine where the latter modifications are possibly related to the intestinal malabsorption associated with cystic fibrosis.

Evidence of mucin secretion in human lung adenocarcinoma cell lines NCIH650 and NCIH2077 and effect of select secretagogues on mucin secretion.

Mucins comprise an important class of tumor-associated antigens. The objectives of the present study were (a) to establish an in vitro model system using human non-small cell lung adenocarcinoma cell lines NCIH650 and NCIH2077 (b) provide evidence that these cell lines secrete mucin in culture conditions and (c) investigate the effects of select secretagogues on mucin secretion. The cell lines were established in ACL-4 medium containing several growth factors and retinoic acid and 5% fetal calf serum. The high molecular weight glycoconjugates secreted in the culture medium were purified by ammonium sulfate precipitation and Superose 6 and Superose 12 FPLC chromatography. The purified high molecular weight glycoconjugate fraction and the carcinoma cells were shown to have mucin by dot blot, Western blot and immunohistochemical analysis, respectively, using specific antibodies to purified major mucin, HTM-1. Also, incorporation experiments with mucin precursor 3H-glucosamine demonstrated that the cells indeed synthesize high molecular weight mucins. The effects of secretagogues such as, 8-bromocyclic AMP, ionomycin, phorbol-12-myristate-13-acetate and neutrophil elastase on mucin secretion were also investigated. Only 8-bromocyclic AMP and neutrophil elastase influenced mucin secretion. These studies provided strong evidence that the lung adenocarcinoma cell lines secrete high molecular weight mucins in culture conditions and only two of the four tested secretagogues significantly increased mucin secretion. Thus, this in vitro model system may be useful in determining alterations in mucin structure, if any, in lung adenocarcinomas as well as in studying the regulation of mucin gene expression.

Anionic glycoconjugates from differentiated and dedifferentiated cultures of bovine articular chondrocytes: modulation by TGF-beta.

Primary, high density bovine articular chondrocyte (BAC) cultures, stimulated with transforming growth factor-beta-1, elaborated a high molecular weight anionic glycoconjugate, kDa 540, which does not contain glycosaminoglycan chains (Chan and Anastassiades, 1996). The effect of exogenously added transforming growth factor-beta-1 on the elaboration of the high molecular weight glycoconjugate and of proteoglycans was studied during dedifferentiation of the chondrocytes, utilizing a serial subculture technique under anchorage-dependent conditions, up to four subcultures. The high molecular weight glycoconjugate was detected in the media of all growth-factor-stimulated chondrocyte subcultures, as well as stimulated primary cultures, but not in unstimulated primary cultures or subcultures. By contrast, a large proteoglycan, was only secreted by primary cultures and first subcultures, whether treated with transforming growth factor-beta-1 or untreated. This proteoglycan contained mostly chondroitin sulfate chains, whose hydrodynamic size was increased by the addition of transforming growth factor-beta-1. Further, the pattern of the proteoglycans appearing in the media of subcultures 2-4 was influenced by the addition of transforming growth factor-beta-1, so that while these control subcultures elaborated both the large and small chondroitin sulfate proteoglycans, the equivalent stimulated subcultures elaborated only intermediate sized chondroitin sulfate proteoglycan(s). These results suggest that while dedifferentiation of articular chondrocytes, achieved by subculturing, strongly modulates the effect of exogenously added transforming growth factor-beta-1 on the type of proteoglycan elaborated, the process of dedifferentiation does not influence the transforming-growth-factor-beta-dependent synthesis of the high molecular weight anionic glycoconjugate.

Establishment and characterization of tracheal epithelial cell lines, TM01 and TM02-3, from transgenic mice bearing temperature-sensitive simian virus 40 large T-antigen gene.

Murine tracheal epithelial cell lines, TM01 and TM02-3, were established from a primary culture of tracheal cells of adult transgenic mice bearing a temperature-sensitive simian virus (SV40) large T-antigen gene. Both TM01 and TM02-3 cells, which grew until confluent monolayers were formed, maintained tight contact with neighboring cells, and retained the characteristics of epithelial cells with microvilli on the surface. These cells grew at a permissive temperature (33 degrees C), but did not at a nonpermissive temperature (39 degrees C), indicating that TM01 and TM02-3 cells undergo temperature-sensitive growth. Large T-antigen was expressed only in the nuclei at 33 degrees C. Sepharose CL-4B column chromatography using a 14C-glucosamine hydrochloride, indicating that both cells produced high molecular weight glycoconjugates, and suggesting that these cells may originate from mucus-producing cells. TM01 cells expressed intercellular adhesion molecular-1 (ICAM-1) in both unstimulated and stimulated (1,000 U/ml tumor necrosis factor-alpha and 500 U/ml interferon-gamma) conditions, whereas TM02-3 cells expressed ICAM-1 only under stimulated conditions. We conclude that these cell lines may serve as a useful model to study the tracheal cell functions under defined in vitro conditions.

Muscarinic-induced mucin secretion and intracellular signaling by hamster tracheal goblet cells.

Hamster tracheal epithelial cell cultures were used to investigate muscarinic regulation of high-molecular-weight glycoconjugate (HMWG) secretion by airway goblet cells. HMWG were radiolabeled with N-acetyl-D-[1-(3)H]glucosamine, precipitated with trichloroacetic acid and phosphotungstic acid, and counted by liquid scintillation. Carbachol (100 microM) increased HMWG secretion (166.6 +/- 18.7%, P < 0.001, n = 20), and this response was blocked by the muscarinic receptor antagonist atropine. Ca2+ may not be essential for carbachol response since 1) carbachol-activated secretion was not inhibited by chelating extracellular Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) or by reducing both extracellular and intracellular Ca2+ with BAPTA-acetoxymethyl ester in low-Ca2+ medium; 2) the carbachol response was only partially blocked in low-Ca2+ medium; and 3) calcium ionophore did not stimulate HMWG secretion. However, carbachol-stimulated secretion was abolished by pertussis toxin (PTX), indicating the involvement of a PTX-sensitive guanine nucleotide-binding regulatory protein (G protein), and by the protein kinase C (PKC) inhibitor chelerythrine chloride. Furthermore, carbachol-stimulated secretion was not inhibited by overnight incubation with phorbol 12-myristate 13-acetate. In conclusion, carbachol-stimulated secretion of HMWG appears to be coupled to a PTX-sensitive G protein and requires the activation of a phorbol ester-insensitive PKC isoform.

Identification of monoclonal antibody At5 as a new member of HNK-1 antibody family: the reactivity with myelin-associated glycoprotein and with two brain-specific proteoglycans, phosphacan and neurocan.

Monoclonal antibody At5 was primarily developed against chordin, a notochord-specific antigen of Acipenseridae (sturgeon fishes). In higher vertebrates the antibody reacted mainly with neural tissue antigens. In this study we have shown that the specificity of monoclonal antibody At5 is similar to that of antibodies of HNK-1 family which react with two glycolipids and with several high molecular weight glycoconjugates of neural tissue. We have demonstrated by protein sequencing and immunoblotting that one of At5 target antigens of human brain is dMAG, a derivative of myelin-associated glycoprotein. In the preparations of At5 antigens proteoglycans phosphacan and neurocan were identified by immunoblotting with specific monoclonal antibodies 6B4 and 1G2, respectively. The distribution of At5 and 6B4 immunoreactivity was studied on sections of mixed oligoastrocytoma. Oligodendroglioma area of this tumor was intensely stained with both antibodies, whereas astrocytoma area did not exhibit any At5 or 6B4 immunoreactivity.

Enhancement of glycosylation of cellular glycoconjugates in the squamous carcinoma cell line MDA886Ln by ?-all-trans retinoic acid

Retinoids have been shown to inhibit the growth and modulate the glycosylation of head and neck squamous cell carcinoma (HNSCC) cells including the MDA886Ln cells. To examine the effects of beta-all-trans retinoic acid (RA) on glycoconjugates in HNSCC MDA886Ln cells, the cells were grown in the absence or presence of 1 microM RA and then labeled with tritiated monosaccharides, extracted and analysed by polyacrylamide gel electrophoresis and fluorography. RA increased markedly the incorporation of [3H]-glucosamine, [3H]-galactose, and [3H]-mannose into numerous cellular glycoconjugates, however, the incorportion of [3H]-fucose and [3H]-leucine was almost unaffected by RA. RA increased the incorporation of glucosamine and galactose but not mannose into high molecular weight (HMW) glycoconjugates of about 220 and 500-600 kDa. To analyse the steady state level of glycoconjugates by lectin blotting, extracts of unlabeled cells were separated by gel electrophoresis and the gels were probed with 125I-labeled wheat germ agglutinin (WGA) and Maackia amurensis (MA) agglutinin. Both lectins were found to bind to numerous glycoconjugates including the HMW glycoconjugates, whereas 125I-peanut agglutinin bound only to the HMW glycoconjugates RA treatment increased the binding of all three lectins to the HMW glycoconjugates. These findings demonstrate that RA enhanced the incorporation of specific monosaccharides into a variety of glycoconjugates and in particular into HMW mucin-like glycoconjugates. This effect of RA may be the result of induction of a more normal differentiation state of the HNSCC cells.