Ganglioside vaccines with emphasis on GM2

OBJECTIVE: To investigate the impact of an educational intervention on knowledge and anxiety level of women scheduled for colposcopy after an abnormal Papanicolaou (Pap) test. DESIGN: Experimental, randomized controlled study. SETTING: An inner-city medical school. PARTICIPANTS: The final sample consisted of 58 women in the intervention group and 55 women in the control group. Exclusion criteria included any previous colposcopy. INTERVENTIONS: The women in the intervention group received in the mail, approximately 1 week before their appointment, a one-page handout about colposcopy. The control group received no mailed handout. After arriving for the visit, women were asked to participate in the study and then were interviewed. MAIN OUTCOME MEASURES: Knowledge of reason for visit and knowledge of colposcopy as measured by content analysis of interview; and anxiety as measured by the Spielberger State/Trait Anxiety Inventory. RESULTS: Women in the intervention group demonstrated significantly more knowledge about the reason for their visit and about colposcopy than did the other women. No significant difference in mean anxiety score was found between the groups. CONCLUSIONS: The intervention increased knowledge about colposcopy for this population. Because patient education is an essential nursing function, these results are encouraging. This intervention can be replicated by nurses in other settings. Further research is necessary to understand how nurses can best help women alleviate anxiety before colposcopy.     

Galectin-1 is a major receptor for ganglioside GM1, a product of the growth-controlling activity of a cell surface ganglioside sialidase, on human neuroblastoma cells in culture

Cell density-dependent inhibition of growth and neural differentiation in the human neuroblastoma cell line SK-N-MC are associated with a ganglioside sialidase-mediated increase of GM1 and lactosylceramide at the cell surface. Because these glycolipids expose galactose residues, we have initiated the study of the potential role of galectins in such cellular events. Using specific antibodies, galectin-1 but not galectin-3 was found to be present at the cell surface. Assessment of carbohydrate-dependent binding revealed a saturable amount of ligand sites approaching 2.6 x 10(6) galectin-1 molecules bound/cell. Presence during cell culture of the sialidase inhibitor 2-deoxy-2,3-dehydro-N-acetylneuraminic acid or of the GM1-binding cholera toxin B subunit effected a decrease of the presentation of galectin-1 ligands by 30-50%. The assumption that GM1 is a major ligand for galectin-1 was reinforced by the correlation between the number of carbohydrate-dependent 125I-iodinated GM1-neoganglioprotein binding sites and the amount of immunoreactive surface galectin-1, the marked sensitivity of probe binding to the presence of anti-galectin-1 antibody, and the inhibition of cell adhesion to surface-immobilized GM1 by the antibody. The results open the possibility that the carbohydrate-dependent interaction between ganglioside GM1 and galectin-1 may relay sialidase-dependent alterations in this cell system.     

Ganglioside GM1 attenuates scopolamine-induced amnesia in rats and mice

We evaluated ethanol- and HCl-induced mucosal damages in developing rats. The degree of damage induced by ethanol and HCl was greatest in 1-week-old rats and decreased significantly with age until 4 weeks; thereafter it increased again. To evaluate the effect of weaning on the maturational changes in mucosal defense, we compared ethanol-induced mucosal damage among three groups of newborn rats: (1) receiving milk only; (2) receiving only rat chow from 14 days of age, and (3) having free access to milk and chow. There were no significant differences at 18 and 21 days of age. The mucus thickness increased with age until 8 weeks and was not affected by weaning. In conclusion, developmental changes occur in gastric mucosal protection in rats. Weaning does not have a significant effect on these changes.     

Ganglioside GM1 activates the mitogen-activated protein kinase Erk2 and p70 S6 kinase in U-1242 MG human glioma cells

Gangliosides are implicated in the regulation of cellular proliferation as evidenced by differences in ganglioside composition associated with malignant transformation and density of cells in culture, as well as their inhibitory effects when added to cells growing in culture. Exogenously added gangliosides have a bimodal effect on proliferation in U-1242 MG glioma cells, inhibiting DNA synthesis in growing cells and stimulating it in quiescent cells. We investigated the mechanisms involved in stimulation of DNA synthesis using [3H]thymidine incorporation and immune complex kinase assays to identify responsible signal transduction pathways. Treatment of quiescent U-1242 MG cells with GM1 caused activation of the mitogen-activated protein (MAP) kinase isoform Erk2. Pretreatment with the specific MAP kinase kinase inhibitor PD98059 prevented the GM1-stimulated Erk2 activation and GM1-stimulated DNA synthesis. GM1 treatment stimulated another distinct signaling pathway leading to activation of p70 S6 kinase (p70s6k), and this was prevented by pretreatment with rapamycin. Rapamycin also inhibited GM1-stimulated DNA synthesis. Activation of both pathways and stimulation of DNA synthesis were inhibited by forskolin treatment; however, GM1 had no effect on cyclic AMP levels. Platelet-derived growth factor also activated both Erk2 and p70s6k but did not cause DNA synthesis, suggesting that GM1 may stimulate additional cascades, which also contribute to GM1-mediated DNA synthesis.     

Ganglioside GM1 enhances induction by nerve growth factor of a putative dimer of TrkA

GM1 enhances nerve growth factor (NGF)-stimulated neuritogenesis and prevents apoptotic death of PC12 cells; both may be due to enhancement of TrkA dimerization. In this study, we examined the effect of GM1 on NGF-induced TrkA dimerization in Trk-PC12 (6-24) cells. NGF increased tyrosine phosphorylation of the 140-kDa protein (TrkA monomer), and preincubation with GM1 potentiated this effect. Adding the protein cross-linker bis(sulfosuccinimidyl) suberate with NGF resulted in the appearance of two major bands (220 and 330 kDa) when probed with antibodies against TrkA or phosphotyrosine, and GM1 also enhanced this effect. We interpret the 330-kDa band as being a homodimer of TrkA. The identity of the 220-kDa band is still not certain but may consist of a posttranslationally modified form of TrkA. Our results suggest that GM1 is augmenting the effects of NGF on PC12 cells by enhancing the dimerization and activation of the TrkA receptor.     

The O-glycosylated stalk domain is required for apical sorting of neurotrophin receptors in polarized MDCK cells

Delivery of newly synthesized membrane-spanning proteins to the apical plasma membrane domain of polarized MDCK epithelial cells is dependent on yet unidentified sorting signals present in the luminal domains of these proteins. In this report we show that structural information for apical sorting of transmembrane neurotrophin receptors (p75(NTR)) is localized to a juxtamembrane region of the extracellular domain that is rich in O-glycosylated serine/threonine residues. An internal deletion of 50 amino acids that removes this stalk domain from p75(NTR) causes the protein to be sorted exclusively of the basolateral plasma membrane. Basolateral sorting stalk-minus p75(NTR) does not occur by default, but requires sequences present in the cytoplasmic domain. The stalk domain is also required for apical secretion of a soluble form of p75(NTR), providing the first demonstration that the same domain can mediate apical sorting of both a membrane-anchored as well as secreted protein. However, the single N-glycan present on p75(NTR) is not required for apical sorting of either transmembrane or secreted forms.     

trans-2-phenylcyclopropylamine is a substrate for and inactivator of horseradish peroxidase

Horseradish peroxidase (HRP) is well known for mediating the electron-transfer oxidation of electron-rich aromatic 'donors' such as phenols and anilines, but has not been described to oxidize aliphatic amines. We here confirm the inability of HRP to oxidize typical aliphatic amines, even those which would exist significantly as free bases at the operative pH. In contrast, trans-2-phenylcyclopropylamine (2-PCPA) is both a substrate (turnover product is cinnamaldehyde) and a time-dependent inactivator of HRP. These activities of 2-PCPA are consistent with either a concerted or rapid sequential one-electron-oxidation/ring-opening to give an intermediate capable of covalent binding to the enzyme. 2-PCPA is the first known example of a simple aliphatic amine which serves as a substrate for HRP under turnover conditions.     

L-DOPA is a substrate for tyrosine hydroxylase

In the presence of thiols, tyrosine hydroxylase (TH) oxidizes L-dihydroxyphenylalanine (L-DOPA) with a specific activity of up to 140 nmol min(-1) mg(-1) at 37 degrees C and pH 7.0, which is approximately 12-50% of its TH activity under similar experimental conditions. Using assay conditions that are optimal for measuring TH activity, the specific DOPA oxidase activity of human TH is similar to that of mushroom tyrosinase, but the two enzymes are clearly different in terms of substrate specificities, cofactor dependencies, and selectivity with respect to the effects of metal chelators and other inhibitors. In the presence of an excess of dithiothreitol, 2-mercaptoethanol, cysteine, or reduced glutathione, the reaction products of the two enzymes are identical and have been identified tentatively as thioether derivatives of DOPA. Theoretically, the oxidation of L-DOPA by TH may contribute to the formation of neuromelanin (pheomelanin) in catecholaminergic neurons and in the metabolism of DOPA to reactive intermediates that can react with free thiol groups in cellular proteins. The DOPA oxidase activity of TH can lead to errors in the estimation of in vivo or in vitro TH activity, and currently used assay protocols may have to be modified to avoid interference from this activity.     

IkappaB is a substrate for a selective pathway of lysosomal proteolysis

In lysosomes isolated from rat liver and spleen, a percentage of the intracellular inhibitor of the nuclear factor kappa B (IkappaB) can be detected in the lysosomal matrix where it is rapidly degraded. Levels of IkappaB are significantly higher in a lysosomal subpopulation that is active in the direct uptake of specific cytosolic proteins. IkappaB is directly transported into isolated lysosomes in a process that requires binding of IkappaB to the heat shock protein of 73 kDa (hsc73), the cytosolic molecular chaperone involved in this pathway, and to the lysosomal glycoprotein of 96 kDa (lgp96), the receptor protein in the lysosomal membrane. Other substrates for this degradation pathway competitively inhibit IkappaB uptake by lysosomes. Ubiquitination and phosphorylation of IkappaB are not required for its targeting to lysosomes. The lysosomal degradation of IkappaB is activated under conditions of nutrient deprivation. Thus, the half-life of a long-lived pool of IkappaB is 4.4 d in serum-supplemented Chinese hamster ovary cells but only 0.9 d in serum-deprived Chinese hamster ovary cells. This increase in IkappaB degradation can be completely blocked by lysosomal inhibitors. In Chinese hamster ovary cells exhibiting an increased activity of the hsc73-mediated lysosomal degradation pathway due to overexpression of lamp2, the human form of lgp96, the degradation of IkappaB is increased. There are both short- and long-lived pools of IkappaB, and it is the long-lived pool that is subjected to the selective lysosomal degradation pathway. In the presence of antioxidants, the half-life of the long-lived pool of IkappaB is significantly increased. Thus, the production of intracellular reactive oxygen species during serum starvation may be one of the mechanisms mediating IkappaB degradation in lysosomes. This selective pathway of lysosomal degradation of IkappaB is physiologically important since prolonged serum deprivation results in an increase in the nuclear activity of nuclear factor kappa B. In addition, the response of nuclear factor kappa B to several stimuli increases when this lysosomal pathway of proteolysis is activated.     

Cloning and expression of sialidase L, a NeuAcalpha2-->3Gal-specific sialidase from the leech, Macrobdella decora

Sialidase L is a NeuAcalpha2-->3Gal linkage-specific sialidase that releases 2,7-anhydro-NeuAc instead of NeuAc from sialoglycoconjugates (Chou, M.-Y., Li, S.-C., Kiso, M., Hasegawa, A., and Li, Y.-T.(1994) J. Biol. Chem. 269, 18821-18826). A 2. 5-kilobase cDNA of sialidase L was cloned by a combination of methods based on polymerase chain reactions. The composite cDNA sequence reveals an open reading frame coding for 762 amino acids, including a putative 28-residue signal peptide at the N terminus that is similar to the signal sequence of the Clostridium septicum sialidase. The result suggests that sialidase L is a secretory enzyme. The coding sequence excluding the putative signal peptide of sialidase L was overexpressed in Escherichia coli. The purified recombinant enzyme was characterized to be as active as the enzyme isolated from the leech. It also possessed the strict NeuAcalpha2-->3Gal linkage specificity and released the unique cleavage product, 2,7-anhydro-NeuAc from sialoglycoconjugates. The deduced amino acid sequence of sialidase L exhibits little similarity with other reported sialidases. However, sialidase L contains a conserved "FRIP region" and four repeating "Asp box" motifs that align well with the corresponding positions of bacterial sialidases. The predicted beta-strand structures near the conserved motifs of sialidase L are similar to those of Salmonella typhimurium sialidase. Several conserved single amino acid residues of bacterial sialidases, including those known to be involved in the active site of Salmonella enzyme, are conserved in the deduced amino acid sequence of sialidase L. This observation suggests that part of the catalytic mechanism of sialidase L may be similar to the ordinary sialidase.     

N-glycolylneuraminic acid-containing GM1 is a new molecule for serum antibody in Guillain-Barré syndrome

To clarify the pathogenesis of Guillain-Barré syndrome (GBS) after parenteral injections of bovine brain gangliosides, we searched for new molecules in bovine brain gangliosides recognized by sera from GBS patients. Gangliosides fractionated in a Q-Sepharose column were used as the antigens, and the binding of serum IgG or IgM was examined by thin-layer chromatography/immunostaining. Fourteen of 175 serum samples from the patients reacted with the monosialoganglioside fraction 2. In the neutral solvent system, a band in this fraction migrated with N-acetylneuraminic acid-containing GM1 [GM1(NeuAc)], whereas in the alkaline solvent system it migrated slower. This suggested that the band was N-glycolylneuraminic acid-containing GM1 [GM1(NeuGc)]. In both solvent systems, its mobility was almost the same as that of authentic GM1(NeuGc) from mouse liver. Secondary ion mass spectrometry showed that the ganglioside's structure was consistent with that of GM1(NeuGc). IgG anti-GM1(NeuGc) antibodies in sera from the GBS patients were significantly absorbed by GM1(NeuAc), indicative that the anti-GM1(NeuGc) antibodies cross-react with GM1(NeuAc). N-Glycolylneuraminic acid-containing gangliosides are so highly immunogenic in humans that the injection of GM1(NeuGc) could induce the production of IgG anti-GM1(NeuGc) antibody, which cross-reacts with GM1(NeuAc).     

An intestinal mucin is the target substrate for a baculovirus enhancin

An invertebrate intestinal mucin (IIM) was identified from a lepidopterous insect, Trichoplusia ni. The IIM is a major protein constituent of the peritrophic membrane that facilitates the digestive process, as well as protecting invertebrate digestive tracts from microbial infections. The IIM demonstrated biochemical characteristics similar to vertebrate mucins, but exhibited strong association with the chitin-containing peritrophic membrane matrix. We have demonstrated that a baculovirus enhancin, which is encoded and carried by specific baculoviruses, has mucin-degrading activity both in vitro and in vivo. The in vivo degradation of IIM by enhancin was correlated with the enhancement of baculovirus infections in insects. These findings have shown that viruses have evolved a novel strategy to overcome intestinal mucinous barriers against microorganisms by utilizing a mucin-degrading enzyme.     

Clostridium perfringens epsilon-toxin acts on MDCK cells by forming a large membrane complex

Epsilon-toxin is produced by Clostridium perfringens types B and D and is responsible for a rapidly fatal enterotoxemia in animals, which is characterized by edema in several organs due to an increase in blood vessel permeability. The Madin-Darby canine kidney (MDCK) cell line has been found to be susceptible to epsilon-toxin (D. W. Payne, E. D. Williamson, H. Havard, N. Modi, and J. Brown, FEMS Microbiol. Lett. 116:161-168, 1994). Here we present evidence that epsilon-toxin cytotoxic activity is correlated with the formation of a large membrane complex (about 155 kDa) and efflux of intracellular K+ without entry of the toxin into the cytosol. Epsilon-toxin induced swelling, blebbing, and lysis of MDCK cells. Iodolabeled epsilon-toxin bound specifically to MDCK cell membranes at 4 and 37 labeled C and was associated with a large complex (about 155 kDa). The binding of epsilon-toxin to the cell surface was corroborated by immunofluorescence staining. The complex formed at 37 degrees C was more stable than that formed at 4 degrees C, since it was not dissociated by 5% sodium dodecyl sulfate and boiling.     

Allopurinol blocks shock-wave-induced rises in cytosolic calcium levels in MDCK cells

OBJECTIVE: To evaluate physicians' probability estimates of acute appendicitis based on structured collection of clinical data. DESIGN: Open prospective study. SETTING: District hospital, Norway. SUBJECTS: 304 patients admitted with suspected acute appendicitis. INTERVENTIONS: Initial diagnostic accuracy of physicians was compared with corresponding results from a computer model. MAIN OUTCOME MEASURES: The estimated probabilities of appendicitis in different testing groups were analysed using receiver operating characteristic (ROC) curves. RESULTS: Physicians' estimates had a mean area under ROC-curve of 0.81 (95% CI 0.79 to 0.82), not significantly different from the computer model. Both correlated well with the actual rate of appendicitis, but the physicians tended to overestimate the probability by 10%. CONCLUSION: Physicians' probability estimates perform rather well. Further attempts to implement a probabilistic approach in the diagnostic process of acute appendicitis therefore seem justified.     

Cdc2 kinase directly phosphorylates the cis-Golgi matrix protein GM130 and is required for Golgi fragmentation in mitosis

Mitotic fragmentation of the Golgi apparatus can be largely explained by disruption of the interaction between GM130 and the vesicle-docking protein p115. Here we identify a single serine (Ser-25) in GM130 as the key phosphorylated target and Cdc2 as the responsible kinase. MEK1, a component of the MAP kinase signaling pathway recently implicated in mitotic Golgi fragmentation, was not required for GM130 phosphorylation or mitotic fragmentation either in vitro or in vivo. We propose that Cdc2 is directly involved in mitotic Golgi fragmentation and that signaling via MEK1 is not required for this process.     

Ganglioside and neurotrophic growth factor interactions in retinal neuronal and glial cells

Ganglioside (GG) and neurotrophic growth factor (GF) interactions in retinal neuronal and glial cells have been very little studied. Rat retinas were mechanically separated into outer (photoreceptor or PR) and inner (other neurons, IR) halves by planar vibratome sectioning and retinal Müller glial (RMG) cells were isolated and cultured according to previously published methods. The distribution on a percent molar basis of individual GG was different between the two halves: PR were dominated by GD3 (48% total GG) and contained only trace amounts (< 4%) of complex species (GT1b, GQ); IR was more typical of mature brain tissue, exhibiting substantial amounts (approximately 25%) of more complex GG. The GG profile of RMG cells was also simple, dominated by GM3 (60%) and GD1a (20%). A single addition to the medium of 500 pM bFGF or EGF for 48 hr to cultured RMG cells led to significant increases in total GG levels of 30-40%. Such treatments by both growth factors induced increases in GM3, whereas longer exposure (96 hr) of confluent RMG to these factors additionally stimulated synthesis of more complex GG. Incubations of RMG with [3H]-glucosamine showed that GG synthesis was 2-fold stimulated by growth factors. We also tested the effect of GM3 on one of the bFGF receptor transduction pathways, namely PI-3 kinase activation. To our knowledge these data constitute the first demonstration of neurotrophic factor stimulation of GG levels in cells of CNS in vitro. Such complex interactions may have particularly important consequences for neural physiopathology.