Effects of daunorubicin on ganglioside expression and neuronal differentiation of mouse embryonic stem cells

Gangliosides are implicated in neuronal development processes. The regulation of ganglioside levels is closely related to the induction of neuronal cell differentiation. In this study, the relationship between ganglioside expression and neuronal cell development was investigated using an in vitro model of neural differentiation from mouse embryonic stem (mES) cells. Daunorubicin (DNR) was applied to induce the expression of gangliosides in embryoid body (EB) (4+). We observed an increase in expression of gangliosides in all stages of EBs by treatment of DNR (2microM). High-performance thin-layer chromatography (HPTLC) showed that gangliosides GD3, GD1a, GT1b, and GQ1b increased in DNR-treated 7-day-old EB (4+) [EB (4+):7]. DNR treatment significantly increased the expression of gangliosides, especially GT1b and GQ1b in comparison to control cells. Interestingly, GQ1b co-localized with microtubule-associated protein 2 (MAP-2) expressing cells in DNR-treated EB (4+):7. The co-localization of GQ1b and MAP-2 was found in neurite-bearing cells in DNR-treated 15-day-old EB (4+) [EB (4+):15], whereas no significant expression of GQ1b and less neurite formation were observed in untreated control. Also, the expression of synaptophysin and NF200, both neuronal markers associated with neruites, was increased by DNR treatment. These results demonstrate that DNR increases expression of gangliosides, especially GQ1b, in differentiating neuronal cells. Further, neurite-bearing neuronal cell differentiation can be facilitated by DNR, possibly through the induction of gangliosides. Thus, the present data suggest that DNR is beneficial for facilitating neuronal differentiation from ES cells and among the gangliosides analyzed in the present study, GQ1b is mainly involved in neurite formation.     

The lipid composition of axons from bovine brain

Abstract— Bovine axons derived from myelinated CNS axons were found to have 13.5% lipid. This lipid was composed of 20.1% cholesterol, 20.1% galactolipid, 14.6% ethanolarhine phosphatides (56.4% in the plasmalogen form), 18.3% choline phosphatides (10.0% in the plasmalogen form), 9.3% sphingomyelin, 5.6% phosphatidyl serine and 3.4% phosphatidyl inositol. The bovine axons had 0.33 μg ganglioside NeuNAc/mg dry weight. The axon gangliosides were found to contain the four major types of bovine gangliosides, as well as gangliosides G_M2 and G_D3. The latter two amounted to 20.9 and 15.8 mole per cent respectively, of total gangliosides. On a molar basis, about one half of the gangliosides were monosialogangliosides, with a decreased contribution by gangliosides G_T1 and GD_1b relative to ganglioside distributions which have been reported for most other CNS components. The relationship of the bovine axonal lipid composition to bovine white matter and its constituents, as well as to other CNS and PNS axonal preparations is discussed.     

Nonmuscle Myosin Heavy Chain B Is Recognized by a Monoclonal Antibody that Inhibits GM1-Enhanced Neuritogenesis

Abstract: Addition of gangliosides to neuroblastoma cells maintained in vitro has been shown to enhance neuritogenesis. Although the mechanism by which they exert this effect is unknown, it has been postulated that they may act by adhering to cell surface proteins. In this article, we describe the isolation and identification of an S20Y murine neuroblastoma cell protein recognized by a monoclonal antibody that was prepared against putative GM1-binding proteins and shown to inhibit GM1-enhanced neuritogenesis. The protein identified was nonmuscle myosin heavy chain B, which appears to function in neurite formation but may not adhere to gangliosides.     

Confirmation of the Cholinergic Specificity of the Chol-1 Gangliosides in Mammalian Brain Using Affinity-Purified Antisera and Lesions Affecting the Cholinergic Input to the Hippocampus

An antiserum raised to Torpedo electromotor synaptosomal membranes (anti-TSM antiserum) induces a cholinergic-specific immune lysis of mammalian brain synaptosomes and recognizes a group of minor gangliosides appeared, therefore, to be specific to the cholinergic neuron and were designated Chol-1. To confirm the cholinergic specificity of the Chol-1 gangliosidic antigens, we have shown that not only does a mammalian ganglioside fraction that is enriched with respect to the Chol-1 gangliosides inhibit the cholinergic-specific immune lysis induced by the anti-TSM antiserum, but also it can be used to affinity-purify a subpopulation of immunoglobulins from the anti-TSM antiserum that also induce a cholinergic-specific lysis. Furthermore, we have demonstrated that fimbrial lesions, which cause a massive degeneration of cholinergic terminals in the ipsilateral hippocampus, lead to a loss of the Chol-1 gangliosides concomitant with that shown by choline acetyltransferase activity and that lesions to the entorhinal cortex, which cause a loss of mainly glutamergic synapses in the ipsilateral dentate gyrus leading to cholinergic sprouting from adjacent hippocampal areas and an increase in cholinergic markers in the dentate gyrus, produce concomitant increases in choline acetyltransferase activity and Chol-1 content. These results provide strong evidence in favour of the cholinergic specificity of the Chol-1 gangliosides.     

Isolated Bovine Spinal Motoneurons Have Specific Ganglioside Antigens Recognized by Sera from Patients with Motor Neuron Disease and Motor Neuropathy

The gangliosides GM1 and GD1b have recently been reported to be potential target antigens in human motor neuron disease (MND) or motor neuropathy. The mechanism for selective motoneuron and motor nerve impairment by the antibodies directed against these gangliosides, however, is not fully understood. We recently investigated the ganglioside composition of isolated bovine spinal motoneurons and found that the ganglioside pattern of the isolated motoneurons was extremely complex. GM1, GD1a, GD1b, and GT1b, which are major ganglioside components of CNS tissues, were only minor species in motoneurons. Among the various ganglioside species in motoneurons, several were immunoreactive to sera from patients with MND and motor neuropathy. One of these gangliosides was purified from bovine spinal cord and characterized as N-glycolylneuraminic acid-containing GM1 [GM1(NeuGc)] by compositional analysis, fast atom bombardment mass spectra, and the use of specific antibodies. Among seven sera with anti-GM1 antibody activities, five sera reacted with GM1(NeuGc) and two did not. Two other gangliosides, which were recognized by another patient's serum, appeared to be specific for motoneurons. We conclude that motoneurons contained, in addition to the known ganglioside antigens GM1 and GD1b, other specific ganglioside antigens that could be recognized by sera from patients with MND and motor neuropathy.     

Studies on the proteolytic activity of γ-globulin preparations

1. The proteolytic activities of several gamma-globulin preparations were tested. These included sulphate-precipitated human and bovine preparations and human and bovine Cohn fraction II preparations as well as purified gamma-globulin preparations. Up to 14mg. of diffusible peptides and glycopeptides/g. of gamma-globulin was liberated after dialysis and up to 10mg. of peptides/g. after incubation and trichloroacetic acid precipitation, as products of the degradation process in incubated gamma-globulin. 2. in-Aminohexanoic acid and p-chloromercuribenzoic acid, as well as heating at 60 degrees for 40min., were shown to inhibit strongly these proteolytic activities. Streptokinase was shown to activate strongly the proteolytic activity of all the human preparations (sulphate-precipitated, Cohn fraction II, and purified gamma-globulin). 3. Two distinct pH optima were shown for human and bovine gamma-globulin preparations: one at pH8, the other at pH3.8 (the latter activity could be demonstrated only in the presence of cysteine). 4. Both (131)I-labelled human Cohn fraction II and bovine fibrinogen were attacked by a sulphate-precipitated preparation of gamma-globulin. Of the synthetic substrates tested toluene-p-sulphonyl-l-arginine methyl ester was hydrolysed by both the sulphate-precipitated and Cohn fraction II preparations, as was benzoyl-l-arginine amide at pH5, but only in the presence of cysteine. 5. These data are interpreted to indicate that at least two enzymes are present in gamma-globulin preparations, one being similar to the plasmin system, the other similar to cathepsin B.     

Cationic amphipathic peptides, derived from bovine and human lactoferrins, with antimicrobial activity against oral pathogens

Peptides derived from the N-terminal domain that comprises an amphipathic alpha-helix in human lactoferrin (LFh 18-31 and LFh 20-38) and bovine lactoferrin (LFb 17-30 and LFb 19-37) were chemically synthesised. Since many positively charged amphipathic alpha-helices contain antimicrobial activity, the peptides were tested for their antimicrobial activity against various oral pathogens. Both peptides from bovine lactoferrin had more potent antimicrobial activities than the human equivalents. Peptide LFb 17-30, containing the largest number of positively charged amino acids, showed the highest antimicrobial activity to both Gram-positive and Gram-negative bacteria. Since native lactoferrin molecules had no killing activity, release of these peptides from the native protein should be investigated to explore the use in oral care products.     

Differential effects of three inhibitors of glycosphingolipid biosynthesis on neuronal differentiation of embryonal carcinoma stem cells

Gangliosides have been implicated in having important roles in neural development. It has been shown that disruption of ganglioside biosynthesis inhibits neurite outgrowth. However, many contradictory results have been reported. The inconsistency of these reports may result from the differential use of neuronal cell lines and inhibitors for ganglioside biosynthesis. In order to clarify the inconsistency in these studies, we utilized an in vitro neuronal differentiation model using an embryonic caricinoma (EC) stem cell line to elucidate the relationship between ganglioside expression and neural development. These cells were exposed to three different inhibitors of glucosylceramide synthase, the first enzyme committed for the biosynthesis of most of the brain gangliosides. All three inhibitors, d-threo-1-phenyl-2-decanoylamino-3-morphlino-1-propanol (D-PDMP), d-threo-1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (D-PPPP), and N-butydeoxynojirimycin (NB-DNJ) can inhibit greater than 90% of ganglioside biosynthesis at certain concentrations, respectively. D-PDMP significantly slowed down cellular proliferation in undifferentiated P19 EC cells, inhibited neurite outgrowth, and eventually caused cell death in differentiated cells. However, no retardation in cell growth, neuronal differentiation, and neurite outgrowth was observed in cultures treated with D-PPPP or NB-DNJ despite the depletion of gangliosides. These results indicate that the effect of D-PDMP on cellular proliferation, neurite outgrowth, and survival of differentiated cells is independent of the inhibition of ganglioside biosynthesis.     

Synthesis, Shedding, and Intercellular Transfer of Human Medulloblastoma Gangliosides: Abrogation by a New Inhibitor of Glucosylceramide Synthase

Abstract: The biological activity of human medulloblastoma tumor gangliosides very likely involves the interaction of these molecules with host cells in the tumor microenvironment. To trace the hypothesized intercellular transfer of shed medulloblastoma gangliosides, we used an in vitro dual-chamber culture system in which the tumor cells, the shed gangliosides, and the target cells to which they might bind would not be perturbed during the transfer process. We observed that under these unmanipulated conditions, gangliosides were shed by the Daoy medulloblastoma cell line (∼300 pmol/10⁸ cells/h), traversed the chamber membrane, and stably bound to the target fibroblasts at the very high density of 10⁷ molecules per cell within 48 h. To determine if this substantial intercellular transfer of shed gangliosides, with its potential of modifying target cell function, could be blocked, we evaluated a new inhibitor of glucosylceramide synthase, dl - threo -1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol (PPPP). PPPP (1.0 µ M ) reduced (90%) Daoy cell ganglioside content strikingly, without causing toxicity or inhibiting cell proliferation. Subsequently, ganglioside shedding by the medulloblastoma cells was diminished significantly (to ∼50 pmol/10⁸/h), and binding of radiolabeled shed medulloblastoma gangliosides to target fibroblasts was consequently almost completely abrogated. We conclude that the shedding and transfer of potentially biologically active human medulloblastoma gangliosides can be diminished effectively by PPPP.     

Effects of Specific Gangliosides on the In Vitro Proliferation of MPTP-Susceptible Cells

Abstract: To determine which portion of a ganglioside molecule might be necessary for the enhancement of recovery from MPTP-induced lesions, the ability of specific gangliosides to stimulate proliferation of MPTP-treated 140–3 cells was investigated. The results indicate that of the gangliosides tested, GM1 was the most effective. Although GD1 a and GT1 b were able to enhance the proliferation of MPTP-treated cells, twice as much GT1b was needed to induce the same effect seen with GM1. In contrast, asialo-GM1, GM2, and GM3 were ineffective at promoting proliferation of MPTP-treated cells. The isolated oligosaccharide of GM1 had little effect. These results indicate that in addition to the sialosyl residue, at least the Gal(β1–3)Gal-NAc portion of the oligosaccharide chain and the ceramide moiety are essential for the induction of proliferation of the MPTP-treated cells. Investigation of the time of addition of GM1 on its ability to counteract the MPTP-induced inhibition of 140–3 cell proliferation indicated that addition of GM1 before or concomitantly with MPTP resulted in a significant reduction in MPTP-induced inhibition of cell proliferation.     

Formation of Ganglioside GD 1b-Lactone in Rat Brain from Intracisternally Administered GD 1b

The presence of ganglioside GD1b, in lactone form GD1b-L, was ascertained in rat brain. The possible formation of GD1b-L from GD1b in brain was explored by the intracisternal injection of GD1b, 3H-labelled at the level of the terminal galactose. This was followed by recognition of the radioactive gangliosides formed at different times (1, 3, and 7 days) after injection. Whereas at 0 time after injection the only radioactive ganglioside was GD1b, after 1, 3, and 7 days other radioactive gangliosides were also found, thus indicating GD1b penetration into the brain tissue, followed by metabolic processing. Besides GD1b, the following radioactive gangliosides were recognized: GM1 and GM2, derived from GD1b degradation; GT1b, formed by the direct sialylation of GD1b; and GD1b-L, produced by metabolic lactonization. The radioactivity carried by GD1b-L was maximal 3 days after injection; its time course was different from that of the other gangliosides, suggesting that the process of lactonization is separate from that of both degradation and glycosylation. Under the same experimental conditions, some radioactive gangliosides also appeared in the liver, although in much smaller amounts than in brain. Radioactive GD1b-L could not be detected in liver, thus indicating that metabolic lactonization is a tissue- or organ-specific process.     

Neurite outgrowth of neuroblastoma cells: dependence on adhesion surface--cell surface interactions

Neurite outgrowth of C 1300 neuroblastoma cells, which were dispersed from adherent cultures or grown in suspension, was studied on different protein-coated surfaces. Of 29 different surface structures studied, including surfaces treated with various fibronectins, lectins, glycosidases, or glycosyltransferases capable of stimulating fibroblast spreading, only the surfaces coated with plasma fibronectin or with a protein mixture secreted by C6 glioma cells displayed an extensive activity in the sprouting assay. Neurite outgrowth was inhibited by brain gangliosides and by colominic acid (a sialic acid polymer). A 50% inhibition of neurite outgrowth of N18 neuroblasts induced by the glioma cell proteins was observed at the following approximate concentration: 100 microM (0.2 mg/ml) GD1A ganglioside, 20 microM (0.04 mg/ml) GT1B ganglioside, and 5 mg/ml colominic acid. Specificity of inhibition was suggested by the finding that a few polyanionic substances tested were not inhibitory in the sprouting assay, and that the type of gangliosides inhibiting sprouting were found to be major sialoglycolipids of the neuroblasts. A hypothesis is discussed, according to which neurite outgrowth of neuroblasts is stimulated by adhesion involving interactions of the adhesion-mediating protein with cell surface carbohydrates characteristic of brain gangliosides.     

Gangliosides do not raise cyclic AMP levels during inhibition of lymphocyte mitogenesis

The effect of bovine brain gangliosides on the intrathymocyte levels of cyclic AMP as a potential mediator of ganglioside action has been studied. Commercial tri-, and disialogangliosides, at 2.5 to 5 μM were found to produce a rapid and profound increase (eg., 10 fold within 2 min by trisialoganglioside). When the preparations were purified on Florisil, this effect on cyclic AMP content was lost, but not the immunoinhibitory potency of the ganglioside (as tested on Concanavalin A induced DNA synthesis). The water soluble “ganglioside associated protein” fractions separated from commercial di- and trisialo gangliosides by Florisil chromatography did not alter the cyclic AMP levels of thymocytes. Previous reports of an effect of commercial gnagliosides on the enzymes of cyclic AMP metabolism in nervous tissue should be re-evaluated.     

Induction of immune response by synthetic fragments of the bovine prion protein and their analogues in mice of various lines

The antibodies to the bovine prion protein were produced by immunizing mice of three lines with five synthetic fragments of the protein and their six analogues. The analogues contained the amino acid substitutions that, according to theoretical calculation, should lead to an increase in the immunogenic activity of peptides. All the peptides, except for one, induced the formation of antibodies. All the sera containing the antipeptide antibodies were tested by an immunohistochemical method. The sera that were effectively bound to the brain preparations from the bovine with spongiform encephalopathy were identified; it was shown that they do not interact with the preparations of normal brain. Therefore, it was shown that the immunization of mice with the synthetic fragments of a prion protein helps obtain specific antibodies suitable for the study and diagnostics of prion diseases.     

Interactions of lactoferricin-derived peptides with LPS and antimicrobial activity

Synthetic peptides derived from human and bovine lactoferricin, as well as tritrpticin sequences, were assayed for antimicrobial activity against wild-type Escherichia coli and LPS mutant strains. Antimicrobial activity was only obtained with peptides derived from the bovine lactoferricin sequence and peptides corresponding to chimeras of human and bovine sequences. None of the peptides corresponding to different regions of native human lactoferricin showed any antimicrobial activity. The results underline the importance of the content of tryptophan and arginine residues, and the relative location of these residues for antimicrobial activity. Results obtained for the same assays performed with LPS mutants suggest that lipid A is not the main binding site for lactoferricin which interacts first with the negative charges present in the inner core. Computer modelling of the most active peptides led to a model in which positively charged residues of the cationic peptide interact with negative charges carried by the LPS to disorganise the structure of the outer membrane and facilitate the approach of tryptophan residues to the lipid A in order to promote hydrophobic interactions.     

A method for the quantitative analysis of nerve growth in vitro

Summary A method is described for the quantitative analysis of the nerve-growth-promoting activity of biological molecules in tissue culture. The criteria used for the evaluation of this activity is based on the neurite length as well as the total number of neurites produced by the explant of whole dorsal root ganglia from 12-d-old chick embryos. A nerve growth index (NGI) is given to each ganglion during each of a 5-d culture period. The NGI is defined as the product of average neurite length in millimeters and the total number of neurites. We report that with increasing concentrations of fetal bovine serum, there was a proportional increase in NGI due to increased neurite density while the neurite length was not greatly affected. The NGI of several proteins with known nerve growth promoting activity, namely nerve growth factor, insulin, transferrin, and fibronectin were investigated for their activity and compared with that of fetal bovine serum. This work was supported in part by grant GM-10374 from the National Institutes of Health, Bethesda, MD.     

Ganglioside-dependent cell attachment and endocytosis of murine polyomavirus-like particles

For murine polyomavirus (Py), previous studies suggest the cellular target is a terminal alpha2,3-linked sialic acid. Here, we investigate the binding and uptake of mouse polyomavirus-like particles (PyVLP) derived from bacterially expressed VP1. We find that in fibroblast 3T6 cells, binding of PyVLP was substantially reduced by sialidase treatment, but only moderately affected by protease treatment, suggesting glycolipids such as the sialic acid-containing gangliosides mediate cell attachment. We further tested the entry requirement of PyVLP using the ganglioside-deficient GM95 murine cell line, and find PyVLP binding and entry were reduced in these cells. Finally, we find that addition of gangliosides G(M1), G(D1a), and G(T1b) to GM95 cells restored cellular PyVLP binding and uptake. Taken together, results indicate that gangliosides function in PyVLP cell attachment and endocytosis.     

Myelin-associated glycoprotein interacts with ganglioside GT1b. A mechanism for neurite outgrowth inhibition

Myelin-associated glycoprotein (MAG) is expressed on myelinating glia and inhibits neurite outgrowth from post-natal neurons. MAG has a sialic acid binding site in its N-terminal domain and binds to specific sialylated glycans and gangliosides present on the surface of neurons, but the significance of these interactions in the effect of MAG on neurite outgrowth is unclear. Here we present evidence to suggest that recognition of sialylated glycans is essential for inhibition of neurite outgrowth by MAG. Arginine 118 on MAG is known to make a key contact with sialic acid. We show that mutation of this residue reduces the potency of MAG inhibitory activity but that residual activity is also a result of carbohydrate recognition. We then go on to investigate gangliosides GT1b and GD1a as candidate MAG receptors. We show that MAG specifically binds both gangliosides and that both are expressed on the surface of MAG-responsive neurons. Furthermore, antibody cross-linking of cell surface GT1b, but not GD1a, mimics the effect of MAG, in that neurite outgrowth is inhibited through activation of Rho kinase. These data strongly suggest that interaction with GT1b on the neuronal cell surface is a potential mechanism for inhibition of neurite outgrowth by MAG.     

Loss of hepatocyte co-operative activity after inhibition of ganglioside GM1 synthesis and shedding

Pretreatment of hepatocyte cultures with 1 microM d-l-threo-1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol-HCL (PPPP) for 24 h decreased the ganglioside GM1 content of the cells by approximately 50% and that of the conditioned medium by 90%. No rhythm in the rate of protein synthesis was detected in dense cultures pretreated with PPPP, but was observed in control dense cultures. Conditioned medium from control dense cultures induced synchrony in sparse cultures, which were non-synchronous in their own medium. In contrast, conditioned medium from dense cultures pretreated with PPPP did not synchronize sparse cultures. Since protein synthesis rhythm is a marker of cell synchronization, i.e. their co-operative activity, then non-oscillatory behavior means loss of cell co-operation. The protein synthesis rhythm was restored 24 h after hepatocytes were transferred to PPPP-free medium. Restoration was more rapid when 0.9 microM gangliosides (standard mixture from bovine brain) were added to the medium just after the withdrawal of PPPP. These novel results concerning the loss of rhythm of protein synthesis in low GM1 ganglioside medium support the conclusion that ganglioside is implicated in the regulation of cell co-operative activity.     

Increased tumor cell reactivity and complement-dependent cytotoxicity with mixtures of monoclonal antibodies against different gangliosides

Melanomas and other cancers of neuroectodermal origin express multiple cell-surface gangliosides in patterns that vary significantly even within the same tumor type. Monoclonal antibodies (mAb) against four of these gangliosides (GM2, GD2, 9-O-acetyl-GD3 and GD3) were tested alone and in combination on 14 tumor cell lines (7 melanomas, 3 neuroblastomas, 3 sarcomas and 1 astrocytoma) using flow cytometry and complement-dependent cytotoxicity (CDC) assays. Increased tumor cell recognition and CDC resulting from the combination of three or four mAb were found in 14/14 tested cell lines, and this was most striking when each mAb was used at suboptimal concentration. At these concentrations, the average mean fluorescence intensity of the 14 cell lines with individual mAb was between 3.0 and 6.8 and increased to 10.8 and 18.8 with the three- and four-mAb mixtures. The average percentage CDC-specific release with individual mAb was 2.0%–8.3%, and 12.3% and 16.6% with the three- and four-mAb combinations. The number of cell lines showing significant mean fluorescence intensity and CDC increased from 2–8/14 with single mAb to 13–14/14 with the mixtures of three or four mAb. Our experimental results support the rationale for active immunization with a polyvalent ganglioside vaccine or passive therapy with a combination of mAb to different gangliosides in patients with tumors of neuroectodermal origin. In addition, our studies have demonstrated that 9-O-acetyl-GD3 is a surprisingly effective target for immune attack, although it is a minor constituent of these cells.