Fluorescent BODIPY-labelled GM1 gangliosides designed for exploring lipid membrane properties and specific membrane-target interactions

New fluorophore-labelled G_M1 gangliosides have been synthesised and spectroscopically characterised. Spectroscopically different BODIPY groups were covalently linked, specifically to either the polar or the hydrophobic part of the ganglioside molecule. The absorption and fluorescence spectroscopic properties are reported for 564/571-BODIPY- and 581/591-BODIPY-labelled G_M1. Each of the different BODIPY groups is highly fluorescent and depolarisation experiments provide molecular information about the spatial distribution in lipid bilayers, as well as order and dynamics. From experiments performed on two spectroscopically different BODIPY:s, specific interactions can be revealed by monitoring the rate/efficiency of donor-acceptor electronic energy transfer. Systems of particular interest for applying these probes are e.g. mixtures of lipids, and peptides/proteins interacting with lipid membranes.     

Treatment with GM1 Ganglioside Restores Striatal Dopamine in the 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine-Treated Mouse

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 30 mg/kg i.p. daily for 7 days, was administered to mice. This dosage regimen resulted in an approximately 50% reduction of striatal dopamine (DA) level. Chronic administration of GM1 ganglioside (II3NeuAc-GgOse Cer), beginning between 1 to 4 days after terminating MPTP dosing, resulted in partial restoration of the striatal DA level. From dose- and time-response studies, it appeared that 30 mg/kg i.p. of GM1 administered daily for approximately 23 days resulted in an approximately 80% restoration of the DA level and complete restoration of the 3,4-dihydroxyphenylacetic acid (DOPAC) content. This dosage of GM1 also restored the turnover rate of DA in the striatum to near normal. Discontinuing GM1 treatment resulted in a fall of DA and DOPAC levels to values found in mice treated with MPTP alone. There was no evidence for regeneration of nerve terminal amine reuptake in the GM1-treated mice as evaluated by DA uptake into synaptosomes. Our biochemical findings in animals suggest that early GM1 ganglioside treatment of individuals with degenerative diseases of dopaminergic nigrostriatal neurons might be fruitful.     

GM3抑制人白血病J6－2细胞肌醇磷脂代谢循环

采用无载体 ³²P和[ ³H]肌醇标记磷脂,观察了促分化剂神经节苷脂GM₃对人单核样白血病J6-2细胞肌醇磷脂代谢的影响.GM₃抑制[ ³²P]Pi和[ ³H]肌醇掺入J6-2细胞磷脂酰肌醇(PI),促进[ ³²P]Pi和[ ³H]肌醇掺入磷脂酰肌醇-4,5-二磷酸(PIP₂),抑制[ ³²P]Pi掺入磷脂酸(PA),抑制[ ³H]肌醇掺入三磷酸肌醇(IP₃).GM₃的上述作用均为浓度依赖性的,随GM₃浓度的提高而增强.上述结果表明,GM₃抑制J6-2细胞的肌醇磷脂代谢循环.     

The different inhibiting effect of cholera toxin on two leukemia cell lines does not correlate with their toxin binding capacity

The murine leukemia cell lines L1210 and WEHI-3B show a very different sensitivity to the cholera toxin (CT).Thein vitro growth of L1210 is completely inhibited by 10^–8 M CT, while WEHI-3B growth shows the same inhibition at 10^–11 M.The analysis of membrane ganglioside pattern of the two cell lines shows that in L1210 cells the major component is the GM1a ganglioside while the monosialoganglioside fraction from WEHI-3B is entirely composed of gangliosides of the b series among which GM1b is the more represented. The total cholera toxin binding capacity of the ganglioside extract from L1210 cells is more than hundred fold higher than that of WEHI-3B and this difference is also confirmed by the number of CT receptors/cell and by the binding of FITC-B subunit of CT on the cells. These surprising data are in conflict with the poor sensitivity to CT evidenced by L1210 compared to WEHI-3B cells.In order to clarify this discrepancy we investigated the cAMP accumulation, the cell viability and the clonogenicity of these two leukemia cell lines following the treatment with CT and forskolin (FRSK).The treatment of WEHI-3B cells with CT induces a dramatic increase of intracellular cAMP which highly correlates with cell death and the decrease of clonogenicity and this result is partially obtained by the treatment with FRSK, L1210 cells do not evidence significant cAMP accumulation neither with CT nor with FRSK treatment.These data suggest that the different inhibiting effect of CT on WEHI-3B and L1210 cells does not correlate with their different pattern of gangliosides and the related toxin binding capacity. Further they indicate that the growth inhibition of WEHI-3B cells is closely related with a cAMP-dependent cell killing mechanism, while the inhibition of L1210 growth (produced by high concentration of CT) is mediated by a cAMP independent mechanism.     

Tay-Sachs disease with atypical chronic course and limited brain storage: Alpha-locus hexosaminidase genetic compound

A 19-year-old Irish-Jewish male had a slow neurologic regression starting at age 4 1/2 years with stuttering. The chronic course resembled that of Spielmeyer-Vogt (juvenile ceroid-lipofuscinosis) disease. The brain was atrophic with neuronal losses and huge compound inclusions in the remaining neurons. Lipid NANA was within normal limits in gray and white matter and G_M2 gangliosides were moderately elevated at 11.5% lipid NANA. Beta-hexosaminidase A activity was lipid composition showed nonspecific abnormalities. Exhaustive tissue extraction ruled out the possibility of tightly bound gangliosides to account for the relatively low G_M2 ganglioside concentration. The extract contained unidentified chromogenic substances interfering with the resorcinol reaction. The similarly affected patient's sister lived to age 26 years and her brain was even more atrophic. No biochemical abnormality to account for progressive neuronal losses and relative lack of G_M2 ganglioside storage was found.Deceased.Special issue dedicated to Dr. Leon S. Wolfe.     

神经节苷脂类抑制BT325细胞系的生长

应用自提的神经节苷脂GM3, 牛脑神经节苷脂(bovine brain gangliosides, BBG)加入低血清培养的人多形成胶质细胞瘤BT325细胞中观察其对该细胞系的影响; 以及GM3、BBG对表皮生长因子(EGF)刺激该细胞系生长的拮抗作用. 结果表明GM3、BBG均能抑制BT325细胞生长,GM3的抑制作用远高于BBG, 其抑制率为60.28% (P＜0.01), BBG为19.33％,在含不同浓度的EGF培养液中分别加入GM3、BBG均可拮抗EGF对BT325生长的刺激作用, GM3的拮抗作用远高于BBG.     

GM3掺入肌质网膜及其对Ca2+-ATP酶的正向调节

用微量提取和高效薄板层析方法研究了外源性神经节苷脂GM3掺入兔肌质网膜的动力学过程.将掺入量分别相对于掺入浓度、时间和温度作图,显示掺入曲线均呈抛物线形式.当掺入体系中GM3浓度为8 μmol／L、掺入时间为90 min、掺入温度为35℃时,其掺入量达到最大值,约为掺入体系中GM3量的50%.上述结果表明外源性GM3对肌质网膜的作用不仅仅是一种简单的水相反应,而是一个依赖于掺入浓度、时间和温度,并具有一定的饱和度的掺入到肌质网膜脂双层中的动力学过程.进一步的实验表明外源性GM3的掺入能明显增加肌质网Ca^2＋-ATP酶的活力.这为从分子水平上研究糖脂对细胞内膜系统的结构与功能的调节作用提供了重要的基础.     

1H NMR Ganglioside Ceramide Resonance Region on the Differential Diagnosis of Low and High Malignancy of Brain Gliomas

1. The high-resolution ¹H NMR (MRS) spectra of human brain tumor homogenates revealed a broad resonance at 5.3–5.4 ppm in glioblastoma multiforme (N = 16) and brain metastases (N = 2). The broad resonance was identified as ceramide, a sphingosine–fatty acid combination portion of ganglioside, indicating an elevated abundance of monounsaturated fatty acids. GLC analysis of gangliosides in the highly malignant glioblastoma multiforme revealed that the elevated monounsaturated fatty acid is oleic acid (C18:1). The resonance at 5.3–5.4 ppm region was not detectable in normal human brain (N = 2), in meningiomas (N = 2), or in low-grade astrocytomas (N = 12). In normal human brain the abundance of monounsaturated fatty acid is minimal.2. This investigation was made possible because the method of producing homogenate resulted in (i) no loss of lipids during the process and (ii) a well-homogenised sample, with (iii) no loss in chemical integrity.3. The properties of tumor gangliosides include antigenic specificity and immunosuppresive activity and the ceramide, a sphingosine–fatty acid combination, noticeably influences the ganglioside immunosuppressive activity.4. The observation of ¹H NMR ceramide resonance in high-malignant brain tumors emphasizes the dramatic role of aberrant gangliosides and ceramide precursors on the grade of malignancy and invasiveness.5. Further insight into the specific nature of the ceramide portion of gangliosides in grading the malignancy of brain tumors should be investigated further.     

Structural characterization andin vivo immunosuppressive activity of neuroblastoma GD2

Shedding of neuroblastoma gangliosides is positively correlated with tumour progression in patients with neuroblastoma. In assessing the biological activity of these ganglioside molecules, we recently found that total human neuroblastoma gangliosides inhibit cellular immune responses. Here, we have studied the major neuroblastoma ganglioside, G_D2. G_D2 was purified by high performance liquid chromatography and structurally characterized by mass spectrometry. Immunoregulatory effects of G_D2 in vivo were then determined in an established murine model. G_D2 significantly downregulated the local cellular immune response to an allogeneic cell challenge; the usual increase in mass of the lymph node draining the injection site was reduced by 88%, from 1.52 to 0.19 mg (control versus G_D2-treated mice;p<0.01). In parallel, lymphocyte recovery from each node was also reduced from 2.4 to 1.2×10⁶ cells, and lymphocyte DNA synthesis was reduced to half of the control level. These results show that certain shed tumour gangliosides, such as G_D2, function as intercellular signalling molecules, downregulate the cellular immune response, and may thereby enhance tumour formation and progression.     

GM1 ganglioside modulates prostaglandin E1 stimulated adenylyl cyclase in neuro-2A cells

This study demonstrates modulation by GM1 ganglioside of prostaglandin E1 (PGE1)-induced cAMP formation in Neuro-2a neuroblastoma cells. Pretreatment of the cells with neuraminidase, an enzyme that increases cell surface GM1, resulted in significant elevation of PGE1-induced cAMP formation, as did preincubation of the cells with nmolar concentrations of GM1. Pretreatment with brain ganglioside mixture lacking GM1 had no effect. Cholera toxin B subunit, a specific GM1-binding ligand, inhibited adenylyl cyclase. When the concentration of exogenous GM1 in which the cells were preincubated was increased from nmolar to molar levels there was a dose-responsive fall off in cAMP elevation, attributed to progressive inhibition of adenylyl cyclase by increasing GM1. These results are interpreted as indicating modulation of this PGE1 receptor in Neuro-2a cells by plasma membrane-localized GM1 in a structure-specific manner.Abbreviations PGE1 prostaglandin E1 - Ctx B B subunit of cholera toxin - BBG bovine brain ganglioside mixture - DMEM Dulbecco's modified Eagle's medium - FBS fetal bovine serum - IBMX 3-isobutyl-1-methylxanthine - N'ase neuraminidase - D-PBS Dulbecco's phosphate-buffered saline