The effect of ganglioside GQ1b on the NMDA receptor signaling pathway in H19-7 cells and rat hippocampus

Gangliosides, sialic acid-containing glycosphingolipids, are related to various synaptic functions in the rat brain. Previously, we investigated the behavioral effects of the ganglioside GQ1b on learning and memory using the Y-maze and Morris water maze test. GQ1b-treated rats showed highly increased memory performance on the Y-maze and the Morris water maze test. In this study, we determined the role of GQ1b on the activation of the N-methyl-d-aspartate (NMDA) receptor signaling pathway in H19-7 rat hippocampal cells and the hippocampus of rats. After 12 h of treatment with GQ1b, the expression levels of NMDA receptor subunit 2A and 2B were increased in H19-7 cells and the hippocampus of rats. In addition, treatment of GQ1b increased the tyrosine phosphorylation of NR2B that may enhance NMDA receptor synaptic activation and enhancement of NMDA receptors. Also, following GQ1b treatment, the phosphorylation of extracellular signal-regulated kinases (ERK1/2) and protein kinase A, a cAMP activated protein kinase (PKA) increased in H19-7 cells and the hippocampus of rats. These increases resulted in an increase in the phosphorylation of cAMP response element binding protein (CREB). These results suggest that GQ1b might facilitate the activation of the NMDA receptor signaling pathway in the hippocampus of rats, an effect which is dependent on ERK1/2, PKA and CREB phosphorylation. Also, these data support our previous result that GQ1b improves the learning and memory of rats.     

Binding of immunoglobulin G antibodies in Guillain-Barré syndrome sera to a mixture of GM1 and a phospholipid: possible clinical implications

Anti-GM1 immunoglobulin G (IgG) antibodies are frequently present in sera from patients with Guillain-Barré syndrome (GBS). A previous report on a patient who had a neuropathy with immunoglobulin M (IgM) M-protein binding to a conformational epitope formed by phosphatidic acid (PA) and gangliosides prompted us to investigate the binding of IgG antibodies in GBS sera to a mixture of GM1 and PA (GM1/PA). Of 121 GBS patients, 32 had anti-GM1 IgG antibodies. All 32 also had antibody activity against GM1/PA. Twenty-five (78%) of 32 patients had greater activity against GM1/PA than against GM1 alone. Twelve patients who had no anti-GM1 IgG antibodies had IgG antibody activity against GM1/PA. No GBS patient had IgG antibody against PA alone. In contrast, two rabbit anti-GM1 antisera had greater activity against GM1 alone than against GM1/PA. IgG antibody with greater binding activity against a mixture of GM1 and a phospholipid than against GM1 alone may have an important role in the pathogenesis of GBS and has implications for diagnosis.     

窖蛋白1在无血清状态下诱导FBJ细胞死亡

目的研究无血清状态下,FBJ-S1细胞较FBJ-LL细胞更易死亡的原因。方法用细胞活性检测法分析细胞的活性,分别用HPTLC法、逆转录PCR法和免疫印迹法检测细胞中GD1a和窖蛋白1(caveolin-1)的含量。结果研究表明,在无血清培养基培养条件下,富含神经节苷脂GM3和GD1a的低转移性的FBJ-S1小鼠骨肉瘤细胞趋向死亡,但对含有几乎相同量GM3而无GD1a表达的高转移性的FBJ-LL细胞的生长却未见明显影响。同时发现:a.FBJ-S1细胞富含窖蛋白1,而FBJ-LL细胞中窖蛋白1表达量则较低;b.利用小干扰基因沉默技术在FBJ-S1细胞中沉默窖蛋白1的表达,结果使细胞存活率升高;c.使用GD1a处理FBJ-LL细胞导致其趋向死亡,通过免疫印迹法检测窖蛋白1发现其蛋白表达量增加。结论在无血清培养条件下,窖蛋白1诱导FBJ细胞的死亡。     

神经节苷脂GM1与亚低温联合应用对新生鼠缺氧缺血性脑损伤的保护作用

目的探讨神经节苷脂GM1与亚低温对新生大鼠缺氧缺血性脑损伤的保护作用。方法用75只新生7 d的SD大鼠制作缺氧缺血(HIBD)模型,随机分为假手术(A)组、模型(B)组、神经节苷脂GM1(C)组、亚低温(D)组、神经节苷脂与亚低温联合干预(E)组共5组,每组15只。观察不同干预方法对新生大鼠缺氧缺血后24h脑含水量、神经细胞死亡率及30d后学习和记忆能力的影响。结果B组脑含水量、神经细胞死亡率显著高于A组,学习、记忆能力显著下降(P<0.05),各干预组脑含水量、神经细胞死亡率下降,学习、记忆能力显著改善(P<0.05),而以E组改变更明显(P<0.05)。结论神经节苷脂GM1和(或)亚低温均可显著减轻HI后脑损伤,联合干预保护作用更佳。     

神经节苷脂对早产儿脑损伤神经行为的影响

【目的】探讨神经节甘脂(GM-1)对早产儿脑损伤保护作用及临床疗效。【方法】82例发生脑室周围一脑室内出血(PVH-IVH)和脑室周围白质软化(PVL)脑损伤的早产儿分为GM-1组(42例)和对照组(40例)。GM-1组在常规治疗基础上加用GM-1治疗。观察与比较两组临床主要症状呼吸暂停发生率和吸吮-吞咽能力,并分别于校正胎龄40周和41周时予新生儿神经行为评分(NBNA)。【结果】GM-1组两次测查均明显高于对照组(P均<0.01)。临床主要症状吸吮-吞咽能力明显优于对照组(P<0.05),呼吸暂停发生率显著低于对照组(P<0.05)。【结论】GM-1为中枢神经系统损伤修复药物,对脑损伤的早产儿神经行为发育有促进作用,可改善近期预后,提高生存质量。     

GM-1对大鼠视网膜缺血再灌注损伤的保护作用

目的 探讨单唾液酸神经节苷脂（monosialoganglioside,GM-1）对大鼠视网膜缺血冉灌注损伤后视网膜组织及超微结构的保护作用.方法 健康成年Wistar大鼠75只,随机分为3组：正常组5只、NS组35只,GM-1组35只.正常对照组不加任何处理因素,NS组和GM-1组通过升高眼压造成视网膜缺血60 min,分别于缺血前12 h、1 h及缺血结束时3次腹腔注射NS 3mL/kg或GM-1 3mL/kg（10 mg/mL）,并于再灌注0 h（单纯缺血后）、1 h、6 h、12 h、24 h、3 d和7 d共7个时间点取双眼眼球,每时间点5只,HE染色观察视网膜组织结构并测量视网膜内层平均厚度（mean thickhess of the inner retinal layers,MTIRL）,透射电镜观察视网膜超微结构变化.结果 缺血再灌注后,内层视网膜依次表现出水肿、凋亡、萎缩的损伤过程.GM-1可明显减轻其水肿和萎缩的程度,并减少凋亡的发生.结论 GM-1对视网膜缺血再灌注损伤具有明确的保护作用,可能是其有效治疗药物.     

A rat model of leptomeningeal human neoplastic xenografts

Leptomeningeal (LM) cancer spread from either a primarybrain tumor or a systemic cancer is rapidlyfatal. Current therapies are ineffective and highly toxicto normal nervous system tissues. A xenograft modelof LM neoplasia in nude rats using adiversity of tumor cell types was established inorder to evaluate new treatment strategies and tostudy the pharmacokinetics and biological effects of treatmentsadministered into the subarachnoid space. Consistent leptomeningeal engraftmentand progressive tumor growth was seen after intrathecalinjection of 9 of 13 tumor cells lines,including 2 melanomas, 2 neuroblastomas, 2 medulloblastomas, 2gliomas, and 1 breast cancer. Clinical signs rangedfrom steady weight loss commencing from the dayafter tumor implantation to absence of any signsfor three weeks until the sudden occurrence ofmajor neurological deficits or death. Pathologic examination showedonly leptomeningeal tumor growth with some cell linesand severe parenchymal invasion with others. CSF cytologyconsistently demonstrated tumor cells in animals with LMdisease. Cranial magnetic resonance (MR) following intravenous (IV)administration of a contrast agent revealed enhancing lesionsone week following melanoma tumor implantation. Reliable ventricularpuncture was demonstrated by radiography following intraventricular (IVent)injection of an iodinated contrast material. IVent instillationof saline, albumin, or antibodies did not provokeclinical toxicity or an inflammatory response.     

In vivo and in vitro effects of melatonin or ganglioside GT1B on L-cysteine-induced brain mitochondrial DNA damage in mice.

The effects of L-cysteine on mitochondrial DNA (mtDNA) in mouse brain were investigated both in vivoandin vitro. An intracerebroventricular (icv) injection of L-cysteine (1.25 micromol/animal) caused mtDNA damage in brain frontal and central portions of the cortex, broad-spectrum limbic and severe sustained seizures in mice, and increased lipid peroxidation in the whole brain. The L-cysteine-mediated effects were prevented by an intraperitoneal (ip) preinjection of melatonin (20 mg/kg) or an intracerebroventricular preinjection of ganglioside GT1b (90 nmol/animal). Furthermore, in in vitroexperiments, L-cysteine (0.05, 0.5, or 1.0 mM) caused damage to brain mtDNA and increased lipid peroxidation in a concentration-dependent manner when incubated at 37 degrees C for 20 or 60 min with a homogenate prepared from whole mouse brains. However, the mtDNA damage and the increased lipid peroxidation were completely abolished by a cotreatment with melatonin (1.5 mM), a potent scavenger of the hydroxyl radical (*OH), or ganglioside GT1b (60 microM), a potent inhibitor of glutamate-receptor-mediated activation and translocation of protein kinase C and lipid peroxidation. These results suggest that reactive oxygen species including the *OH may be involved in l-cysteine-induced brain mtDNA damage, lipid peroxidation, and development of seizures in mice. Therefore, we concluded that *OH scavengers, such as the pineal hormone melatonin and ganglioside GT1b, can protect against brain mtDNA damage, seizures, and lipid peroxidation induced by reactive oxygen species producers such as L-cysteine.     

Cholesterol reduction by methyl-beta-cyclodextrin attenuates the delta opioid receptor-mediated signaling in neuronal cells but enhances it in non-neuronal cells

Opioid receptors have been shown to be located in and regulated by lipid rafts/caveolae in caveolin-rich non-neuronal cells. Here, we found that caveolin-1 level was very low in rat brain and undetectable in NG108-15 cells, which endogenously express delta opioid receptors (DOR). Rat caudate putamen (CPu) membranes, NG108-15 cells and CHO cells stably transfected with FLAG-mouse-DOR (CHO-FLAG-mDOR) were homogenized, sonicated in a detergent-free 0.5M Na(2)CO(3) buffer and fractionated through discontinuous or continuous sucrose density gradients. About 70% of opioid receptors in CPu and DOR in both cell lines were present in low-density (5-20% sucrose) membrane domains enriched in cholesterol and ganglioside M1 (GM1), characteristics of lipid rafts in plasma membranes. In both cells, stimulation with permeable or non-permeable full agonists, but not with partial or inverse agonists, for 30min shifted approximately 25% of DORs out of rafts, by a naloxone-reversible and pertussis toxin-insensitive mechanism, which may undergo internalization. Methyl-beta-cyclodextrin (MCD) treatment greatly reduced cholesterol and shifted DOR to higher density fractions and decreased DPDPE affinities. MCD treatment attenuated DPDPE-induced [(35)S]GTPgammaS binding in CPu and NG108-15 cells, but enhanced it in CHO-FLAG-mDOR cells. In CHO-FLAG-mDOR cells, G(alphai) co-immunoprecipitated with caveolin-1, which was shown to inhibit G(alphai/o), and MCD treatment dramatically reduced the association leading to disinhibition. Thus, although localization in rafts and agonist-induced shift of DOR are independent of caveolin-1, lipid rafts sustain DOR-mediated signaling in caveolin-deficient neuronal cells, but appear to inhibit it in caveolin-enriched non-neuronal cells. Cholesterol-dependent association of caveolin-1 with and the resulting inhibition of G proteins may be a contributing factor.     

Suppression of lung metastasis of mouse Lewis lung cancer P29 with transfection of the ganglioside GM2/GD2 synthase gene

Ganglioside functions in tumor metastasis were analyzed by carbohydrate remodeling of a mouse Lewis lung cancer (subline P29) by introducing beta1,4GalNAc-T cDNA. Although P29 was originally a low-metastatic subline in the s.c. injection system, it showed high potential in lung metastasis when i.v.-injected via the tail vein. Two lines of GM(2)(+) transfectants showed markedly reduced metastatic potential to the lung compared to 2 control lines. However, cell proliferation rates and expression levels of various cell adhesion molecules, e.g., integrin family members, SLe(x) and CD44, were essentially unchanged after transfection of the cDNA. Then, cell adhesion to fibronectin-coated dishes was examined, showing that GM(2) (+) transfectants attached to the plates much more slowly than controls, suggesting functional modulation of integrins with newly expressed GM(2). Phosphorylation of the FAK located at downstream of integrin molecules was markedly reduced in GM(2)(+) transfectants, suggesting that GM(2) suppressed cell adhesion signals via fibronectin-integrin interaction.