Two Compounds Isolated From Ganglioside GM1 Promote Angiogenesis in Zebrafish.

Ganglioside has been implicated to play important roles in modulating various cell signaling and biological functions. However, the functional analysis of a single ganglioside in a zebrafish model is so far lacking. In this study, we investigated the angiogenic effects of 2 monosialoganglioside compounds isolated from GM1 in zebrafish embryos. First, we showed the tested compounds are adequate safe. Then, we found that these compounds exhibited significant proangiogenic effect through enhancement of endothelial cell proliferation, migration, and differentiation. Furthermore, the 2 compounds were proved to promote angiogenesis through, at least partially, modulating the level of Notch signaling. This study provides the novel insights into the clinical application of the 2 ganglioside compounds and GM1.

Biochemical characterization of GM1 micelles-Amphotericin B interaction.

In this work a thorough characterization of the GM1 micelle-Amphotericin B (AmB) interaction was performed. The micelle formation as well as the drug loading occurs spontaneously, although influenced by the physicochemical conditions, pH and temperature. The chromatographic profile of GM1-AmB complexes at different molar ratios shows the existence of two populations. The differential absorbance of GM1, monomeric and aggregate AmB, allowed us to discriminate the presence of all of them in both fractions. Thus, we noted that at higher proportion of AmB in the complex, increases the larger population which is composed mainly of aggregated AmB. The physical behavior of these micelles shows that both GM1- AmB complexes were stable in solution for at least 30 days. However upon freeze-thawing or lyophilization-solubilization cycles, only the smallest population, enriched in monomeric AmB, showed a complete solubilization. In vitro, GM1-AmB micelles were significantly less toxic on cultured cells than other commercial micellar formulations as Fungizone, but had a similar behavior to liposomal formulations as Ambisome. Regarding the antifungal activity of the new formulation, it was very similar to that of other formulations. The characterization of these GM1-AmB complexes is discussed as a potential new formulation able to improve the antifungal therapeutic efficiency of AmB.

Neuronal differentiation of human mesenchymal stromal cells increases their resistance to Aß42 aggregate toxicity.

Cell therapy is a promising approach for the treatment of neurodegenerative conditions such as Alzheimer's and Parkinson's diseases. However, the presence of toxic aggregates in tissue raises the question of whether grafted stem cells are susceptible to amyloid toxicity before they differentiate into mature neurons. To address this question, we investigated the relative vulnerability of human mesenchymal stromal cells and their neuronally differentiated counterparts to Aß(42) oligomers and whether susceptibility correlates with membrane GM1 content, a key player in oligomer toxicity. We found that our cell model was highly susceptible to aggregate toxicity, whereas neuronal differentiation induced resistance to amyloid species. This data correlated well with the content of membrane GM1, levels of which decreased considerably in differentiated cells. These findings extend our knowledge of stem cell vulnerability to amyloid species, which remains a controversial issue, and confirm that amyloid-GM1 interactions play an important role in cell impairment.

GD1alpha-replica peptides functionally mimic GD1alpha, an adhesion molecule of metastatic tumor cells, and suppress the tumor metastasis.

A novel peptide technology to produce mimicking peptides of carbohydrate moiety (which we propose to name glyco-replica peptides) is a useful tool to elucidate the functions of glycoconjugate. Carbohydrate moiety of ganglioside GD1alpha functions as a molecule involved in the adhesion between murine highly metastatic lymphoma RAW117-H10 cells and hepatic sinusoidal endothelial (HSE) cells. To prepare peptides which mimic the carbohydrate structure of GD1alpha, phage clones expressing peptides which bound to a monoclonal antibody against GD1alpha (KA17) were isolated from a phage-displayed random peptide library. Four phage clones having affinity to the monoclonal antibody KA17 were isolated, and these clones showed inhibitory effect on the binding of KA17 to GD1alpha. The amino acid sequences of the displayed pentadecamers were determined, and one of the phages displaying sequence WHWRHRIPLQLAAGR bound to HSE cells directly and showed the highest inhibitory effect on the adhesion between RAW117-H10 cells and HSE cells. The synthesized peptides having the same sequences to the displayed 15mers in the four isolated phage clones also showed the inhibitory effect on the adhesion of RAW117-H10 cells to HSE cells, and, again, the WHWRHRIPLQLAAGR peptide showed the highest inhibitory effect. Furthermore, intravenous injection of the peptide brought almost complete inhibition of the metastasis of RAW117-H10 cells to lung and spleen, and about 50% inhibition of the liver metastasis. These results indicate that GD1alpha plays an important role for metastasis of RAW117-H10 cells, and the peptides obtained by the present procedure are able to mimic the functional role of the glycoconjugate.

Potential efficacy and toxicity of GM1 ganglioside against trimethyltin-induced brain lesions in rats: comparison with protracted food restriction.

GM1 ganglioside (one week each at 10, 5, and 2.5 mg GM1/kg per day, ip) or gradual food restriction leading to a reduction in body weight to 75% of control were tested for their ability to block or reverse histopathologic and behavioral effects of trimethyltin (TMT) poisoning in rats. TMT (a single oral gavage of 6.0 mg TMT HCI/kg body weight) reduced hippocampal weight, decreased hippocampal cell counts, decreased autoshaped learning measures, and suppressed progressive fixed ratio (PFR) lever pressing without affecting stable lever pressing. Neither GM1 nor greater food restriction affected hippocampal weight. Greater food restriction prevented TMT's effects on autoshaping but not on PFR behavior, was without behavioral effects in animals not treated with TMT, and did not affect hippocampal histology. GM1 prevented certain TMT-induced decrements in autoshaping and PFR behavior but also suppressed autoshaping and stimulated stable fixed ratio behavior in animals not treated with TMT. GM1 also reduced hippocampal serotonin concentration, another "lesion-like" change. GM1 blocked TMT-induced hippocampal CA3b cell loss, but did not protect CA3c cells, the main locus of TMT hippocampal damage. The results support the idea that exogenous GM1 is a potent neuroactive agent with complex actions in intact organisms, potentially beneficial and potentially toxic. Like GM1, food restriction induces complex and potentially beneficial effects, but it lacked GM1's biochemical and behavioral "side effects" (i.e. toxicity) in these experiments.

Initiated stem cells in murine intestinal carcinogenesis: prolonged survival, control by NK cells, and progression.

Weekly injections of dimethylhydrazine (DMH) (25 mg/kg), or azoxymethane (AOM) (8 mg/kg) to young adult male CDI mice for 1-2 months produced generalized intestinal crypt hyperplasia, which we measured in duodenum in terms of number of interphase and mitotic cells present in crypts. As shown earlier, the crypts expanded because of the presence of a hyperproliferative "initiated" crypt subpopulation which was also sensitive to natural killer (NK) cells. Hyperplasia was thus present as long as NK activity was suppressed by the carcinogen treatment. After interruption of the treatment for periods of 1, 2, 3, 6 and 10 months in the various groups, hyperplasia soon regressed as a result of elimination of the subpopulation by the recovering NK cells. When NK activity was once again eliminated during the terminal days of these "interruption periods" (by injections of anti-asialo GM-I antibody, alpha AGM-I), the original hyperplasia was fully reconstituted, apparently from stem cells of the subpopulation which survived up to 10 months in their crypt base location. These "initiated stem cells" represented, then, the original carcinogenic insult during the pre-cancerous period. They also appeared to be the source of the eventual neoplasia, as treating the animals with mutagens during the interruption periods produced specific changes in crypt base histology: new "crypt base basophilic" (CBB) cells appeared which produced large accumulations as well as microscopic tumors when NK activity was suppressed (by alpha AGM-I). Some of the initiated stem cells were apparently transformed into neoplastic ones which remained under NK control, the NK cells preventing the establishment of their progeny. Further experiments indicated that, although the initiated stem cells are not eliminated by normal NK activity, activated NK cells can kill them, thereby eliminating the potential source of neoplasia.

Neurotoxicology of chronic infusion of the ganglioside GM1 in the ewe: phase I. intrathecal administration.

Gangliosides, including GM1, provide a measure of improved functional recovery after ischemic, toxic, and traumatic brain injuries in animal studies. Since systemically injected GM1 has provided equivocal results in a variety of human neurodegenerative conditions, the possibility exists that intrathecal or intracerebroventricular delivery might provide more effective concentrations along the neuroaxis. In preparation to consider clinical trials, the potential neurotoxicologic effects of chronic intrathecal GM1 were studied in ewes. Preliminary in vitro tests first demonstrated the stability and compatibility of GM1 in implanted pumps. Two groups of adult ewes were then implanted with either Therex or Infusaid continuous flow implantable pumps and chronic intrathecal catheters. Ewes were infused intrathecally with either preservative-free normal saline (n = 5) or GM1 (n = 7) 100 micrograms-10,000 micrograms/d for up to 24 wk. No abnormal behavioral responses were noted. Cerebrospinal fluid analyzed for GM1 concentrations by thin layer chromatography revealed no evidence of GM1 accumulation. After the animals were killed, spinal cords were removed, fixed, sectioned, and stained. Histologic analysis revealed no generalized pattern of neuronal damage, demyelination, gliosis, or axonopathy to distinguish intrathecal normal saline or GM1. In both treated and control groups, the only consistent finding was a pericatheter-associated compression of white matter with axonal dilation, vacuolation, and occasional neuronal loss. Catheter tracts in both groups were also associated with variable leptomeningeal fibroproliferative changes in adjacent dura and pia, at times in conjunction with more generalized duromeningeal thickening. In summary, chronic intrathecal GM1 in doses up to 10 mg/d had no definable neuropathologic consequences.