Glucosylceramide synthase inhibitors D-PDMP and D-EtDO-P4 decrease the GM3 ganglioside level, differ in their effects on insulin receptor autophosphorylation but increase Akt1 kinase phosphorylation in human hepatoma HepG2 cells.

Gangliosides function as modulators of several cell growth related receptors. It was shown for caveolin-rich adipocytes, that GM3 ganglioside binds to insulin receptor (IR), dissociates its complex with caveolin, and thus lowers IR autophosphorylation following insulin treatment. We extended those studies into human hepatocyte-derived HepG2 cells, characterized by a high level of IR but low of caveolin. To lower the glycosphingolipid content, estimated by GM3 concentration, two glucosylceramide synthase inhibitors d-threo-1-pheny-2-decanoylamino-3-morpholino-1-propanol (d-PDMP) and d-threo-1-(3,4,-ethylenedioxy)phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (d-EtDO-P4) were used. d-PDMP at 40 µM or d-EtDO-P4 at 1 µM concentrations in culture medium decreased the GM3 content to 22.3% (17.8-26.1%) and 18.1% (13.7-24.4%), respectively, of the control value. The reduction of GM3 obtained with d-PDMP was accompanied by a 185.1% (153.5-423.8%) significant increase in the level of IR autophosphorylation following cell stimulation with 100 nM insulin. The effect of d-EtDO-P4 on IR autophosphorylation was smaller amounting to an increase by 134.8% (111.3-167.8%) of the control level and statistically non-significant. The effects of d-PDMP and d-EtDO-P4 could also be detected at the level of Akt1 kinase. In cells grown in the presence of d-PDMP the level of phosphorylated Akt1 was 286.0% (151.4%-621.1%) of that in the control. In this case the effect of d-EtDO-P4 was similar: 223.0% (181.4-315.4%) significant increase in phosphorylated Akt1. We assume that glycosphingolipid depletion in HepG2 cells may affect not only IR autophosphorylation but also, independently, the phosphorylation of Akt1, by modifying the membrane microenvironment of this kinase.

Highly selective inhibition of butyrylcholinesterase by a novel melatonin-tacrine heterodimers.

Novel inhibitors of cholinesterases, especially butyrylcholinesterase (BuChE), were obtained by coupling melatonin-tacrine heterodimers via the carbamate bond. Compounds 14a-i possessed potent cholinesterase inhibitory activity (with IC50 values as low as 1.18 nM for acetylcholinesterase (AChE) and 0.24 nM for butyrylcholinesterase (BuChE)). These heterodimers exhibit selectivity toward BuChE, being from 4- to 256-fold more active toward BuChE than AChE, but still acting as better AChE inhibitors than tacrine 4.

Selective inhibition of butyrylcholinesterase by singlet oxygen-generated melatonin derivatives.

The inhibition of cholinesterases plays a crucial role in a therapy of neurodegenerative diseases, including Alzheimer's disease. Especially, butyrylcholinesterase (BChE) has recently gained special interest. On the other hand, compounds having antioxidative properties may have a beneficial role in slowing down neurodegeneration processes. To combine these two effects, we synthesized a series of new derivatives of melatonin, which is a strong antioxidant, possessing structural elements essential for the inhibitory activity against cholinesterase. The structure of the new compounds was confirmed by NMR spectroscopy and mass spectrometry, and their activity against cholinesterases was measured in vitro using modified Ellman's method. The compounds obtained showed a high inhibitory activity, together with a strong selectivity against BChE. These results may point at new area of interest in a research on cholinesterase inhibitors.

Preparation of Alexa Fluor 350-conjugated nonradioactive or 3H-labeled GM1 ganglioside derivatives with different ceramides.

Alexa Fluor 350 hydrazide (AF) was coupled to the aldehyde group at C-6 of terminal galactose of oxidized GM1 gangliosides containing different fatty acid residues (GM1s). The AF-GM1 hydrazones obtained were reduced with NaBH(4) or [3H]NaBH(4) and purified by high-performance thin layer chromatography (HPTLC) and high-performance liquid chromatography (HPLC). Final yields of AF-GM1s exceeded 30%, purity was better than 97%, and radiochemical purity of 3H-labeled AF-GM1s was more than 94.5%. Structures of AF-GM1s were confirmed by electrospray ionization-mass spectrometry (ESI-MS). When added to HL-60 cell culture media, more than 81.6 or 78.9% of the AF-[3H]GM1s were taken up by cells in a bovine serum albumin- or trypsin-resistant manner, respectively. Approximately 70% of the AF-[3H]GM1s were recovered in HL-60 total plasma membrane fraction.

The ceramide structure of GM1 ganglioside differently affects its recovery in low-density membrane fractions prepared from HL-60 cells with or without triton-X100.

Gangliosides are characteristically enriched in various membrane domains that can be isolated as low density membrane fraction insoluble in detergents (detergent-resistant membranes, DRMs) or obtained after homogenization and sonication in 0.5 M sodium carbonate (low-density membranes, LDMs). We assessed the effect of the ceramide structure of four [(3)H]-labeled GM1 ganglioside molecular species (GM1s) taken up by HL-60 cells on their occurrence in LDMs, and compared it with our previous observations for DRMs. All GM1s contained C18 sphingosine, which was acetylated in GM1(18:1/2) or acylated with C(14), C(18) or C(18:1) fatty acids (Fas).

The oxidation products of melatonin derivatives exhibit acetylcholinesterase and butyrylcholinesterase inhibitory activity.

It is already well documented that melatonin exhibits strong antioxidant properties. It traps several reactive oxygen species including singlet oxygen, peroxyl and hydroxyl radicals. Also, peroxynitrite-induced reactions are inhibited by melatonin. The oxidation of melatonin by singlet molecular oxygen [O(2) ((1)Delta(g))] may produce cyclic 3-hydroxymelatonin whose structure we have already studied. In this investigation we report on the synthesis of several melatonin analogues having a carbamate substituent instead of the methoxy group at 5 position of the indole ring. These compounds behave analogously to melatonin with respect to singlet oxygen and produce the corresponding cyclic 3-hydroxymelatonin analogues. The structures of the products were investigated with spectral methods and X-ray crystallography. The compounds obtained possess the 2,3,8,8a-tetrahydropyrrolo[2,3-b]indole heterocyclic system which is a structural motif characteristic of alkaloids, physostigmine and phenserine, that are potent acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitors used in the Alzheimer's disease treatment. We measured the inhibitory activity of the obtained compounds against AChE and BChE from human erythrocytes and serum. In the case of the compounds having a phenylcarbamate and methoxyphenylcarbamate substituents, the inhibitory activity (IC(50)) ranged from 0.252 +/- 0.033 to 3.804 +/- 0.581 microM. Other compounds were less active and showed rather complex interactions with the structure-activity relationship in need of further investigation.

Structure of the ceramide moiety of GM1 ganglioside determines its occurrence in different detergent-resistant membrane domains in HL-60 cells.

To investigate the effect of the ceramide moiety of GM1 ganglioside on its association with detergent resistant membrane domains (DRMs) in human leukemia HL-60 cells, [(3)H] labeled GM1 molecular species (GM1s) with ceramides consisting of C18 sphingosine acetylated or acylated with C(8), C(12), C(14), C(16), C(18), C(22), C(24), C(18:1), C(22:1), or C(24:1) fatty acids (FAs), or C20 sphingosine acetylated or acylated with C(8) or C(18) FA were prepared and added to culture media. GM1s uptake by HL-60 cells was affected by the structure of their ceramides. Resistance to removal with trypsin and the stoichiometry of [(125)I] cholera toxin (CT) binding indicated that the added GM1s were incorporated into the membranes of the cells used for the isolation of DRMs in a manner resembling endogenous gangliosides. The ceramide moieties of the GM1s determined their occurrence in DRMs and the dependence of their recovery in this membrane fraction on the amount of Triton X-100 (TX) used for extraction as well as on cholesterol depletion. The GM1s with sphingosine acylated with C(14), C(16), C(18) C(22), or C(24) FAs were similarly abundant in DRMs. GM1s acylated with C(18:1), C(22:1), or C(24:1) were less abundant than those acylated with saturated FA of the same length. GM1s acetylated or acylated with C(8) FA were detected in DRMs in the lowest proportion. Depletion of 73% of cell cholesterol with methyl-beta-cyclodextrin significantly affected the recovery in DRMs of GM1s acetylated or acylated with C(8) or unsaturated FAs but not of GM1 acylated with C(18), C(22), or C(24) FAs. After cross-linking with CT B subunit, all GM1s were recovered in DRMs in a similarly high proportion irrespective of their ceramide structure or cholesterol depletion. DRMs prepared with low TX concentration at the TX/cell protein ratio of 0.3:1 were separated by multistep sucrose density gradient centrifugation into two fractions. The GM1s with sphingosine acetylated or acylated with C(18) or C(18:1) FAs occurred in these fractions in different proportions.

Ganglioside reactive antibodies of IgG and IgM class in sera of patients with differentiated thyroid cancer.

We searched for the presence of ganglioside reactive antibodies in sera of patients with differentiated thyroid cancer (DTC). Sera were screened by ELISA with plates coated with GM3(NeuAc), GM3(NeuGc), GM2, GM1, FucGM1, GD3, GD1a or GD1b gangliosides. Ganglioside reactive antibodies were detected more frequently in sera of patients with DTC than in sera of healthy persons, in keeping with the possibility of autoimmunization during carcinogenesis. Antibodies of IgM and IgG classes reactive with FucGM1 occurred most often. However, the infrequent occurrence of ganglioside reactive antibodies, their low titer and lack of correlation between their presence and clinical condition of the patients indicate that determination of these antibodies has no diagnostic value in DTC.