Exogenously administered gangliosides fail to increase in vivo metastatic frequency or in vitro growth of murine neoplastic cells.

The influence of gangliosides on tumor growth and frequency of metastasis in vivo, as well as growth of neoplastic cells in vitro, was tested utilizing the mouse fibrosarcoma cell line MN4. In mice receiving intramuscular tumor transplants, injections of a ganglioside mixture twice daily did not influence the tumor volume or the number of spontaneous metastases per animal. Furthermore, in mice receiving the cells by tail vein injection, injections of a ganglioside mixture once or twice daily did not affect the number of metastatic foci in the lungs. Preincubation of neoplastic cells with the ganglioside mixture decreased the number of metastatic foci in the lungs of mice receiving the cells by tail i.v. injection. The addition of ganglioside mixture to the culture medium for up to a 48-h incubation period had no effect, independently of the cell density utilized, on either the rate of DNA synthesis or the relative numbers of neoplastic cells as compared to controls; at higher ganglioside concentrations, growth was actually reduced. These results are interpreted to indicate that gangliosides, under the present conditions, do not influence tumor growth and metastatic neoplastic capacity in vivo, and growth in vitro.

Effect of parenteral administration of GM1 on cytokines and anti-ganglioside antibody patterns. Preliminary report in normal human individuals.

To assess the effects of monosialoganglioside GM1 on some immunological parameters, 12 healthy men were treated with 100 mg GM1 i.m. daily for 15 days. Before and after treatment, the following were studied: (1) serum levels of antibodies against GM1, asialo-GM1 (aGM1), GM2 and GD1b; (2) serum levels of interleukin (IL)-1 beta, IL-2, soluble IL-2 receptor (sIL-2R), tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma); (3) IL-1 beta and TNF-alpha production by peripheral blood monocytes (PBMO). Anti-ganglioside antibody and cytokine serum levels were not affected by exogenous GM1 administration with the exception of a transient increase in anti-GM1 antibody titer observed in one subject. In addition, no inhibition of IL-1 beta and TNF-alpha production by PBMO was observed. These preliminary data do not support a potential immunogenic or immunomodulatory function for in vivo administered GM1.

Monosialoganglioside internal ester stimulates the dopaminergic reinnervation of the striatum after unilateral hemitransection in rat.

The effects of the administration of GM1 monosialoganglioside internal ester, AGF2, on the dopaminergic reinnervation of the striatum in rats with unilateral hemitransection has been studied. AGF2 increases the apparent Vmax and the density of tyrosine-hydroxylase-positive nerve terminals in the striatum of the lesioned side, without modification of the tyrosine-hydroxylase activity in the unlesioned side. AGF2, at lower doses, is more active than its parent natural molecule GM1. AGF2 has a larger half-life and a higher distribution volume than GM1, and undergoes a slow hydrolysis in the serum releasing the original natural compound GM1. Mannitol and dexamethazone, often used to prevent swelling of the brain after injury, or isoaxonine, proposed to stimulate neurite growth are unable to reproduce the effects of AGF2 on the recovery of striatal tyrosine-hydroxylase activity after hemitransection. The data are compatible with the view that AGF2, through its conversion into GM1, facilitates the collateral sprouting of the nigro-striatal dopaminergic neurons.

Antimicrobial susceptibility test of Helicobacter pylori isolated from Jos, Nigeria.

Fifty-five strains of Helicobacter pylori isolated from November 1997 until October 1998 from 33 female and 22 male adults attending for endoscopy at the Evangel Hospital, Jos, Nigeria were assayed for antibiotic susceptibility to amoxycillin, clarithromycin, metronidazole and tetracycline by the E-test strip method. Minimum inhibitory concentration (MIC) within the attainable peak serum concentrations for each drug was used as the parameter to determine the susceptibility of H. pylori. The results showed 100% susceptibility for amoxycillin, 89.0% for tetracycline, 87.3% for clarithromycin and 60% for metronidazole. The MIC50 and MIC90 values were: 0.016 microgram/mL and 0.75 microgram/mL for amoxycillin, 0.016 microgram/mL and 2 micrograms/mL for clarithromycin, 0.094 microgram/mL and 12 micrograms/mL for tetracycline, and 2 micrograms/mL and > 48 micrograms/mL for metronidazole. The MIC90 values for metronidazole (> 48 micrograms/mL) and tetracycline (12 micrograms/mL) were in each case higher than the break-point value (peak serum concentrations) of 8 micrograms/mL for metronidazole and 3 micrograms/mL for tetracycline. This pattern of resistance to metronidazole and tetracycline has to be considered when therapeutic regimens against H. pylori contain either or both drugs.

Semisynthetic preparation of N-glycolylneuraminic acid containing GM1 ganglioside: chemical characterization, physico-chemical properties and some biochemical features.

N-Glycolylneuraminic acid containing GM1, GM1(NeuGc), was prepared by semisynthetic procedure. The procedure makes use of GM1 ganglioside deacetylated at the level of sialic acid residue (deAc-GM1) and of 1,3-dioxalan-2,4-dione. DeAc-GM1 is prepared from GM1 by alkaline hydrolysis in the presence of tetramethylammonium hydroxide and the glycolylating compound by reaction of glycolic acid with phosgene in dioxane, followed by cyclization under vacuum. Mass spectrometric and nuclear magnetic resonance spectroscopy analyses clearly indicated the presence, in the neosynthesized ganglioside of a glycolic group in the sialic acid residue. Laser-light scattering measurements show that GM1(NeuGc) aggregates in aqueous media being present in solution as micelles with a molecular weight of 576,000 and a hydrodynamic radius of 62.4 A as determined at 25 degrees C. GM1(NeuGc) promotes neurite outgrowth in N-2a cells to a similar degree as GM1(NeuAc), but shows different behaviour under treatment with sialidase from Arthrobacter ureafaciens.

Aggregation properties of semisynthetic GM1 ganglioside (II3Neu5AcGgOse4Cer) containing an acetyl group as acyl moiety.

The aggregative properties of GM1 ganglioside containing an acetyl group as acyl moiety [GM1(acetyl)] in aqueous solution have been studied by static and dynamic light scattering measurements and surface tension experiments. GM1 (acetyl) spontaneously aggregates as small micelles showing a hydrodynamic radius and molecular weight of 34 A and 102 kDa, respectively, down to a concentration of 2.0 x 10(-5) M.

New chemical trends in ganglioside research.

A report is given of recent progress in the methodology for isolation of gangliosides from natural sources, for the preparation of molecular species of gangliosides homogeneous in both the oligosaccharide and ceramide portions of the molecule, for chemical manipulation and derivatization of gangliosides, and for the preparation of gangliosides radiolabelled in different parts of the molecule. Particular emphasis has been given to: high performance liquid chromatographic procedures capable to separate gangliosides on the basis of their oligosaccharide or ceramide moieties and yielding completely homogeneous compounds, that is gangliosides with a single oligosaccharide, a single long chain base and a single fatty acid; two-dimensional thin-layer chromatographic procedures, provided with a fully computerized quantification system, particularly suitable to identifying gangliosides containing alkali-labile linkages, including ganglioside lactones; chemical procedures of high yield for reducing gangliosides at the double bond of long chain base, for selective removal of the fatty acyl moiety and replacement with a novel fatty acid, and for the synthesis of ganglioside lactones; chemical procedures for inserting fluorescent, paramagnetic or photoreactive probes at the fatty acyl part of the ganglioside molecule; procedures for chemical isotopic radiolabelling of gangliosides at the level of sialic acid acetyl group and at the fatty acid moiety. Examples are provided evidencing the significance and potential use of a variety of ganglioside derivatives in the study of ganglioside metabolism and functional implications.

Synthesis, structure, and conformation of the dilactone derivative of GD1b ganglioside.

Treatment of DG1b, beta-Gal-(1----3)-beta-GalNAc-(1---- 4)-[alpha-Neu5Ac-(2----8)-alpha-Neu5Ac-(2---- 3)]-beta-Gal-(1----4)-beta-Glc-(1----1)-Cer, with dicyclohexylcarbodi-imide in anhydrous methyl sulfoxide affords 95-98% of GD1b-dilactone. The carboxyl groups of the two sialic acid units are involved in ester linkages, as proved by ammoniolysis and reduction which gave derivatives containing the amide of sialic acid and N-acetylneuraminulose, respectively. 1H-N.m.r. spectroscopy showed that the lactone rings involved position 9 of the inner sialic acid and position 2 of the inner galactose and that the disialosyl chain is extended toward the -beta-Gal-(1 ----4)-beta-Glc- portion of the ganglioside moiety.

Synthesis and structural characterization of the dilactone derivative of GD1a ganglioside.

Treatment of GD1a [alpha-Neu5Ac-(2----3)-beta-GalNAc-(1----4)-[alpha- Neu5Ac-(2----3)]-beta-Gal-(1----4)-beta-Glc-(1----1)-Cer] with dicyclohexylcarbodi-imide in anhydrous methyl sulfoxide affords 94-98% of GD1a-dilactone. The involvement of the carboxyl groups of the two sialic acid residues in the lactone rings was proved by ammoniolysis and reduction experiments, which gave ganglioside derivatives containing the amide of sialic acid and N-acetylneuraminulose, respectively. 1H-N.m.r. spectroscopy showed that the lactone rings involved position 2 of each galactose residue in the ester linkages.

Competitive binding assay for quantitative determination of GM1 ganglioside in plasma and cerebrospinal fluid.

A competitive binding assay for the quantitative determination of GM1 ganglioside is described. After extraction from biological fluids, GM1 was incubated with a known amount of cholera toxin B-subunit conjugated with horseradish peroxidase, and exposed to GM1 adsorbed onto polystyrene microwells. Since GM1 in solution blocks the binding of toxin B-subunit to GM1 adsorbed onto the solid phase, enzyme activity serves as a reciprocal measure of GM1 concentration in the sample. The assay was used to determine the basal level of GM1 in plasma and cerebrospinal fluid in different populations.

Pharmacokinetics of GM1 ganglioside following parenteral administration.

The pharmacokinetic parameters of monosialotetrahexosylganglioside (GM1) have been determined in healthy volunteers at 3 dose levels: 100, 200, 300 mg. Each dose was administered to separate groups of 12 volunteers. GM1 levels were determined in plasma, urine, and faeces by a method based on the property of the cholera toxin beta subunit to react specifically with GM1 ganglioside. A non-compartmental model was applied to determine standard pharmacokinetic parameters. The average AUC increased with dose (1002 +/- 121.2, 1306 +/- 146.1, 3155 +/- 121.6 micrograms mL-1 h after 100, 200, 300 mg, respectively). Plasma clearance was less than 3 mL min-1 and the distribution volume was close to the plasma volume (on average between 4.3 and 7.2 L). Mean residence time was about 43 h for all doses. GM1 was not detected in urine, while in faeces the amount of GM1 determined was similar to the baseline values obtained before dosing.

"If you scream, I'll kill you".

A quiet night in a nursing home is interrupted by an unwelcome visitor. This harrowing, first-person account is followed by an article giving expert advice on handling assaults.

Monosialoganglioside GM1 blood levels in maternal and newborn umbilical cord blood at birth.

Blood levels of the monosialoganglioside GM1 (nomenclature according to Svennerholm) were tested at birth in the umbilical cord of 37 neonates and their mothers. Comparisons were made based on gestational age and modality of delivery. GM1 blood levels at birth were significantly higher in mothers than in newborns (373.66 +/- 56.83 ng/ml vs 217.95 +/- 21.24 ng/ml; P < 0.01). Newborns delivered by cesarean section showed levels of GM1 significantly higher than those delivered vaginally (298.97 +/- 38.55 ng/ml vs 169.62 +/- 12.62 ng/ml; P < 0.01), and preterm newborns had significantly higher levels of GM1 than full-term newborns (253.50 +/- 40.83 ng/ml vs 193.71 +/- 21.74 ng/ml; P < 0.01). No differences in blood levels of GM1 were observed in the mothers in relation to length of pregnancy or modality of delivery. The higher levels of GM1 observed in preterm newborns indicate an increased turnover and/or enhanced bioavailability of the monosialoganglioside GM1 for the developing central nervous system. Further data are required to evaluate the significance of the increased cord levels of GM1 in neonates after cesarean section.

Positive ion fast atom bombardment mass spectrometric analysis of the molecular species of glycerophosphatidylserine.

The structure of 1,2-dipalmitoyl-sn-glycero-3-phosphoserine was analyzed by positive ion fast atom bombardment mass spectrometry and collisional activation mass-analyzed ion kinetic energy spectroscopy. The molecular weight, the polar-head group, and the fatty acid composition of this species were identified by the appearance of protonated and solvated protonated species ions, diglyceride and monoglyceride fragment ions. After purification of glycerophosphatidylserine from bovine brain and rat kidney by normal phase high-performance liquid chromatography, molecular species were identified by either positive or negative ion fast atom bombardment mass spectrometry. The study suggests that negative ion fast atom bombardment ionization is a more powerful tool for the identification of the molecular species of glycerophosphatidylserine from biological samples. Positive ion fast atom bombardment represents a useful alternative for analysis of major molecular species in natural glycerophosphatidylserine.

Actions of morphine and enkephalins on the internal anal sphincter of the cat: relevance for the physiological role of opiates.

The effects of Leu-enkephalin, Met-enkephalin and morphine on the electrical activity of the internal anal sphincter were studied in anesthetized spinalized cats and in vitro on sphincteric muscle strips. All the effects of enkephalins and morphine were antagonized by naloxone (2 mg/kg, i.v. in vivo and 10(-6)M in vitro). In vivo, the enkephalins (0.01 mg/kg i.v.) and morphine (2 mg/kg, i.v.) decreased the amplitude of the excitatory responses evoked in the sphincter by stimulation of the hypogastric nerves. Opiates presumably act on the sympathetic nerve endings by reducing the release of noradrenaline. In vitro, the enkephalins (10(-6)M) and morphine (10(-6)M) had a similar inhibitory effect, indicating that opiates act, at least partly, at intramural level. In vivo, the enkephalins and morphine produced an inhibition of the spontaneous electrical activity of the internal anal sphincter. This inhibition occurs also in vitro; it is thus due to a peripheral effect of opiates acting either directly on the sphincteric smooth muscle cells, or through the nervous structures controlling sphincteric motility. In addition, the distribution of nerves containing enkephalin-like immunoreactivity, using whole mount preparations of cat internal anal sphincter, indicates that this area is supplied with a dense Leu- and Met-enkephalinergic innervation. Met- and Leu-enkephalin-like immunoreactive axons were detected within the circular and longitudinal muscles.

A procedure for the preparation of GM3 ganglioside from GM1-lactone.

A simple procedure is described for preparing GM3 ganglioside, from a few milligrams to grams, from GM1-lactone (Sonnino et al., (1985) Glycoconjugate J 2: 343-54) [1]. The synthesis was carried out under the following optimal conditions: 30 mM GM1-lactone in 0.25 M H2SO4 in DMSO, 30 min, 70 degrees C, nitrogen atmosphere, strong stirring. The yield of GM3 was 55%. The procedure applied to milligram amounts of GD1b-dilactone gave GD3 ganglioside.

Brain content of glycosphingolipids after oral administration of monosialogangliosides GM1 and LIGA20 to rats.

Natural (GM1) and semisynthetic [113-Neu-5-AcGgOse4-2-D-erythro-1,3- dihydroxy-2-dichloroacetylamide-4-trans-octadecene (LIGA20)] glycosphingolipids, given parenterally, protect neurones against glutamate-induced death without producing the side effects typical of glutamate receptor antagonists. Chronic glutamate-related neurotoxicity (e.g., in recurring strokes in elderly hypertensive patients, and in Parkinson disease) could be prevented also by glycosphingolipids treatment, but this therapeutic intervention will require a protracted administration of orally active glycosphingolipids. Here we demonstrate that 3-6 h after oral administration of 68 mumol/kg of LIGA20 and GM1 to rats, the brain content of LIGA20 is 50-fold higher than that of GM1. The brain concentration of LIGA20 remains elevated for at least 12-24 h. Because the LIGA20 that reaches the brain is slowly metabolized, repeated oral administrations of this glycosphingolipid can yield to its accumulation in brain, and can yield various brain levels depending on the dose and frequency of drug administration. In contrast this is not possible with GM1, which given orally for 7 d, cannot accumulate in brain in pharmacologically significant concentrations.