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.     

A clarification of the effects of DCCD on the electron transfer and antimycin binding of the mitochondrialbc 1 complex

We have studied in detail the effects of dicyclohexylcarbodiimide (DCCD) on the redox activity of the mitochondrialbc ₁ complex, and on the binding of its most specific inhibitor antimycin. An inhibitory action of the reagent has been found only at high concentration of the diimide and/or at prolonged times of incubation. Under these conditions, DCCD also displaced antimycin from its specific binding site in thebc ₁ complex, but did not apparently change the antimycin sensitivity of the ubiquinol-cytochromec reductase activity. On the other hand, using lower DCCD concentrations and/or short times of incubation, i.e., conditions which usually lead to the specific inhibition of the proton-translocating activity of thebc ₁ complex, no inhibitory effect of DCCD could be detected in the ubiquinol-cytochromec reductase activity. However, a clear stimulation of the rate of cytochromeb reduction in parallel to an inhibition of cytochromeb oxidation has been found under these conditions. On the basis of the present work and of previous reports in the literature about the effects of DCCD on thebc ₁ complex, we propose a clarification of the various effects of the reagent depending on the experimental conditions employed.     

Occurrence of glycosylation and deglycosylation of exogenously administered ganglioside GM1 in mouse liver.

Ganglioside GM1, 3H-labelled at the level of terminal galactose or of sphingosine, was intravenously injected into Swiss albino mice and some steps in its metabolic fate in the liver were investigated. After administration of [3H]sphingosine-labelled GM1 all major liver gangliosides [GM3, GM2, GM1, GD1a-(NeuAc,NeuGl)] became radioactive, the radioactivity residing in all cases on the sphingosine moiety. The specific radioactivity was highest in GM1, which carried about 53% of the radioactivity incorporated into gangliosides, followed by GM2, with 34.5% of incorporated radioactivity, GM3 and GD1a-(NeuAc,NeuGl), both with about 5% of incorporated radioactivity. After administration of [3H]galactose-labelled GM1 the only radioactive gangliosides present in the liver were GM1 and GD1a-(NeuAc,NeuGl), the former carrying about 95% of the total ganglioside-incorporated radioactivity, the latter about 3%. Both gangliosides were radioactive exclusively in the terminal galactose residue. According to these results exogenously administered GM1, after being taken up by the liver, is mainly degraded to GM2 and GM3, a part being, however, sialylated to GD1a-(NeuAc,NeuGl). All this suggests that exogenous GM1 may be involved in the metabolic routes of endogenous liver gangliosides.     

Changes in the Ceramide Composition of Rat Forebrain Gangliosides with Age

Five major gangliosides (GM1, GD1a, GD1b, GT1b, and GQ1b) were extracted and isolated by normal-phase HPLC from the forebrain of Sprague-Dawley rats of ages ranging from 3 days to 24 months. Each ganglioside was fractionated by reverse-phase HPLC into the molecular species carrying a single long-chain base moiety. At all ages, the C18:1 and C20:1 long-chain base species predominated, whereas the C18:0 and C20:0 ones represented 1-3% of the total. The C18:1 long-chain base species, predominant at 3 days (91-96%), diminished with age and reached, at 2 years, 73%, 65%, 61%, 59%, and 45% of the total for GD1a, GM1, GT1b, GD1b, and GQ1b, respectively. The content of the C20:1 long-chain base species, low at birth (4-9%), increased with age in all gangliosides and reached, at 2 years, 27-55% of the total. The developmental behavior of the ganglioside species containing the C18:1 long-chain base was characterized by the following: (a) a biphasic profile with a maximum around 15 days for GD1a, the most abundant ganglioside at all ages; (b) an increase until 6 months for GM1; (c) a sharp decrease until 30 days, followed by leveling for GT1b; and (d) a low, constant level for GD1b and GQ1b. All the ganglioside species containing the C20:1 long-chain base showed a constant increase during development, the increase being more marked in the first 30 days.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of Asp-235-->Asn substitution on the absorption spectrum and hydrogen peroxide reactivity of cytochrome c peroxidase.

The spectroscopic properties of a mutant cytochrome c peroxidase, in which Asp-235 has been replaced by an asparagine residue, were examined in both nitrate and phosphate buffers between pH 4 and 10.5. The spin state of the enzyme is pH dependent, and four distinct spectroscopic species are observed in each buffer system: a predominantly high-spin Fe(III) species at pH 4, two distinct low-spin forms between pH 5 and 9, and the denatured enzyme above pH 9.3. The spectrum of the mutant enzyme at pH 4 is dependent upon specific ion effects. Increasing the pH above 5 converts the mutant enzyme to a predominantly low-spin hydroxy complex. Subsequent conversion to a second low-spin form is essentially complete at pH 7.5. The second low-spin form has the distal histidine, His-52, coordinated to the heme iron. To evaluate the effect of the changes in coordination state upon the reactivity of the enzyme, the reaction between hydrogen peroxide and the mutant enzyme was also examined as a function of pH. The reaction of CcP(MI,D235N) with peroxide is biphasic. At pH 6, the rapid phase of the reaction can be attributed to the bimolecular reaction between hydrogen peroxide and the hydroxy-ligated form of the mutant enzyme. Despite the hexacoordination of the heme iron in this form, the bimolecular rate constant is approximately 22% that of pentacoordinate wild-type yeast cytochrome c peroxidase. The bimolecular reaction of the mutant enzyme with peroxide exhibits the same pH dependence in nitrate-containing buffers that has been described for the wild-type enzyme, indicating a loss of reactivity with the protonation of a group with an apparent pKa of 5.4. This observation eliminates Asp-235 as the source for this heme-linked ionization and strengthens the hypothesis that the pKa of 5.4 is associated with His-52. The slower phase of the reaction between peroxide and the mutant enzyme saturates at high peroxide concentration and is attributed to conversion of unreactive to reactive forms of the enzyme. The fraction of enzyme which reacts via the slow phase is dependent upon both pH and specific ion effects.     

Some events of mitosis and cytokinesis in the generative cell of Ornithogalum virens L.

The organization of the microtubule (Mt) cytoskeleton during mitosis and cytokinesis of the generative cell (GC) in Ornithogalum virens L. (bicellular pollen type, chromosome number, n = 3) from prophase to telophase/sperm formation was investigated by localization of -tubulin immunofluorescence using a conventional fluorescence microscope and a confocal laser scanning microscope. Chromosomes were visualized with DNA-binding fluorochrome dyes (ethidium bromide and 46-diamino-2-phenyl-indole). The GC of O. virens is characterized by G2/M transition within the pollen grain and not in the pollen tube as occurs in the majority of species with bicellular pollen. It was found that prophase in the GC starts before anthesis and prometaphase takes place after 10 min of pollen germination. The prophase Mts are organized into three prominent bundles, located near the generative nucleus. The number of these Mt bundles is the same as the number of GC chromosomes, a relation which has not previously been considered in other species. The most evident feature in the prophase/ prometaphase transition of O. virens GC is a direct rapid rearrangement of Mt bundles into a network which appears to interact with kinetochores and form a typical prometaphase Mt organization. The metaphase chromosomes are arranged into a conventional equatorial plate, and not in tandem as is thought to be characteristic of GC metaphase. The metaphase spindle consists of kinetochore fibres and a few interzonal fibres which form dispersed poles. Anaphase is characterized by a significant elongation of the mitotic spindle concomitant with the extension of the distance between the opposite poles. At anaphase the diffuse poles converge. Cytokinesis is realized by cell plate formation in the equatorial plane of the GC. The phragmoplast Mts between two future sperm nuclei appear after Mts of the mitotic spindle have disappeared.Abbreviations DAPI 46-diamino-2-phenyl-indole - GC generative cell - GN generative nucleus - Mt microtubule This research was made possible in part due to TEMPUS Programme and Global Network for Cell and Molecular Biology UNESCO grants to Magorzata Bana. The experimental part of the work was done in Siena University. M. Banas is very grateful to Prof. Mauro Cresti and his group for scientific interest, offering the excellent laboratory facilities, and kind reception.     

Large DNA inversions,deletions, and TaqI site mutations in severe haemophilia A

Large DNA inversions caused by an intrachromosomal recombination between homologous regions located in intron 22 and 5 of the factor VIII gene have recently been identified in patients with severe haemophilia A. To evaluate better the prevalence of this large inversion and to estimate the overall sensitivity of the Southern blot/hybridization method we analysed the factor VIII gene of 49 unrelated patients with severe haemophilia A. All patients were screened for the inversion mutation, TaqI site mutations, and deletions. Mutations were identified in 31 (63%) patients, and comprised 24 large inversions, 4 partial deletions, and 3 point mutations. Three different haplotypes were characterised in the patients presenting the inversion mutation, confirming its independent origin. Two novel deletions are reported: a large one spanning from intron 14 to intron 22 and a deletion of 86 bp comprising the 3 region of exon 1 and 39–41 bp of intron 1. DNA sequencing of the deletion junction showed no significant homology between normal 5 and 3 sequences around the breakpoints. A novel missense mutation is also reported: CGAGGA, Arg-2209 to Gly. These results confirm that the inversion mutation is the most common cause of severe haemophilia A and indicate that the Southern blot/hybridization assay should be used as the first method for screening of mutations in severe haemophilia A.