Bleeding diathesis and gastro-duodenal ulcers in inherited cytosolic phospholipase-A2 alpha deficiency.

Arachidonic acid (AA), when cleaved from phospholipids by cytosolic phospholipase A2 alpha (cPLA2a), generates eicosanoids, with pro-hemostatic, pro-inflammatory, vasoactive and gastro-protective functions. We describe a patient (27-year-old man) and his twin-sister with early-onset bleeding diathesis and recurrent gastro-intestinal (GI) ulcers. Platelet aggregation/δ-granules secretion by collagen was impaired, but normal by AA; serum levels of thromboxane (Tx) B2 and 12-hydroxyeicosatetraenoic acid, and urinary levels of 11-dehydro-TxB2 were extremely low. Patients were homozygous for 1723G>C transition in PLA2G4A gene, which changed the codon for Asp575 to His. GI ulcers affected 5/14 heterozygous (< 40 years) and 1/16 wild-type homozygous (> 60 years) family members; none had bleeding diathesis. The proband, his sister and mother also had mildly reduced factor XI levels. Platelet messenger RNA expression did not differ among subjects with different PLA2G4A genotypes. Conversely, platelet cPLA2a was undetectable by Western Blotting in the proband and his sister, and decreased in 1723G>C heterozygous subjects, suggesting that the variant is transcribed, but not translated or translated into an unstable protein. We described a syndromic form of deficiency of cPLA2a , characterised by recurrent GI ulcers and bleeding diathesis, associated with mild inherited deficiency of factor XI. Unlike other reported patients with cPLA2a deficiency, these patients had extremely low levels of platelet TxA2 biosynthesis.

Protein kinase C modulates NMDA receptors in the myenteric plexus of the guinea pig ileum during in vitro ischemia and reperfusion.

BACKGROUND: Ischemic episodes lead to profound functional and structural alterations of the gastrointestinal tract which may contribute to disorders of intestinal motility. Enhancement of glutamate overflow and the consequent activation of NMDA (N-methyl-D-aspartate) receptors may participate to such changes by modulating different enteric neurotransmitter systems, including cholinergic motor pathways. METHODS: The molecular mechanism/s underlying activation of NMDA receptors in the guinea pig ileum were investigated after glucose/oxygen deprivation (in vitro ischemia) and during reperfusion. KEY RESULTS: The number of ileal myenteric neurons positive for NR1, the functional subunit of NMDA receptors, and its mRNA levels were unchanged after in vitro ischemia/reperfusion. In these conditions, the protein levels of NR1, and of its phosphorylated form by protein kinase C (PKC), significantly increased in myenteric neurons, whereas, the levels of NR1 phosphorylated by protein kinase A (PKA) did not change, with respect to control values. Spontaneous glutamate overflow increased during in vitro ischemia/reperfusion. In these conditions, the NMDA receptor antagonists, D(-)-2-amino-5-phosphonopentanoic acid [(D)-AP5] (10 µmol L(-1)) and 5,7-dichlorokynurenic acid (5,7-diClKyn acid) (10 µmol L(-1)) and the PKC antagonist, chelerythrine (1 µmol L(-1)), but not the PKA antagonist, H-89 (1 µmol L(-1)), were able to significantly depress the increased glutamate efflux. CONCLUSIONS & INFERENCES: The present data suggest that in the guinea pig ileum during in vitro ischemia/reperfusion, NR1 protein levels increase. Such event may rely upon posttranscriptional events involving NR1 phosphorylation by PKC. Increased NR1 levels may, at least in part, explain the ability of NMDA receptors to modulate a positive feedback on ischemia/reperfusion-induced glutamate overflow.

beta 1,3-Galactosyltransferase beta 3Gal-T5 acts on the GlcNAcbeta 1-->3Galbeta 1-->4GlcNAcbeta 1-->R sugar chains of carcinoembryonic antigen and other N-linked glycoproteins and is down-regulated in colon adenocarcinomas.

We attempted to determine whether beta1,3-galactosyltransferase beta3Gal-T5 is involved in the biosynthesis of a specific subset of type 1 chain carbohydrates and expressed in a cancer-associated manner. We transfected Chinese hamster ovary (CHO) cells expressing Fuc-TIII with beta3Gal-T cDNAs and studied the relevant glycoconjugates formed. beta3Gal-T5 directs synthesis of Lewis type 1 antigens in CHO cells more efficiently than beta3Gal-T1, whereas beta3Gal-T2, -T3, and -T4 are almost unable to direct synthesis. In the clone expressing Fuc-TIII and beta3Gal-T5 (CHO-FT-T5), sialyl-Lewis a synthesis is strongly inhibited by swainsonine but not by benzyl-alpha-GalNAc, and sialyl-Lewis x is absent, although it is detected in the clones expressing Fuc-TIII and beta3Gal-T1 (CHO-FT-T1) or Fuc-TIII and beta3Gal-T2 (CHO-FT-T2). Endo-beta-galactosidase treatment of N- glycans prepared from clone CHO-FT-T5 releases (+/-NeuAcalpha2-->3)Galbeta1-->3[Fucalpha1-->4]GlcNAcbeta1-->3Gal but not GlcNAcbeta1-->3Gal or type 2 chain oligosaccharides, which are found in CHO-FT-T1 cells. This result indicates that beta3Gal-T5 expression prevents poly-N-acetyllactosamine and sialyl-Lewis x synthesis on N-glycans. Kinetic studies confirm that beta3Gal-T5 prefers acceptors having the GlcNAcbeta1-->3Gal end, including lactotriosylceramide. Competitive reverse transcriptase mediated-polymerase chain reaction shows that the beta3Gal-T5 transcript is expressed in normal colon mucosa but not or poorly in adenocarcinomas. Moreover, recombinant carcinoembryonic antigen purified from a CHO clone expressing Fuc-TIII and beta3Gal-T5 reacts with anti-sialyl-Lewis a and carries type 1 chains on oligosaccharides released by endo-beta-galactosidase. We conclude that beta3Gal-T5 down-regulation plays a relevant role in determining the cancer-associated glycosylation pattern of N-glycans.

Differential expression of beta1,3galactosyltransferases in human colon cells derived from adenocarcinomas or normal mucosa.

Two beta1,3galactosyltransferases are detected in human colon cells: one corresponds to beta3GalT1, the other (beta3GalTx) is found to be different from any cloned beta3GalT since in vitro it utilizes GlcNAc very efficiently under specific reaction conditions. Expression of beta3GalT1 transcript is high in normal colon mucosa and control neuroectodermal cells, which do not express sialyl-Lewis a antigen, and low in colon adenocarcinoma cells, as assessed by competitive RT-PCR. beta3GalTx activity is high in adenocarcinoma cells expressing sialyl-Lewis a and undetectable in all other cells, suggesting differential involvement and opposite regulation of such enzymes during carcinogenesis.

Mouse C127 cells transfected with fucosyltransferase fuc-TIII express masked Lewisx but not Lewisx antigen.

To study human alpha1,3/1,4fucosyltransferase (Fuc-TIII) as an alpha1,3 fucosyltransferase, we constructed two cell clones, C127-FT and C127-T-FT, by transfecting cDNA in parental (C127) or Polyoma T antigen expressing (C127-T) mouse cells, respectively. Both C127-FT and C127-T-FT clones express high levels of a fucosyltransferase activity kinetically similar to Fuc-TIII and an RNA that is amplified by a Fuc-TIII-specific oligonucleotide primer pair after reverse transcription. Clone C127-FT is Lewisxpositive, by flow cytometry, only after alpha-galactosidase or sialidase treatment, and releases [3H]Fuc N-glycans which efficiently bind to immobilized Griffonia simplicifolia I and Sambucus nigra lectins. Immunoblotting confirms that C127-FT glycoproteins acquire Lewisxreactivity only after specific deglycosylation, and shows that a small subset of Griffonia simplicifolia I isolectin B4reactive glycoproteins bears masked Lewisx, suggesting fine substrate recognition by Fuc-TIII. Moreover, transient transfection of H type alpha1, 2fucosyltransferase in clone C127-T-FT directs synthesis of Lewisyantigen, as detected by flow cytometry. Results indicate that Fuc-TIII expressed in C127 cells synthesizes masked Lewisxantigen while Lewisxantigen is not detectable.

Beta-1,3-galactosyltransferase and alpha-1,2-fucosyltransferase involved in the biosynthesis of type-1-chain carbohydrate antigens in human colon adenocarcinoma cell lines.

We studied the beta-1,3-galactosyltransferase (GalT) and alpha-1,2-fucosyltansferase (FT) involved in the biosynthesis of type-1-chain carbohydrate antigens in human colon adenocarcinoma cell lines. We detected a GalT activity able to use GlcNAc as acceptor and found that lacto-N-biose I (Galbeta1-3GlcNAc) is the only reaction product. Such beta1,3GalT is kinetically similar to a pig trachea enzyme involved in mucin synthesis. The specific activity is high in cells that react strongly with anti-Lewis a and anti-Lewis b antibodies, and undetectable in a cell line that lacks antibody reaction. Reverse-transcriptase-mediated PCR analysis followed by DNA sequencing indicated that secretor-type alpha1,2FT is expressed in the cells, while the H type alpha1,2FT is not. The apparent Km values for donor and acceptor substrates determined for alpha1,2FT are similar to those of secretor-type alpha1,2FT and the specific activity measured correlates with Lewis b antigen expression on the cell surface. Moreover, some of the cell lines express Lewis y and H type 2 antigens, indicating that secretor type alpha1,2FT is responsible for their synthesis. Results suggest that biosynthesis of type-1-chain tumor-associated antigens in human colon carcinoma cells is operated by secretor-type alpha1,2FT, as reported in normal mucosa, and that beta1,3GalT activity may play a relevant role in its control.

Dictyostelium cytosolic fucosyltransferase synthesizes H type 1 trisaccharide in vitro.

A fucosyltransferase activity has been detected using lacto-N-biose I as acceptor in the lower eukaryote Dictyostelium discoideum. This transferase requires divalent cations and is inhibited by N-ethylmaleimide and detergent treatment. Apparent calculated Km values for GDP-Fuc and lacto-N-biose I are 1.27 microM and 2.80 mM, respectively. The activity is quantitatively recovered in the supernatant after centrifugation at 100000 x g for 1 h. The reaction product, as determined by gel permeation chromatography, sensitivity to fucosidases, and analysis of partially methylated derivatives, is Fucalpha1-2Galbeta1-3GlcNAc (H type 1 trisaccharide).

Molecular cloning, expression, chromosomal assignment, and tissue-specific expression of a murine alpha-(1,3)-fucosyltransferase locus corresponding to the human ELAM-1 ligand fucosyl transferase.

Terminal Fuc alpha 1-3GlcNAc moieties are displayed by mammalian cell surface glycoconjugates in a tissue-specific manner. These oligosaccharides participate in selectin-dependent leukocyte adhesion and have been implicated in adhesive events during murine embryogenesis. Other functions for these molecules remain to be defined, as do the tissue-specific expression patterns of the corresponding alpha-(1-3)-fucosyltransferase (alpha 1-3FT) genes. This report characterizes a murine alpha 1-3FT that shares 77% amino acid sequence identity with human ELAM ligand fucosyltransferase (ELFT, also termed Fuc-TIV). The corresponding gene maps to mouse chromosome 9 in a region of homology with the Fuc-TIV locus on human chromosome 11q. In vitro, the murine alpha 1-3FT can efficiently fucosylate the trisaccharide Gal alpha 1-3Gal beta 1-4GlcNAc (apparent Km of 0.71 mM) to form an unusual tetrasaccharide (Gal alpha 1-3Gal beta 1-4[Fuc alpha 1-3]GlcNAc) described in periimplantation mouse tissues. The enzyme can also form the Lewis x determinant from Gal beta 1-4GlcNAc (Km = 2.05 mM), and the sialyl Lewis x determinant from NeuNAc alpha 2-3Gal beta 1-4GlcNAc (Km = 1.78mM). However, it does not yield sialyl Lewis x determinants when expressed in a mammalian cell line that maintains sialyl Lewis x precursors. Transcripts from this gene accumulate to low levels in hematopoietic organs, but are unexpectedly abundant in epithelia that line the stomach, small intestine, colon, and epididymus. Epithelial cell-specific expression of this gene suggests function(s) in addition to, and distinct from, its proposed role in selectin ligand synthesis.

Topography of glycosyltransferases involved in the initial glycosylations of gangliosides.

We attempted to establish within which organelle UDP-Glc:ceramide beta 1----1'glucosyltransferase (GlcT) is located and moreover to obtain information about its orientation on intracellular membranes as well as that of UDP-Gal:glucosylceramide beta 1----4galactosyltransferase (GalT-2) and CMP-NeuAc:lactosylceramide alpha 2----3sialyltransferase (SAT-1). An extremely purified Golgi apparatus fraction was the only liver fraction where a ceramide-dependent formation of glucosylceramide could be demonstrated. This Golgi fraction, mainly constituted by stacks of intact cisternae which retained the same topographical orientation as in vivo, was then incubated with liposomal dispersions of glycosphingolipid-glycosyltransferase acceptors in reaction mixtures containing all the requirements for enzyme activity but no detergent. Under such conditions, SAT-1 and other late acting glycosyltransferases were over 90% latent, while both GlcT and GalT-2 were just as active as in the detergent-containing assay; they were still inhibited by EDTA. Sepharose-immobilized ceramide and Sepharose-immobilized glucosylceramide were found to be suitable acceptors for GlcT and GalT-2, respectively, still using intact Golgi cisternae as the enzyme source. Moreover, a part of GlcT and GalT-2 activity was released from intact Golgi cisternae upon cathepsin D treatment. These results provide strong evidence that GlcT and GalT-2 face the cytoplasmic side of the Golgi apparatus, whereas SAT-1 and the other late acting enzymes face the luminal side.

A part of glucosylceramide formed from exogenous lactosylceramide is not degraded to ceramide but re-cycled and glycosylated in the Golgi apparatus.

The subcellular fate of glucosylceramide (GlcCer) formed from exogenous lactosylceramide (LacCer) in rat liver is investigated. LacCer radiolabeled on different positions of the molecule was intravenously administered to rats as a liposomal dispersion. A Golgi apparatus fraction 140-fold enriched in specific markers and constituted by intact cisternal stacks, as well as the lysosomal and plasma membrane fractions concurrently prepared from the same homogenate, were then studied in order to determine the time course of radioactive glycosphingolipids. LacCer quickly decreased with time in the plasma membrane, whereas in the lysosomes it increased up to 4 h and decreased thereafter. In both fractions results were regardless of the labeling position. In the Golgi apparatus, LacCer increased up to 12 h and then decreased. In this fraction, the radioactivity values of [Glc-3H]LacCer were over twice those of [Gal-3H]LacCer. GlcCer was found only after [Glc-3H]LacCer administration. In the lysosomes, its time course provided a peak similar in shape but delayed in timing with respect to that of LacCer. Conversely, in the Golgi apparatus GlcCer was earlier formed, but earlier consumed, than LacCer. Gangliosides increased in the Golgi apparatus until 4 h and then decreased after 12 h, whereas in the plasma membrane they were progressively accumulated. In both fractions the amount of [Glc-3H]gangliosides was over twice that of [Gal-3H]gangliosides was over twice that of [Gal-3H]gangliosides. Since we demonstrated that the sugars released in the course of LacCer degradation (LacCer----galactose + GlcCer----glucose + ceramide) are not incorporated into glycoconjugates, we conclude that a part of GlcCer formed during the lysosomal degradation of LacCer actually reaches the Golgi apparatus where it undergoes successive glycosylation.

Localization in the Golgi apparatus of rat liver UDP-Gal:glucosylceramide beta 1----4galactosyltransferase.

The presence and subcellular localization of UDP-Gal:glucosylceramide beta 1----4galactosyltransferase (GalT-2) was investigated in rat liver. For this purpose, purified Golgi apparatus, endoplasmic reticulum, and plasma membrane fractions were prepared from the liver and used as the enzyme source for detecting GalT-2. A pure Golgi apparatus, highly enriched in many glycosyltransferases, was the only fraction where GalT-2 was measurable. The reaction product formation rate under appropriate assay conditions, which requires high detergent concentration and Mn2+, was low but comparable with that of other glycosyltransferases. The product formation was stimulated by exogenously added acceptor GlcCer, donor UDP-Gal, and Golgi protein. The reaction product was a single spot that was identified by chromatographic behavior, sensitivity to beta-galactosidase, and permethylation studies as Gal beta 1----4Glc beta 1----1'Cer (lactosylceramide). A metabolic experiment, performed by determining the glycosphingolipids which became radioactive in the above subcellular fractions prepared from the liver of animals treated with glucose-labeled glucosylceramide, further indicated that the in vivo glycosylation of glucosylceramide takes place in the Golgi apparatus.

Sub-Golgi distribution in rat liver of CMP-NeuAc GM3- and CMP-NeuAc:GT1b alpha 2----8sialyltransferases and comparison with the distribution of the other glycosyltransferase activities involved in ganglioside biosynthesis.

Using a sucrose density gradient fractionation of a highly purified Golgi apparatus from rat liver, we determined the sub-Golgi distribution of CMP-NeuAc:GM3 ganglioside alpha 2----8sialyltransferase (GM3-SAT) and CMP-NeuAc:GT1b ganglioside alpha 2----8sialyltransferase (GT1b-SAT), in comparison with that of the other glycosyltransferase activities involved in ganglioside biosynthesis. While GM3-SAT was recovered in several density fractions, GT1b-SAT was mainly found on less dense sub-Golgi membranes; this indicates that these two activities are physically separate. Moreover, with regard to the monosialo pathway, CMP-NeuAc:lactosylceramide alpha 2----3sialyltransferase, UDP-GalNAc:GM3 ganglioside beta 1----4N-acetylgalactosaminyltransferase, UDP-Gal:GM2 ganglioside beta 1----3galactosyltransferase, and CMP-NeuAc:GM1 ganglioside alpha 2----3sialyltransferase were resolved from more dense to less dense fractions, respectively. In the disialo pathway, UDP-GalNAc:GD3 ganglioside beta 1----4N-acetylgalactosaminyltransferase, UDP-Gal:GD2 ganglioside beta 1----3galactosyltransferase and CMP-NeuAc:GD1b ganglioside alpha 2----3sialyltransferase co-distributed with the corresponding activities of the monosialo pathway. These last results indicate that many Golgi glycosyltransferases involved in ganglioside biosynthesis are localized in the order in which they act.

Recycling of glucosylceramide and sphingosine for the biosynthesis of gangliosides and sphingomyelin in rat liver.

It was previously shown that sphingomyelin and gangliosides can be biosynthesized starting from sphingosine or sphingosine-containing fragments which originated in the course of GM1 ganglioside catabolism. In the present paper we investigated which fragments were specifically re-used for sphingomyelin and ganglioside biosynthesis in rat liver. At 30 h after intravenous injection of GM1 labelled at the level of the fatty acid ([stearoyl-14C]GM1) or of the sphingosine ([Sph-3H]) moiety, it was observed that radioactive sphingomyelin was formed almost exclusively after the sphingosine-labelled-GM1 administration. This permitted the recognition of sphingosine as the metabolite re-used for sphingomyelin biosynthesis. Conversely, gangliosides more complex than GM1 were similarly radiolabelled after the two treatments, thus ruling out sphingosine re-utilization for ganglioside biosynthesis. For the identification of the lipid fragment re-used for ganglioside biosynthesis, we administered to rats neutral glycosphingolipids (galactosylceramide, glucosylceramide and lactosylceramide) each radiolabelled in the sphingosine moiety or in the terminal sugar residue. Thereafter we compared the formation of radiolabelled gangliosides in the liver with respect to the species administered and the label location. After galactosylceramide was injected, no radiolabelled gangliosides were formed. After the administration of differently labelled glucosylceramide, radiolabelled gangliosides were formed, regardless of the position of the label. After lactosylceramide administration, the ganglioside fraction became more radioactive when the long-chain-base-labelled precursors were used. These results suggest that glucosylceramide, derived from glycosphingolipid and ganglioside catabolism, is recycled for ganglioside biosynthesis.

Precursor-product relationship between GM1 and GD1a biosynthesized from exogenous GM2 ganglioside in rat liver.

The demonstration of a precursor-product relationship in the course of GM1 and GD1a biosynthesis is described in the present paper. We injected rats with GM2 gangliosides [GalNAc beta 1----4(NeuAc alpha 2----3)Gal beta 1----4Glc beta 1----1'Cer] of brain origin, which were isotopically radiolabeled on the GalNAc ([GalNAc-3H]GM2) or sphingosine ([Sph-3H]GM2) residue. We then compared the time-courses of GM1 and GD1a biosynthesis in the liver after the administration of each radiolabeled GM2 derivative. After the administration of [GalNAc-3H]GM2, GM1, and GD1a were both present as doublets, that could be easily resolved on TLC. The lower spot of each doublet was identified as a species having the typical rat brain ceramide moiety and represented gangliosides formed through direct glycosylation of the injected GM2. The upper spot of each doublet was identified as a species having the typical rat liver ceramide moiety and represented gangliosides formed through recycling of the [3H]GalNAc residue, released during ganglioside catabolism. After the administration of [Sph-3H]GM2, only ganglioside with the rat brain ceramide moiety were found, that represented the sum of ganglioside formed through direct glycosylation and those formed through recycling of some sphingosine-containing fragments. In each case, the time-course of GM1 and GD1a biosynthesis exhibited a precursor-product relationship. The curve obtained from the direct glycosylation showed a timing delay with respect to those obtained from recycling of GM2 fragments. These results are consistent with the hypothesis that the sequential addition of activated sugars to a sphingolipid precursor is a dissociative process, catalyzed by physically independent enzymatic activities.

Subcellular biosynthesis and transport of gangliosides formed from exogenous lactosylceramide in rat liver.

In order to clarify the mechanisms of ganglioside biosynthesis and transport we intravenously administered a liposomal dispersion of radiolabelled lactosylceramide (LacCer) to rats and then followed the time course of the individual gangliosides which became radioactive in the Golgi-apparatus and plasma-membrane fractions prepared from the liver. After administration of radiolabelled LacCer the liver retained a substantial amount of radioactivity, which was distributed among an organic phase (mainly residual LacCer), a fraction containing low-Mr substances (mainly 3H2O) and a ganglioside fraction. The hepatocytes were found to provide the bulk of gangliosides biosynthesized from exogenous LacCer. After subcellular fractionation, the total radioactive gangliosides increased in the Golgi apparatus up to 8 h, to then decrease and practically disappear at 24 h; in the plasma membranes they were progressively concentrated, accounting for high absolute values. Ganglioside patterns were greatly modified with time in both the Golgi apparatus and plasma membrane, but without significant differences between them. Biosynthesis in the Golgi apparatus and accumulation in the plasma membrane of each individual ganglioside followed a precursor-product relationship. The obtained results indicated that once a ganglioside is biosynthesized in the Golgi apparatus, it is in part made available for translocation to the plasma membrane, which rapidly occurs, and is in part retained in the Golgi apparatus, where it acts as a precursor for the biosynthesis of more glycosylated gangliosides.

The N-acetylgalactosamine residue of exogenous GM2 ganglioside is recycled for glycoconjugate biosynthesis in rat liver.

A metabolic recycling of N-acetylgalactosamine (GalNAc), liberated from exogenous GM2 ganglioside [nomenclature of Svennerholm (1964) J. Lipid Res. 5, 145-155; IUPAC-IUB recommendations (1977) Lipids 12, 455-468], is demonstrated in rat liver. After the injection of a GM2 ganglioside isotopically radiolabelled on the terminal GalNAc residue ([GalNAc-3H]GM2), the liver retained a large amount of radioactivity distributed among: (1) a glycoprotein/glycosaminoglycan fraction, (2) a ganglioside fraction; and (3) a free-sugar fraction. Furthermore, volatile radioactivity was also found. The relative incorporation in the above fractions was time-dependent. The glycoprotein/glycosaminoglycan fraction contained radioactivity that was located on the GalNAc and GlcNAc residues. The ganglioside fraction was composed of two main families: gangliosides formed by a recycling of the liberated GalNAc, and gangliosides derived by direct utilization of the administered GM2. The free-sugar fraction contained mainly GalNAc. We suggest that GalNAc, after being released in the course of intra-lysosomal ganglioside catabolism, crosses the lysosomal membrane and passes into the cytosol, where the part not degraded is re-utilized for the biosynthesis of the different glycoconjugate classes.

Extensive precursor-product relationship between gangliosides formed from exogenous glucosylceramide in rat liver.

Glucosylceramide, radiolabelled on the glucose residue, was administered to rats and the radioactive gangliosides formed at different time points were chemically characterized. They were identified as GM3, GM1, GD1a and GD1b, each one carrying only radioactive glucose. The time course of each individual ganglioside showed that the simpler gangliosides were formed earlier but were consumed earlier than the more complex ones, resulting in radioactivity patterns that were different at each time point. Only 30 h after injection did it resemble that of endogenous rat liver gangliosides. These results indicate that an extensive precursor-product relationship actually exists in the course of ganglioside biosynthesis.

Inhibition of cholesterol biosynthesis in freshly isolated blood mononuclear cells from normolipidemic subjects and hypercholesterolemic patients treated with bezafibrate.

Bezafibrate was given for 15 days at a dose of 200 mg t.i.d. to 4 normolipidemic subjects, to 5 patients with putative heterozygous familial hypercholesterolemia, and to 6 patients with primary hypercholesterolemia of the non-familial type. At the end of the treatment, the rate of incorporation of labelled acetate into non-saponifiable lipids in freshly isolated blood mononuclear cells decreased in all subjects. On the average, acetate incorporation decreased by 31% in cells from normolipidemic subjects, 41% in cells from familial, and 45% in cells from non-familial hypercholesterolemia patients. Results of the present study suggest that the lowering effect of bezafibrate on serum cholesterol is mainly due to the inhibition of cholesterol synthesis through the suppression of HMG-CoA reductase as was demonstrated in rat hepatocytes and in cultured human blood mononuclear cells.

Two glycosphingolipid sialyltransferases are localized in different sub-Golgi compartments in rat liver.

A highly purified Golgi preparation from rat liver was fractionated on a sucrose density gradient and the activity of two sialyltransferases, CMP-NeuAc: Gal beta 1----4Glc-Cer (lactosylceramide) alpha-2----3sialyltransferase; Sat-1), and CMP-NeuAc:Gal beta 1----3GalNAc beta 1----4(NeuAc alpha 2----3) Gal beta 1----4Glc-Cer (GM1 ganglioside) alpha 2----3sialyltransferase; SAT-4), involved in the biosynthesis of gangliosides were assayed in the collected fractions. These two activities were recovered in different regions of the gradient; SAT-1 was found in a more dense region than SAT-4. This distribution coincided with that of two N-Asn linked oligosaccharide processing enzymes (UDP-GlcNAc:lysosomal enzyme precursor GlcNAc-1-phosphotransferase and UDP-Gal:ovalbumin galactosyltransferase), assumed as putative markers of cis- and trans-Golgi cisternae, respectively. These findings are consistent with the assembly of ganglioside oligosaccharide chains occurring in different sub-Golgi compartments.

Uptake, cell penetration and metabolic processing of exogenously administered GM1 ganglioside in rat brain.

GM1 ganglioside, after intravenous injection into rats, is absorbed and taken up by various organs and tissues, including brain. The capacity of brain to take up gangliosides, referred to weight unit, is comparable to that of kidney and muscle. After injection of [Gal-(3)H]GM1 a relevant portion of brain associated radioactivity resided in the soluble fraction and was of a volatile nature. After brain subcellular fractionation, the lysosomal, plasma membrane and Golgi apparatus fractions carried the highest specific radioactivity. In addition, an enriched fraction of brain capillaries was highly labelled, suggesting that GM1 ganglioside is also tightly bound to the vessel walls. The metabolic events encountered in brain by exogenous gangliosides were investigated, in detail, after intracisternal injection of [Sph-(3)H]GM1. The results obtained demonstrate that GM1 is extensively metabolized in brain. Besides the degradation products (GM2, GM3, lactosylceramide, glucosylceramide, ceramide), compounds of a biosynthetic origin were also found to be formed: these include GD1a, GD1b and sphingomyelin. All the above results could indicate that gangliosides, after intravenous administration to rats, are taken up by brain, bind to the capillary network, penetrate into neural cells, associate to both plasma membranes and intracellular structures and undergo metabolic processing with formation of a number of products of both catabolic and biosynthetic origin.