Attachment and multiplication,morphology and protein production of human fetal primary liver cells cultured in hormonally defined media

In Vitro Cellular &; Developmental Biology - Plant -     

Structure of a ganglioside with Cad blood group antigen activity

The Cad antigen is a rare erythrocyte blood group antigen expressed on both sialoglycoprotein and ganglioside structures. It is related both serologically and biochemically to the Sda blood group antigen expressed on over 90% of Caucasian erythrocytes. We reported previously that Cad erythrocytes contain a novel ganglioside that binds Helix pomatia lectin and inhibits human anti-Sda antibody. We have now purified the Cad ganglioside and determined its structure. The ganglioside contained Glc-Gal-GlcNAc-GalNAc-NeuAc in a molar ratio of 1.00:1.94:0.95:0.93:1.05. Its chromatographic mobility was between that of GM1 and GD3. After treatment with beta-hexosaminidase (human placenta Hex A), the product migrated with 2-3-sialosylparagloboside (IV3NeuAcnLc4OseCer), it no longer bound H. pomatia lectin, and it acquired the ability to bind an antibody to sialosylparagloboside. Treatment of this material with neuraminidase (Vibrio cholerae) yielded a product with the mobility of paragloboside (nLc4OseCer) that bound monoclonal antibody 1B2, which is specific for terminal N-acetyllactosaminyl structures. Treatment of the Cad ganglioside with Arthrobacter ureafaciens neuraminidase yielded a product reactive with monoclonal antibody 2D4, which is specific for terminal GalNAc beta (1-4)Gal structures. These data provide strong evidence that the Cad ganglioside structure is GalNAc beta (1-4)[NeuAc alpha (2-3)]Gal beta (1-3)Gal beta (1-4)GlcCer. 1H NMR analysis also supports the conclusion that the terminal GalNAc is linked beta (1-4) to Gal. High-performance thin-layer chromatographic ganglioside patterns from three blood group Cad individuals showed a direct correlation between the quantity of Cad ganglioside and the strength of Cad antigen expression on the erythrocytes, as measured by hemagglutination.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural analysis of glycosyl-phosphatidylinositol membrane anchor of the Toxoplasma gondii tachyzoite surface glycoprotein gp23

In this study we describe the biochemical features of the Toxoplasma gondii tachyzoite surface glycoprotein, gp23, demonstrating that it is attached to the parasite membrane by a glycosyl-phosphatidyl inositol anchor. Gp23 was metabolically labeled with tritiated palmitate, myristate, ethanolamine, inositol, glucosamine, mannose and galactose, as expected for a GPI-anchor structure. Gp23 was released from the surface of living parasites after treatment with phosphatidyl inositol-specific phospholipase C (PI-PLC) and the resulting water-soluble protein was immunoprecipitated with a monoclonal antibody specific for gp23. The GPIcore glycan was generated after aqueous-HF dephosphorylation followed by nitrous acid deamination and its carbohydrate structure was analyzed using selective exo- and endoglycosidase treatments. Finally, the phosphatidylinositol moiety of gp23 was characterized using PI-PLC and phospholipase A₂ (PLA₂) digestions. Our cumulative data suggest that gp23 of T gondii tachyzoites contains a modified GPI-backbone similar to the mammalian Thy-1 anchor, consisting of a conserved core structure (ethanolaminePO₄-6-Manαl-2-Manαl-6-Manαl-4-GIcNαl-6-PI) bearing β-linked N-acetylgalactosamine residue(s).     

Developmental changes of monohexosylceramide and free ceramide in the large intestine of the rat

Neutral glycosphingolipids were isolated from the colon of rats between birth and adulthood. The glycolipid concentration was stable during this period. Epithelial cells of the adult colon contained three times more glycolipids than the whole organ. The distribution pattern underwent only minor modifications during development. Free ceramide contributed for 23-27% of the total neutral sphingolipids at all ages. In 6-day-old rats, it was constituted of nonhydroxylated fatty acids linked to C18-sphingenine (57.3% of the bases), C18- and C20-4D-hydroxysphinganine (24.2 and 14.0% of the bases, respectively). This composition was essentially maintained during development. Glucosylceramide was the major glycolipid at all ages (40-50% of the total neutral sphingolipid content). At birth, 40% of its fatty acids were 2-hydroxylated and 93% of the bases were C18-4D-hydroxysphinganine. In adult epithelial cells, 75% of the fatty acids were 2-hydroxylated and C18- and C20-4D-hydroxysphinganine contributed for 66 and 25% of the bases, respectively. A transient increase of the contribution of nonhydroxylated fatty acids and C18-sphingenine was observed during the first week of life. C20-4D-hydroxysphinganine, which was characterized by gas-liquid chromatography of its aldehydes after periodate oxidation and of its N-acetyl O-trimethylsilyl derivatives, appeared after birth and reached 20% of the bases after two weeks. These findings are another example of the specificity of the lipidic part of glucosylceramide during the ontogenic differentiation.     

Glutamine Supplementation Alleviates Vasculopathy and Corrects Metabolic Profile in an In Vivo Model of Endothelial Cell Dysfunction

Endothelial Cell Dysfunction (ECD) is a recognized harbinger of a host of chronic cardiovascular diseases. Using a mouse model of ECD triggered by treatment with L-Nω-methylarginine (L-NMMA), we previously demonstrated that renal microvasculature displays a perturbed protein profile, including diminished expression of two key enzymes of the Krebs cycle associated with a Warburg-type suppression of mitochondrial metabolism. We hypothesized that supplementation with L-glutamine (GLN), that can enter the Krebs cycle downstream this enzymatic bottleneck, would normalize vascular function and alleviate mitochondrial dysfunction. To test this hypothesis, mice with chronic L-NMMA-induced ECD were co-treated with GLN at different concentrations for 2 months. Results confirmed that L-NMMA led to a defect in acetylcholine-induced relaxation of aortic rings that was dose-dependently prevented by GLN. In caveolin-1 transgenic mice characterized by eNOS inactivation, L-NMMA further impaired vasorelaxation which was partially rescued by GLN co-treatment. Pro-inflammatory profile induced by L-NMMA was blunted in mice co-treated with GLN. Using an LC/MS platform for metabolite profiling, we sought to identify metabolic perturbations associated with ECD and offset by GLN supplementation. 3453 plasma molecules could be detected with 100% frequency in mice from at least one treatment group. Among these, 37 were found to be differentially expressed in a 4-way comparison of control vs. LNMMA both with and without GLN. One of such molecules, hippuric acid, an “uremic toxin” was found to be elevated in our non-uremic mice receiving L-NMMA, but normalized by treatment with GLN. Ex vivo analysis of hippuric acid effects on vasomotion demonstrated that it significantly reduced acetylcholine-induced vasorelaxation of vascular rings. In conclusion, functional and metabolic profiling of animals with early ECD revealed macrovasculopathy and that supplementation GLN is capable of improving vascular function. Metabolomic analyses reveal elevation of hippuric acid, which may further exacerbate vasculopathy even before the development of uremia.     

Disruption of the mouse mTOR gene leads to early postimplantation lethality and prohibits embryonic stem cell development

The mammalian target of rapamycin (mTOR) is a key component of a signaling pathway which integrates inputs from nutrients and growth factors to regulate cell growth. Recent studies demonstrated that mice harboring an ethylnitrosourea-induced mutation in the gene encoding mTOR die at embryonic day 12.5 (E12.5). However, others have shown that the treatment of E4.5 blastocysts with rapamycin blocks trophoblast outgrowth, suggesting that the absence of mTOR should lead to embryonic lethality at an earlier stage. To resolve this discrepancy, we set out to disrupt the mTOR gene and analyze the outcome in both heterozygous and homozygous settings. Heterozygous mTOR (mTOR(+/-)) mice do not display any overt phenotype, although mouse embryonic fibroblasts derived from these mice show a 50% reduction in mTOR protein levels and phosphorylation of S6 kinase 1 T389, a site whose phosphorylation is directly mediated by mTOR. However, S6 phosphorylation, raptor levels, cell size, and cell cycle transit times are not diminished in these cells. In contrast to the situation in mTOR(+/-) mice, embryonic development of homozygous mTOR(-/-) mice appears to be arrested at E5.5; such embryos are severely runted and display an aberrant developmental phenotype. The ability of these embryos to implant corresponds to a limited level of trophoblast outgrowth in vitro, reflecting a maternal mRNA contribution, which has been shown to persist during preimplantation development. Moreover, mTOR(-/-) embryos display a lesion in inner cell mass proliferation, consistent with the inability to establish embryonic stem cells from mTOR(-/-) embryos.     

Stereospecific synthesis of "para-hydroxymexiletine" and sodium channel blocking activity evaluation

Both enantiomers of "para-hydroxymexiletine" (PHM), one of the main metabolites of mexiletine, were synthesized and fully characterized. Properties of (R)- and (S)-PHM, in terms of blocking potency and stereoselectivity on frog skeletal muscle Na(+) channels, were evaluated. The presence of a hydroxy group on the aryloxy moiety in the 4-position, as in PHM, reduced potency with respect to mexiletine in reducing I(Na max). However, PHM showed clear use-dependent behavior similar to that of mexiletine and, in contrast with what is observed with the parent compound, maintained its stereoselectivity during the use-dependent block. Chirality 16:72-78, 2004.