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K Sandhoff

Publications and source records attributed to K Sandhoff.

At least 73 records · Page 4Linked to original sources
PubMed1997

Bcl-2 antagonizes apoptotic cell death induced by two new ceramide analogues.

Ceramides which arise in part from the breakdown of sphingomyelin comprise a class of antiproliferative lipids and have been implicated in the regulation of programmed cell death better known as apoptosis. In the present study, two new synthetic ceramide analogues, N-thioacetylsphingosine and FS-5, were used in Molt 4 cells to induce cell death. Besides their cytotoxic effects at concentrations > or = 14 microM the data obtained clearly show that both analogues induced apoptosis at concentrations below this critical concentration as assessed by trypan blue exclusion and cleavage of the death substrate poly-(ADP-ribose) polymerase (PARP). Additional experiments in bcl-2-transfected Molt 4 cells revealed that the apoptotic but not the lytic effects of the analogues were antagonized by the apoptosis inhibitor Bcl-2. Furthermore, neither N-thio-acetylsphingosine nor FS-5 induced PARP cleavage in bcl-2-transfected Molt 4 cells indicating that the induction of apoptotic cell death by cell permeable ceramides is not due to unspecific disturbance of the cell membrane.

Amides↗
PubMed1997

cis-4-Methylsphingosine decreases sphingolipid biosynthesis by specifically interfering with serine palmitoyltransferase activity in primary cultured neurons.

The effect of six different structurally modified sphingosine analogues on biosynthesis of sphingolipids was studied in primary cultured murine cerebellar neurons. Treatment of cells with cis-4-methylsphingosine at micromolar levels resulted in a markedly decreased sphingolipid biosynthesis, whereas the other compounds examined, trans-4-methylsphingosine, cis-5-methylsphingosine, trans-5-methylsphingosine, cis-sphingosine, and 1-deoxysphingosine, inhibited sphingolipid biosynthesis less efficiently. The inhibition of sphingolipid biosynthesis by the various compounds was paralleled by a decrease of serine palmitoyltransferase activity in situ. For cis-4-methylsphingosine the inhibitory effect on serine palmitoyltransferase activity was shown to be concentration- and time-dependent. Half-maximal reduction of enzyme activity occurred after 24 h of treatment with 10 microM of the compound. The activity of other enzymes of sphingolipid biosynthesis as well as phospholipid and protein biosynthesis was not affected. Analysis of the sphingoid moiety of cellular sphingolipids suggests that the sphingosine analogues listed above were subject to degradation rather than being utilized as precursors for sphingolipid biosynthesis by cultured neurons. Except of 1-deoxysphingosine, the other five sphingosine analogues were shown to be substrates for sphingosine kinase in vitro. After 24 h of treatment of primary cerebellar neurons with the various sphingosine analogues the relative percentage of the respective intracellular 1-phosphate derivatives paralleled exactly the inhibitory effect on serine palmitoyltransferase activity observed when cells were treated with the unphosphorylated compounds. In contrast to the respective 1-phosphate derivatives of the other methyl-branched sphingosine analogues examined, cis-4-methylsphingosine 1-phosphate showed an intracellular accumulation suggesting a delayed turnover rate in cultured murine neurons for this compound. These results suggest that the inhibitory effect of the sphingosine analogues on serine palmitoyltransferase is mediated by their respective 1-phosphate derivatives and that the pronounced effect of cis-4-methylsphingosine is caused by a high intracellular concentration of cis-4-methylsphingosine 1-phosphate. cis-4-Methylsphingosine, in addition, caused drastic changes in cell morphology of primary cerebellar neurons, which were not observed when these cells were treated with one of the other sphingosine analogues examined.

Acyltransferases↗
PubMed1997

Synthesis and mass spectrometric characterization of digoxigenin and biotin labeled ganglioside GM1 and their uptake by and metabolism in cultured cells.

Selective acylation of mono-deacetyl lyso-GM1, i.e. beta-D-galactopyranosyl-(1-->3)-2-acetamido-2-deoxy-beta-D-galactopyr ano syl -(1-->4)-(alpha-D-neuraminyl-(2-->3))-beta-D-galactopyranosyl- (1-->4)-beta-D-glucopyranosyl-(1-->1)-(2S,3R,4E)-2-amino-4-octa decen-1,3-diol, with N-succinimidyl-[1-14C]stearate afforded labeled mono-deacetyl GM1, i.e. beta-D-galactopyranosyl-(1-->3)-2-acetamido-2-deoxy-beta-D-galactopyr ano syl- (1-->4)-(alpha-D-neuraminyl-(2-->3)-beta-D-galactopyranosyl-(1-->4)-beta -D- glucopyranosyl-(1-->1)-(2S,3R,4E)-2-[1-14C]octadecanamido-4- octadecen-1, 3-diol, in good yield. Its condensation with either N-succinimidyl-digoxigenyl-3-O-methyl carbonyl-epsilon-amino caproate or N-succinimidyl-D-biotinyl-epsilon-aminocaproate led to radioactive GM1 derivatives carrying a tag for immuno-electron microscopy at the sialic acid residue. These GM1 derivatives could be hydrolyzed to the corresponding GM3 derivatives by treatment with GM1-beta-galactosidase and beta-hexosaminidases. There was no further degradation by sialidases due to the bulky tag in the sialic acid residue. The uptake of biotin labeled GM1 by human skin fibroblasts, rat neuroblastoma cells B104 and human neuroblastoma cells SHSY5Y was 0.85, 0.58 and 1.62 nmol lipid/mg cellular protein, respectively, after an incubation for 66 h at 37 degrees C and was similar to that of untagged GM1. The uptake of digoxigenin labeled GM1 by these cell types was, however, significantly higher (3.1, 6.8, and 20.0 nmol lipid/mg cellular protein, respectively). Both the biotin and digoxigenin labeled GM1 analogs were catabolized to the corresponding GM2 and GM3 derivatives in lysosomes of cultured cells. This demonstrates that these synthetic analogues are suitable for studying, by immuno-electron microscopy, their endocytosis and distribution in intralysosomal membranes.

Biotin↗
PubMed1997

Evidence for the involvement of Glu-355 in the catalytic action of human beta-hexosaminidase B.

In a previous study the photoactivable affinity probe, 3-azi-1-[([6-3H]2-acetamido-2-deoxy-1-beta-D-galactopyranosyl)thio ]-b utane, was used to identify the active site of beta-hexosaminidase B, a beta-subunit dimer (Liessem, B., Glombitza, G. J., Knoll, F., Lehmann, J., Kellermann, J., Lottspeich, F., and Sandhoff, K. (1995) J. Biol. Chem. 270, 23693-23699). The probe predominately labeled Glu-355, a highly conserved residue among hexosaminidases. To determine if Glu-355 has a role in catalysis, beta-subunit mutants were prepared with the Glu-355 codon altered to either Ala, Gln, Asp, or Trp. After expression of mutant proteins using recombinant baculovirus, the enzyme activity associated with the beta-subunits was found to be reduced to background levels. Although catalytic activity was lost, the mutations did not otherwise affect the folding or assembly of the subunits. The mutant beta-subunits could be isolated using substrate affinity chromatography, indicating they contained intact substrate binding sites. As shown by cross-linking with disuccinimidyl suberate, the mutant beta-subunits were properly assembled. They could also participate in the formation of functional beta-hexosaminidase A activity as indicated by activator-dependent GM2 ganglioside degradation activity produced by co-expression of the mutant beta-subunits with the alpha-subunit. Finally, the mutant subunits showed normal lysosomal processing in COS-1 cells, demonstrating that a transport-competent protein conformation had been attained. Collectively the results provide strong support for the intimate involvement of Glu-355 in beta-hexosaminidase B-mediated catalysis.

Affinity Labels↗
PubMed1997

Biosynthesis, processing, and intracellular transport of GM2 activator protein in human epidermal keratinocytes. The lysosomal targeting of the GM2 activator is independent of a mannose-6-phosphate signal.

The processing, intracellular transport, and endocytosis of the GM2 activator protein (GM2AP), an essential cofactor of beta-hexosaminidase A for the degradation of ganglioside GM2, was investigated in human epidermal keratinocytes. The GM2AP precursor is synthesized as an 18-kDa peptide, which is singly glycosylated, resulting in 22-kDa high mannose and 24-27-kDa complex glycoforms. A small portion of the 22-kDa form bears phosphomannosyl residues. About 30% of the GM2AP precursor is secreted during 12 h after synthesis, consisting almost exclusively of complex glycoforms. In a post-Golgi compartment, the intracellular remainder is converted to a 20-kDa mature form within 24 h, bearing a heavily trimmed N-glycan on a 17-kDa backbone. Interestingly, even nonglycosylated GM2AP is delivered to the lysosome, as shown by tunicamycin treatment and subcellular fractionation. Also, its endocytosis is independent of carbohydrate-linked signals and is even more effective for nonglycosylated GM2AP. We conclude that a mannose-6-phosphate-independent pathway for the lysosomal delivery of GM2AP exists in cultured human keratinocytes.

Biological Transport↗
PubMed1997

Functional characterization of the N-glycosylation sites of human acid sphingomyelinase by site-directed mutagenesis.

Most soluble lysosomal enzymes require a mannose-6-phosphate recognition marker present on asparagine-linked oligosaccharides for proper targeting to lysosomes. We have determined the influence of the six potential N-linked oligosaccharide chains of human acid sphingomyelinase (ASM) on catalytic activity, targeting, and processing of the enzyme. Each N-glycosylation site was modified by site-directed mutagenesis and subsequently expressed in COS-1 cells. Evidence is presented that five of these sites are used. Elimination of the four N-terminal glycosylation sites does not disturb lysosomal targeting, processing, or enzymatic activity. However, removal of the two C-terminal N-glycosylation sites inhibits the formation of mature enzyme. Absence of glycosylation site five resulted in rapid cleavage of the primary translation product to an enzymatically inactive protein which accumulated inside the endoplasmic reticulum/Golgi, whereas deletion of glycosylation site six led to the formation of an inactive ASM precursor, also retained inside the endoplasmic reticulum/Golgi. Our results also provide evidence that the site of early proteolytic cleavage of newly synthesized ASM must be located between the second and third glycosylation sites.

Animals↗
PubMed1997

Accumulation of sphingolipids in SAP-precursor (prosaposin)-deficient fibroblasts occurs as intralysosomal membrane structures and can be completely reversed by treatment with human SAP-precursor.

The degradation of glycosphingolipids takes place in lysosomes by action of specific exohydrolases, with the assistance of sphingolipid activator proteins (SAPs). Four of the SAPs, SAP-A to -D (also called saposins A to D), are synthesized from a single protein, the SAP-precursor (prosaposin). Deficiency in this precursor protein, a rare inherited disease in humans, results in the storage of sphingolipids with short oligosaccharide head groups within the patients' tissues, and electron microscopy revealed the accumulation of large multivesicular storage organelles. In this study we analyze the multivesicular storage organelles in cultivated fibroblasts from these patients. The results support our hypothesis that endocytosis of plasma membrane-derived lipids occurs via small intraendosomal and intralysosomal vesicles and membrane structures that are then digested within the lysosomes (Sandhoff, K., T. Kolter, Trends in Cell Biol. 6, 98-103 (1996). First, we show that the storage compartment consists of late endosomes and lysosomes by immunogold labeling for marker proteins of these organelles. The transport of endocytosed bovine serum albumin-colloidal gold or cationized ferritin into the compartment occurs with the timing expected for transport to late endocytic organelles. Second, complementation of the medium of the SAP-precursor-deficient fibroblasts with only nanomolar concentrations of purified SAP-precursor nearly completely reversed the aberrant accumulation of multivesicular structures, thereby abolishing most of the intralysosomal membrane structures. Analysis of the sphingolipid pattern of the cells after metabolic labeling with [14C]serine reveals that the cells' ability to degrade glycosphingolipids is completely restored by feeding of SAP-precursor at the same concentrations. This is the first demonstration in vivo that endocytosed SAP-precursor is processed into functional active SAPs A,- B,- C, and D and that the degradation of the vesicular structures within the lysosomes depends on the presence of the SAPs. Moreover, these studies suggest that a therapy program based on feeding purified SAP-precursor may be valuable in treating the entire family of diseases which result from the loss of one or more of the SAPs.

Endosomes↗
PubMed1997

Turnover of endogenous ceramide in cultured normal and Farber fibroblasts.

De novo synthesis and turnover of endogenous ceramide in cultured skin fibroblasts from patients affected with Farber lipogranulomatosis were studied by biosynthetical labeling of cellular sphingolipids with [14C]serine. The cellular uptake of [14C]serine and incorporation into de novo synthesized ceramide was similar in normal and Farber fibroblasts, with a half life of newly synthesized ceramide of 2.7 h in normal and diseased cells. Newly synthesized ceramide was found to be channeled directly into biosynthesis of complex sphingolipids rather than contributing to the pool of accumulated ceramide in Farber fibroblasts. The degradation of ceramide generated by the catabolism of complex sphingolipids in Farber cells was greatly delayed compared with control fibroblasts, with differences in the amount of radiolabeled cellular ceramide becoming evident after 6 h chase time. Individual Farber cell lines differed from each other in the amount of accumulated ceramide; however, no correlation was found between ceramide accumulation and residual acid ceramidase activity as determined in vitro. In addition, the amount of radiolabeled sphingomyelin was significantly increased in Farber fibroblasts suggesting a delayed degradation of this compound in this ceramide storage disorder. We propose biosynthetical labeling of endogenous ceramide with [14C]serine, in addition to other established methods, as a highly sensitive and reliable method for the diagnosis of Farber disease, allowing semiquantitative measurement of ceramide accumulation in cultured skin fibroblasts of patients affected with Farber lipogranulomatosis.

Acid Ceramidase↗
PubMed1996

Molecular cloning and characterization of a full-length complementary DNA encoding human acid ceramidase. Identification Of the first molecular lesion causing Farber disease.

Human acid ceramidase ((AC) N-acylsphingosine amidohydrolase, EC 3.5. 1.23) hydrolyzes the sphingolipid ceramide into sphingosine and free fatty acid. Ceramide is an essential component of all sphingolipids and an important cell-signaling molecule. Moreover, an inherited deficiency of AC activity leads to the lysosomal storage disorder known as Farber disease. Human AC was purified from urine, and 117 amino acid residues were determined by microsequencing. Degenerative oligonucleotide probes were then constructed and used to screen for human fibroblast and pituitary cDNA libraries. Several partial cDNA clones were obtained, and two of these were combined to construct a full-length cDNA containing a 17-base pair (bp) 5'-untranslated sequence, a 1185-bp open reading frame encoding 395 amino acids, a 1110-bp 3'-untranslated sequence, and an 18-bp poly(A) tail. Transient expression of the full-length cDNA in COS-1 cells led to a 10-fold increase in AC activity. In addition, biosynthetic studies carried out in the transfected cells demonstrated that 13-kDa (alpha) and 40-kDa (beta) AC subunits were derived from a common 55-kDa precursor encoded by the full-length cDNA. This protein pattern was identical to that seen in normal human skin fibroblasts. A homoallelic point mutation (T222K) was also identified in the AC gene of a patient suffering from Farber disease, further confirming the authenticity of the full-length cDNA.

Acid Ceramidase↗
PubMed1996

Purification of acid sphingomyelinase from human placenta: characterization and N-terminal sequence.

Human placental acid sphingomyelinase (ASM) was purified by sequential chromatography on Con A-Sepharose, octyl-Sepharose and Matrex gel red A. Final purification to apparent homogeneity was achieved by immunoaffinity chromatography employing polyclonal anti-ASM antibodies. The antibodies also allowed specific detection of ASM by Western blotting at various stages of purification. The ASM activity was enriched about 110,000-fold over that of the crude extract, yielding an enzyme preparation with a specific activity of about 1 mmol/h per mg protein in a detergent-containing assay system. Analysis of the final preparation by SDS-PAGE resulted in a single protein band with a molecular mass of approximately 75 kDa, which was reduced to approximately 60 kDa after complete deglycosylation. Microsequencing of the purified ASM revealed the N-terminal amino acid sequence of the mature placental enzyme.

Amino Acid Sequence↗
PubMed1996

Biosynthesis, processing, and targeting of sphingolipid activator protein (SAP )precursor in cultured human fibroblasts. Mannose 6-phosphate receptor-independent endocytosis of SAP precursor.

Sphingolipid activator proteins (SAPs) are essential cofactors for the lysosomal degradation of glycosphingolipids with short oligosaccharide chains by acidic exohydrolases. SAP-A, -B, -C, and -D derive from proteolysis of a 73-kDa glycoprotein, the SAP precursor. In the present publication, we studied the intracellular transport and the endocytosis of SAP precursor in human skin fibroblasts. Our data indicate that SAP precursor bears phosphate residues on noncomplex carbohydrate chains linked to the SAP-C and the SAP-D domain and sulfate residues on complex carbohydrate chains located within the SAP-A, -C, and possibly the SAP-D domain. Treatment of fibroblasts with either bafilomycin A1 or 3-methyladenine indicates that proteolytic cleavage of SAP precursor begins as early as in the late endosomes. To determine whether targeting of SAP precursor depends on mannose 6-phosphate residues, we analyzed the processing of SAP precursor in I-cell disease fibroblasts. In these cells nearly normal amounts of newly synthesized SAP-C were found, although secretion of SAP precursor was enhanced 2-3-fold. Moreover, SAP-C could be localized to lysosomal structures by indirect immunofluorescence in normal and in I-cell disease fibroblasts. Mannose 6-phosphate was not found to interfere significantly with endocytosis of SAP precursor. Normal fibroblasts internalized SAP precursor secreted from I-cells nearly as efficiently as the protein secreted from normal cells. To our surprise, deglycosylated SAP precursor was taken up by mannose 6-phosphate receptor double knock out mouse fibroblasts more efficiently than the glycosylated protein. We propose that intracellular targeting of SAP precursor to lysosomes is only partially dependent on mannose 6-phosphate residues, whereas its endocytosis occurs in a carbohydrate-independent manner.

Animals↗
PubMed1996

Only sphingolipid activator protein B (SAP-B or saposin B) stimulates the degradation of globotriaosylceramide by recombinant human lysosomal alpha-galactosidase in a detergent-free liposomal system.

The degradation of globotriaosylceramide (GbO-se3Cer) by insect-cell derived recombinant human alpha-galactosidase (EC 3.2.1.22) was carried out in a detergent-free liposomal system in order to mimic intralysosomal conditions. GbOse3Cer incorporated into unilamellar liposomes was used as the substrate, and naturally occurring sphingolipid activator proteins, rather than detergents, were used to stimulate the enzyme reaction. The degradation of GbOse3Cer was dependent on the presence of both alpha-galactosidase and sphingolipid activator protein B (SAP-B or saposin B). It proceeded optimally at pH 4.6, and was enhanced by increasing amounts of both alpha-galactosidase (0.24-24 mU/50 microliters assay) and SAP-B (0-5 micrograms/50 microliters assay). The enzyme reaction was not affected by SAP-A, SAP-C, or SAP-D. Therefore, our results indicate that only SAP-B is essential for the degradation of GbOse3Cer by alpha-galactosidase.

Animals↗
PubMed1996

Differential regulation of Src-family protein tyrosine kinases in GPI domains of T lymphocyte plasma membranes.

The association of glycosylphosphatidylinositol (GPI)-anchored cell surface glycoproteins with Src-family protein tyrosine kinases was analysed in intact T lymphocyte plasma membranes. Following subcellular fractionation without detergent, 25% of the recovered plasma membranes were light density vesicles enriched in GPI-anchored glycoproteins and sphingolipids (GPI domains), while the remainder behaved as heavier density vesicles containing equal amounts of lipids and proteins. Qualitatively similar lipids were found in both vesicle types, but only light density vesicles made of 65-75% lipids yielded a Triton X-100 resistant, sedimentable fraction containing GPI-linked glycoproteins and sphingolipids. The GPI-rich vesicles phosphotyrosylated an exogenous substrate as efficiently as the denser vesicles, despite a low Lck and Fyn kinase content. Likewise, these kinases were more efficiently phosphorylated in GPI domains than in denser vesicles. GPI domains thus could constitute plasma membrane "hot spots" where associated Src kinases assume an optimally active conformation that contributes to signaling via GPI-anchored cell surface glycoproteins.

Animals↗
PubMed1996

Identification of domains in human beta-hexosaminidase that determine substrate specificity.

The lysosomal beta-hexosaminidases are dimers composed of alpha and beta subunits. beta-Hexosaminidase A (alphabeta) is a heterodimer, whereas hexosaminidase B (betabeta) and S (alphaalpha) are homodimers. Although containing a high degree of amino acid identity, each subunit expresses a unique active site that can be distinguished by a differential ability to hydrolyze charged substrates. The site on the beta-subunit primarily degrades neutral substrates, whereas the alpha-subunit site is, in addition, active against sulfated substrates. Isozyme specificity is also exhibited with glycolipid substrates. Among human isozymes, only beta-hexosaminidase A together with the GM2 activator protein can degrade the natural substrate, GM2 ganglioside, at physiologically significant rates. To identify the domains of the human beta-hexosaminidase subunits that determine substrate specificity, we have generated chimeric subunits containing both alpha- and beta-subunit sequences. The chimeric constructs were expressed in HeLa cells to screen for activity and then selected constructs were produced in the baculovirus expression system to assess their ability to degrade GM2 ganglioside in the presence of GM2 activator protein. Generation of activity against the sulfated substrate required the substitution of two noncontinuous alpha-subunit sequences (amino acids 1-191 and 403-529) into analogous positions of the beta-subunit. Chimeric constructs containing only one of these regions linked to the beta-subunit sequence showed either neutral substrate activity only (amino acids 1-191) or lacked enzyme activity entirely (amino acids 403-529). Neither the chimeras nor the wild-type subunits displayed activator-dependent GM2-hydrolyzing activity when expressed alone. However, one chimeric subunit containing alpha amino acids 1-191 fused with beta amino acids 225 to 556, when co-expressed with the wild-type alpha-subunit, showed activity comparable with that of recombinant beta-hexosaminidase A formed by the co-expression of the alpha- and beta-subunits. This result indicates that the beta-subunit amino acids 225-556 contribute an essential function in the GM2-hydrolyzing activity of beta-hexosaminidase A.

Amino Acid Sequence↗
PubMed1996

Expression of the three alternative forms of the sphingolipid activator protein precursor in baby hamster kidney cells and functional assays in a cell culture system.

Sphingolipid activator proteins (SAPs) are non-enzymatic glycoproteins required for lysosomal degradation of various sphingolipids with short oligosaccharide chains by their respective exohydrolases. Four of these (SAP-A to SAP-D or saposins A to D) are derived from a common precursor by proteolytic processing. Alternative splicing of the SAP-precursor gene results in insertion of additional 6 or 9 bases of exon 8' or 8, respectively, into the SAP-B coding region of the transcribed mRNAs. To examine the features of the three different SAP-precursor proteins (prosaposins), the respective cDNAs were stably expressed in baby hamster kidney cells. Pulse-chase experiments with transfected cells and endocytosis studies on human fibroblasts showed that synthesis, transport, and maturation of all SAP-precursor led to formation of the four mature SAPs (SAP-A to SAP-D). In order to determine the biological function of the three different SAP-B isoforms, SAP-precursor-deficient human fibroblasts were loaded with recombinant SAP-precursor proteins with or without 2- and 3-amino acid insertions, respectively, purified from the medium of the baby hamster kidney cells. They were found to stimulate at nanomolar concentrations the turnover of biosynthetically labeled ceramide, glucosylceramide, and lactosylceramide. Since the physiological function of SAP-B is to stimulate the degradation of sulfatide by arylsulfatase A (EC 3.1.6.1) and globotriaosylceramide by beta-galactosidase (EC 3.2.1.23) loading studies with the respective exogenously labeled lipids on SAP-precursor-deficient fibroblasts were performed. Addition of different purified SAP-precursors to the medium of the lipid-loaded fibroblasts showed positive stimulation of the lipid degradation by all three SAP-B isoforms derived from the SAP-precursors. These findings establish that all three forms of the SAP-B can function as sulfatide/globotriaosylceramide activator.

Alternative Splicing↗
PubMed1996

The effect of fumonisin B1 on developing chick embryos: correlation between de novo sphingolipid biosynthesis and gross morphological changes.

Fumonisins, mycotoxins produced by Fusarium moniliforme and a number of other fungi, are potent inhibitors of the sphinganine-N-acyltransferase, a key enzyme of sphingolipid biosynthesis, and cause neuronal degeneration, liver and renal toxicity, cancer and other injury to animals. In this study we investigated the effect of fumonisin B1 on the sphingolipids of developing chick embryos. After yolk sac injection of fumonisin B1 a concentration and time dependent increase of the sphinganine-over-sphingosine ratio of the embryos could be demonstrated. Studies were done to evaluate the effect of fumonisin B1 on the glycophingolipid pattern of the chick embryos. In the presence of 72 micrograms fumonisin B1 per egg the incorporation of [14C]galactose and of [14C]serine into embryonic glycosphingolipids was reduced by about 70%, although the mass of glycosphingolipids was not affected by the toxin. However, a reduction of the wet weight of the treated embryos was observed. Additionally, histological examinations of whole embryo sections of control and fumonisin B1 treated embryos are presented. Fumonisin B1 caused haemorrhages under the skin as well as in the liver of treated embryos. A close correlation between disruption of sphingoid metabolism and light microscopic detectable tissue lesions could be observed.

Animals↗
PubMed1996

Oriented 1,2-dimyristoyl-sn-glycero-3-phosphorylcholine/ganglioside membranes: a Fourier transform infrared attenuated total reflection spectroscopic study. Band assignments; orientational, hydrational, and phase behavior; and effects of Ca2+ binding.

Fourier transform infrared (FTIR) attenuated total reflection (ATR) spectroscopy was used to elucidate the hydration behavior and molecular order of phospholipid/ganglioside bilayers. We examined dry and hydrated films of the gangliosides GM1, deacetyl-GM1, lyso-GM1, deacetyllyso-GM1, and GM3 and oriented mixed films of these gangliosides with 1,2-dimyristoyl-sn-glycero-3-phosphorylcholine (DMPC) using polarized light. Analysis of the amide I frequencies reveals that the amide groups are involved in intermolecular interactions via hydrogen bonds of varying strengths. The tilt angle of the acyl chains of the lipids in mixed films was determined as a function of ganglioside structure. Deacetylation of the sialic acid in the headgroup has a stronger influence on the tilt angle than the removal of the ganglioside fatty acid. The phase behavior was examined by FTIR ATR spectroscopy and by differential scanning calorimetry (DSC) measurements on lipid suspensions. At the same molar concentration, lyso-gangliosides have less effect on changes of transition temperature compared to the double-chain analogs. Distinct differences in the amide band shapes were observed between mixtures with lyso-gangliosides and normal double-chain gangliosides. Determined from the dicroic ratio RATR, the orientation of the COO- group in all DMPC/ganglioside mixtures was found to be relatively fixed with respect to the membrane normal. In 4:1 mixtures of DMPC with GM1 and deacetyl-GM1, the binding of Ca2+ leads to a slight decrease in chain tilt in the gel phase, probably caused by a dehydration of the membrane-water interface. In mixtures of DMPC with GM3 and deacetyl-lyso-GM1, a slight increase in chain tilt is observed. The chain tilt in DMPC/lyso-GM1 mixtures is unchanged. Analysis of the COO- band reveals that Ca2+ does not bind to the carboxylate group of the sialic acid of GM1 and deacetyl-GM1, the mixtures in which a decrease in chain tilt was observed. Binding to the sialic acid was only observed for mixtures of DMPC with GM3, lyso-GM1, and deacetyl-lyso-GM1. Ca2+ obviously accumulates at the bilayer-water interface and leads to partial dehydration of the headgroup region in the gel as well as in the liquid-crystalline phase. This can be concluded from the changes in the amide I band shapes. With the exception of DMPC/deacetyl-GM1, the effects on the ester C==O bands are small. The addition of Ca2+ has minor effects on the phase behavior, with the exception of the DMPC/GM1 mixture.

Animals↗