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At least 721 records · Page 40Linked to original sources
PubMed1993

Regional localization of the gene coding for the GM2 activator protein (GM2A) to chromosome 5q32-33 and confirmation of the assignment of GM2AP to chromosome 3.

The gene coding for the GM2 activator protein (GM2A) was previously mapped by us to chromosome 5 by an ELISA-based technique. Here we confirm this assignment using a PCR analysis of somatic cell hybrids and describe a regional localization to chromosome 5q32-33 by in situ hybridization. We also confirm the assignment of a pseudogene GM2AP to chromosome 3.

Animals↗
PubMed1982

Melittin stimulates incorporation and degradation of sphingomyelin in synaptosomal plasma membranes.

Melittin enhanced sphingomyelin (SPM) degradation by the neutral membrane-bound sphingomyelinase from calf brain synaptosomal plasma membranes (SYM) up to 20-fold. Melittin in concentrations as high as 100 microM did not significantly alter membrane fluidity of SYM as measured by fluorescence depolarization and electron spin resonance (ESR) using diphenylhexatriene and a doxyl derivative of SPM, respectively. In the concentration range 100--1000 microM, melittin was observed to rigidify SYM. The incorporation of SPM derivatives into the lipid bilayer of SYM was demonstrated by ESR measurements. Melittin enhanced the uptake of SMP-derivatives into SYM.

Animals↗
PubMed1989

Modulation of ganglioside biosynthesis in primary cultured neurons.

Murine cerebellar cells were pulse labeled with [14C]galactose, and the incorporation of radioactivity into gangliosides and neutral glycosphingolipids was examined under different experimental conditions. In the presence of drugs affecting intracellular membrane flow, as well as at 15 degrees C, labeled GlcCer was found to accumulate in the cells, whereas the labeling of higher glycosphingolipids and gangliosides was reduced. Monensin and modulators of the cytoskeleton effectively blocked biosynthesis of the complex gangliosides GM1, GD1a, GD1b, GT1b, and GQ1b, whereas incorporation of radioactivity into neutral glycosphingolipids, such as glucosylceramide and lactosylceramide, as well as GM3, GM2, and GD3 was either increased or unaltered. As monensin has been reported to interfere with the flow of molecules from the cis to the trans stacks of the Golgi apparatus, this result highlights at least one subcompartmentalization of ganglioside biosynthesis within the Golgi system. Inhibitors of energy metabolism affected, predominantly, the biosynthesis of the b-series gangliosides, whereas a reduced temperature (15 degrees C) more effectively blocked incorporation of radiolabel into the a-series gangliosides, a result suggesting the importance of GM3, as the principal branching point, for the regulation of ganglioside biosynthesis.

Animals↗
PubMed1992

Fractionation of primary cultured cerebellar neurons: distribution of sialyltransferases involved in ganglioside biosynthesis.

Primary cultured neurons were fractionated using sucrose density gradients. The activities of four sialyltransferases (GM3, GD3, GD1a, and GT1a synthase) involved in ganglioside biosynthesis were assayed in the collected fractions. The distribution of GM3 synthase coincided with that of mannosidase II, an enzyme assumed to be a cis-Golgi marker. Both enzymes were mainly associated with the more dense fraction. GD1a and GT1a synthase activities, on the other hand, were mainly recovered in the less dense fraction. Moreover, they were colocalized with thiamine pyrophosphatase, an enzyme assumed to be a marker of the late Golgi (trans-Golgi and trans-Golgi network). GD3 synthase activity was equally distributed between both fractions. These results are integrated in a model of ganglioside biosynthesis.

Animals↗
PubMed1998

Induction of apoptosis by synthetic ceramide analogues in the human keratinocyte cell line HaCaT.

In contrast to extracellular, long chain ceramides which comprise a structural component of the epidermal water barrier, intracellular ceramides originating from sphingomyelin hydrolysis have been shown to inhibit proliferation and to induce apoptosis in different cell populations. To further elucidate the possible role of intracellular ceramides in human epidermis, two new cell-permeable ceramide analogues, N-thioacetylsphingosine (C2-Cer=S) and 4-dodecanoylamino-decan-5-ol (FS-5), were synthesized and tested for their ability to suppress cell growth and to induce apoptosis in immortalized human keratinocytes. It was shown that the well-investigated ceramide analogue N-acetylsphingosine (C2-Cer=O), as well as the new compound C2-Cer=S inhibited proliferation of HaCaT cells with half-inhibitory concentrations (IC50) of 20 microg/ml and 10 microg/ml, respectively, whereas FS-5 has been potent with an IC50>40 microg/ml. Overall, all three ceramide analogues induced apoptosis in HaCaT cells as assessed by DNA-fragmentation using ELISA technique and in situ nick end labelling, thereby confirming the importance of ceramide signalling in keratinocytes.

Amides↗
PubMed2003

Metachromatic leukodystrophy: consequences of sulphatide accumulation.

UNLABELLED: Metachromatic leukodystrophy is a lysosomal lipid storage disorder. It is caused by mutations in the gene for arylsulphatase A, an enzyme involved in the degradation of the sphingolipid 3'-O-sulphogalactosylceramide (sulphatide). This membrane lipid can be found in various cell types, but in particularly high concentrations in the myelin of the nervous system. Patients suffer from progressive, finally lethal, demyelination due to accumulation of sulphatide. In the nervous system, lipid storage not only affects oligodendrocytes but also neurons and, in addition, leads to astrogliosis and activation of microglia. At the cellular level, lysosomal sulphatide storage also affects the lipid composition of myelin itself and has consequences for the amount and localization of particular myelin membrane-associated proteins. Here we review data, largely based on an arylsulphatase A knock-out mouse model of metachromatic leukodystrophy. CONCLUSION: The knock-out mouse model of metachromatic leukodystrophy has provided insights into the histopathological and cellular consequences of sulphatide storage.

Animals↗
PubMed2006

The enzyme-binding region of human GM2-activator protein.

The GM2-activator protein (GM2AP) is an essential cofactor for the lysosomal degradation of ganglioside GM2 by beta-hexosaminidase A (HexA). It mediates the interaction between the water-soluble exohydrolase and its membrane-embedded glycolipid substrate at the lipid-water interface. Functional deficiencies in this protein result in a fatal neurological storage disorder, the AB variant of GM2 gangliosidosis. In order to elucidate this cofactor's mode of action and identify the surface region of GM2AP responsible for binding to HexA, we designed several variant forms of this protein and evaluated the consequences of these mutations for lipid- and enzyme-binding properties using a variety of biophysical and functional studies. The point mutants D113K, M117V and E123K showed a drastically decreased capacity to stimulate HexA-catalysed GM2 degradation. However, surface plasmon resonance (SPR) spectroscopy showed that the binding of these variants to immobilized lipid bilayers and their ability to solubilize lipids from anionic vesicles were the same as for the wild-type protein. In addition, a fluorescence resonance energy transfer (FRET)-based assay system showed that these variants had the same capacity as wild-type GM2AP for intervesicular lipid transfer from donor to acceptor liposomes. The concentration-dependent effect of these variants on hydrolysis of the synthetic substrate 4-methylumbelliferyl-2-acetamido-2-deoxy-6-sulfo-beta-D-glucopyranoside (MUGS) indicated a weakened association with the enzyme's alpha subunit. This identifies the protein region affected by these mutations, the single short alpha helix of GM2AP, as the major determinant for the interaction with the enzyme. These results further confirm that the function of GM2AP is not restricted to a biological detergent that simply disrupts the membrane structure or lifts the substrate out of the lipid plane. In contrast, our data argue in favour of the critical importance of distinct activator-hexosaminidase interactions for GM2 degradation, and corroborate the view that the activator/lipid complex represents the true substrate for the degrading enzyme.

Animals↗
PubMed2005

Saposin C stimulates growth and invasion, activates p42/44 and SAPK/JNK signaling pathways of MAPK and upregulates uPA/uPAR expression in prostate cancer and stromal cells.

AIM: To determine the effect of saposin C (a known trophic domain of prosaposin) on proliferation, migration and invasion, as well as its effect on the expression of urokinase plasmonogen activator (uPA), its receptor (uPAR) and matrix metalloproteinases (MMP)-2 and -9 in normal and malignant prostate cells. In addition, we tested whether saposin C can activate p42/44 and stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) signal transduction pathways of the mitogen-activated protein kinase (MAPK) superfamily. METHODS: We employed Western blot analysis, phospho-specific antibodies, cell proliferation assay, reverse transcriptase-polymerase chain reaction, in vitro kinase assays and migration and invasion to determine the effect of saposin C on various biological behaviors of prostate stromal and cancer cells. RESULTS: Saposin C, in a cell type-specific manner, upregulates uPA/uPAR and immediate early gene c-Jun expression, stimulates cell proliferation, migration and invasion and activates p42/44 and SAPK/JNK MAPK pathways in prostate stromal and cancer cells. Normal prostate epithelial cells were not responsive to saposin C treatment in the above studies. CONCLUSION: Saposin C functions as a multipotential modulator of diverse biological activities in prostate cancer and stromal cells. These results strongly suggest that saposin C functions as a potent growth factor for prostatic cells and may contribute to prostate carcinogenesis and/or the development of hormone-refractory prostate cancer.

Cell Division↗
PubMed1998

Sphingolipid metabolism. Sphingoid analogs, sphingolipid activator proteins, and the pathology of the cell.

Sphingolipid metabolism and function was investigated using sphingoid analogs, cells from human sphingolipidoses patients, and knockout animals. Treatment of primary cultured murine cerebellar cells with the structurally modified sphingosine base cis-4 methylsphingosine resulted in decreased sphingolipid biosynthesis accompanied by significant morphological changes. Plasma-membrane-derived glycosphingolipids (GSLs) destined for digestion are internalized through the endocytic pathway and delivered to lysosomes. There, GSLs are degraded by the action of exohydrolases, which are supported, in the case of GSLs with short oligosaccharide chains, by sphingolipid activator proteins (SAPs or saposins). The inherited deficiency of activators give rise to sphingolipid storage diseases. The analysis of cultured fibroblasts from corresponding patients suggests a new model for the topology of endocytosis and lysosomal digestion. Mice with disrupted genes for activator proteins and for GM2 degrading hexosaminidases turned out to be useful models for human diseases.

Animals↗
PubMed1998

Recent advances in the biochemistry of sphingolipidoses.

Glycosphingolipids are ubiquitous membrane components of eukaryotic cells. They participate in various cell recognition events and can regulate enzymes and receptors within the plasma membrane. Sphingolipidoses are due to an impaired lysosomal digestion of these substances. Glycosphingolipids are degraded by the action of exohydrolases, which are supported, in the case of glycosphingolipids with short oligosaccharide chains, by sphingolipid activator proteins. Five sphingolipid activator proteins are known so far, the GM2-activator and the SAPs, SAP-A to D (also called saposins). Degradation of glycosphingolipids requires endocytic membrane flow of plasma membrane derived glycosphingolipids into the lysosomes. Recent research focused on the topology of this process and on the mechanism and physiological function of sphingolipid activator proteins. Limited knowledge is available about enzymology and topology of glycosphingolipid biosynthesis. Recently, intermediates of this metabolic pathway have been identified as novel signalling molecules. Inhibition of glycosphingolipid biosynthesis has been shown to be beneficial in the animal model of Tay-Sachs disease. Mice with disrupted genes for lysosomal hydrolases and activator proteins are useful models for known human diseases and are valuable tools for the study of glycosphingolipid metabolism, the pathogenesis of sphingolipidoses and novel therapeutic approaches.

Animals↗
PubMed1995

Muscle computed tomography features of motor neuron disease in late-onset GM2 gangliosidosis.

Late-onset GM2 gangliosidosis, a rare inherited neuronal storage disease, is characterized by a variety of clinical manifestations. The common clinical picture comprises neuromuscular, spinocerebellar, extrapyramidal, cognitive, and psychiatric abnormalities. Details of the extent of muscle involvement have never been reported. Eight patients with this syndrome were evaluated for the existence and extent of motor neuron disease using routine electrodiagnosis and systematic evaluation of skeletal musculature by computed tomography. Motor neuron disease was present in each and every patient regardless of the clinical manifestations and to a degree beyond that suspected on neurological examination. Muscle imaging disclosed a diffuse wasting and fatty replacement of muscles with predilection of pelvic and thigh muscles, and especially the quadriceps group. It seems that progressive motor disability in this syndrome is mainly due to motor neuron disease, as manifested by muscle atrophy, which can be easily demonstrated by muscle computed tomography.

Adipose Tissue↗