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At least 577 records · Page 32Linked to original sources
PubMed1997

Acidic sphingomyelinase mediates entry of N. gonorrhoeae into nonphagocytic cells.

Invasion of human mucosal cells by N. gonorrhoeae via the binding to heparansulfate proteoglycan receptors is considered a crucial event of the infection. Using different human epithelial cells and primary fibroblasts, we show here an activation of the phosphatidylcholine-specific phospholipase C (PC-PLC) and acidic sphingomyelinase (ASM) by N. gonorrhoeae, resulting in the release of diacylglycerol and ceramide. Genetic and/or pharmacological blockade of ASM and PC-PLC cause inhibition of cellular invasion by N. gonorrhoeae. Complementation of ASM-deficient fibroblasts from Niemann-Pick disease patients restored N. gonorrhoeae-induced signaling and entry processes. The activation of PC-PLC and ASM, therefore, is an essential requirement for the entry of N. gonorrhoeae into distinct nonphagocytic human cell types including several epithelial cells and primary fibroblasts.

Bacterial Proteins↗
PubMed1999

The human homolog of Drosophila cornichon protein is differentially expressed in alloactivated T-cells.

To identify novel genes induced in the early stage of T-cell activation, mRNA expression in alloactivated human lymphocytes was examined. Differential display-reverse transcription PCR analysis revealed a 207-bp cDNA fragment which was upregulated 24 h after allostimulation of a human T-cell line. The corresponding complete 1396 bp cDNA, named TGAM77, encodes a predicted 134 amino acid protein which shares 63% homology with the cornichon (cni) protein of Drosophila melanogaster. Upregulation of TGAM77 mRNA in the early phase of T-cell activation was confirmed by Northern blot and RT-PCR analysis of activated human lymphocytes. TGAM77 mRNA is expressed in a variety of human tissues with various expression levels. In analogy to cni which is involved in an epidermal growth factor-like signaling pathway inducing cellular asymmetry in Drosophila oogenesis, TGAM77 might function in similar signaling establishing vectorial re-localization and concentration of signaling events in T-cell activation.

Amino Acid Sequence↗
PubMed1998

Expression of recombinant human acid sphingomyelinase in insect Sf21 cells: purification, processing and enzymatic characterization.

Biochemical and structural studies on human acid sphingomyelinase (haSMase) depend on the access to homogeneous biologically active enzyme. Due to the low abundance of native haSMase (n-haSMase) in human tissue, conventional purification strategies are not suitable for the isolation of preparative amounts of the enzyme. We describe a novel approach to the functional expression and purification of haSMase employing the baculovirus expression vector system. Infection of Spodoptera frugiperda 21 cells with recombinant baculovirus encoding haSMase leads to the expression of a glycosylated 75 kDa precursor protein, which is subsequently processed to an enzymatically active secreted 72 kDa haSMase. Variations in N-glycosylation and proteolytic maturation account for the difference in molecular mass between mature recombinant (72 kDa) and human placental haSMase (75 kDa). N-terminal amino acid sequencing of recombinant haSMase (r-haSMase) reveals a 23-residue N-terminal extension compared to the placental enzyme. The apparent K(m) and Vmax values for sphingomyelin degradation by r-haSMase in a micellar assay system are 32 microM and 0.56 mmol h-1 mg-1, respectively. In conclusion, the established baculovirus expression vector system provides an efficient tool for the expression and functional characterization of haSMase.

Animals↗
PubMedDate not supplied

Physiological relevance of sphingolipid activator proteins in cultured human fibroblasts.

The physiological degradation of several membrane-bound glycosphingolipids (GSLs) by water-soluble lysosomal exohydrolases requires the assistance of sphingolipid activator proteins (SAPs). Four of these SAPs are synthesized from a single precursor protein (prosaposin). Inherited deficiency of this precursor results in a rare disease in humans with an accumulation of ceramide (Cer) and glycolipids such as glucosylceramide and lactosylceramide (LacCer). In a previous study, we have shown that human SAP-D stimulates the lysosomal degradation of Cer in precursor deficient cells. In order to study the role of SAPs (or saposins) A-D in cellular GSL catabolism, we recently investigated the catabolism of exogenously added [(3)H]labeled ganglioside GM1, Forssman lipid, and endogenously [(14)C]labeled GSLs in SAP-precursor deficient human fibroblasts after the addition of recombinant SAP-A, -B, -C and -D. We found that activator protein deficient cells are still able to slowly degrade gangliosides GM1 and GM3, Forssman lipid and globotriaosylceramide to a significant extent, while LacCer catabolism critically depends on the presence of SAPs. The addition of either of the SAPs, SAP-A, SAP-B or SAP-C, resulted in an efficient hydrolysis of LacCer.

Base Sequence↗
PubMed2002

Transcriptional repression of the rat steroidogenic acute regulatory (StAR) protein gene by the AP-1 family member c-Fos.

PGF2alpha, working via protein kinase C, may inhibit transcription of the StAR gene through negative regulatory factors. Administration of PGF2alpha increased c-Fos mRNA with a corresponding reduction in StAR mRNA. A search of the rat StAR promoter revealed three putative AP-1 elements at bp positions -85, -187 and -1561, which demonstrated specific binding of c-Fos by mobility shift assays. Co-transfection of c-Fos with the p-1862 StAR promoter caused a reduction in luciferase activity in the presence or absence of cAMP. Mutation of all three AP-1 sites in the p-1862 StAR promoter abolished c-Fos repression. Mutation of the proximal AP-1 site in the p-1862 StAR promoter reduced SF-1 mediated induction. This study is the first to demonstrate that c-Fos represses StAR gene transcription and adds to the current knowledge on the complex relationship that exists between SF-1 and c-Fos in the regulation of StAR activity.

Animals↗
PubMed2003

Detection and identification of Vav1 protein in primary cultured murine cerebellar neurons and in neuroblastoma cells (SH-SY5Y and Neuro-2a).

Vav1 was detected in neuronal cells during a screening for 1-methylthiodihydroceramide (1-MSDH-Cer) binding proteins. 1-MSDH-Cer is a metabolically stable analogue of dihydroceramide that was reported to strongly interfere with the formation of ceramide and hence the biosynthesis of all sphingolipids in neuronal cells. To identify target proteins that function as putative mediators of this molecule, a 1-MSDH-Cer affinity chromatography was utilised. When the cytosolic fraction of human neuroblastoma SH-SY5Y cells was subjected to 1-MSDH-Cer affinity chromatography, the sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the eluted protein fraction revealed an about 2-fold enrichment of the 98 kD protein band. Tryptic digestion of the excised band in combination with MALDI mass spectrometry strongly suggested that this band contained Vav1 protein. This was surprising since Vav1 in contrast to the other two isoforms Vav2 and Vav3 is believed to be exclusively expressed in hematopoietic cells and has not been detected in neuronal cells until now. The expression of Vav1 was confirmed in human SH-SY5Y neuroblastoma cells and additionally in murine Neuro-2A neuroblastoma cells as well as in primary cultured murine cerebellar neurons by Western blot analysis and reverse transcription polymerase chain reaction.

Animals↗
PubMed2003

Characterization of two Turkish beta-hexosaminidase mutations causing Tay-Sachs disease.

Two homoallelic mutations have recently been identified in the alpha-subunit of hexosaminidase A (EC 3.2.1.52) causing the infantile form of Tay-Sachs disease in Turkish patients. Both of these mutations, a 12 bp deletion (1096-1107 or 1098-1108 or 1099-1109) in exon 10 and a point mutation (G1362 to A, Gly454 to Asp) in exon 12, are located in the catalytic domain of the hexosaminidase alpha-chain. In order to determine whether these mutations affect the function of the catalytic domain or result in an instable protein, both mutant cDNAs were overexpressed in COS-1 cells. As judged by Western blotting, transfections of wild-type cDNA produced pro-alpha-chain and mature alpha-chain in parallel with a fivefold increase in cellular hexosaminidase activity using the synthetic substrate 4-methylumbelliferyl beta-N-acetylglucosamine 6-sulfate (MUGS). However, both mutants produced only pro-alpha-chains, although no mature form or detectable hexosaminidase activity towards two different synthetic substrates was observed. These data are consistent with the biochemical phenotype of infantile Tay-Sachs disease. We conclude that the overexpressed mutant pro-alpha-chains were misfolded and could not undergo further proteolytic processing to the active form of the enzyme in the lysosome.

Animals↗
PubMed2003

A new point mutation (G412 to A) at the last nucleotide of exon 3 of hexosaminidase alpha-subunit gene affects splicing.

We report the sixth mutation associated with the infantile form of Tay-Sachs disease in the Turkish population. The mutation is a single nucleotide transition (G to A) at the last nucleotide of exon 3 of hexosaminidase A (HEX A) alpha-subunit gene. The 14 exons and their flanking sequences of the HEX A gene were amplified and analyzed by polymerase chain reaction-single stranded conformational polymorphism (PCR-SSCP). Sequencing of exon 3 showed a homozygous mutation. Cultured patient's fibroblasts produced no detectable mRNA for HEX A alpha-subunit gene by Northern blot analysis. We speculate that abnormal mRNA was rapidly degraded following transcription. Our data are consistent with the idea that the severe infantile form of Tay-Sachs disease is associated with a total lack of Hex A activity in the patient. A similar mutation (G to T) had been observed at the 5'-donor splice site of exon 3. It resulted in abnormal splicing and skipping of exon 3. The other acceptor and donor splice site mutations described in the HEX A gene ablate normal mRNA splicing. Identification of multiple mutant HEX A alleles shows molecular heterogeneity of infantile Tay-Sachs disease in our population.

Blotting, Northern↗
PubMed2000

Characterization of two alpha-galactosidase mutants (Q279E and R301Q) found in an atypical variant of Fabry disease.

The mutant products Q279E ((279)Gln to Glu) and R301Q ((301)Arg to Gln) of the X-chromosomal inherited alpha-galactosidase (EC 3.2.1. 22) gene, found in unrelated male patients with variant Fabry disease (late-onset cardiac form) were characterized. In contrast to patients with classic Fabry disease, who have no detectable alpha-galactosidase activity, atypical variants have residual enzyme activity. First, the properties of insect cell-derived recombinant enzymes were studied. The K(m) and V(max) values of Q279E, R301Q, and wild-type alpha-galactosidase toward an artificial substrate, 4-methylumbelliferyl-alpha-D-galactopyranoside, were almost the same. In order to mimic intralysosomal conditions, the degradation of the natural substrate, globotriaosylceramide, by the alpha-galactosidases was analyzed in a detergent-free-liposomal system, in the presence of sphingolipid activator protein B (SAP-B, saposin B). Kinetic analysis revealed that there was no difference in the degradative activity between the mutants and wild-type alpha-galactosidase activity toward the natural substrate. Then, immunotitration studies were carried out to determine the amounts of the mutant gene products naturally occurring in cells. Cultured lymphoblasts, L-57 (Q279E) and L-148 (R301Q), from patients with variant Fabry disease, and L-20 (wild-type) from a normal subject were used. The 50% precipitation doses were 7% (L-57) and 10% (L-148) of that for normal lymphoblast L-20, respectively. The residual alpha-galactosidase activity was 3 and 5% of the normal level in L-57 and L-148, respectively. The quantities of immuno cross-reacting materials roughly correlated with the residual alpha-galactosidase activities in lymphoblast cells from the patients. Compared to normal control cells, fibroblast cells from a patient with variant Fabry disease, Q279E mutation, secreted only small amounts of alpha-galactosidase activity even in the presence of 10 mM NH(4)Cl. It is concluded that Q279E and R301Q substitutions do not significantly affect the enzymatic activity, but the mutant protein levels are decreased presumably in the ER of the cells.

Ammonium Chloride↗