Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: UMLS:C0027819 (neuroblastoma)
27,800 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A common feature of scrapie and related transmissible spongiform encephalopathies is the accumulation of an abnormal protease-resistant form of PrP which may be the major component of the infectious agent. While it is known that both the normal (protease-sensitive) PrP and protease-resistant PrP are encoded by the same endogenous gene, the nature of the disease-associated modification of PrP is not understood. To study the cellular events leading to the formation of protease-resistant PrP, we have compared its biosynthesis to that of its normal isoform in scrapie-infected mouse neuroblastoma cells. In pulse-chase labeling experiments, the protease-resistant PrP was synthesized and degraded much more slowly than the normal PrP, suggesting that protease-resistant PrP is made from a protease-sensitive precursor. More significantly, we found that the precursor of protease-resistant PrP was eliminated from intact cells by treatments with phosphatidylinositol-specific phospholipase C and trypsin. This demonstrated that, unlike the protease-resistant PrP itself, the precursor is phospholipase- and protease-sensitive and at least transiently found on the cell surface. By these criteria, the precursor of protease-resistant PrP is indistinguishable from the normal PrP isoform. These results indicate that the conversion of PrP to the protease- and phospholipase-resistant state is a post-translational event that occurs after the precursor reaches the cell surface.
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PMID:The scrapie-associated form of PrP is made from a cell surface precursor that is both protease- and phospholipase-sensitive. 168 Aug 59

The cellular prion protein (PrPC) is a sialoglycoprotein anchored to the external surface of cells by a glycosyl phosphatidylinositol moiety. During scrapie, an abnormal PrP isoform designated PrPSc accumulates, and much evidence argues that it is a major and necessary component of the infectious prion. Based on the resistance of native PrPSc to proteolysis and to digestion with phosphatidylinositol-specific phospholipase C as well as the enhancement of PrPSc immunoreactivity after denaturation, we devised in situ immunoassays for the detection of PrPSc in cultured cells. Using these immunoassays, we identified the sites of PrPSc accumulation in scrapie-infected cultured cells. We also used these immunoassays to isolate PrPSc-producing clones from a new hamster brain cell line (HaB) and found an excellent correlation between their PrPSc content and prion infectivity titers. In scrapie-infected HaB cells as well as in scrapie-infected mouse neuroblastoma cells, most PrPSc was found to be intracellular and most localized with ligands of the Golgi marker wheat germ agglutinin. In one scrapie-infected HaB clone, PrPSc also localized extensively with MG-160, a protein resident of the medial-Golgi stack whereas this colocalization was not observed in another subclone of these cells. Whether the sites of intracellular accumulation of PrPSc are limited to a few subcellular organelles or they are highly variable remains to be determined. If the intracellular accumulation of PrPSc is found in the cells of the central nervous system, then it might be responsible for the neuronal dysfunction and degeneration which are cardinal features of prion diseases.
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PMID:Scrapie prion proteins accumulate in the cytoplasm of persistently infected cultured cells. 169 23

Previous studies have indicated that scrapie infection results in the accumulation of a proteinase K-resistant form of an endogenous brain protein generally referred to as prion protein (PrP). The molecular nature of the scrapie-associated modification of PrP accounting for proteinase K resistance is not known. As an approach to understanding the cellular events associated with the PrP modification in brain tissue, we sought to identify proteinase K-resistant PrP (PrP-res) in scrapie-infected neuroblastoma cells in vitro and to compare properties of PrP-res with those of its normal proteinase K-sensitive homolog, PrP-sen. PrP-res was detected by immunoblot in scrapie-infected but not uninfected neuroblastoma clones. Densitometry of immunoblots indicated that there was two- to threefold more PrP-res than PrP-sen in one infected clone. Metabolic labeling and membrane immunofluorescence experiments indicated that PrP-sen was located on the cell surface and could be removed from intact cells by phosphatidylinositol-specific phospholipase C and proteases. In contrast, PrP-res was not removed after reaction with these enzymes. Thus, either the scrapie-associated PrP-res was not on the cell surface or it was there in a form that is resistant to these hydrolytic enzymes. Attempts to detect intracellular PrP-res by immunofluorescent staining of fixed and permeabilized cells revealed that PrP was present in discrete perinuclear Golgi-like structures. However, the staining pattern was similar in both scrapie-infected and uninfected clones, and thus the intracellular staining may have represented only PrP-sen. Analysis of scrapie infectivity in cells treated with extracellular phospholipase, proteinase K, and trypsin indicated that, like PrP-res, the scrapie agent was not removed from the infected cells by any of these enzymes.
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PMID:Normal and scrapie-associated forms of prion protein differ in their sensitivities to phospholipase and proteases in intact neuroblastoma cells. 196 4

Both the cellular and scrapie isoforms of the prion protein (PrP) designated PrPc and PrPSc are encoded by a single-copy chromosomal gene and appear to be translated from the same 2.1-kb mRNA. PrPC can be distinguished from PrPSc by limited proteolysis under conditions where PrPC is hydrolyzed and PrPSc is resistant. We report here that PrPC can be released from the surface of both normal-control and scrapie-infected murine neuroblastoma (N2a) cells by phosphatidylinositol-specific phospholipase C (PIPLC) digestion and it can be selectively labeled with sulfo-NHS-biotin, a membrane impermeant reagent. In contrast, PrPSc was neither released by PIPLC nor labeled with sulfo-NHS-biotin. Pulse-chase experiments showed that [35S]methionine was incorporated almost immediately into PrPC while incorporation into PrPSc molecules was observed only during the chase period. While PrPC is synthesized and degraded relatively rapidly (t1/2 approximately 5 h), PrPSc is synthesized slowly (t1/2 approximately 15 h) and appears to accumulate. These results are consistent with several observations previously made on rodent brains where PrP mRNA and PrPC levels did not change throughout the course of scrapie infection, yet PrPSc accumulated to levels exceeding that of PrPC. Our kinetic studies demonstrate that PrPSc is derived from a protease-sensitive precursor and that the acquisition of proteinase K resistance results from a posttranslational event. Whether or not prolonged incubation periods, which are a cardinal feature of prion diseases, reflect the slow synthesis of PrPSc remains to be established.
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PMID:Scrapie and cellular prion proteins differ in their kinetics of synthesis and topology in cultured cells. 196 66

Numerous studies have indicated that a modified proteinase K-resistant form of an endogenous brain protein, prion protein (PrP), is associated with scrapie infection in animals. This scrapie-associated PrP modification appears to occur posttranslationally in brain, but its molecular nature is not known. To learn about the normal PrP biosynthesis and whether it is altered by scrapie infection in vitro, we did metabolic labeling experiments with uninfected and scrapie-infected mouse neuroblastoma tissue culture cells. Pulse-chase labeling experiments indicated that, in both cell types, two major PrP precursors of 28 and 33 kilodaltons (kDa) were processed to mature 30- and 35- to 41-kDa forms. Endoglycosidase H, tunicamycin, and phospholipase treatments revealed that the 28- and 33-kDa precursors resulted from the addition of high-mannose glycans to a 25-kDa polypeptide containing a phosphatidylinositol moiety and that maturation of the precursors involved the conversion of the high-mannose glycans to hybrid or complex glycans. Treatments of the live cells with trypsin and phosphatidylinositol-specific phospholipase C indicated that the mature PrP species were expressed solely on the cell surface, where they were anchored by covalent linkage to phosphatidylinositol. Once on the cell surface, the major PrP forms had half-lives of 3 to 6 h. No differences in PrP biosynthesis were observed between the scrapie-infected versus uninfected neuroblastoma cells.
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PMID:Prion protein biosynthesis in scrapie-infected and uninfected neuroblastoma cells. 256 14

The ability of beta-amyloid peptides to activate the classical complement cascade and the presence of various complement proteins including the membrane attack complex (C5b-9) on dystrophic neurites in Alzheimer's disease brains, raises the possibility that the complement system may contribute to this neurodegenerative disorder. To address this issue, we have studied the effect of complement activation on nerve growth factor (NGF)-differentiated rat pheochromocytoma PC12 cells, and on retinoic acid (RA)-differentiated human neuroblastoma SH-SY5Y cells. Although incubation of both cell types with human serum resulted in activation of complement, as indicated by iC3b formation, only PC12 but not SH-SY5Y cells were killed by human serum treatment. In contrast, heat-inactivated serum (56 degrees C, 45 min) was not neurotoxic. On SH-SY5Y cells, both PCR amplification and immunocytochemistry demonstrated the presence of CD59, a glycosylphosphatidylinositol-anchored protein that restricts homologous complement activation by inhibiting the formation of the membrane attack complex. The presence of CD59 probably accounts for the inability of human complement to lyse the human cell lines. Indeed, removal of glycosylphosphatidylinositol (GPI)-anchored proteins with phosphatidylinositol-specific phospholipase C (PI-PLC) rendered SH-SY5Y cells vulnerable to complement attack and eventually led to serum-medicated cell death. Reconstituted C5b-9 was also toxic to both PC12 and PI-PLC-pretreated SH-SY5Y cells. These observations suggest that complement activation can cause neuronal cell death and that this process is regulated by homologous restriction.
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PMID:Complement-mediated neurotoxicity is regulated by homologous restriction. 774 16

Information on the transmembrane signaling events and subsequent biochemical processes initiated by ciliary neurotrophic factor (CNTF) receptor activation in neurons is lacking. SH-SY5Y cells, a human neuroblastoma cell line expressing CNTF receptors, were used to study metabolic changes associated with functional ligand-receptor interactions. Real-time measurements quantifying the rate of extracellular acidification by SH-SY5Y cells (a measure of metabolic activity) were made using a silicon-based cytosensor. Application of recombinant human CNTF (rhCNTF) to resting SH-SY5Y cells increased their acidification rate in a concentration and time-dependent manner with an apparent EC50 of 60 ng/ml. Pretreatment of cells with phosphatidylinositol-specific phospholipase C (PI-PLC) prevented the CNTF, but not an NGF-stimulated increase in acidification rate. Collectively, these results demonstrate that: (1) SH-SY5Y cells express functional CNTF receptors; and (2) the initial signal transduction mechanism activated by the CNTF receptor in SH-SY5Y cells is distinct from that activated by the NGF receptor; however, both may ultimately stimulate the same downstream biochemical messengers to increase cellular metabolism.
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PMID:Recombinant human ciliary neurotrophic factor stimulates the metabolic activity of SH-SY5Y cells as measured by a cytosensor microphysiometer. 806 84

ChPrP is the chicken homologue of PrPC, the cellular isoform of the mammalian prion protein. We have used sequence-specific antibodies to immunoprecipitate and immunoblot chPrP derived from stably transfected cultures of neuroblastoma cells, as well as from chicken brain and cerebrospinal fluid. We have also used mass spectrometry to characterize fragments of the protein purified from conditioned medium. The majority of chPrP protein present in neuroblastoma cells and on isolated brain membranes can be released by incubation with phosphatidylinositol-specific phospholipase C, indicating that these molecules are attached to the cell surface by a glycosylphosphatidylinositol anchor. Surprisingly, most of the surface-anchored molecules are truncated at their N-terminus distal to the proline/glycine-rich repeats. The corresponding N-terminal fragments are found in medium conditioned by neuroblastoma cells, as well as in cerebrospinal fluid and a postmicrosomal supernatant of brain. One of these fragments extends from Lys25 to Phe116. 35-45-kDa forms of chPrP that can be metabolically labeled with [3H]ethanolamine can also be found in extracellular media. We propose that the chPrP molecule undergoes at least two cleavages as part of its normal metabolism: one within the glycosylphosphatidylinositol anchor and one within or just N-terminal to the central hydrophobic domain. The second cleavage lies within a region of 24 amino acids that is identical in chPrP and mammalian PrP, and represents a major processing event that may have physiological as well as pathological significance.
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PMID:Processing of a cellular prion protein: identification of N- and C-terminal cleavage sites. 809 41

High-molecular-mass alkaline phosphatase (H-Mr AP) was detected in sera from children with solid tumors without liver metastases. H-Mr AP activities were determined by a liquid chromatographic and an electrophoretic method. In 5 out of 10 cases with solid tumors--Ewing sarcoma (n = 2), neuroblastoma (n = 2), and rhabdoid tumor (n = 1)--H-Mr AP activities ranged from 3.1-40.4 U/L and 3.1-16% of total serum AP activity. In sera of patients with leukemia (n = 18) H-Mr AP was not detectable. After the treatment of the sera with papain and phosphatidylinositol-specific phospholipase C, which release membrane-associated AP from membrane particles, H-Mr AP was no longer detectable. These results indicate that H-Mr AP in the sera of patients with solid tumors may derive from increasing cell shedding of the tumor cells with elevated levels of membrane fragments in serum, which is a well known phenomenon in liver tumors. H-Mr AP was not more detectable in the serum after successful tumor treatment. These data suggest that H-Mr AP was produced by the tumors and that this parameter may be a serological marker for some solid tumors even in the presence of normal total AP serum activity.
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PMID:High-molecular-mass or macromolecular alkaline phosphatase in sera of children with solid tumors. 815 5

We report that upon muscarinic stimulation of SK-N-BE(2) human neuroblastoma cells, the extent of phosphoinositide-derived diacylglycerol (DG) conversion to phosphatidic acid (PA), operated by a DG kinase, is dependent on the potency of receptor stimulation and correlates with the reduction of phosphatidylinositol 4,5-bisphosphate mass. Evidence is provided that agonist-evoked Ca2+ mobilisation or protein kinase activation are not key events in triggering receptor-generated DG conversion to PA; furthermore, the phenomenon is compartmentalized, namely it occurs within a topologically restricted area that is poorly accessible to DG artificially generated by cell treatment with bacterial phosphatidylinositol-specific phospholipase C. Possible mechanisms driving regulation of the DG kinase operating in the transduction system investigated are discussed.
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PMID:Phosphoinositide-derived diacylglycerol conversion to phosphatidic acid is a receptor-dependent and compartmentalized phenomenon in human neuroblastoma. 897 96


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