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)

Nicotinamide deamidase (nicotinamide amidohydrolase, EC 3.5.1.19) has been demonstrated in the conditioned growth medium of the M1 clonal cell line of mouse C1300 neuroblastoma. The enzyme has been purified 1200-1500-fold by Sephadex G25, hydroxyapatite, DEAE-cellulose, Sephadex G200 and NAD-Sepharose column chromatographies. The purified protein was characterized by polyacrylamide gel electrophoresis under non-denaturing and denaturing conditions. The apparent molecular weight has been estimated to be 230,000, and the subunits had respective molecular weights of 65,000 and 50,000. Histidine was the only NH2-terminal amino acid found. The enzyme is a glycoprotein; mannose and N-acetyl-glucosamine have been identified. The effects of various ions on its activity have been investigated. The enzyme has a Km for nicotinamide in the order of 10(-6) M, a pH optimum of 7.2 and a pHi of 5.4. It is inhibited by heating and by sulfhydryl reagents. The existence of a nicotinamide deamidase with a high affinity for nicotinamide favors the operation of the Preiss-Handler pathway in M1 cells cultured in vitro. We found an induction of nicotinamide deamidase and a cellular increase of NAD with a higher nicotinamide supply and a repression of the released enzyme with supplying NAD in the nutrition medium of M1 cell cultures.
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PMID:Purification and characterization of a nicotinamide deamidase released into the growth medium of neuroblastoma in vitro. 737 17

We synthesized a fluorescent derivative of the tridecapeptide neurotensin (NT), with the aim of providing a new tool for the pharmacological characterization and anatomic localization of NT receptors in mammalian brain. Fluoresceinylated NT (N alpha-fluoresceinyl thiocarbamyl (FTC)-[Glu1]NT; fluo-NT) was synthesized using solid-phase methodology and purified to 99% homogeneity by preparative high-pressure liquid chromatography (HPLC). Analytical HPLC, acidic and carboxypeptidase Y hydrolysis, and fast atom bombardment-mass spectroscopy confirmed that the purified compound was selectively labeled on the [Glu1] terminus and that a single FTC moiety was coupled to each molecule of [Glu1]NT. Flow cytometric analysis of the binding of fluo-NT to SN17 septal neuroblastoma cells indicated that the fluorescent derivative bound neural NT receptors with an affinity comparable to that of monoiodinated NT([125I]-NT). Competition experiments on mouse brain membrane preparations showed fluo-NT to inhibit specific [125I]-NT binding with a coefficient of inhibition (KI) virtually identical to that of the native peptide (0.67 vs 0.55 nM). Conventional epifluorescence and confocal microscopic analysis of specific fluo-NT binding to sections of the rat midbrain revealed a topographic distribution of the bound fluorescent ligand similar to that previously observed with autoradiography using [125I]-NT. However, fluo-NT provided markedly higher cell resolution and enabled, in particular, the detection of hitherto unnoted intracytoplasmic receptor clusters. Binding of fluo-NT to live SN17 hybrid cells indicated that the fluorescent ligand had retained its ability to internalize in vivo and confirmed that this internalization process was both time- and temperature-dependent. In sum, the present study demonstrates that fluo-NT is applicable to both the pharmacological study of NT binding sites using flow cytometry and to the regional and cellular localization of these sites by conventional epifluorescence and confocal microscopy.
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PMID:Synthesis of a biologically active fluorescent probe for labeling neurotensin receptors. 818 37

Human neuroblastoma GOTO cell lines were established that stably express recombinant human lysosomal protective protein/cathepsin A (PPCA) cDNA by transfection. Intracellular cathepsin A (acid serine carboxypeptidase) activity increased four-fold compared with in those of the parent and mock-transfected cell lines. The immunoreactive 54 kDa precursor/zymogen and mature 32/20 kDa two-chain forms were produced in the cells. The amount of the latter form expressed in the GOTO cells was significantly larger than those in the PPCA-overexpressing CHO cell lines previously established. The intracellular proteins showed a typical lysosomal granular distribution and the glycosylated 54 kDa precursor was secreted into the culture medium without the addition of an alkalizing agent. The PPCA-overexpressing cell lines also retained the ability to differentiate bi-directionally as well as the parent cells; into neuronal cells on induction by dibutyryl cAMP in serum-free medium and into Schwannian cells on induction by bromodeoxyuridine. During the course of differentiation into neuronal and Schwannian cells, the intracellular cathepsin A activity further increased two and five times, respectively, which was associated with an increase in the expression of the 32/20 kDa two-chain form. The glycosylated precursor proteins were taken up via the mannose 6-phosphate receptors, and the cathepsin A, alpha-neuraminidase and beta-galactosidase (beta-Gal) activities deficient in the fibroblasts derived from a patient with PPCA deficiency (galactosialidosis) were restored. These results suggest that the bi-directional differentiation of GOTO cell lines stably expressing the recombinant human PPCA gene could be a model system for analyzing the functions of PPCA in peripheral neuronal cells and Schwannian cells as well as the recombinant PPCA could be a useful source for enzyme replacement therapy (ERT) for galactosialidosis patients.
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PMID:Expression of lysosomal protective protein/cathepsin A in a stably transformed human neuroblastoma cell line during bi-directional differentiation into neuronal and Schwannian cells. 1468 10