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)

We previously reported the inhibition of cell-growth in Neuro-2A cells, mouse neuroblastoma, by Zn2+ chelation with EDTA. This paper describes the purification of a factor that prevents EDTA-induced cell-growth inhibition from chick embryo brain. The purified factor has a molecular mass of 16 kDa on SDS-polyacrylamide gel electrophoresis under reducing conditions. This factor prevents the cell-growth inhibition in a dose-dependent manner and also binds thyroxine. Analysis of the N-terminal amino acid sequence revealed that 40 residues coincide with the sequence of chicken liver transthyretin.
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PMID:A factor that prevents EDTA-induced cell-growth inhibition: purification of transthyretin from chick embryo brain. 1050 89

Pituitary adenylate cyclase activating polypeptide type I receptor (PACAPr) belongs to the novel subfamily of the G-protein coupled receptors with a long extracellular N-terminus, which functions as a major binding site for the PACAP. Three different N-terminal fragments of rat PACAPr were overexpressed in Escherichia coli and purified using His-tags or maltose-binding protein as anchors for affinity chromatography. The purified and refolded proteins were used for the production and screening of monoclonal antibodies (MAbs) to PACAPr. Fifteen hybridoma cell lines producing MAbs specific to PACAPr were generated and characterized. Epitope analysis by competitive enzyme-linked immunoadsorbent assay (ELISA) indicated the presence of two groups of overlapping epitopes in the N-terminal fragment of PACAPr. Reactivity of MAbs with SDS-denaturated and native rat PACAPr was demonstrated by immunoblotting and flow cytometric analysis using transiently transfected COS cells and stably transfected CHO cells expressing rat PACAPr. Each antibody was examined by immunoblotting for the ability to cross react with the human PACAPr in human neuroblastoma NB-OK cells and most of them were shown to recognize human PACAPr as effectively as rat PACAPr. MAbs against the N-terminal extracellular domain of PACAPr can be used for the immunochemical study of the receptor-ligand interaction and for the investigation of PACAPr distribution in normal and tumor tissues.
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PMID:Production and characterization of monoclonal antibodies to pituitary adenylate cyclase activating polypeptide type I receptor. 1057 Dec 63

We have previously reported that 4-tert-butyl-[3-(2-chloroethyl)ureido] benzene (4-tBCEU), a potent cytotoxic agent, modulates the synthesis of tubulins, suggesting that its cytotoxicity may be mediated through an antimicrotubule mechanism. Indeed, 4-tBCEU and its 4-iso-propyl (4-isopropyl [3-(2-chloroethyl)ureido] benzene) and 4-sec-butyl (4-sec-butyl [3-(2-chloroethyl)ureido] benzene) homologues induced disruption of the cytoskeleton and arrest of the cell cycle in G2 transition and mitosis. To better understand the mechanisms responsible for microtubule disruption by 1-aryl-3-(2-chloroethyl)ureas (CEU), we first examined their cytotoxicity on Chinese hamster ovary cells resistant to vinblastine and colchicine due to the expression of mutated tubulins (CHO-VV 3-2). These cells showed resistance to CEU, e.g., 4-tBCEU having an IC50 of 21.3+/-1.1 microM as compared with an IC50 of 11.6+/-0.7 microM for wild-type cells, suggesting a direct effect of the drugs on tubulins. Western blot analysis confirmed the disruption of microtubules and evidenced the formation of an additional immunoreactive beta-tubulin with an apparent lower molecular weight on SDS polyacrylamide gel. Incubation of MDA-MB-231 cells with [urea-14C]-4-tBCEU revealed the presence of a radioactive protein that coincided with the additional beta-tubulin band, indicating that CEU could covalently bind to the beta-tubulin. The 4-tBCEU-binding site on beta-tubulin was identified by competition of the CEU with colchicine, vinblastine, and iodoacetamide, a specific alkylating agent of sulfhydryl groups of cysteine residues. Colchicine, but not vinblastine, prevented the formation of the additional beta-tubulin band, suggesting that 4-tBCEU alkylates either Cys239 or Cys354 residues near the colchicine-binding site. To determine the cysteine residue alkylated by 4-tBCEU, we incubated the radiolabeled drug with human neuroblastoma cells (SK-N-SH) that overexpress the betaIII-tubulin, an isoform where Cys239 is replaced by a serine residue. The results clearly showed that betaIII-tubulin is not alkylated by [urea-14C]-4-tBCEU, suggesting that cysteine 239 residue is essential for the reactivity of 4-tBCEU with beta-tubulin. Taken together, these findings indicate that the mechanism of cytotoxicity of CEU involves microtubule depolymerization through alkylation of beta-tubulin.
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PMID:Microtubule disruption induced in vivo by alkylation of beta-tubulin by 1-aryl-3-(2-chloroethyl)ureas, a novel class of soft alkylating agents. 1070 14

A mutant of Bacillus subtilis IMR-NK1, which is used for the production of domestic "natto" in Taiwan, produced high fibrinolytic enzyme activity by solid-state fermentation using wheat bran as medium. In addition, a strong fibrinolytic enzyme was purified from the cultivation media. The purified enzyme was almost homogeneous, as examined by SDS-PAGE and capillary electrophoresis. The enzyme had an optimal pH of 7.8, an optimal temperature of 55 degrees C, and a K(m) of 0.15% for fibrin hydrolysis. The molecular mass estimated by gel filtration was 31.5 kDa, and the isoelectric point estimated by isoelectric focusing electrophoresis was 8.3. The enzyme also showed activity for hydrolysis of fibrinogen, casein, and several synthetic substrates. Among the synthetic substrates, the most sensitive substrate was N-succinyl-Ala-Ala-Pro-Phe-pNA. PMSF and NBS almost completely inhibited the activity of the enzyme. These results indicate that the enzyme is a subtilisin-like serine protease, similar to nattokinase from Bacillus natto.
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PMID:Potent fibrinolytic enzyme from a mutant of Bacillus subtilis IMR-NK1. 1095 93

2,5-Hexanedione (2,5-HD), the neurotoxic metabolite of n-hexane, can structurally modify neurofilaments (NF) by pyrrole adduct formation and subsequent covalent cross-linking. 2,5-HD also induces accumulations of NF within the pre-terminal axon. We examined whether exposure of NF to 2,5-HD affected NF degradation. Two different models were used: (1) NF-enriched cytoskeletons isolated from human sciatic nerve were incubated with 2,5-HD in vitro and (2) differentiated human neuroblastoma cells (SK-N-SH) were exposed to 2, 5-HD in culture prior to isolation of cytoskeletal proteins. The cytoskeletal preparations were subsequently incubated with calpain II. The amount of NF-H and NF-L remaining after proteolysis was determined by SDS-PAGE and quantitative immunoblotting. NF-M proteolysis could not be quantified. Incubation of sciatic nerve cytoskeletal preparations with 2,5-HD resulted in cross-linking of all three NF proteins into high molecular weight (HMW) material with a range of molecular weights. Proteolysis of the NF-H and NF-L polypeptides was not affected by 2,5-HD-exposure. Degradation of the HMW material containing NF-H or NF-L was retarded when comparing with degradation of the NF-H and NF-L polypeptides, respectively, from control samples, but not as compared to the corresponding NF polypeptides from 2,5-HD-treated samples. Exposure of SK-N-SH cells to 2,5-HD also resulted in considerable cross-linking of NF. No differences were found between the proteolytic rates of NF-L and NF-H from exposed cells as compared with those subunits from control cells. Moreover, degradation of cross-linked NF-H was not different from monomeric NF-H. In conclusion, whether 2,5-HD affects calpain-mediated degradation of cross-linked NF proteins will depend on which model better reflects NF cross-linking as occurring in 2, 5-HD-induced axonopathy. However, with both models it was demonstrated that exposure of NF proteins to 2,5-HD without subsequent cross-linking is not adequate to inhibit NF proteolysis in vitro by added calpain.
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PMID:Effects of 2,5-hexanedione on calpain-mediated degradation of human neurofilaments in vitro. 1113 63

The nuclear envelope separates the nucleoplasm from the rest of the cell. Throughout the cell cycle, its structural integrity is controlled by reversible protein phosphorylation. Whereas its phosphorylation-dependent disassembly during mitosis is well characterized, little is known about phosphorylation events at this structure during interphase. The few characterized examples cover protein phosphorylation at serine and threonine residues, but not tyrosine phosphorylation at the nuclear envelope. Here, we demonstrate that tyrosine phosphorylation and dephosphorylation occur at the nuclear envelope of intact Neuro2a mouse neuroblastoma cells. Tyrosine kinase and phosphatase activities remain associated with purified nuclear envelopes. A similar pattern of tyrosine-phosphorylated nuclear envelope proteins suggests that the same tyrosine kinases act at the nuclear envelope of intact cells and at the purified nuclear envelope. We have also identified eight tyrosine-phosphorylated nuclear envelope proteins by 2D BAC/SDS/PAGE, immunoblotting with phosphotyrosine-specific antibodies, tryptic in-gel digestion, and MS analysis of tryptic peptides. These proteins are the lamina proteins lamin A, lamin B1, and lamin B2, the inner nuclear membrane protein LAP2beta, the heat shock protein hsc70, and the DNA/RNA-binding proteins PSF, hypothetical 16-kDa protein, and NonO, which copurify with the nuclear envelope.
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PMID:Identification of tyrosine-phosphorylated proteins associated with the nuclear envelope. 1116 78

The cause of Huntington's disease (HD) is a pathological expansion of the polyglutamine domain within the NH(2)-terminal region of huntingtin. Neuronal intranuclear inclusions and cytoplasmic aggregates composed of the mutant huntingtin within certain neuronal populations are a characteristic hallmark of HD. Because in vitro expanded polyglutamine repeats are glutaminyl-donor substrates of tissue transglutaminase (tTG), it has been hypothesized that tTG may contribute to the formation of these aggregates in HD. Therefore, it is of fundamental importance to establish whether tTG plays a significant role in the formation of mutant huntingtin aggregates in the cell. Human neuroblastoma SH-SY5Y cells were stably transfected with truncated NH(2)-terminal huntingtin constructs containing 18 (wild type) or 82 (mutant) glutamines. In the cells expressing the mutant truncated huntingtin construct, numerous SDS-resistant aggregates were present in the cytoplasm and nucleus. Even though numerous aggregates were present in the mutant huntingtin-expressing cells, tTG did not coprecipitate with mutant truncated huntingtin. Further, tTG was totally excluded from the aggregates, and significantly increasing tTG expression had no effect on the number of aggregates or their intracellular localization (cytoplasm or nucleus). When a YFP-tagged mutant truncated huntingtin construct was transiently transfected into cells that express no detectable tTG due to stable transfection with a tTG antisense construct, there was extensive aggregate formation. These findings clearly demonstrate that tTG is not required for aggregate formation, and does not facilitate the process of aggregate formation. Therefore, in HD, as well as in other polyglutamine diseases, tTG is unlikely to play a role in the formation of aggregates.
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PMID:Tissue transglutaminase does not contribute to the formation of mutant huntingtin aggregates. 1128 71

The vanilloid receptor VR1 is an ion channel predominantly expressed by primary sensory neurons involved in nociception. Here we describe its biochemical properties and assess the subcellular localization, the glycosylation state and the quaternary structure of VR1 expressed in HEK293 cells and in the DRG-derived cell line F-11 (N18TG2 mouse neuroblastoma x rat dorsal root ganglia, hybridoma). VR1 was found to be glycosylated in both cell types. Of the five potential N-glycosylation sites, the predicted transient receptor potential channel-like transmembrane folding proposes N604 is localized extracellularly. We used site-directed mutagenesis to mutate the Asn at position 604 to Thr. This mutated VR1 was not glycosylated, confirming the extracellular location of N604 and its role as the exclusive site of glycosylation of the VR1 protein. VR1 occured in high molecular mass complexes as assessed by blue native PAGE. In the presence of limited amounts of SDS dimers, trimers and tetramers of VR1 were observed, consistent with the predicted tetrameric quaternary structure of the receptor. Cross-linking with dimethyladipimidate yielded almost exclusively dimers. Whereas VR1 localized both to the plasma membrane and to intracellular membranes in HEK293 cells, it localized predominantly to the plasma membrane in F-11 cells. Using confocal laserscanning microscopy, we observed an enrichment of anti-VR1 immunoreactivity in neurite-like structures of F-11 cells. In the light of conflicting literature data on biochemical characteristics of VR1, our data suggest that dorsal root ganglion-derived F-11 cells provide a powerful experimental system for the study of VR1 biochemistry.
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PMID:Biochemical characterization of the vanilloid receptor 1 expressed in a dorsal root ganglia derived cell line. 1168 72

An alpha-2,8-sialyltransferase (ST8), the enzyme involved in the biosynthesis of polysialic acid chains, has been purified and partly characterized from undifferentiated human erythroleukemia K562 cells. Purification, based on a key step of affinity chromatography utilizing immobilized colominic acid, was greater than 1000-fold. The enzyme molecular weight determined by SDS-PAGE was estimated to be about 40 kDa, in good agreement with literature data. For the determination of the main kinetic parameters (Vmax and K(M)), fetuin turned out to be the unique substrate acceptor. In fact, other compounds such as asialofetuin, transferrin, alpha1-acid glycoprotein, and G(M3), routinely used to explore the different ST8 isoforms' activities, did not serve as substrate acceptors. In all cases, contrary to the routinely adopted protocol where a radioactive substrate donor is employed, for our purpose a non-radioactive, fluorescent substrate donor such as cytidine-5'-monophospho-9-(3-fluoresceinylthioureido)-9-deoxy-N-acetyl-neuraminic acid (CMP-9-fluoresceinyl-NeuAc) was used. Thus, under our experimental conditions, by using fetuin, data reported in a typical Lineweaver-Burk plot gave a Vmax value of about 4 nkatal/mg of protein and a K(M) value around 0.61 mM. Just as with the estimated molecular weight, these kinetic data were also in good agreement with those already reported for the ST8 purified from human neuroblastoma CHP-134 cells. In particular, in both cases, Vmax values were almost similar (4 nkatal/mg of protein for our ST8 purified from K562 cells and 4.35 nkatal/mg of protein for ST8 purified from CHP-134 cells); conversely, the K(M) value we found was about 3.25-fold lower than that found by Stoykova and Glick (0.61 mM vs. 2 mM). Then, although our purification was lower than that obtained by Stoykova and Glick (1080-fold vs. 2910-fold), the enzyme we purified showed a greater apparent affinity.
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PMID:Purification and partial characterization of an alpha-2,8-sialyltransferase from human erythroleukemia K562 cells. 1176

The absolute levels of cAMP-dependent protein kinase (cAMP-dPK) subunits (R-I, R-II and C) and cGMP-dependent protein kinase (cGMP-dPK) holoenzyme were studied in neuroblastoma-glioma hybrid cells before and after dibutyryl-cAMP (Bt2cAMP) treatment which results in differentiation of these cells. The levels were determined by two different techniques utilizing antibodies which had been raised against each individual purified protein kinase subunit (or the holoenzyme in the case of the cGMP-dPK). Electrophoretic transfer of samples from SDS-polyacrylamide gels to nitrocellulose paper, followed by immunolabeling of protein kinase subunits with their respective antibodies and [125I]Protein A, demonstrated the monospecific nature of the antibodies, and a selective, several-fold increase in the R-I subunit in Bt2cAMP-treated cells, with no change in the level of R-II or C subunits. A simple enzyme-linked immunosorbent assay (ELISA) capable of measuring nanogram amounts of the various subunits confirmed the selective increase in the R-I subunit. ELISA assay results also indicated that the R-I subunits present before and after Bt2cAMP treatment are antigenically homologous. In conclusion, the specific, sensitive immunological methods described here demonstrate the capacity of neuroblastoma-glioma hybrid cells to regulate separately the levels of the two distinct subunits (R-I and C) of the Type I cAMP-dPK.
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PMID:Dibutyryl cAMP treatment of neuroblastoma-glioma hybrid cells results in selective increase in cAMP-receptor protein (R-I) as measured by monospecific antibodies. 1189 19


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