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)

Pooled lymphocytes collected from cancer patients were mixed with a biotinylated murine MAb specific to human IgG4. To this were added streptavidin-conjugated magnetic beads. After magnetically separating the bead-lymphocyte complex, the B cells were washed and fused with the WIL-2 derived human fusion partner, SHFP-1. Subsequently derived human-human hybridomas were screened for IgG4 immunoreactivity to target tumor cell lines. Several hybridomas reacted with a variety of malignant cell types, including melanoma, neuroblastoma, and pancreatic tumor cells. One hybridoma in particular, designated SC-GM4, recognized an antigen by Western blot with an apparent molecular weight of 57 kDa. This facile approach of magnetically separating selected populations of lymphocytes should be relatively simple to apply to other antigens and antibodies to preselect the type, class, and properties of the desired MAb.
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PMID:Isotype-directed enrichment of B cells by magnetic beads in the generation of immunoreactive human monoclonal antibodies. 888 Feb 12

We modified the method of somatic cell fusion for neurons to improve the efficiency of hybrid production. C1300 neuroblastoma cells were incubated with emetine and actinomycin D before fusion. After fusion between C1300 cells and adult mouse dorsal root ganglia neurons with polyethyleneglycol, we were able to select the hybrids from non-fused cells. We obtained hybrid clones at high efficiency (7.2 clones/10(4) neurons).
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PMID:Improved method for producing neuronal hybrids using emetine and actinomycin D. 889 46

Alzheimer's disease (AD) is associated with defects in mitochondrial function. Mitochondrial-based disturbances in calcium homeostasis, reactive oxygen species (ROS) generation, and amyloid metabolism have been implicated in the pathophysiology of sporadic AD. The cellular consequences of mitochondrial dysfunction, however, are not known. To examine these consequences, mitochondrially transformed cells (cybrids) were created from AD patients or disease-free controls. Mitochondria from platelets were fused to rho0 cells created by depleting the human neuroblastoma line SH-SY5Y of its mitochondrial DNA (mtDNA). AD cybrids demonstrated a 52% decrease in electron transport chain (ETC) complex IV activity but no difference in complex I activity compared with control cybrids or SH-SY5Y cells. This mitochondrial dysfunction suggests a transferable mtDNA defect associated with AD. ROS generation was elevated in the AD cybrids. AD cybrids also displayed an increased basal cytosolic calcium concentration and enhanced sensitivity to inositol-1,4, 5-triphosphate (InsP3)-mediated release. Furthermore, they recovered more slowly from an elevation in cytosolic calcium induced by the InsP3 agonist carbachol. Mitochondrial calcium buffering plays a major role after this type of perturbation. beta-amyloid (25-35) peptide delayed the initiation of calcium recovery to a carbachol challenge and slowed the recovery rate. Nerve growth factor reduced the carbachol-induced maximum and moderated the recovery kinetics. Succinate increased ETC activity and partially restored the AD cybrid recovery rate. These subtle alterations in calcium homeostasis and ROS generation might lead to increased susceptibility to cell death under circumstances not ordinarily toxic.
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PMID:Calcium homeostasis and reactive oxygen species production in cells transformed by mitochondria from individuals with sporadic Alzheimer's disease. 916 22

Two transcription initiation sites have been identified in the mouse mu-opioid receptor (MOR) gene at approximately -793 and -268 upstream of the translation start site. To test if the MOR gene contains two functional promoters, chimeric constructs of mouse MOR deletion fragments, fused to a luciferase reporter gene, were transiently transfected into the neuroblastoma cell line SK-N-SH, a MOR-expressing cell line, and two MOR-non-expressing cell lines. Results from transient transfection assays confirmed the existence of two functional independent promoters in the mouse MOR gene, and also revealed that the region from -1337 to -93 does not contain all the elements necessary to confer tissue-specific expression of the MOR gene.
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PMID:Transcription of the mouse mu-opioid receptor gene is regulated by two promoters. 937 9

Neuronal hybrid cells established by somatic cell fusion are useful for studies of neuronal properties at the molecular level (Hammond, D.N., Lee, H.J., Tonsgard, J.H. and Wainer, B.H., Development and characterization of clonal cell lines derived from septal cholinergic neurons, Brain Res., 512 (1990) 190-200; Wainwright, M.S., Perry, B.D., Won, L.A., O'Malley, K.L., Wang, W.Y., Ehrlich, M.E. and Heller, A., Immortalized murine strial neuronal cell lines expressing dopamine receptors and cholinergic properties, J. Neurosci., 15 (1995) 676-688). The somatic cell fusion method requires a fusion partner which is unable to survive in the selection medium if it does not fuse with primary cells to isolate the hybrid cells. Hypoxanthine guanine phosphoribosyltransferase (HPRT)-deficient partner cells and hypoxanthine, aminopterin and thymidine (HAT) selection medium are commonly used for this procedure (Harlow, E. and Lane, D. (Eds.), Antibodies: a Laboratory Manual, Cold Spring Harbor Laboratory Publications, New York, 1988, pp. 139-243). The present method requires neither HPRT-deficient cells nor HAT medium. Primary neurons are fused with the C1300 neuroblastoma cells pretreated with emetine (Grollman, A.P., Inhibitors of protein biosynthesis, J. Biol. Chem., 243 (1968) 4089-4094), an inhibitor of ribosomes and actinomycin D (Perry, R.P., Selective effects of actinomycin D on the intracellular distribution of RNA synthesis in tissue culture cells, Exp. Cell Res., 29 (1963) 400-406), an inhibitor of ribosomal RNA (rRNA) synthesis, before fusion. By this treatment, we are able to isolate hybrid cells after fusion because non-fused C1300 cells die due to the loss of active ribosomes and protein synthesis, whereas C1300 cells fusing with primary cells survive due to the supply of intact ribosomes and rRNA from primary cells. This method produces neuronal hybrids at high efficiency.
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PMID:Method for production of neuronal hybridoma using emetine and actinomycin D. 938 57

Protein kinase C (PKC) activation after treatment of human neuroblastoma SK-N-BE(2)C cells with phorbol 12-myristate 13-acetate (PMA) was found to enhance transcription of the human dopamine beta-hydroxylase (DBH) in those cells. To identify which cis-acting element is responsive to the PMA treatment during DBH gene expression, we employed transient transfection assays with serially deleted constructs of the human DBH gene's 5' upstream region fused to the chloramphenicol acetyltransferase (CAT) gene. Treatment of transfected cells with PMA resulted in an approximate threefold increase in CAT expression for all deletion constructs ranging from -978 bp to -262 bp, while the enhancement did not occur with a construct shortened to -114 bp. The region between -262 and -114 bp from the initiation site of transcription contains several cis-regulatory elements including a cyclic AMP response element (CRE) and putative AP1 and YY1 sequences. Site-directed mutagenesis of those cis-acting elements were performed to identify which of the elements mediated the PMA-induced transcriptional enhancement. Substitution of bases in the putative AP1 site containing in part a putative YY1 sequence did not effect the PMA inducibility. However, specific mutations in the CRE sequence abolished the PMA-inducible effect. Changing the CRE sequence into an authentic AP1 sequence (TGACGTCC --> TGACTCA) did not affect the PMA inducibility, suggesting that AP1 factors might interact with the new AP1 site upon PKC activation. A specific PKC inhibitor, GF109203X, completely inhibited the stimulatory effect of PMA on the expression of the human DBH gene. PMA induced an increase in the DBH mRNA level as detected by Northern blot analysis. Gel retardation showed that the binding of nuclear factors to CRE, putative YY1, and AP1 was sequence specific. Our data suggest that the enhancement of the human DBH gene expression by PMA treatment is mediated by the CRE motif in the 5' upstream region of the gene, and occurs via a PKC-dependent pathway.
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PMID:A protein kinase C-activating phorbol ester enhances transcription of the human DBH gene through a cyclic AMP response element in SK-N-BE(2)C cells. 942 17

The 121-kDa pore membrane protein (POM121) is a bitopic integral membrane protein specifically located in the pore membrane domain of the nuclear envelope with its short N-terminal tail exposed on the luminal side and its major C-terminal portion adjoining the nuclear pore complex. In order to locate a signal for targeting of POM121 to the nuclear pores, we overexpressed selected regions of POM121 alone or fused to the green fluorescent protein (GFP) in transiently transfected COS-1 cells or in a stably transfected neuroblastoma cell line. Microscopic analysis of the GFP fluorescence or immunostaining was used to determine the intracellular distribution of the overexpressed proteins. The endofluorescent GFP tag had no effect on the distribution of POM121, since the chimerical POM121-GFP fusion protein was correctly targeted to the nuclear pores of both COS-1 cells and neuroblastoma cells. Based on the differentiated intracellular sorting of the POM121 variants, we conclude that the first 128 amino acids of POM121 contains signals for targeting to the continuous endoplasmic reticulum/nuclear envelope membrane system but not specifically to the nuclear pores and that a specific nuclear pore targeting signal is located between amino acids 129 and 618 in the endoplasmically exposed portion of POM121.
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PMID:Intracellular distribution of an integral nuclear pore membrane protein fused to green fluorescent protein--localization of a targeting domain. 946 6

Prostaglandin E2 (PGE2) enhances transcription of the human dopamine beta-hydroxylase (DBH) gene in human neuroblastoma SK-N-BE(2)C cells. To identify a PGE2-responsive cis-acting element in the human DBH gene, serial deletion constructs of the human DBH 5'-upstream region fused to the chloramphenicol acetyltransferase (CAT) reporter gene were transiently transfected into SK-N-BE(2)C cells. Treatment of the transformed cells with PGE2 increased CAT expression two- to threefold in all constructs except where the promoter region was shortened beyond position -114 bp. There are several cis-regulatory elements in the region between -262 and -114 bp from the transcription initiation site that include a cyclic AMP response element (CRE) and a putative AP1 sequence. We presupposed that the CRE and AP1 might be candidates for PGE2 stimulation, and therefore, used site-directed mutagenesis to change the CRE and AP1 motives and test which of the two elements mediated the transcriptional enhancement. Only a specific mutation within the CRE sequence abolished the PGE2 effect. In addition, cotransfection with an expression vector expressing PKA inhibitor resulted in the specific blockage of the PGE2 effect on DBH gene expression. Northern blot analysis revealed that the increase in DBH gene transcription caused by PGE2 results in elevated DBH mRNA levels. Gel-retardation and competition assays confirmed that the binding of nuclear factors to the CRE site is sequence specific. Our data, therefore, indicate that PGE2 enhances the transcription of the human DBH gene. The effect is mediated by the CRE motif through activation of PKA.
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PMID:Stimulation of human DBH gene expression by prostaglandin E2 in human neuroblastoma SK-N-BE(2)C cells. 948 16

The mouse gene encoding ST8Sia IV/PST, one of two polysialic acid synthases, was isolated and characterized. The mST8Sia IV/PST gene was found to comprise over 60 kilobases and to be composed of five exons. Primer extension analysis revealed that transcription started from 333 nucleotides upstream of the translational initiation site. Transfection with nested deletion mutants of the 5'-flanking region fused to the luciferase reporter gene revealed that the promoter activity of the -107/+145 region was correlated with the gene expression of mST8Sia IV/PST in embryonal carcinoma P19 and neuroblastoma F11 cells. This proximal promoter region lacks an apparent TATA box but has putative binding sites for transcription factors Sp1 and NF-Y (CCAAT binding protein) at nucleotide positions -66/-57 and -47/-37, respectively. Individual deletions and mutations of the inverted Sp1 binding site or inverted NF-Y binding site caused significant reduction of the promoter activity, indicating that each binding site was involved in essential transcription control. Mobility shift assaying also revealed that Sp1 and NF-Y in a nuclear extract of P19 cells bind to the promoter region of the mST8Sia IV/PST gene. Deletion of the region from -60 to -40, which contains parts of both the Sp1 and NF-Y binding sites, completely abolished the promoter activity, suggesting that both Sp1 and NF-Y are synergetically involved in transcription regulation of the mST8Sia IV/PST gene in P19 and F11 cells. Although the overall structures of the two polysialic acid synthase genes (ST8Sia II/STX and IV/PST) are very similar, there is no extensive sequence homology between the 5'-flanking regions of the ST8Sia II/STX and IV/PST genes, suggesting that these two genes are expressed under different regulatory systems.
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PMID:Genomic structure and promoter activity of the mouse polysialic acid synthase (mST8Sia IV/PST) gene. 951 73

The expression of the neuropeptide galanin (GAL) is elevated in vivo upon nerve stimulation, injury, and in vitro by phorbol 12-myristate-13-acetate (PMA), suggesting that a signal pathway involving protein kinase C activation may be involved in GAL-gene activation. When plasmids containing a different length of the bovine GAL-promoter fused to luciferase were transfected into the human neuroblastoma cell line (SK-N-SH subclone SH-SY5Y), a PMA-responsive element was identified in the promoter-region -68 to -46 base pairs (bp). Co-transfection experiments with plasmids expressing cJun and cFos revealed that they could act alone, as well as synergistically with PMA to induce luciferase activity. Electrical mobility shift assays revealed that a cAMP response element (CRE)-like sequence (TGACGCGG; -59 to -52 bp) bound PMA-inducible nuclear proteins present in SH-SY5Y cells. These proteins appear to bind mainly as CRE-binding protein/activating-transcription-factor (CREB/ATF) and Jun/ATF heterodimers. In addition, an apparent PMA-inducible protein(s) not recognized by CREB/ATF and Jun antibodies bound to the CRE-like containing probe.
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PMID:Characterization of phorbolester-inducible human neuronal factors involved in trans-activation of the galanin gene. 960 91


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