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: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have previously shown that ZNF74, a candidate gene for DiGeorge syndrome, encodes a developmentally expressed zinc finger gene of the Kruppel-associated box (KRAB) multifinger subfamily. Using RACE, RT-PCR, and primer extension on human fetal brain and heart mRNAs, we here demonstrate the existence of six mRNA variants resulting from alternative promoter usage and splicing. These transcripts encode four protein isoforms differing at their N terminus by the composition of their KRAB motif. One isoform, ZNF74-I, which corresponds to the originally cloned cDNA, was found to be encoded by two additional mRNA variants. This isoform, which contains a KRAB motif lacking the N terminus of the KRAB A box, was devoid of transcriptional activity. In contrast, ZNF74-II, a newly identified form of the protein that is encoded by a single transcript and contains an intact KRAB domain with full A and B boxes, showed strong repressor activity. Deconvolution immunofluorescence microscopy using transfected human neuroblastoma cells and nonimmortalized HS68 fibroblasts revealed a distinct subcellular distribution for ZNF74-I and ZNF74-II. In contrast to ZNF74-I, which largely colocalizes with SC-35 in nuclear speckles enriched in splicing factors, the transcriptionally active ZNF74-II had a more diffuse nuclear distribution that is more characteristic of transcriptional regulators. Taken with the previously described RNA-binding activity of ZNF74-I and direct interaction with a hyperphosphorylated form of the RNA polymerase II participating in pre-mRNA processing, our results suggest that the two ZNF74 isoforms exert different or complementary roles in RNA maturation and in transcriptional regulation.
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PMID:Alternative promoter usage and splicing of ZNF74 multifinger gene produce protein isoforms with a different repressor activity and nuclear partitioning. 1131 19

The cell density-dependent growth inhibition of human SK-N-MC neuroblastoma cells is initiated by increased ganglioside sialidase activity leading to elevated cell surface presentation of ganglioside GM1, a ligand of galectin-1. We herein show that the extent of the cell surface expression of the galectin coincides with marked increases of the sialidase activity. Reverse transcriptase-polymerase chain reaction analysis excludes a regulation at the transcriptional level. Exposure of cells to purified galectin-1 reveals its carbohydrate-dependent activity to reduce cell proliferation. Assays to detect DNA fragmentation biochemically and cytometrically and to block caspases render it unlikely that galectin-1 acts as a classical proapoptotic factor on these cells. Because the chimeric galectin-3 shares binding sites and binding parameters with galectin-1 for these cells, we tested whether this galectin will elicit the same response as the homodimeric cross-linking galectin-1. Evidently, galectin-3 fails to affect cell growth by itself but interferes with galectin-1 upon coincubation. Its proteolytically truncated variant, the C-terminal lectin domain with impaired capacity to form aggregates when surface bound, has only weak binding properties. Thus, the way in which the galectin-1 interacts topologically with an apparently common set of ligands relative to galectin-3 is crucial for eliciting post-binding events. We conclude that galectin-1 is a probable effector in the sialidase-dependent growth control in this system. Moreover, the experiments with galectin-3 reveal functional divergence, most probably based on different topologies of presentation of homologous carbohydrate-binding sites.
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PMID:Negative regulation of neuroblastoma cell growth by carbohydrate-dependent surface binding of galectin-1 and functional divergence from galectin-3. 1145 61

In order to clone candidate tumor suppressor genes whose loss contributes to the pathogenesis of neuroblastoma (NB), we performed polymerase chain reaction (PCR) screening using a high-density sequence tagged site-content map within a commonly deleted region (chromosome band 1p36) in 24 NB cell lines. We found a approximately 480 kb homozygously deleted region at chromosome band 1p36.2 in one of the 24 NB cell lines, NB-1, and cloned the human homologue (KIF1B-beta) of the mouseKif1B-beta gene in this region. The KIF1B-beta gene had at least 47 exons, all of which had a classic exon-intron boundary structure. Mouse Kif1B is a microtubule-based putative anterograde motor protein for the transport of mitochondria in neural cells. We performed mutational analysis of the KIF1B-beta gene in 23 cell lines using 46 sets of primers and also an allelic imbalance (AI) analysis of KIF1B-beta in 50 fresh NB samples. A missense mutation at codon 1554, GTG (Gly) to ATG (Met), silent mutations at codon 409 (ACG to ACA) and codon 1721 (ACC to ACT), and polymorphisms at codon 170, GAT (Asp) to GAA (Glu), and at codon 1087, TAT (Tyr), to TGT (Cys), were all identified, although their functional significances remain to be determined. The AI for KIF1B-beta was slightly higher (38%) than those for the other two markers (D1S244, D1S1350) (35 and 32%) within the commonly deleted region (1p36). Reverse transcriptase-PCR analysis of the KIF1B-beta gene revealed obvious expression in all NB cell lines except NB-1, although decreased expression of the KIF1B-beta gene was found in a subset of early- and advanced-stage NBs. These results suggest that the KIF1B-beta gene may not be a candidate for tumor suppressor gene of NB.
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PMID:Genomic structure and mutational analysis of the human KIF1B gene which is homozygously deleted in neuroblastoma at chromosome 1p36.2. 1152 94

In this study the expression of uncoupling protein 3 (UCP3) and its regulation by insulin-like growth factor 1 (IGF-I) and insulin in human neuroblastoma SH-SY5Y cells were characterized. Reverse transcriptase-PCR, Western blot, and immunofluorescence analysis showed that SH-SY5Y cells express UCP3 natively. IGF-I induced a time- and concentration-dependent induction of UCP3 protein reaching a twofold expression after 72 h with 10 nM IGF-I. Extremely high insulin concentrations (860 nM) and 10 nM trIGF-I, a truncated form of IGF-I with the same affinity for the IGF-I receptor as the full-length IGF-I, but with lower activity on the insulin receptor, also upregulated UCP3. We conclude that SH-SY5Y cells express UCP3 natively and that the expression is regulated by IGF-I via the IGF-I receptor.
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PMID:Insulin-like growth factor type 1 upregulates uncoupling protein 3. 1158 36

Fractalkine has been identified as a novel chemokine that exhibits cell adhesion and chemoattractive properties in the central nervous system (CNS), and the fractalkine receptors, CX3CR1, are also expressed in the CNS. In the present study, the expression of fractalkine and fractalkine receptors was investigated in enriched populations of human CNS neurons, astrocytes, and microglia. In addition, the regulatory role played by protein kinase C (PKC) in fractalkine secretion in neurons was determined in A1 human hybrid neuronal cell line produced between a human cerebral neuron and a human neuroblastoma cell. Human neurons and astrocytes expressed fractalkine mRNA as determined by the revserse transcriptase-polymerase chain reaction (RT-PCR) analysis, while human microglia preparation did not express the fractalkine message. Human neurons and microglia expressed CX3CR1 mRNA, but astrocytes did not. These results suggest that fractalkine secreted by CNS neurons and astrocytes produce biological effects in neurons and microglia. Although phorbol ester did not change the expression of fractalkine mRNA level in A1 hybrid neurons, it did upregulate fractalkine secretion over unstimulated controls. This upregulation of fractalkine production was suppressed by the treatment with Ro32-0432, a PKC inhibitor. These results indicate that intracellular signals transduced by PKC play an important role in the regulation of soluble fractalkine at the post-transcriptional level in human neurons. As for the biological function of fractalkine, extracellularly applied fractalkine increased the number of bromodeoxyuridine-labeled microglia 3-fold over the untreated controls, indicating fractalkine induces proliferation of human microglia. These observations suggest that fractalkine released by injured neurons could induce proliferation, activation and/or migration of microglia at the injured brain sites.
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PMID:Fractalkine and fractalkine receptors in human neurons and glial cells. 1212 82

Evidence has accumulated showing that vasoactive peptides, such as endothelin-1, adrenomedullin and urotensin-II, are expressed in various kinds of tumour cells. In the present study, the expression of endothelin-1 and endothelin receptors was studied in eight human tumour cell lines: T98G (glioblastoma), IMR-32 and NB69 (neuroblastoma), BeWo (choriocarcinoma), SW-13 (adrenocortical carcinoma), DLD-1 (colonic carcinoma), HeLa (cervical carcinoma) and VMRC-RCW (renal carcinoma). Reverse transcriptase-PCR showed expression of endothelin-1 mRNA in seven out of the eight cell lines, the exception being BeWo cells. ET(A) receptor mRNA was expressed in T98G, IMR-32 and NB69 cells, but weakly in the other cells. ET(B) receptor mRNA was expressed in IMR-32, NB69 and BeWo cells, but only weakly in T98G and HeLa cells. Immunoreactive endothelin was detected in the culture media of six out of the eight cell lines, but not in that of IMR-32 or BeWo cells. Treatment of T98G cells with an anti-endothelin-1 antibody or an anti-adrenomedullin antibody for 24 h decreased cell numbers to approx. 84% and 90% of control respectively. Treatment with the ET(A) receptor antagonist BQ-610 (1 microM) significantly decreased cell number to about 90% of control, whereas the ET(B) receptor antagonist BQ-788 had no significant effect. On the other hand, exogenously added endothelin-1, adrenomedullin or urotensin-II (0.1 microM) had no significant effects on cell number. These results suggest that endothelin-1 acts as a paracrine or autocrine growth stimulator in tumours. The effect of endothelin-1 on tumour growth appears to be mediated by the ET(A) receptor.
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PMID:Three vasoactive peptides, endothelin-1, adrenomedullin and urotensin-II, in human tumour cell lines of different origin: expression and effects on proliferation. 1219 50

In this study, the mouse neuroblastoma cell line Neuro-2a was analyzed for expression of angiotensin II receptors. Reverse-transcriptase polymerase chain reaction (RT-PCR) showed that Neuro-2a cells express mRNA of angiotensin II (AngII) receptor subtypes AT1A, AT1B, and AT2. Analysis of Neuro-2a cells by Western blotting revealed AT1 and AT2 receptor protein expression. The predominant molecular weights were determined to be 50.4 kDa for the AT1 receptor and 62.4 kDa for the AT2 receptor. Observation of AT1 and AT2 receptor localization within Neuro-2a cells using immunocytochemistry showed distribution similar to other G-protein coupled receptors with diffuse distribution in the cytosol, perinuclear enrichment and accumulation of receptors on the outer cellular periphery with extension into the neurites. Furthermore, we observed InsP3 formation following AngII induction that could be abolished in presence of the AT1A receptor antagonist losartan. The results clearly show expression of the AngII receptor types AT1A and AT2 in the Neuro-2a cell line. We conclude that Neuro-2a cells represent an interesting model cell line for study of mechanisms that control the interplay between these receptors.
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PMID:Angiotensin II receptor types 1A, 1B, and 2 in murine neuroblastoma Neuro-2a cells. 1268 May 93

Sphingosine 1-phosphate (S1P) has assumed great importance within neuroscience research because of putative links between S1P-sensitive Edg receptors and neuroregeneration, cell survival, and alterations in neurite outgrowth. In the present study, we examined the mechanisms by which the endogenous complement of S1P-sensitive human Edg receptors can elevate Ca(2+) in the human neuroblastoma cell line, SH-SY5Y. Reverse transcriptase-polymersase chain reaction (RT-PCR) confirmed the expression of mRNA for Edg 3, 5, and 8 subtypes of S1P-responsive Edg receptors in SH-SY5Y cells. Neither S1P nor the muscarinic agonist methacholine were able to cause a change in SH-SY5Y cell morphology, whereas retinoic acid caused a range of changes, including an increase in neurite outgrowth, under similar test conditions. Stimulation with S1P resulted in a slowly rising increase in cytosolic Ca(2+) levels. These responses were dependent upon inositol-1,4,5-trisphosphate receptors, thapsigargin-sensitive endoplasmic reticulum, and also intact functional mitochondria. S1P-evoked Ca(2+) responses were similar in mechanism to those of methacholine, which activated a much faster responding, larger amplitude Ca(2+) response. These studies indicate that in an endogenous human expression system, S1P appears to be an efficacious agonist of Edg receptors. Despite its slow time course of response, S1P appears to activate the same single Ca(2+) store in SH-SY5Y cells as is activated by methacholine and other G protein coupled receptors.
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PMID:Characterisation of a sphingosine 1-phosphate-activated Ca2+ signalling pathway in human neuroblastoma cells. 1283 64

Amplification of the MYCN gene, resulting in overexpression of MYCN, distinguishes a subset of neuroblastomas with poor prognosis. We recently identified MYCN as a target gene of the E2F transcription factors. Here we show that Sp1 and Sp3 cooperate with E2F-1 to activate the MYCN promoter. However, in a neuroblastoma cell line that does not express MYCN, overexpression of E2F-1 was not sufficient to activate the MYCN promoter even in the presence of trichostatin A and 5-aza-cytidine. This was because of a failure of E2F-1 to bind to the MYCN promoter in these cells, although access of E2F-1 to the inactive MYCN promoter was not blocked by a nucleosome. Differences in nucleosomal organization of the MYCN promoter in different cell lines did not correlate with gene activation per se but with the switch from basal to activated transcription. Binding of E2F and Sp1/Sp3 to the MYCN promoter in vivo correlated with acetylation of histones H3 and H4 and recruitment of RNA polymerase II and the protein acetyltransferase Tip60 but not with nucleosome remodeling. Our results define distinct chromatin states of the MYCN promoter, indicate that factors in addition to E2F and Sp1/Sp3 are required to activate MYCN in neuroblastomas, and provide evidence for a novel mechanism of controlling access of E2F to selected target genes.
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PMID:E2F and Sp1/Sp3 Synergize but are not sufficient to activate the MYCN gene in neuroblastomas. 1464 38

An orphan G protein-coupled receptor from rat has recently been discovered to be activated by the nucleobase adenine (Proc Natl Acad Sci USA 99:8573-8578, 2002). In the present study, we show for the first time that the adenine receptor is expressed in membrane preparations of native tissues and cell lines in high density, including rat brain cortex, rat brain striatum, and the mouse neuroblastoma x rat glioma hybrid cell line NG108-15. Saturation analysis with [3H]adenine at rat brain cortical membranes exhibited a single high-affinity binding site with a KD value of 27.2 nM, and a binding capacity of 2.28 pmol/mg of protein. Kinetic studies revealed unusual binding kinetics of [3H]adenine with rapid association and slow dissociation. A series of compounds were investigated in [3H]adenine competition experiments at rat brain cortex. Only minor substitution of the adenine structure was tolerated, the most potent compounds of the present series being 2-fluoroadenine (Ki value of 620 nM), 8-thioadenine (Ki value of 2.77 microM), N6-methyladenine (Ki value of 3.64 microM), and 7-methyladenine (Ki value of 4.13 microM), all of which were partial agonists (40-60% intrinsic activity). Adenine dose dependently inhibited forskolin-stimulated adenylate cyclase in membrane preparations of NG108-15 cells as well as in intact cells, showing that the receptor is functional in NG108-15 cells. Reverse transcriptase-polymerase chain reaction experiments followed by sequencing indicate that the NG108-15 cells express the murine ortholog of the adenine receptor. Moreover, preliminary radioligand binding studies with [3H]adenine at membranes of human astrocytoma 1321N1 cells suggest that a human ortholog of the rat adenine receptor exists.
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PMID:Evidence for the functional expression and pharmacological characterization of adenine receptors in native cells and tissues. 1560 13


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