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)

Onconase is a cytotoxic ribonuclease with antitumor properties. A semisynthetic gene encoding the entire protein sequence was constructed by fusing oligonucleotides coding for the first 15 and the last 6 of the 104 amino acids to a genomic clone that encoded the remaining amino acid residues [Newton, D. L., et al. (1997) Protein Eng. 10, 463-470]. The resulting protein product expressed in Escherichia coli exhibited little enzymatic or cytotoxic activity due to the unprocessed N-terminal Met amino acid residue. In this study, we demonstrate that modification of the 5'-region of the gene to encode [Met-(-1)]Ser or [Met-(-1)]Tyr instead of the native pyroglutamate results in recombinant onconase derivatives with restored activities. [Met-(-1)]rOnc(E1S) was more active than [Met-(-1)]rOnc(E1Y) in all assays tested. Consistent with the action of native onconase, [Met-(-1)]rOnc(E1S) was a potent inhibitor of protein synthesis in the cell-free rabbit reticulocyte lysate assay, degrading tRNA at concentrations that correlated with inhibition of protein synthesis. An interesting difference between the recombinant onconase derivatives and the native protein was their susceptibility to inhibition by the major intracellular RNase inhibitor, PRI (onconase is refractory to PRI inhibition). [Met-(-1)]rOnc(E1S) and [Met-(-1)]rOnc(E1Y) inhibited protein synthesis in intact SF539 neuroblastoma cells with IC50's very similar to that of onconase (IC50 3.5, 10, and 10 microg/mL after 1 day and 0.16, 0.35, and 2.5 microg/mL after 5 days for onconase, [Met-(-1)]rOnc(E1S), and [Met-(-1)]rOnc(E1Y), respectively). Similar to that of onconase, cytotoxic activity of the recombinant derivatives was potentiated by monensin, NH4Cl, and retinoic acid. Brefeldin A completely blocked the enhancement of cytotoxicity caused by retinoic acid with all three proteins. Thus, drug-induced alterations of the intracellular trafficking of the recombinant derivatives also resembles that of onconase. Stability studies as assessed in serum-containing medium in the presence or absence of cells at 37 degreesC showed that the recombinant proteins were as stable to temperature and cell culture conditions as the native protein. Therefore, exchanging the Glu amino acid residue at the amino terminus of onconase with an amino acid residue containing a hydroxyl group produces recombinant proteins with ribonuclease and cytotoxic properties similar to native onconase.
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PMID:Single amino acid substitutions at the N-terminus of a recombinant cytotoxic ribonuclease markedly influence biochemical and biological properties. 954 48

Tau isoforms migrating at 46-68 and 97-115 kDa were prominent within heat-stable Triton-soluble material, and were present in lesser concentration with Triton-insoluble cytoskeletons, derived from undifferentiated SH-SY-5Y human neuroblastoma cells. Conversely, a 26-30 kDa tau isoform was enriched in the cytoskeleton and detected at relatively minor levels within cytosolic fractions. Pulse labeling with 35S-methionine indicated that this 26-30 kDa "small tau" did not represent a breakdown product of larger isoforms. Since the nucleus is retained within the Triton-insoluble cytoskeleton, additional cultures were fractionated onto sucrose to obtain purified nuclei. The vast majority of small tau was recovered within purified nuclei. Small tau was reactive with tau antibodies directed towards N-terminal, C-terminal and central epitopes, further confirming that this small isoform was not derived from proteolytic cleavage of larger tau isoforms. Small tau demonstrated alkaline phosphatase-sensitive reactivity with multiple phospho-dependent tau antibodies. Small tau was depleted within 3 days of retinoic acid-induced differentiation, suggesting that the putative function of this isoform may be obsolete following terminal differentiation of neurons.
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PMID:A 26-30 kDa developmentally-regulated tau isoform localized within nuclei of mitotic human neuroblastoma cells. 966 21

Alanyl aminopeptidase (AAP-S) was purified to homogeneity from rat liver cytosol. The molecular weight of the purified enzyme was calculated to be approximately 100,000 on Sephacryl S-200 HR and to be 90,000 on SDS-PAGE in the presence of beta-mercaptoethanol. These findings suggested that the enzyme exists as a monomeric form in rat liver cytosol. The enzyme rapidly hydrolyzed the substrates Ala-, Tyr- and Met-MCAs, and moderately hydrolyzed Arg-, Lys-, Leu-, Phe- and Lys-Ala-MCAs at pHs ranging from 7.5to 8.0. The enzyme also hydrolyzed several amino acid 4-methyl-coumaryl-7-amide (MCA) substrates. The order for k(cat)/Km values of AAP-S at the optimal pH (pH 7.5) was Lys->Met->Arg->Ala->Leu->Phe->Tyr->Lys-Ala-MCAs. It was strongly inhibited by bestatin, leuhistin, actinonin, amastatin, 1, 10-phenanthroline, PCMBS, Zn2+, Cd2+, Co2+, Cu2+ and Hg2+, and puromycin. The amino acid sequence of the first 43 residues of the enzyme was determined as Pro1-Glu-Lys-Arg-Pro5-Phe-Glu-Arg-Leu-Pro10-Thr-Glu-Val-Ser-Pro 15-Ile-Asn-Tyr-Ser-Leu20-(Cys)-Leu-Lys-Pro-Asp25-Leu-Leu- Asp-Phe-Thr30-Phe-Glu-Gly-Lys-Leu35-Glu-Ala-Ala-Ala-Gln40 -Val-Arg-Gln-. This N-terminal amino acid sequence is almost identical with those of puromycin-sensitive enkephalin-degrading aminopeptidases in rat and human brains, and the mouse neuroblastoma cell line Neuro2A. These findings suggest that the AAP-S from rat liver cytosol is a puromycin-sensitive aminopeptidase. Furthermore, with immunohistochemistry the enzyme was strongly stained in the cytosol of the rat liver cells.
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PMID:Isolation and characterization of an alanyl aminopeptidase from rat liver cytosol as a puromycin-sensitive enkephalin-degrading aminopeptidase. 968 21

The ability of action-potential-like waveforms (APWs) to attenuate opioid-induced inhibition of N-type Ca2+ channels was investigated in the neuroblastoma x glioma cell line NG108-15 using whole-cell voltage clamp methods. In in vitro differentiated NG108-15 cells, the opioid agonist [d-ala2]-methionine-enkephalin (DAME) reversibly decreased omega-conotoxin-GVIA-sensitive Ba2+ currents (N-type currents). Agonist-mediated inhibition of N-type currents could be transiently relieved by strong unphysiological depolarizing prepulses to +80 mV (facilitation). Significant facilitation was also achieved by conditioning the cell with a train of 15 APWs, which roughly mimicked physiological action potentials (1- to 6-ms-long depolarizations to +30 mV from a holding potential of -40 mV). The APW-induced facilitation depended on both conditioning pulse frequency and duration. Summation of the disinhibition produced by each APW was possible because reinhibition following repolarization to -40 mV was a much slower process (tau=88 ms) than the onset of facilitation at +80 mV (tau=7 ms). These results provide evidence that N-type Ca2+ channel facilitation may be a physiologically relevant process, and suggest that neuronal firing may relieve agonist-induced inhibition of N-type currents to an extent depending on both the shape of action potentials and the frequency of firing.
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PMID:Action-potential-like depolarizations relieve opioid inhibition of N-type Ca2+ channels in NG108-15 cells. 991 1

S-Adenosylmethionine (AdoMet) synthetase (EC 2.5.1.6), which catalyzes the synthesis of AdoMet from methionine and ATP, is the major methyl donor for transmethylation reactions and propylamino donor for the biosynthesis of polyamines in biological systems. We have reported previously that wild-type C-1300 murine neuroblastoma (wMNB) cells, made resistant to the nucleoside analogue (Z)-5'-fluoro-4',5'-didehydro-5'-deoxyadenosine (MDL 28,842), an irreversible inhibitor of S-adenosylhomocysteine (AdoHcy) hydrolase (EC 3.3.1.1), express increased AdoMet synthetase activity (M. R. Hamre et al., Oncol. Res., 7: 487-492, 1995). In the present study, immunoblot analyses of AdoMet Synthetase with isoform-specific (MATII) antibodies demonstrated an elevation in the AdoMet synthetase immunoprotein in nucleoside analogue-resistant MNB cells (rMNB-MDL) when compared to wild-type, nonresistant MNB cells. An increase of 2.1-fold was observed in the alpha2/alpha2' catalytic subunit, which differed significantly from the much smaller increment in the noncatalytic beta-subunit of AdoMet synthetase. Densitometric analyses revealed that an increased expression of AdoMet synthetase in rMNB-MDL cells was due to overexpression of the alpha2 (Mr 53,000; 2.6-fold) and alpha2' (Mr 51,000; 1.8-fold) subunits. AdoMet synthetase mRNA expression in rMNB-MDL cells was remarkably greater than wMNB cells, as determined by quantitative competitive reverse transcription-PCR (QC-PCR) analysis. DNA (cytosine) methyl transferase expression, measured by reverse transcription-PCR analysis, was also elevated significantly in rMNB-MDL cells. In contrast, Western blot analyses demonstrated down-regulation (1.6-fold) of AdoMet synthetase in doxorubicin-resistant human leukemia cells (HL-60-R) expressing multidrug resistance protein when compared with wild-type, nonresistant HL-60 cells. The resistance of rMNB-MDL cells to nucleoside analogue inhibitors of S-adenosylhomocysteine hydrolase correlates directly with overexpression of the alpha2/alpha2' subunits of AdoMet synthetase. Cellular adaptation allows sufficient AdoMet to be synthesized, so that viability of the MNB cells can be maintained even in the presence of high AdoHcy concentrations. This novel mechanism of drug resistance does not appear to require multidrug resistance protein (P-glycoprotein) overexpression.
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PMID:S-adenosylmethionine synthetase is overexpressed in murine neuroblastoma cells resistant to nucleoside analogue inhibitors of S-adenosylhomocysteine hydrolase: a novel mechanism of drug resistance. 1021 91

Previous studies have shown D2-like dopamine receptor involvement in the regulation of phospholipid methylation (PLM), while others have documented impaired methionine and folate metabolism in schizophrenia. Utilizing [14C]formate labeling in cultured neuroblastoma cell lines, we now show that D4 dopamine receptors (D4R) mediate the stimulatory effect of dopamine (DA) on PLM. The effect of DA was potently blocked by highly D4R-selective antagonists and stimulated by the D4R-selective agonist CP-226269. DA-stimulated PLM was dependent upon the activity of methionine cycle enzymes, but DA failed to increase PLM in [3H]methionine labeling studies, indicating that a methionine residue in the D4R might be involved in mediating PLM. A direct role for MET313, located on transmembrane helix No. 6 immediately adjacent to phospholipid headgroups, was further suggested from adenosylation, site-directed mutagenesis and GTP-binding results. A comparison of PLM in lymphocytes from schizophrenia patients vs control samples showed a four-fold lower activity in the schizophrenia group. These findings reveal a novel mechanism by which the D4R can regulate membrane composition. Abnormalities in D4R-mediated PLM may be important in psychiatric illnesses such as schizophrenia.
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PMID:D4 dopamine receptor-mediated phospholipid methylation and its implications for mental illnesses such as schizophrenia. 1039 13

The primary structure of sheep brain pyridoxal kinase has been determined by direct chemical and physical methods. The enzyme contains 312 amino acid residues with an acetylated methionine at the N-terminus, yielding a molecular mass of 34,861 Da. The functional role played by the two tryptophanyl residues in positions 52 and 244 of the polypeptide chain has been investigated by fluorescence spectroscopy. The tryptophanyl residues are not completely exposed to the rapidly relaxing solvent and they are poorly accessible to collisional quenchers. Chemical modification with NBS abolishes the catalytic activity of the kinase. The amino acid sequence of the sheep brain enzyme shows high similarity (86.2% identity) with the human pyridoxal kinase recently reported [Hanna, Turner, and Kirkness, (1997), J. Biol. Chem. 272, 10756-10760]. Comparison of the mammalian proteins with bacterial and yeast putative pyridoxal kinases retrieved from the Swiss-Prot data bank shows a low degree of overall similarity. In particular, the putative ATP-binding domain is conserved, whereas the region that appears to be crucial in the binding of the pyridoxal substrate is not. Thus, the assignment of the bacterial and yeast cDNA-deduced proteins as pyridoxal kinases should be taken with caution.
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PMID:Structure of pyridoxal kinase from sheep brain and role of the tryptophanyl residues. 1039 44

1. The carboxyl terminus of the amyloid precursor protein (APP) has several identified regions that may potentially contribute to the pathogenic effects of Alzheimer's disease (AD). To examine these effects, the authors iodinated a short synthetic peptide corresponding to amino acids 679-687 of APP695. They also produced a [35S]-Methionine labeled peptide corresponding to the entire carboxyl 100 amino acids of APP695 via a reticulocyte lysate coupled in vitro transcription/translation system. 2. Human neuroblastoma cells (SK-N-SH) and non-neuronal epithelial cells (RK-13) were cultured, harvested, and lysed. The S1 cell extract fractions were combined with either of the labeled peptides and incubated at different temperatures to allow for interaction and binding of cellular proteins with the peptides. These interactions were identified as gel mobility shift patterns on native PAGE gels. The presence of distinct bands on the gels indicate that the APP C-terminus interacts with several intracellular proteins, some of which may be detrimental to the cell. The authors have tested the possibility that the accumulation of C-terminal proteins may result in apoptosis. 3. Apoptosis in neural cells is one detrimental effect that has been attributed to APP. The authors examined hippocampal tissue sections from Alzheimer's disease (AD) and age-matched normal control patients for a difference in the number of apoptotic nuclei present using an in situ apoptosis detection kit that labels the numerous free DNA ends present in apoptotic nuclei. The number of apoptotic nuclei found in AD neuronal tissue was significantly higher than in normal tissue.
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PMID:Examination of the interactions of the amyloid precursor protein carboxyl terminus to intracellular protein: possible role in apoptosis. 1050 80

Estrogen exerts complex physiologic effects on brain functions which could partly be mediated through modulation of the dopaminergic system. Transcription control of the human D1A dopamine receptor gene by estrogenic stimulation was studied in the D1A expressing neuroblastoma cell line SK-N-MC. Transient co-transfection of D1A gene promoter-CAT constructs along with expression vectors for steroid hormone receptors indicated that estrogen, but not progesterone or glucocorticoid, receptors up-regulate transcription of this gene by about 1.7-fold. Serial 5' deletion mutants of the D1A gene upstream region localized the estrogen responsive segment between nucleotides -1472 and -1342 relative to the initiator methionine. This region contains a half palindrome (TGACC) for the consensus estrogen responsive element (ERE). Additional co-transfection experiments revealed that estrogen receptors specifically activate the upstream D1A promoter but not the downstream promoter located in the intron of this gene. Consistent with transient co-transfection experiments, 17beta-estradiol treatment of SK-N-MC cells transfected with an estrogen receptor expression vector resulted in an approximately 20% increase in steady-state levels of long D1A transcripts derived from the upstream promoter but not of short transcripts originating from the intron promoter. These observations demonstrate a molecular basis for estrogen induced up-regulation of D1A gene transcription and provide a mechanism for modulation of central dopaminergic functions by this hormone.
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PMID:Up-regulation of D1A dopamine receptor gene transcription by estrogen. 1061 33

The candidate tumor-suppressor gene ING1 encodes p33(ING1), a nuclear protein which physically interacts with TP53. It has been shown that p33(ING1) acts in the same biochemical pathway as TP53, leading to cell growth inhibition. Interestingly, a rearrangement of the ING1 gene was found in a neuroblastoma cell line, supporting its involvement in tumor development. Because ING1 resides on the long arm of chromosome 13 (13q34) (a region frequently deleted in many tumor types), we sought to characterize its role in head and neck squamous-cell carcinoma (HNSCC). We first analyzed 44 primary tumors for loss of heterozygosity (LOH) at 13q, using four widely spaced microsatellite markers (13q14, 13q14.3-q22, 13q22, and 13q34). Twenty (48%) of the tumor samples showed LOH in all of the informative markers tested, including D13S1315 at 13q34. Two of the tumors displayed partial losses restricted to one marker (D13S118 at 13q14 in tumor 1164, and D13S135 at 13q14.3-q22 in tumor 1398). We then determined the genomic structure of the ING1 gene and sequenced the entire coding region in 20 primary tumors showing 13q LOH and in five head and neck cancer cell lines. A single germline polymorphism was detected in 10 of the tumors analyzed (T to C change) located 110 nucleotides upstream of the starting methionine. No somatic mutations were found in any of the samples, suggesting that ING1 is not a tumor suppressor gene target in head and neck cancer. Genes Chromosomes Cancer 27:319-322, 2000.
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PMID:Molecular analysis of the candidate tumor suppressor gene ING1 in human head and neck tumors with 13q deletions. 1067 22


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