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Results for “RNA, Competitive Endogenous”

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PubMed1996

Effects of modifying the tRNA(3Lys) anticodon on the initiation of human immunodeficiency virus type 1 reverse transcription.

tRNA(3Lys) is a primer for reverse transcription in human immunodeficiency virus type 1 (HIV-1), and the anticodon of tRNA(3Lys) has been implicated in playing a role in both its placement onto the HIV-1 genome and its interaction with HIV-1 reverse transcriptase (RT). In this work, the anticodon in a tRNA(3Lys) gene was changed from UUU to CUA (tRNA(3Lys)Su+) or, in addition, G-73 was altered to A (tRNA(3Lys)Su+G73A). COS-7 cells were transfected with either wild-type or mutant tRNA(3Lys) genes, and both the wild-type and mutant tRNA(3Lys) produced were purified by using immobilized tRNA-specific hybridization probes. Each mutant tRNA(3Lys) was tested for its ability to prime reverse transcription in vitro, either alone or in competition with wild-type tRNA(3Lys). Short RT extensions of wild-type and mutant tRNALys could be distinguished from each other by their different mobilities in one-dimensional single-stranded conformation polymorphism polyacrylamide gel electrophoresis. These reverse transcription products show that heat-annealed tRNA(3Lys)Su+ has the same ability as heat-annealed wild-type tRNA(3Lys) to prime RT and competes equally well with wild-type tRNA(3Lys) for priming RT. tRNA(3Lys)Su+G73A has 60% of the wild-type ability to prime RT but competes poorly with wild-type tRNA(3Lys) for priming RT. However, the priming abilities of wild-type and mutant tRNA(3) are quite different when in vivo-placed tRNA is examined. HIV-1 produced in COS cells transfected with a plasmid containing both the HIV-1 proviral DNA and DNA coding for tRNA(3Lys)Su+ contains both endogenous, cellular wild-type tRNA(3Lys) and mutant tRNA(3Lys). When total viral RNA is used as the source of primer tRNA placed onto the genomic RNA in vivo, only wild-type tRNA(3Lys) is used as a primer. If the total viral RNA is first heated and exposed to hybridizing conditions, then both the wild-type and mutant tRNA(3Lys) act as primers for RT. These results indicate that the tRNA(3Lys)Su+ packaged into the virions is unable to act as a primer for RT, and a model is proposed to explain the disparate results between heat-annealed and in vivo-placed primer tRNA.

Anticodon↗

PubMed1973

Effects of polyadenylic acids on functions of murine RNA tumor viruses.

Single-stranded polyribonucleotides, which competitively inhibit murine RNA tumor virus reverse transcriptase in vitro, were tested as inhibitors of various virus functions in cell culture. The compounds had two concentration-dependent effects. At high concentrations (100 mug/ml), both poly(adenylic acid) [poly(A)] and poly(2'-O-methyladenylic acid) [poly(Am)] inhibited the uptake of radioactively labeled leukemia virus by Swiss mouse embryo cells, but neither had a similar effect on Sindbis virus adsorption. At low concentrations (10 mug/ml), poly(Am) did not inhibit the uptake of leukemia virus but did inhibit virus replication by 85%; in contrast, the replication of Sendai virus and Sindbis virus was not inhibited significantly at this concentration. Both compounds were effective only when added prior to or early during virus infection. Poly(Am) was a much more effective inhibitor than poly(A), probably due to the nuclease resistance of the former compound. Poly(Am) at 5 mug/ml also inhibited transformation of 3T3 cells by Moloney sarcoma virus. However, neither poly(A) nor poly(Am) at high concentrations inhibited the activation of endogenous leukemia virus by iododeoxyuridine in AKR mouse embryo cells. These results suggest that virus reverse transcriptase plays an essential role in both the replication of exogenous murine leukemia viruses and the transformation of cells by murine sarcoma viruses but probably has no role in the activation of endogenous leukemia virus.

Adenosine Monophosphate↗

PubMed2002

Competition between SsrA tagging and translational termination at weak stop codons in Escherichia coli.

SsrA is a tmRNA involved in tagging polypeptides on stalled ribosomes. The resulting fusion proteins are then degraded. We purified endogenous SsrA-tagged proteins by means of a genetically engineered SsrA and identified some of them. Analysis of the proteins suggested that they are tagged at their C-terminal extremities. One of them, ribokinase, is expressed from a messenger with a poorly efficient stop codon, leading to translational recoding events. A change in the ribokinase coding sequence from a weak to a strong translational stop sequence (UGAc to UAAu) annihilated SsrA tagging. Translational termination by UGA recruits the translational release factor (RF) 2. We observed that SsrA tagging of ribokinase was inversely correlated with RF2 activity, revealing a dynamic competition between translational termination and SsrA tagging.

Codon, Terminator↗

PubMed1982

Nuclear ligation of RNA polynucleotide kinase products.

RNA polynucleotide kinase has been shown to transfer [gamma 32P] from ATP to 5-OH termini of endogenous nuclear RNA. The products of this reaction have been isolated in RNA larger than 125 after in vitro incubation of mouse L cell nuclei. About 20%-30% of these 5'-OH kinase products are polyadenylated. A sizeable fraction of the [gamma 32P] label from ATP is also found in internal phosphodiester bonds after 30-minute nuclear incubation in vitro. The possibility of substantial [32P] 1) 2mM nucleoside triphosphates in the incubation medium, 2) limited nearest-neighbor distribution 3' and 5' to the phosphodiester bond compared with that from [alpha 32P] UTP, 3) different nearest-neighbor distribution for RNA molecules greater than 125 and 12-3S, 4) relative insensitivity of the [gamma 32P] incorporation to alpha-amanitin as compared with total RNA synthesis, 5) internal [32P] appearance in RNA greater than 12S in less than five minutes of incubation, and 6) less than 0.03% to 0.6% of the total [32P] in the alpha position of nucleoside triphosphates after 30 minutes of incubation. The [gamma 32P] incorporation was dependent on high ATP concentration and was insensitive to competition by inorganic phosphate. These results are consistent with the levels of 5' RNA polynucleotide kinase activity in L cell nuclei and suggest the presence of an RNA ligase that can utilize the termini generated by the 5'-OH RNA kinase in a ligation reaction.

Amanitins↗

PubMed2000

A C-terminal carbohydrate-binding domain in the endothelial cell regulatory protein, pigpen: new function for an EWS family member.

The potential for encoding information in carbohydrate (CHO) structures has long been recognized. Selective CHO-binding proteins known as lectins and the biological events they mediate are well known. However, many lectins were originally discovered for biological activities other than saccharide binding, and only subsequently was it realized that one or more of their key functions were mediated by specific CHO recognition. Our previous observations suggested that the nuclear protein pigpen had an affinity for CHO structures. This would represent a new attribute for proteins of the EWS (Ewing's sarcoma) family, of which pigpen is a member. In this study we demonstrate that a CHO-binding domain resides in the C-terminus of the molecule and can be preferentially inhibited by saccharides, most notably N-acetyl-d-galactosamine (GalNAc) and the GalNAc-containing polysaccharide, chondroitin sulfate. Ligand blotting experiments were subsequently performed with fractionated, [(3)H]galactose-labeled cells to demonstrate the presence of chondroitin sulfate-inhibitable endogenous CHO ligands for pigpen in endothelial nuclei. Finally, microinjection of polysaccharide competitor into the nucleus of cultured endothelial cells resulted in a loss of pigpen focal accumulations, suggesting that the CHO-binding activity may be instrumental in subcellular localization of the protein. In summary, our results show ligand preference and domain specificity for pigpen's CHO affinity and provide initial evidence for physiological ligands and function. They may also shed new light on the mechanisms of oncogenic transformation involving EWS proteins.

Animals↗

PubMed2006

Naturally occurring glucagon-like peptide-2 (GLP-2) receptors in human intestinal cell lines.

Although clinical trials with GLP-2 receptor agonists are currently ongoing, the mechanisms behind GLP-2-induced intestinal epithelial growth remain to be understood. To approach the GLP-2 mechanism of action this study aimed to identify intestinal cell lines endogenously expressing the GLP-2 receptor. Here we report the first identification of a cell line endogenously expressing functional GLP-2 receptors. The human intestinal epithelial cell line, FHC, expressed GLP-2 receptor encoding mRNA (RT-PCR) and GLP-2 receptor protein (Western blot). In cultured FHC cells, GLP-2 induced concentration dependent cAMP accumulation (pEC(50)=9.7+/-0.04 (mean+/-S.E.M., n=4)). In addition, a naturally occurring human intestinal fibroblast cell line, 18Co, endogenously expressing GLP-2 receptor encoding mRNA (RT-PCR) and protein (Western blot) was identified. No receptor functionality (binding or G-protein signalling) could be demonstrated in 18Co cells. The identified gut-relevant cell lines provide tools for future clarification of the mechanisms underlying GLP-2-induced epithelial growth.

Animals↗

PubMed2006

DMT1: which metals does it transport?

DMT1-Divalent Metal (Ion) Transporter 1 or SLC11A2/DCT1/Nramp2 - transports Fe2+ into the duodenum and out of the endosome during the transferrin cycle. DMTI also is important in non-transferrin bound iron uptake. It plays similar roles in Mn2+ trafficking. Voltage clamping showed that six other metals evoked currents, but it is unclear if these metals are substrates for DMT1. This report summarizes progress on which metals DMT1 transports, focusing on results from the authors' labs. We recently cloned 1A/+IRE and 2/-IRE DMT1 isoforms to generate HEK293 cell lines that express them in a tetracycline-inducible fashion, then compared induced expression to uninduced expression and to endogenous DMT1 expression. Induced expression increases approximately 50x over endogenous expression and approximately 10x over uninduced levels. Fe2+, Mn2+, Ni2+ and Cu1+ or Cu2+ are transported. We also explored competition between metal ions using this system because incorporation essentially represents DMT1 transport and find this order for transport affinity: Mn>?Cd>?Fe>Pb-Co-Ni>Zn. The effects of decreased DMT1 also could be examined. The Belgrade rat has diminished DMT1 function and thus provides ways of testing. A series of DNA constructs that generate siRNAs specific for DMT1 or certain DMT1 isoforms yield another way to test DMT1-based transport.

Animals↗

PubMed2001

Differential regulation of the human sodium/iodide symporter gene promoter in papillary thyroid carcinoma cell lines and normal thyroid cells.

The absence of TSH-stimulated radioiodide uptake in differentiated thyroid cancer is associated with a high recurrence rate and reduced survival. We studied regulation of the sodium/iodide symporter gene in human papillary thyroid cancer cell lines (BHP) and primary human thyroid cells. BHP cells expressed very low levels of sodium/iodide symporter mRNA and did not concentrate iodide, but iodide uptake was restored to levels seen in FRTL-5 rat thyroid cells by stable transfection of a sodium/iodide symporter cDNA. Sodium/iodide symporter gene expression, therefore, was necessary and sufficient for iodide uptake in BHP cells. We cloned the human sodium/iodide symporter gene 5'-flanking region and analyzed progressive 5'-deletions in transient transfections. We identified a region, -596 to -268, essential to confer full promoter activity in primary normal human thyroid cells. Sodium/iodide symporter promoter activity in four BHP cell lines, however, was markedly reduced, consistent with down-regulation of the endogenous sodium/iodide symporter gene. Nuclear extracts from BHP 2-7 cells had reduced or absent binding to regions of the sodium/iodide symporter promoter shown to be critical for expression, compared with nuclear extracts from FRTL-5 cells. Competition studies indicated that these nuclear proteins were not known thyroid transcription factors. Modifications of the sodium/iodide symporter promoter with demethylation or histone acetylation did not increase sodium/iodide symporter expression, and no deletions of the critical regulatory region were identified in the endogenous gene in BHP cells. Regulation of the sodium/iodide symporter 5'-flanking region in transient transfection paralleled endogenous sodium/iodide symporter expression. Reduced expression of potential novel nuclear factor(s) in these cell lines may contribute to reduced sodium/iodide symporter expression resulting in absence of iodide uptake in some papillary thyroid cancers.

Base Sequence↗

PubMed2001

Coilin forms the bridge between Cajal bodies and SMN, the spinal muscular atrophy protein.

Spinal muscular atrophy (SMA) is a genetic disorder caused by mutations in the human survival of motor neuron 1 gene, SMN1. SMN protein is part of a large complex that is required for biogenesis of various small nuclear ribonucleoproteins (snRNPs). Here, we report that SMN interacts directly with the Cajal body signature protein, coilin, and that this interaction mediates recruitment of the SMN complex to Cajal bodies. Mutation or deletion of specific RG dipeptide residues within coilin inhibits the interaction both in vivo and in vitro. Interestingly, GST-pulldown experiments show that coilin also binds directly to SmB'. Competition studies show that coilin competes with SmB' for binding sites on SMN. Ectopic expression of SMN and coilin constructs in mouse embryonic fibroblasts lacking endogenous coilin confirms that recruitment of SMN and splicing snRNPs to Cajal bodies depends on the coilin C-terminal RG motif. A cardinal feature of SMA patient cells is a defect in the targeting of SMN to nuclear foci; our results uncover a role for coilin in this process.

Amino Acid Sequence↗

PubMed1992

Transcriptional regulation of the thrombomodulin gene.

The transcriptional start sites of the endogenous human thrombomodulin (TM) gene and transiently expressed TM promoter/CAT gene constructs were defined by nuclease S1 mapping which showed two closely spaced sites at +1 and +6, respectively. Transient expression and in vitro transcription assays of 5' and internal deletion mutants of the TM promoter/CAT gene constructs reveal that the region from -72 to -29 exhibits a positive acting domain which is essential for transcriptional activity, whereas the region from -373 to -225 possesses two positive acting subdomains, -343 to -277 and -245 to -225, which together augment transcriptional activity by about 40%. Electrophoretic mobility shift assays with a duplex oligonucleotide corresponding to -72 to -29 and DNase I footprinting experiments show two specific interaction products which individually or cooperatively protect the DNA sequence from about -60 to -30. These components are essential for TM gene transcription since affinity fractionation of nuclear extracts with a duplex oligonucleotide corresponding to -72 to -29 depletes the above interaction products and specifically inhibits in vitro transcription activity of the promoter, whereas addition of the eluted components specifically restores in vitro transcription activity of the promoter. Electrophoretic mobility shift assays with duplex oligonucleotides corresponding to -294 to -215, as well as -373 to -295 and DNase I footprinting experiments show two specific interaction products which individually bind to the two subdomains but not -72 to -29 and protect the coding and noncoding strands from -245 to -225, and the noncoding strand from -337 to -314, respectively. Transient expression studies reveal that the TM promoter construct starting at -51 and including the TATA box is responsive to TNF only in cell lines exhibiting sensitivity of the endogenous receptor gene to cytokine, whereas other promoter constructs possessing a TATA box sequence are insensitive to TNF in all cell types. Based upon the above data, the regulatory events involved in TNF-dependent transcriptional regulation of the TM gene can be defined with the experimental tools and conceptual framework developed by the present investigation.

Animals↗

PubMed1996

Regulation of the mouse inducible-type nitric oxide synthase gene promoter by interferon-gamma, bacterial lipopolysaccharide and NG-monomethyl-L-arginine.

Cytokines and bacterial lipopolysaccharides (LPSs) stimulate nitric oxide production in macrophages by inducing transcription of the gene coding for the inducible isoform of nitric oxide synthase (iNOS). We have cloned the mouse iNOS gene promoter and analysed its structural features and its response to interferon-gamma (IFN-gamma) and Escherichia coli LPS in RAW 264.7 mouse macrophage-like cells. Transcription of a recombinant reporter gene including the promoter and 4 kb of its 5'-flanking DNA, linked to the bacterial chloramphenicol acetyltransferase (CAT) reporter gene, is stimulated by IFN-gamma and, more efficiently, by LPS upon transient transfection in RAW 264.7 cells. Two upstream DNA regions are required for maximal promoter activation of LPS: the first maps between positions -1541 and -775 and the other between -420 and -47, with respect to the major transcriptional start site of the iNOS gene. The upstream-most region also mediates promoter trans-activation by IFN-gamma. As reported earlier for transcription of the endogenous iNOS gene, combined stimulation of RAW 264.7 cells with IFN-gamma and LPS results in lower activation of the transfected promoter, when compared with LPS alone. NG-Monomethyl-L-arginine, a competitive inhibitor of nitric oxide synthase activity, enhances iNOS gene mRNA induction and promoter activation by IFN-gamma and LPS, indicating that nitric oxide can influence negatively the reponsiveness of this gene to inducers. These results suggest the possibility of a negative regulatory feedback exerted by iNOS on the transcriptional activation of its own gene.

Animals↗

PubMed1993

Interaction of a redox-sensitive DNA-binding factor with the 5'-flanking region of the micF gene in Escherichia coli.

The product of the micF gene is an endogenous antisense RNA which down-regulates the expression of a major outer membrane protein, OmpF, in E. coli. We report here that two DNA-binding factors compete for the same site in the promoter region of the micF gene: RSBF, a high-affinity redox-sensitive DNA-binding factor that responds to an active oxygen species other than hydrogen peroxide or superoxide anions; and HRBF a heat-resistant DNA-binding factor. Both RSBF and HRBF bind to the same DNA sequence, 5'-TTAAAATCAATAACTTATTCTTAA3-', located upstream of the transcription start site of the micF gene. We present evidence that RSBF could be the controlling factor of a novel regulon involved in the response to oxidative stress in E. coli.

Bacterial Outer Membrane Proteins↗

PubMed2000

Cardiac aldosterone production in genetically hypertensive rats.

Aldosterone is synthesized in extra-adrenal tissues, both blood vessels and brain. We undertook the present study to determine whether the rat heart produces aldosterone and to investigate the effects of adrenalectomy, ACE inhibition, and angiotensin II on aldosterone synthesis in the heart. To clarify the pathophysiological role of cardiac aldosterone in the hypertensive heart, we compared the synthesis of aldosterone in the hearts of stroke-prone spontaneously hypertensive rats (SHRSP) with that in Wistar-Kyoto rats. The effects of the aldosterone antagonist spironolactone on myocardial hypertrophy in adrenalectomized SHRSP were also studied. Isolated rat hearts were perfused for 2 hours, and the perfusate was analyzed with HPLC and mass spectrometry. The activity of aldosterone synthase was estimated on the basis of the conversion of [(14)C]deoxycorticosterone to [(14)C]aldosterone. The levels of aldosterone synthase gene (CYP11B2) mRNA were determined with competitive polymerase chain reaction. Aldosterone production, the activity of aldosterone synthase, and the expression of CYP11B2 mRNA were increased in hearts from adrenalectomized rats and rats treated with angiotensin II. ACE inhibitors decreased cardiac aldosterone synthesis. Cardiac aldosterone, aldosterone synthase activity, and CYP11B2 mRNA levels in hearts from 2- and 4-week-old SHRSP were significantly greater than those of age-matched Wistar-Kyoto rats. Spironolactone prevented cardiac hypertrophy in adrenalectomized SHRSP. These results suggest that the rat heart produces aldosterone and that endogenous cardiac aldosterone may affect cardiac function and hypertrophy in hypertension in rats.

Adrenalectomy↗

PubMed2004

Role of Ets-2 in the regulation of heme oxygenase-1 by endotoxin.

Ets proteins play a vital role in the regulation of mammalian immunity, and family members Ets-1 and Ets-2 regulate a variety of genes that participate in the propagation of an inflammatory response. Heme oxygenase (HO)-1, although acutely induced by inflammatory stimuli, has cytoprotective properties and prevents an exaggerated inflammatory response. Ets-1 and Ets-2 both induce HO-1 promoter activity; however, Ets-2 was a more potent transactivator of HO-1 in macrophages. A potent inflammatory mediator, bacterial lipopolysaccharide (LPS), induced Ets-2 at the mRNA and protein level, and this induction preceded the up-regulation of HO-1. To further delineate the role of Ets-2 in regulating HO-1 transcription, we performed HO-1 promoter analysis studies in macrophages. Deletion mutants down to -137/+74 maintained an activity analogous to that of the largest construct, -4045/+74. Further deletion constructs (starting with -117/+74) showed a significant reduction in promoter activity when co-transfected with Ets-2 or exposed to LPS. Promoter sequence analysis revealed two putative Ets binding sites (EBSs) in this region, and mutation of these sites showed that EBS -93, more than EBS -125, was critical for full HO-1 promoter activity. Additional studies showed that EBS -93 binds Ets-2 and that mutation of the DNA binding domain of Ets-2 entirely prevented transactivation of HO-1. Finally, overexpression of a dominant negative form of Ets-2 blunted HO-1 promoter induction by LPS, and kinase inhibitors (PI3K more than JNK) that reduced Ets-2 expression markedly decreased endogenous HO-1 expression. Our data provide evidence that Ets-2 contributes to the up-regulation of HO-1 by the potent inflammatory stimulus LPS in macrophages.

Animals↗

PubMed1996

Demonstration and characterization of a transport system capable of lysine and leucine absorption that is encoded for in porcine jejunal epithelium by expression of mRNA in Xenopus laevis oocytes.

Defolliculated Xenopus laevis oocytes were injected with size-fractionated poly(A)+ RNA (RNA) isolated from the jejunal epithelium of growing pigs (average BW 33.8 kg) to identify proteins capable of Na+ -independent amino acid transport. The ability of oocytes to absorb L-lysine (lysine) or L-leucine (leucine) from Na+- free media was quantified in oocytes after injection of RNA fractions or water. Specific RNA fractions were identified that induced saturable uptake of lysine (Kt = 52 microM) and leucine (Kt = 97 microM), whereas endogenous oocyte uptake was not saturable. Induced uptake of .05 mM lysine by oocytes was inhibited (P < .05) 68.1% by 5 mM leucine and 38.9% by .2 mM L-cystine (cystine). Induced uptake of .05 mM leucine was inhibited (P < .05) 83.1% by 5 mM lysine and 23.2% by .2 mM cystine. Although not significant (P > .05), 5 mM L-glutamate (glutamate) quantitatively stimulated the induced uptake of .05 mM lysine by 18.8% and the induced uptake of .05 mM leucine by 60%. To identify mRNA species responsible for this bo,+ transporter-like activity, oocytes were co-injected with the RNA fractions and degenerate DNA oligomers complementary (antisense) to the cloned human kidney bo,+ amino acid transporter, or (as a negative control) with a DNA oligomer complementary to the rabbit intestinal Na+/glucose cotransporter, or with water. Only those oocytes injected with two specific RNA fractions and the antisense DNA oligomer complementary to the bo,+ transporter displayed reduced (P < .05) uptake of lysine (45.7, 55.4%) and leucine (44.1, 65.9%). These results indicate that messenger RNA encoding for a protein capable of stimulating the competitive absorption of lysine and leucine is expressed by the jejunal epithelia of growing pigs.

Animals↗

PubMed2006

Arginine supplementation induces myoblast fusion via augmentation of nitric oxide production.

The semi-essential amino acid, L-arginine (L-Arg), is the substrate for endogenous synthesis of nitric oxide, a molecule that is involved in myoblast proliferation and fusion. Since L-Arg supply may limit nitric oxide synthase (NOS) activity in endothelial cells, we examined L-Arg supplementation in differentiating mouse myoblasts and tested the hypothesis that L-Arg exerts direct effects on myoblast fusion via augmentation of endogenous nitric oxide production. C(2)C(12) myoblasts in differentiation media received one of the following treatments for 120 h: 1 mM L-Arg, 0.1 mM N-nitro-L-arginine methyl ester (L-NAME), L-Arg + L-NAME, 10 mM L-Lysine, or no supplement (Control). Cultures were fixed and stained with hematoxylin and eosin for microphotometric image analysis of myotube density, nuclear density, and fusion index (% of total nuclei in myotubes). Endogenous production of nitric oxide during the treatment period peaked between 24 and 48 h. L-Arg amplified nitric oxide production between 0 and 24 h and increased myotube density, total nuclei number, and nuclear fusion index. These L-Arg effects were prevented by the NOS inhibitor, L-NAME. Further, L-Lysine, a competitive inhibitor of L-Arg uptake, repressed nitric oxide production and reduced myotube density and fusion index. In summary, L-Arg augments myotube formation and increases nitric oxide production in a process limited by cellular L-Arg uptake.

Animals↗

PubMedDate not supplied

Antisense tumor therapy (a dream under construction).

Cancer, as a genetic disease, is a logical target for gene-oriented therapy--either by replacing the missing/nonfunctioning gene or by depressing the activity of an unwanted gene. The latter is really the inhibition of gene expression using oligonucleotide-based or "antisense" treatment. There are several strategies to achieve this: anti-gene or anti-code with triplex formation; ribozyme with endogenous catalytic RNase activity; antisense with oligonucleotides through steric inhibition or RNaseH activation; and sense strategy to inhibit or trap proteins by nucleic acids. There are two essential partners of the approach: targeted sequence in the unwanted gene/molecule and the complementary antisense oligo (-ribo- or -deoxyribonucleotide). The antisense sequences require chemical modifications (mostly on the phosphodiester backbone, less in the sugar or in the bases) to avoid nucleases, to form complexes for better delivery (in the organism and also in the cell). The activity of the unwanted target should be non-randomly associated with cancer (e.g. abl/ber). Both aspects of antisense treatment require further improvements to get longer lasting and real sequence-specific antitumor effect which could be competitive with the available therapeutic modalities.

DNA, Antisense↗

PubMed1989

Functional vasoactive intestinal polypeptide (VIP) receptors in human neuroblastoma subclones that contain VIP precursor mRNA and release VIP-like substances.

Three phenotypically distinct subclones (SH-SY-5Y, SH-EP, SH-IN) of the human neuroblastoma cell line SK-N-SH were found to possess vasoactive intestinal polypeptide (VIP) precursor mRNA, release immunoreactive VIP, and express high-affinity VIP receptors coupled to adenylate cyclase. The apparent molecular mass for the receptor polypeptide, as determined by covalent cross-linking of 125I-VIP, was 49 kDa. After 2 days in culture, a concentration of immunoreactive VIP equivalent to the binding affinity of VIP to its receptor was found in the medium in two of these clones (SH-IN and SH-EP). Conditioned medium from SH-IN cells competitively displaced 125I-VIP binding and increased cAMP levels in SH-EP cells, indicating that all of the necessary components for a potential autocrine action of VIP exist in SK-N-SH cells. After numerous cell passages, the SH-EP subclone converted to a distinct phenotype in which VIP precursor mRNA and VIP immunoreactivity in the cell and medium were no longer detectable. In correlation, the VIP receptor number increased, and the EC50 for VIP stimulation of cAMP production shifted to a lower concentration. This points to the possibility that the continuous presence of endogenous VIP in earlier passage SH-EP cells causes a modification in VIP receptor number and cell responsiveness to VIP.

Blotting, Northern↗