Investigations of antisense oligonucleotides targeted against bcl-2 RNAs

The purpose of this article is to describe how nurse instructors understand nursing and the necessary conditions for high quality nursing care. The approach of the study was inductive and based on phenomenology and a sociological field research method, i.e. grounded theory. It emerged that the core of nursing is a process, termed here caring, with three stages. Three types of caring emerged. Understanding the client, the art of nursing, co-operation between the nurse and other health care professionals were found to be necessary conditions for a high quality nursing care and it was also found that it is not possible to separate caring from the society and its history.     

Phosphorothioate antisense oligonucleotides induce the formation of nuclear bodies

Antisense oligonucleotides are powerful tools for the in vivo regulation of gene expression. We have characterized the intracellular distribution of fluorescently tagged phosphorothioate oligodeoxynucleotides (PS-ONs) at high resolution under conditions in which PS-ONs have the potential to display antisense activity. Under these conditions PS-ONs predominantly localized to the cell nucleus where they accumulated in 20-30 bright spherical foci designated phosphorothioate bodies (PS bodies), which were set against a diffuse nucleoplasmic population excluding nucleoli. PS bodies are nuclear structures that formed in cells after PS-ON delivery by transfection agents or microinjection but were observed irrespectively of antisense activity or sequence. Ultrastructurally, PS bodies corresponded to electron-dense structures of 150-300 nm diameter and resembled nuclear bodies that were found with lower frequency in cells lacking PS-ONs. The environment of a living cell was required for the de novo formation of PS bodies, which occurred within minutes after the introduction of PS-ONs. PS bodies were stable entities that underwent noticeable reorganization only during mitosis. Upon exit from mitosis, PS bodies were assembled de novo from diffuse PS-ON pools in the daughter nuclei. In situ fractionation demonstrated an association of PS-ONs with the nuclear matrix. Taken together, our data provide evidence for the formation of a nuclear body in cells after introduction of phosphorothioate oligodeoxynucleotides.     

Triple helix stabilization properties of oligonucleotides containing 8-amino-2'-deoxyguanosine

Analysis of the Hoogsteen base pairs of guanine and 8-aminoguanine with cytosine in the gase phase shows a strong stabilization of the 8-aminoguanine.cytosine base pair due to the formation of an extra H-bond. Melting profiles of oligonucleotides carrying 8-amino-2'-deoxyguanosine confirm the triple helix stabilization properties of 8-aminoguanine.     

In vivo administration of c-Fos antisense oligonucleotides accelerates amygdala kindling

Following the subcutaneous (s.c.) administration of nicotinamide (10 mmol/kg), the brain and CSF levels of nicotinamide were increased to millimolar concentrations, but the concentrations of N-methylnicotinamide (NMN) in the CSF, and of NMN and NAD+ in brain tissue were not significantly altered. Concomitantly, nicotinamide caused increases of the choline levels in the venous brain blood. In hippocampal slices, nicotinamide (1-10 mM) induced choline release in a calcium- and mepacrine-sensitive manner and, in [3H]choline-labelled slices, increased the levels of [3H]lyso-phosphatidylcholine and [3H]glycerophosphocholine. We conclude that nicotinamide enhances brain choline concentrations by mobilising choline from choline-containing phospholipids, presumably via activation of phospholipase A2, while the formation of NMN does not contribute to this effect.     

Elucidation of gene function using C-5 propyne antisense oligonucleotides

Identification of human disease-causing genes continues to be an intense area of research. While cloning of genes may lead to diagnostic tests, development of a cure requires an understanding of the gene's function in both normal and diseased cells. Thus, there exists a need for a reproducible and simple method to elucidate gene function. We evaluate C-5 propyne pyrimidine modified phosphorothioate antisense oligonucleotides (ONs) targeted against two human cell cycle proteins that are aberrantly expressed in breast cancer: p34cdc2 kinase and cyclin B1. Dose-dependent, sequence-specific, and gene-specific inhibition of both proteins was achieved at nanomolar concentrations of ONs in normal and breast cancer cells. Precise binding of the antisense ONs to their target RNA was absolutely required for antisense activity. Four or six base-mismatched ONs eliminated antisense activity confirming the sequence specificity of the antisense ONs. Antisense inhibition of p34cdc2 kinase resulted in a significant accumulation of cells in the Gap2/mitosis phase of the cell cycle in normal cells, but caused little effect on cell cycle progression in breast cancer cells. These data demonstrate the potency, specificity, and utility of C-5 propyne modified antisense ONs as biological tools and illustrate the redundancy of cell cycle protein function that can occur in cancer cells.     

Sustained reduction of neointima with c-myc antisense oligonucleotides in saphenous vein grafts

BACKGROUND: Treatment of saphenous veins with c-myc antisense oligomers during preparation for grafting reduces medial cellular proliferation and macrophage infiltration, and preserves medial smooth muscle content at 3 days. Accordingly, the purpose of this study was to examine whether c-myc antisense oligomers have an impact on late vein graft remodeling. METHODS: Sixty-two pigs underwent unilateral saphenous vein-carotid artery interposition grafting. Harvested veins were incubated either in saline (control group) or 20-micromol/L or 200-micromol/L concentrations of c-myc antisense oligomers (treated groups) for 30 minutes intraoperatively. Three months after surgery, vein graft histology was assessed. RESULTS: Forty-five of 62 randomized animals survived the experiment; no differences in animal survival or graft patency among the groups were observed (p = NS, chi2). C-myc antisense oligomers significantly decreased neointimal and wall thickness, as well as increased lumenal index, in treated groups (p<0.04, p<0.03, and p<0.001, respectively, analysis of variance). In contrast, there was no difference in medial thickness or perivascular wound healing. CONCLUSION: Intraoperative treatment of saphenous veins with c-myc antisense oligomers decreased neointimal formation at 3 months after grafting. In conjunction with our previous reports, these findings suggest that early inhibition of cellular proliferation and inflammatory infiltration results in a sustained reduction in neointimal formation and favorable graft remodeling.     

Regulation of in vitro gene expression using antisense oligonucleotides or antisense expression plasmids transfected using starburst PAMAM dendrimers

Starburst polyamidoamine (PAMAM) dendrimers are a new type of synthetic polymer characterized by a branched spherical shape and a high density surface charge. We have investigated the ability of these dendrimers to function as an effective delivery system for antisense oligonucleotides and 'antisense expression plasmids' for the targeted modulation of gene expression. Dendrimers bind to various forms of nucleic acids on the basis of electrostatic interactions, and the ability of DNA-dendrimer complexes to transfer oligonucleotides and plasmid DNA to mediate antisense inhibition was assessed in an in vitro cell culture system. Cell lines that permanently express luciferase gene were developed using dendrimer mediated transfection. Transfections of antisense oligonucleotides or antisense cDNA plasmids into these cell lines using dendrimers resulted in a specific and dose dependent inhibition of luciferase expression. This inhibition caused approximately 25-50% reduction of baseline luciferase activity. Binding of the phosphodiester oligonucleotides to dendrimers also extended their intracellular survival. While dendrimers were not cytotoxic at the concentrations effective for DNA transfer, some non-specific suppression of luciferase expression was observed. Our results indicate that Starburst dendrimers can be effective carriers for the introduction of regulatory nucleic acids and facilitate the suppression of the specific gene expression.     

Studies on the synthesis of oligonucleotides containing photoreactive nucleosides: 2-azido-2'-deoxyinosine and 8-azido-2'-deoxyadenosine

Oligonucleotides containing the photoreactive nucleosides 2-azido-2'-deoxyinosine and 8-azido-2'-deoxyadenosine have been prepared using protected 2-fluoro-2'-deoxyinosine and 8-bromo-2'-deoxyadenosine phosphoramidites. After the assembly of the oligonucleotides, the nucleoside derivatives are converted to the corresponding azido derivatives by treatment with lithium azide in dry DMF. Deprotection of oligonucleotides carrying these azidonucleosides is performed with concentrated ammonia at room temperature.     

In vivo suppression of the renal Na+/Pi cotransporter by antisense oligonucleotides

A 20-mer phosphorothioate oligonucleotide (AS1) was designed to hybridize to the message for the rat kidney sodium phosphate cotransporter NaPi-2 close to the translation initiation site. Single intravenous doses of this oligonucleotide were given to rats maintained on a low phosphorus diet to increase NaPi-2 expression. At 3 days after oligonucleotide infusion, rats receiving 2.5 micromol of AS1 exhibited a reduction in renal NaPi-2 to cyclophilin mRNA ratio by 40% +/- 17%, and rats receiving 7.5 micromol of AS1 exhibited a reduction in NaPi-2 to cyclophilin mRNA ratio by 46% +/- 21%. Reversed-sequence AS1 was without effect. The higher dose of 7.5 micromol of AS1 also reduced the rate of phosphate uptake into renal brush border membrane vesicles and the expression of NaPi-2 protein detected by Western blotting in these vesicles. Reversed sequence AS1 was again without effect on these parameters. These results suggest that systemically infused oligonucleotides can exert antisense effects in the renal proximal tubule.     

Selective inhibition of cell-free translation by oligonucleotides targeted to a mRNA hairpin structure

Using an in vitro selection approach we have previously isolated oligodeoxy aptamers that can bind to a DNA hairpin structure without disrupting the double-stranded stem. We report here that these oligomers can bind to the RNA version of this hairpin, mostly through pairing with a designed 6 nt anchor. The part of the aptamer selected against the DNA hairpin did not increase stability of the RNA-aptamer complex. However, it contributed to the binding site for Escherichia coli RNase H, leading to very efficient cleavage of the target RNA. In addition, a 2'- O -methyloligoribonucleotide analogue of one selected sequence selectively blocked in vitro translation of luciferase in wheat germ extract by binding to the hairpin region inserted upstream of the initiation codon of the reporter gene. Therefore, non-complementary oligomers can exhibit antisense properties following hybridization with the target RNA. Our study also suggests that in vitro selection might provide a means to extend the repertoire of sequences that can be targetted by antisense oligonucleotides to structured RNA motifs of biological importance.     

Effect of cytokine-specific antisense oligonucleotides on the immunoglobulin production by rat spleen cells in vitro

We examined in this study the regulation of immunoglobulin (IgM, IgG, IgE) production by spleen cells from N brasiliensis infected rats following addition of antisense oligodeoxynucleotides (ODNs). The ODNs were selected near the AUG initiation codon of mRNA specific for interleukin 4 (IL-4) or interleukin 2 (IL-2). Results show that addition of antisense to IL-4 inhibited IgE production, while the production of IgG and IgM increased. The use of sense IL-4 sequence did not affect immunoglobulin production. In contrast, the use of antisense IL-2 ODN induced an enhancement of IgE as well as of IgM and IgG responses. Both the Ig secretion in culture supernatants and the number of Ig secreting cells, as detected by an Elispot assay, were influenced by the presence of antisense IL-4 ODNs. These results clearly show the involvement of IL-2 and IL-4 in the regulation of isotype selection during antibody synthesis and that IL-2 and IL-4 do operate differently on IgE production. They also argue that antisense strategy represents a useful tool for the antibody regulation.     

Analysis of antisense oligonucleotides by on-capillary isotachophoresis and capillary polymer sieving electrophoresis

An attempt was made to evaluate the stability of an antisense oligonucleotide against nucleases present in HBL 100ras cells. To detect nanomolar concentrations of the oligonucleotide, a sensitive detection system was required. A combination of capillary electrophoresis/laser-induced fluorescence (CE-LIF) with fluorescence derivatization did not improve the sensitivity significantly and also resulted in loss of separation of the derivatized sample. On-column isotachophoresis for the preconcentration of oligonucleotide samples in DB-17 coated capillaries filled with hydroxyethyl cellulose solution could be an alternative. The isotachophoresis (ITP) step allows injection of up to 40% of the capillary volume without loss in peak resolution and peak efficiency. Using ITP-capillary polymer sieving electrophoresis (CPSE), the limit of quantitation at a signal-to-noise ratio of 10 was 73 ng/mL for a 12-mer oligonucleotide. Using these conditions, the gain in sensitivity was 125.     

Efficient 5''-end labeling of oligonucleotides containing self-complementary sequences

BACKGROUND: Auxiliary liver transplantation offers an alternative method to conventional transplantation in acute liver failure. It is especially challenging for children because lifelong immunosuppression may be avoided. However, experience with this procedure is rare and there is controversy about whether to place the graft orthotopically or heterotopically. METHODS: We present the case of a 3-year-old boy with acute liver failure due to non-ABC hepatitis complicated by aplastic anemia who underwent auxiliary liver transplantation. Segments 2 and 3 of the graft were implanted heterotopically in the right lower abdomen. RESULTS: Good liver function was immediately restored. Aplastic anemia resolved 3 weeks after transplantation. Immunosuppressive therapy was discontinued after 14 months, and the graft was left to atrophy. Thirty-nine months after transplantation the boy is alive and well with normal liver function tests and normal blood cell counts. CONCLUSIONS: Heterotopic auxiliary liver transplantation allowed recovery of the native liver in a child with acute liver failure and aplastic anemia due to non-ABC hepatitis.     

8-Methoxypsoralen photoadduct formation in complementary oligonucleotides containing a cross-linkable site

The complete profile of 8-methoxypsoralen photoadduct formation in complementary oligonucleotides (5'-GAGTATGAG and 5'-CATAC) has been determined. Equimolar solutions of the oligonucleotides were irradiated at 4 degrees C in order to stabilize the mini-double helix. Photomodified oligonucleotides were separated by reversed phase chromatography on a Vydac C4 column. Photoadduct formation favored the 5'TAT site in the 9mer over the 5'ATA site in the 5mer by a factor of two. Split-dose studies showed that the monoadducts formed on GAGTATGAG were preferentially converted to cross-links by an additional UVA exposure.     

Structures of oligonucleotides containing 5-(methoxymethyl)-2'-deoxyuridine determined by NMR spectroscopy

Base pairing of 5-(methoxymethyl)-2'-deoxyuridine (MMdU) opposite either adenine or guanine in a seven base pair oligonucleotide duplex has been studied by NMR spectroscopy. When paired with A, we observe that the MMdU.A base pair adopts Watson-Crick geometry. The methoxymethyl substituent is not held in a fixed conformation and may rotate around the C5-CH2 and CH2-O bonds. Examination of the potential energy as a function of rotation around these bonds indicates the presence of four low energy conformations. No hydrogen bonding is indicated for the methoxymethyl substituent, and the four potential minima result from reduced steric clash. For the MMdU.G base pair, the two bases adopt a wobble geometry which does not change with increasing solvent pH. Similarly, we find four low energy conformations for the methoxymethyl substituent in the major groove of the DNA helix.     

Enhanced downregulation of the p75 nerve growth factor receptor by cholesteryl and bis-cholesteryl antisense oligonucleotides

The effects of conjugating cholesterol to either or both ends of a phosphorothioate (PS) oligonucleotide were analyzed in terms of cellular uptake and antisense efficacy. The oligo sequence was directed against the p75 nerve growth factor receptor (p75), and was tested in differentiated PC12 cells, which express high levels of this protein. The addition of a single cholesteryl group to the 5'-end significantly increased cellular uptake and improved p75 mRNA downregulation compared with the unmodified PS oligo. However, only a minor degree of downregulation of p75 protein was obtained with 5' cholesteryl oligos. Three different linkers was used to attach the 5' cholesteryl group but were found not to have any impact on efficacy. Addition of a single cholesteryl group to the 3'-end led to greater p75 mRNA downregulation (31%) and p75 protein downregulation (28%) than occurred with the 5' cholesteryl oligos. The biggest improvement in antisense efficacy, both at the mRNA and protein levels, was obtained from the conjugation of cholesterol to both ends of the oligo. One of the bischolesteryl oligos was nearly as effective as cycloheximide at decreasing synthesis of p75. The bis-cholesteryl oligos also displayed significant efficacy at 1 microM, whereas the other oligos required 5 microM to be effective. The enhanced efficacy of bis-cholesteryl oligos is likely to be due to a combination of enhanced cellular uptake and resistance to both 5' and 3' exonucleases.     

Putative roles for the inducible transcription factor c-fos in the central nervous system: studies with antisense oligonucleotides

Although immediate-early genes such as c-fos are widely believed to play an important role in neuroplasticity, there is limited evidence to support involvement in the initiation of molecular events leading to medium- and long-term changes in brain function following a stimulus. Results using techniques such as transgenic knockout of the gene are often difficult to interpret. Antisense oligonucleotide technology offers an alternative. Infusion of antisense oligonucleotide to modify the expression of c-fos in the brain results in dramatic changes in rotation behaviour in animals challenged with psychostimulant drugs such as amphetamine. Similarly, the knockdown of c-fos expression using antisense oligonucleotides can also alter the rate of amygdala kindling in response to electrical stimulation of the brain. While studies using antisense oligonucleotides to knockdown c-fos expression provide evidence that the expression of c-fos plays an important role in regulating neuronal function, the use of antisense nucleotides has limitations and experiments must be very carefully controlled. Many details of antisense oligonucleotide actions remain unknown.     

A competitive enzyme hybridization assay for plasma determination of phosphodiester and phosphorothioate antisense oligonucleotides

An enzyme competitive hybridization assay was developed and validated for determination of mouse plasma concentrations of a 15mer antisense phosphodiester oligodeoxyribonucleotide and of two phosphorothioate analogs. Assays were performed in 96-well microtiter plates. The phosphodiester sense sequence was covalently bound to the microwells. The 5'-biotinylated antisense sequence was used as tracer. The principle of the assay involves competitive hybridization of tracer and antisense nucleotide to the solid phase-immobilized sense oligonucleotide. Solid phase- bound tracer oligonucleotide was assayed after reaction with a streptavidin-acetylcholinesterase conjugate, using the colorimetric method of Ellman. As in competitive enzyme immunoassays, coloration was inversely related to the amount of analyte initially present in the sample. The limit of quantification was 900 pM for phosphodiester antisense oligonucleotide using a 100 microl volume of plasma without extraction. Cross-reactivity was negligible after a four base deletion in either the 3'or 5'position. The assay was simple and sensitive, suitable for in vitro screening of oligonucleotide hybridization potency in biological fluids and for measuring the plasma pharmacokinetics of phosphorothioate and phosphodiester sequences.