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Biomedical subjects

J Summerton

Publications and source records attributed to J Summerton.

At least 19 recordsLinked to original sources
PubMed2001

Nuclear antisense effects of neutral, anionic and cationic oligonucleotide analogs.

The antisense activity of oligomers with 2'-O-methyl (2'-O-Me) phosphorothioate, 2'-O-methoxyethyl (2'-O-MOE) phosphorothioate, morpholino and peptide nucleic acid (PNA) backbones was investigated using a splicing assay in which the modified oligonucleotides blocked aberrant and restored correct splicing of modified enhanced green fluorescent protein (EGFP) precursor to mRNA (pre-mRNA), generating properly translated EGFP. In this approach, antisense activity of each oligomer was directly proportional to up-regulation of the EGFP reporter. This provided a positive, quantitative readout for sequence-specific antisense effects of the oligomers in the nuclei of individual cells. Nuclear localization of fluorescent labeled oligomers confirmed validity of the functional assay. The results showed that the free uptake and the antisense efficacy of neutral morpholino derivatives and cationic PNA were much higher than that of negatively charged 2'-O-Me and 2'-O-MOE congeners. The effects of the PNA oligomers were observed to be dependent on the number of L-lysine (Lys) residues at the C-terminus. The experiments suggest that the PNA containing Lys was taken up by a mechanism similar to that of cell-penetrating homeodomain proteins and that the Lys tail enhanced intracellular accumulation of PNA oligomer without affecting its ability to reach and hybridize to the target sequence.

Anions↗
PubMed2000

Restoration of hemoglobin A synthesis in erythroid cells from peripheral blood of thalassemic patients.

Mononuclear cells from peripheral blood of thalassemic patients were treated with morpholino oligonucleotides antisense to aberrant splice sites in mutant beta-globin precursor mRNAs (pre-mRNAs). The oligonucleotides restored correct splicing and translation of beta-globin mRNA, increasing the hemoglobin (Hb) A synthesis in erythroid cells from patients with IVS2-654/beta(E), IVS2-745/IVS2-745, and IVS2-745/IVS2-1 genotypes. The maximal Hb A level for repaired IVS2-745 mutation was approximately 30% of normal; Hb A was still detectable 9 days after a single treatment with oligonucleotide. Thus, expression of defective beta-globin genes was repaired and significant level of Hb A was restored in a cell population that would be targeted in clinical applications of this approach.

Cell Nucleus↗
PubMed2000

Antiproliferative effects of steric blocking phosphorodiamidate morpholino antisense agents directed against c-myc.

The phosphorodiamidate Morpholino oligomers (PMO) are a new class of antisense agents that inhibit gene expression by binding to RNA and sterically blocking processing or translation. In a search for a Morpholino agent that would inhibit cell proliferation, it was found that oligomers directed against c-myc, a gene involved in control of the cell cycle, were effective. The sequence specificity and mechanism of action of one agent were determined. The 20-mer 126 lowers c-myc protein levels in treated cells and arrests cells in G0/G1 of the cell cycle. It also acts at the RNA level to inhibit normal pre-mRNA splicing and instead produces an aberrantly spliced mRNA. Irrelevant and mispair control oligomers indicated that the observed antiproliferative effect was sequence specific. This was confirmed in a reporter gene model system using a c-myc 5'-untranslated region (5'-UTR) fused to a cDNA copy of the insect luciferase gene. We conclude that 126 is acting through an antisense mechanism involving Watson-Crick hydrogen bonding to its target RNA. A specific antisense agent directed against a cell cycle-associated gene mRNA may be useful as a therapeutic in diseases characterized by excess cell proliferation, such as restenosis following balloon angioplasty or cancer.

Animals↗
PubMed1999

Morpholino antisense oligomers: the case for an RNase H-independent structural type.

RNase H-competent phosphorothioates (S-DNAs) have dominated the antisense field in large part because they offer reasonable resistance to nucleases, they afford good efficacy in cell-free test systems, they can be targeted against sites throughout the RNA transcript of a gene, and they are widely available from commercial sources at modest prices. However, these merits are counterbalanced by significant limitations, including: degradation by nucleases, poor in-cell targeting predictability, low sequence specificity, and a variety of non-antisense activities. In cell-free and cultured-cell systems where one wishes to block the translation of a messenger RNA coding for a normal protein, RNase H-independent morpholino antisense oligos provide complete resistance to nucleases, generally good targeting predictability, generally high in-cell efficacy, excellent sequence specificity, and very preliminary results suggest they may exhibit little non-antisense activity.

Alternative Splicing↗
PubMed1997

A specificity comparison of four antisense types: morpholino, 2'-O-methyl RNA, DNA, and phosphorothioate DNA.

Cell-free translation studies were carried out to compare the efficacy and specificity of four antisense structural types: DNA, phosphorothioate DNA (S-DNA), 2'-O-methyl RNA, and Morpholino oligos, a novel antisense structural type. In these studies, translational inhibition was assessed for two 20-mers of each structural type, where one 20-mer was complementary to its target sequence in rabbit alpha-globin mRNA and the other 20-mer contained three mispairs to that same target sequence. It is shown that at low concentration of antisense oligomer (50 nM) all four types provide high specificity, but the Morpholino oligos and 2'-O-methyl RNA afford better efficacy. At high oligomer concentration (3.5 microM), all four types provide high efficacy, but the Morpholino oligos and 2'-O-methyl RNA provide substantially better specificity than the DNA and S-DNA.

Animals↗
PubMed1997

Morpholino antisense oligomers: design, preparation, and properties.

Antisense promised major advances in treating a broad range of intractable diseases, but in recent years progress has been stymied by technical problems, most notably inadequate specificity, ineffective delivery into the proper subcellular compartment, and unpredictable activity within cells. Herein is an overview of the design, preparation, and properties of Morpholino oligos, a novel antisense structural type that solves the sequence specificity problem and provides high and predictable activity in cells. Morpholino oligos also exhibit little or no nonantisense activity, afford good water solubility, are immune to nucleases, and are designed to have low production costs.

Animals↗
PubMed1997

Morpholino and phosphorothioate antisense oligomers compared in cell-free and in-cell systems.

Morpholino and phosphorothioate (S-DNA) antisense oligos were compared in both cell-free and in-cell translation systems. In the most stringent test of specificity in the cell-free system, a globin-targeted S-DNA oligo was found to inhibit its target sequence at concentrations of 10 nM and above, but the sequence-specific component of this inhibition dropped below 50% at concentrations of 100 nM and above. A corresponding Morpholino oligo achieved even higher inhibition at 10 nM, but in contrast to the S-DNA, with the Morpholino, the sequence-specific component of this inhibition remained above 93% at a concentration of 3000 nM. In this same cell-free test system, several S-DNA oligos exhibited substantial undesired nonantisense effects at concentrations of 300 nM and above, whereas corresponding Morpholino oligos exhibited little or no nonantisense activity through a concentration of 3000 nM. In scrape-loaded HeLa cells, both globin-targeted and HBV-targeted S-DNAs (both antisense and control oligos) generally failed to achieve significant translational inhibition at extracellular concentrations up to 3000 nM. In contrast, the Morpholino oligos achieved effective and specific translational inhibition at extracellular concentrations ranging from 30 nM to 3000 nM.

Animals↗
PubMed1997

Affinity purification of RNA: sequence-specific capture by nonionic morpholino probes.

Nucleic acid isolation for amplification-based diagnostics requires techniques that do not co-purify inhibitors of DNA polymerases. Also, other requirements for an ideal sample preparation technology include ease of use, capability for automation, high recovery and the use of nontoxic reagents. Affinity purification techniques provide high purification factor with minimal sample processing. Hybridization is the affinity interaction specific to nucleic acids and thus provides a uniquely advantageous method for purifying DNA or RNA for subsequent manipulation. Nonionic (morpholino) probes (Neu-Probes, AntiVirals, Corvallis, OR, USA) have several unique hybridization properties, including resistance to nucleases and the ability to hybridize independently of salt concentration. Therefore, such probes provide advantages over DNA probes for sample preparation by hybridization capture. Three formats for hybridization-based purification of human immunodeficiency virus (HIV) RNA were evaluated using RNA transcripts spiked into crude lysates of normal human plasma. Indirect capture used streptavidin-coated microparticles to capture hybrids of biotinylated capture probes and HIV RNA. Direct capture used particles precoated with probes. In addition, a novel method for acceleration of sequence-specific hybridization was developed and shown to give consistently high recoveries.

DNA-Directed DNA Polymerase↗
PubMed1996

A simple method for delivering morpholino antisense oligos into the cytoplasm of cells.

We report a simple and effective means for delivering Morpholino antisense oligos into the cytosol of cultured anchorage-dependent animal cells. This method, referred to as scrape-loading, is carried out in a matter of seconds, uses a common inexpensive laboratory implement, and has minimal detrimental impact on the cells. Using this delivery method, a Morpholino oligo present at 0.1 microM and 1 microM in the extracellular medium inhibited its targeted genetic sequence within cultured Hela cells at levels of 56% and 85%, respectively. Lack of inhibition by two control Morpholino oligos at concentrations up to 3 microM indicates good sequence specificity by this structural type. Also described is a test system for simple, rapid, and sensitive quantitation of antisense activity in cultured cells.

Animals↗
PubMed1991

Antibacterial activity and inhibition of protein synthesis in Escherichia coli by antisense DNA analogs.

Protein synthesis, which takes place within ribosomes, is essential for the survival of any living organism. Ribosomes are composed of both proteins and RNA. Specific interaction between the 3' end CCUCC sequence of prokaryotic 16S rRNA and a partially complementary sequence preceding the initiating codon of mRNA is believed to be a prerequisite for initiation of protein synthesis. Here we report the use of short (three to six nucleotides) synthetic DNA analogs complementary to this sequence to block protein synthesis in vitro and in vivo in Escherichia coli. In the DNA analogs the normal phosphodiester bond in the antisense DNA was replaced by methylcarbamate internucleoside linkages to enhance transport across plasma membranes. Of the analogs tested, those with the sequence AGG and GGA inhibit protein synthesis and colony formation by E. coli strains lacking an outer cell wall. Polyethylene glycol 1000 (PEG 1000) was attached to the 5' end of some of the test methylcarbamate DNAs to enhance solubility. Analogs of AGG and GGAG with PEG 1000 attached inhibited colony formation in normal E. coli. These analogs may be useful food additives to control bacterial spoilage and biomedically as antibiotics.

3T3 Cells↗
PubMed1988

Styles of coping with threat: implications for health.

We explored individual differences in health-seeking behavior and health status in a primary care population. Specifically, we compared high monitors (those who typically scan for threat-relevant information) with low monitors (those who typically ignore threat-relevant information), while controlling for depression. Overall, high monitors came to the physician with less severe medical problems than did low monitors. Nevertheless, high monitors reported equivalent levels of discomfort, dysfunction, and distress compared with low monitors. Furthermore, during the week following their visit, high monitors expressed less symptom improvement in both physical and psychological problems than did low monitors. Finally, high monitors demanded more tests, information, and counseling during their visit than did their low monitoring counterparts, yet desired a less active role in their own care. The theoretical and practical implications of these findings are discussed.

Adaptation, Psychological↗
PubMed1985

Effect of deoxycholic acid on the tumour incidence, distribution, and receptor status of colorectal cancer in the rat model.

It has previously been observed that 25% of human colorectal cancers contain specific receptors to deoxycholic acid (DCA). In the present study, the effect of intrarectal instillation of DCA on tumour number, distribution, size, and DCA receptor status was measured in rats receiving the colorectal carcinogen, azoxymethane. Rats treated with azoxymethane and intrarectal DCA developed significantly more colorectal cancers than rats receiving azoxymethane and intrarectal saline (median 11.5, range 8-17 vs. median 6.0, range 3-9 tumours/rat, respectively, p less than 0.01). This reflected a significantly higher number of tumours in the distal colon of the DCA-treated group (median 8.0, range 5-10 tumours/rat) compared to the saline-treated group (p less than 0.01). In those rats receiving DCA and azoxymethane, 5 of 12 tumours tested were found to be DCA receptor-positive, compared with only 1 of 11 in the saline and azoxymethane group. These results confirm the belief that DCA acts as a tumour promoter, and suggest a possible role for DCA receptors.

Animals↗
PubMed1984

Chimeric univalent antibodies for treating lymphoid malignancies.

A more effective use of antibody in treating cancer appears to require derivatives with enhanced cytotoxic potential. Working with anti-idiotype antibodies directed against neoplastic lymphocytes, we have shown previously that univalent antibody derivatives with intact Fc-regions can avoid antigenic modulation while retaining the ability to recruit cytotoxic effectors such as complement. Chimeric univalent antibodies represent an extension of this approach. To prepare them Fab' gamma from antibody is linked by thioether bonds to half-cystine in normal Ig of the species to undergo immunotherapy. The derivatives FabIgG and FabFc utilize IgG and Fc gamma respectively as the effector partners of the antibody Fab' gamma. They appear superior to parent antibody in their ability to invoke complement and K-cell killing of target lymphocytes. They show promise of being minimally immunogenic and, because they present homologous Fc, should prove efficient recruiters of host effector functions.

Animals↗
PubMed1983

Bile acid receptors in colorectal cancer.

Bile acids are thought to be involved in both the aetiology and development of colorectal cancer. In this study the existence of specific bile acid receptor proteins has been postulated. A receptor assay which involved labelling with 14C-deoxycholic acid was performed as well as autoradiography using 3H-deoxycholic acid. In an initial study resected colorectal cancer and adjacent histologically normal colorectal mucosa from 39 patients were studied, as were samples of normal gastric mucosa, cancers and benign colorectal tumours. Specific receptors to deoxycholic acid were detected in 12 (30.8 per cent) of the colorectal cancers, but in only 1 (2.6 per cent) of the samples from normal colorectal mucosa (X2 = 11.16, P less than 0.005). No deoxycholic acid receptors were detected in any other tissue studied. Autoradiographs of colorectal cancers showed binding of 3H-deoxycholic acid in receptor-positive tumour tissue. These findings might provide some explanation for the evidence linking bile acids with the disease.

Autoradiography↗
PubMed1983

A rapid method for preparation of bacterial plasmids.

A method for isolating plasmids from Escherichia coli which requires less than 8 h from cell pellet to purified plasmid essentially free of protein, RNA, and chromosomal DNA is presented. By this procedure, amplified plasmid pBR322 was isolated from E. coli strain RR1. The final product had no detectable protein or RNA, and plasmid comprised approximately 99% of the total DNA. The procedure includes lysozyme treatment in hypertonic solution followed by lysis with a mild detergent in the presence of high salt and an RNase inhibitor--conditions which prevent unfolding of the bacterial nucleoid. After centrifuging out the nucleoid and cell debris, the nucleic acids are selectively precipitated with a neutral solution of sodium trichloroacetate and ethanol. RNA is degraded with RNase and the degradation products and RNase are eliminated through a second trichloroacetate/ethanol precipitation. Finally, the plasmid is resuspended and passed through a nitrocellulose filter to remove aggregates and any residual protein and single-stranded DNA--giving a plasmid preparation suitable for electrophoretic fractionation or cleavage with restriction nucleases.

Bacteria↗