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Dwaine A Braasch

Publications and source records attributed to Dwaine A Braasch.

11 recordsLinked to original sources
PubMed2005

Lumenal adenosine and AMP rapidly increase glucose transport by intact small intestine.

Adenosine modulates the intestinal functions of secretion, motility, and immunity, yet little is known about the regulation of nutrient absorption. Therefore, we measured the carrier-mediated uptake of tracer D-[(14)C]glucose (2 microM) by everted sleeves of the mouse intestine after a lumenal exposure to adenosine and a disodium salt of AMP. Rates of glucose uptake by intact tissues increased almost twofold after a 7-min exposure to 5 mM adenosine (a physiological dose). The response was slightly more pronounced for AMP and could be induced by forskolin. The response to adenosine was blocked by theophylline and the A(2) receptor antagonist 3,7-dimethyl-1-proparglyxanthine but not by the A(1) receptor antagonist 8-phenyltheophylline. Glucose uptake by control and AMP-stimulated tissues was inhibited by phloridzin, implying that sodium-dependent glucose transporter 1 (SGLT1) is the responsive transporter, but the involvement of glucose transporter 2 (GLUT2) cannot be excluded. Of clinical relevance, AMP accelerated the systemic availability of 3-O-methylglucose after an oral administration to mice. Our results indicate that adenosine causes a rapid increase in carrier-mediated glucose uptake that is of clinical relevance and acts via receptors linked to a signaling pathway that involves intracellular cAMP production.

3-O-Methylglucose↗
PubMed2004

Biodistribution of phosphodiester and phosphorothioate siRNA.

Short interfering RNAs (siRNAs) are valuable tools for analyzing protein function in mammalian cell culture. This success has led to high expectations for in vivo and therapeutic applications. However, the pharmacokinetic properties of siRNA are not known. Here we report the biodistribution of a phosphodiester (PO) siRNA duplex and examine the effect of phosphorothioate (PS) linkages. Our findings indicate that biodistribution of siRNA is similar to that for single-stranded antisense oligonucleotides and offer insights for use of siRNA in vivo.

Animals↗
PubMed2004

Efficient and isoform-selective inhibition of cellular gene expression by peptide nucleic acids.

Peptide nucleic acids (PNAs) are a potentially powerful approach for the recognition of cellular mRNA and the inhibition of gene expression. Despite their promise, the rules for using antisense PNAs have remained obscure, and antisense PNAs have been used sparingly in research. Here we investigate the ability of PNAs to be effective antisense agents inside mammalian cells, to inhibit expression of human caveolin-1 (hCav-1), and to discriminate between its alpha and beta isoforms. Many human genes are expressed as isoforms. Isoforms may play different roles within a cell or within different tissues, and defining these roles is a challenge for functional genomics and drug discovery. PNAs targeted to the translation start codons for the alpha and beta isoforms inhibit expression of hCav-1. Inhibition is dependent on PNA length. The potency and duration of inhibition by PNAs are similar to inhibition of gene expression by short interferring RNA (siRNA). Expression of the alpha isoform can be blocked selectively by a PNA. Cell proliferation is halted by inhibition of expression of both hCav-1 isoforms, but not by inhibition of the alpha hCav-1 isoform alone. Efficient antisense inhibition and selective modulation of isoform expression suggest that PNAs are versatile tools for controlling gene expression and dissecting the roles of closely related protein variants. Potent inhibition by PNAs may supply a "knock down" technology that can complement and "cross-check" siRNA and other approaches to antisense gene inhibition that rely on oligomers with phosphate or phosphorothioate backbone linkages.

Caveolin 1↗
PubMed2004

Validating bioluminescence imaging as a high-throughput, quantitative modality for assessing tumor burden.

Bioluminescence imaging (BLI) is a highly sensitive tool for visualizing tumors, neoplastic development, metastatic spread, and response to therapy. Although BLI has engendered much excitement due to its apparent simplicity and ease of implementation, few rigorous studies have been presented to validate the measurements. Here, we characterize the nature of bioluminescence output from mice bearing subcutaneous luciferase-expressing tumors over a 4-week period. Following intraperitoneal or direct intratumoral administration of luciferin substrate, there was a highly dynamic kinetic profile of light emission. Although bioluminescence was subject to variability, strong correlations (r >.8, p <.001) between caliper measured tumor volumes and peak light signal, area under light signal curve and light emission at specific time points were determined. Moreover, the profile of tumor growth, as monitored with bioluminescence, closely resembled that for caliper measurements. The study shows that despite the dynamic and variable nature of bioluminescence, where appropriate experimental precautions are taken, single time point BLI may be useful for noninvasive, high-throughput, quantitative assessment of tumor burden.

Animals↗
PubMed2003

RNA interference in mammalian cells by chemically-modified RNA.

RNA interference (RNAi) is proving to be a robust and versatile technique for controlling gene expression in mammalian cells. To fully realize its potential in vivo, however, it may be necessary to introduce chemical modifications to optimize potency, stability, and pharmacokinetic properties. Here, we test the effects of chemical modifications on RNA stability and inhibition of gene expression. We find that RNA duplexes containing either phosphodiester or varying numbers of phosphorothioate linkages are remarkably stable during prolonged incubations in serum. Treatment of cells with RNA duplexes containing phosphorothioate linkages leads to selective inhibition of gene expression. RNAi also tolerates the introduction of 2'-deoxy-2'-fluorouridine or locked nucleic acid (LNA) nucleotides. Introduction of LNA nucleotides also substantially increases the thermal stability of modified RNA duplexes without compromising the efficiency of RNAi. These results suggest that inhibition of gene expression by RNAi is compatible with a broad spectrum of chemical modifications to the duplex, affording a wide range of useful options for probing the mechanism of RNAi and for improving RNA interference in vivo.

Blotting, Western↗
PubMed2002

Antisense inhibition of gene expression in cells by oligonucleotides incorporating locked nucleic acids: effect of mRNA target sequence and chimera design.

Use of antisense oligonucleotides is a versatile strategy for achieving control of gene expression. Unfortunately, the interpretation of antisense-induced phenotypes is sometimes difficult, and chemical modifications that improve the potency and specificity of antisense action would be useful. The introduction of locked nucleic acid (LNA) bases into oligonucleotides confers exceptional improvement in binding affinity, up to 10 degrees C per substitution, making LNAs an exciting option for the optimization of antisense efficacy. Here we examine the rules governing antisense gene inhibition within cells by oligonucleotides that contain LNA bases. LNA- containing oligomers were transfected into cells using cationic lipid and accumulated in the nucleus. We tested antisense gene inhibition by LNAs and LNA-DNA chimeras complementary to the 5'-untranslated region, the region surrounding the start codon and the coding region of mRNA, and identified effective antisense agents targeted to each of these locations. Our data suggest that LNA bases can be used to develop antisense oligonucleotides and that their use is a versatile approach for efficiently inhibiting gene expression inside cells.

5' Untranslated Regions↗
PubMed2002

Enhanced strand invasion by peptide nucleic acid-peptide conjugates.

Efficient and selective recognition of DNA by proteins is due to sequence-specific interactions with a target site and nonselective electrostatic interactions that promote the target's rapid location. If synthetic molecules could mimic these functions, they would render a wide range of chromosome sequences accessible to rationally designed probes. Here we describe conjugates between bispeptide nucleic acids (bisPNAs) designed to specifically recognize duplex DNA and peptides that have been designed to promote rapid sequence recognition. Peptide design was based on the surface of staphylococcal nuclease, a cationic DNA binding protein with low sequence selectivity. We observe that attachment of the designed peptide increases rates of strand invasion by 100-fold relative to unmodified bisPNA. The peptide can contain D-amino acids, increasing the likelihood that it will be stable in cell extract and inside cells. Binding of the conjugate containing the D-amino acid peptide occurred over a broad range of experimental conditions and was sensitive to a single mismatch. Strand invasion was efficient at neutral to basic pH, a wide range of temperatures (0-65 degrees C), and in the presence of up to 7 mM Mg(2+) and 100 mM Na(+) or K(+). Our data suggest that attachment of peptides that mimic cationic protein surfaces to PNAs can afford conjugates that mimic the rapid and selective binding that characterizes native DNA binding proteins. Rapid strand invasion over a wide range of experimental conditions should further expand the utility of strand invasion by PNAs.

Amino Acid Sequence↗
PubMed2002

Implications of high-affinity hybridization by locked nucleic acid oligomers for inhibition of human telomerase.

Oligonucleotides that contain locked nucleic acid (LNA) bases have remarkably high affinity for complementary RNA and DNA sequences. This increased affinity may facilitate the recognition of nucleic acid targets inside cells and thus improve our ability to use synthetic oligonucleotides for controlling cellular processes. Here we test the hypothesis that LNAs offer advantages for inhibiting human telomerase, a ribonucleoprotein that is critical for tumor cell proliferation. We observe that LNAs complementary to the telomerase RNA template are potent and selective inhibitors of human telomerase. LNAs can be introduced into cultured tumor cells using cationic lipid, with diffuse uptake throughout the cell. Transfected LNAs effectively inhibited intracellular telomerase activity up to 40 h post-transfection. Shorter LNAs of eight bases in length are also effective inhibitors of human telomerase. The melting temperatures of these LNAs for complementary sequences are superior to those of analogous peptide nucleic acid oligomers, emphasizing the value of LNA bases for high-affinity recognition. These results demonstrate that high-affinity binding by LNAs can be exploited for superior recognition of an intracellular target.

Animals↗
PubMed2002

Novel antisense and peptide nucleic acid strategies for controlling gene expression.

Antisense oligonucleotides have the potential to make revolutionary contributions to basic science and medicine. Oligonucleotides can bind mRNA and inhibit translation. Because they can be rapidly synthesized to be complementary to any sequence, they offer ideal tools for exploiting the massive amount of genome information now available. However, until recently, this potential was largely theoretical, and antisense experiments often produced inconclusive or misleading outcomes. This review will discuss the chemical and biological properties of some of the different types of oligomers now available and describe the challenges confronting in vitro and in vivo use of oligonucleotides. Oligomers with improved chemical properties, combined with advances in cell biology and success in clinical trials, are affording powerful new options for basic research, biotechnology, and medicine.

Animals↗
PubMed2002

Imaging gene expression in the brain in vivo in a transgenic mouse model of Huntington's disease with an antisense radiopharmaceutical and drug-targeting technology.

UNLABELLED: Disease-specific genes of unknown function can be imaged in vivo with antisense radiopharmaceuticals, providing the transcellular transport of these molecules is enabled with drug-targeting technology. The current studies describe the production of 16-mer peptide nucleic acid (PNA) that is antisense around the methionine initiation codon of the huntingtin gene of Huntington's disease (HD). METHODS: The PNA is biotinylated, which allows for rapid capture by a conjugate of streptavidin and the rat 8D3 monoclonal antibody (mAb) to the mouse transferrin receptor (TfR), and contains a tyrosine residue, which enables radiolabeling with 125I. The reformulated PNA antisense radiopharmaceutical that is conjugated to the 8D3 mAb is designated 125I-PNA/8D3. This form of the PNA is able to access endogenous transferrin transport pathways at both the blood-brain barrier and the brain cell membrane and undergoes both import from the blood to the brain and export from the brain to the blood through the TfR. RESULTS: The ability of the PNA to hybridize to the target huntingtin RNA, despite conjugation to the mAb, was shown both with cell-free translation assays and with ribonuclease protection assays. The 125I-PNA/8D3 conjugate was administered intravenously to either littermate control mice or to R6/2 transgenic mice, which express the exon 1 of the human HD gene. The mice were sacrificed 6 h later for frozen sectioning of the brain and quantitative autoradiography. The studies showed a 3-fold increase in sequestration of the 125I-PNA/8D3 antisense radiopharmaceutical in the brains of the HD transgenic mice in vivo, consistent with the selective expression of the HD exon-1 messenger RNA in these animals. CONCLUSION: These results support the hypothesis that gene expression in vivo can be quantitated with antisense radiopharmaceuticals, providing these molecules are reformulated with drug-targeting technology. Drug targeting enables access of the antisense agent to endogenous transport pathways, which permits passage across the cellular barriers that separate blood and intracellular compartments of target tissues.

Animals↗