pH Biosensing by PI4P Regulates Cargo Sorting at the TGN

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Bifunctional small molecule-oligonucleotide hybrid as microRNA inhibitor

miRNAs are key regulators of various biological processes. Dysregulation of miRNA is linked to many diseases. Development of miRNA inhibitor has implication in disease therapy and study of miRNA function. The biogenesis pathway of miRNA involves the processing of pre-miRNA into mature miRNA by Dicer enzyme. We previously reported a proximity enabled approach that employs bifunctional small molecules to regulate miRNA maturation through inhibiting the enzymatic activity of Dicer. By conjugating to an RNA targeting unit, an RNase inhibitor could be delivered to the cleavage site of specific pre-miRNA to deactivate the complexed Dicer enzyme. Herein, we expanded this bifunctional strategy by showing that antisense oligonucleotides (ASOs), including morpholinos and γPNAs, could be readily used as the RNA recognition unit to generate bifunctional small molecule-oligonucleotide hybrids as miRNA inhibitors. A systematic comparison revealed that the potency of these hybrids is mainly determined by the RNA binding of the targeting ASO molecules. Since the lengths of the ASO molecules used in this approach were much shorter than commonly used anti-miRNA ASOs, this may provide benefits to the specificity and cellular delivery of these hybrids. We expect that this approach could be complementary to traditional ASO and small molecule based miRNA inhibition and contribute to the study of miRNA.     

Prostate Apoptosis Response-4 Mediates Trophic Factor Withdrawal-Induced Apoptosis of Hippocampal Neurons

Prostate apoptosis response-4 (Par-4) is the product of a gene up-regulated in prostate cancer cells undergoing apoptosis. We now report that Par-4 mRNA and protein levels rapidly and progressively increase 4-24 h following trophic factor withdrawal (TFW) in cultured embryonic rat hippocampal neurons. The increased Par-4 levels follow an increase of reactive oxygen species, and precede mitochondrial membrane depolarization, caspase activation, and nuclear chromatin condensation/fragmentation. Pretreatment of cultures with 17beta-estradiol, vitamin E, and uric acid largely prevented Par-4 induction and cell death following TFW, demonstrating necessary roles for oxidative stress and membrane lipid peroxidation in TFW-induced neuronal apoptosis. Par-4 antisense oligonucleotide treatment blocked Par-4 protein increases and attenuated mitochondrial dysfunction, caspase activation, and cell death following TFW. Collectively, our data identify Par-4 as an early and pivotal player in neuronal apoptosis resulting from TFW and suggest that estrogen and antioxidants may prevent apoptosis, in part, by suppressing Par-4 production.     

Interferon γ and antisense transforming growth factor β transgenes synergize to enhance the immunogenicity of a murine mammary carcinoma

Transforming growth factor β (TGFβ) is an immunosuppressive cytokine that contributes to the immunological escape of tumor cells. In a previous study we demonstrated that inhibition of TGFβ production by EMT6 murine mammary tumor cells expressing an antisense TGF-β transgene reduces their tumorigenicity. On the basis of this observation we hypothesized that down-regulation of TGFβ production coupled with interferon γ (IFNγ) stimulation would induce an immune response superior to that generated by either strategy alone. In this study, EMT6 tumor cells expressing antisense TGFβ were transduced with the murine IFNγ gene. Tumor cells expressing either or both transgenes grew more slowly than mock-transduced tumors. Dual-transgene-expressing tumor cells were more immunogenic than tumor cells expressing either transgene alone. Studies in mice depleted of T cell subsets indicated that CD8⁺ T cells are the primary effectors of the antitumor activity observed. These results suggest that down-regulation of immunosuppression combined with cytokine-mediated immune augmentation is a useful strategy to improve antitumor immunity. Received: 6 October 1998 / Accepted: 15 January 1999     

The Influence of the Non‐Nucleotide Insert on the Hybridization Properties of Oligonucleotides

The effect of different non‐nucleotide inserts incorporated into oligonucleotide chains on their hybridization properties was studied by the method of thermal denaturation. Various types of alkyldiols and oligoethylene glycols were used as inserts modifying oligonucleotide backbone. Such modification of oligonucleotides caused the destabilization of their complementary complexes. It was shown that the hybridization properties of the modified oligonucleotides depend on several features of inserts: the type, number, length of insertions, and positions of interrupted dinucleotide steps in oligonucleotide chain.     

Determination of the Absolute P-Configuration of a Phthalidyl-Phosphonate Thymidine-Thymidine Dimer

Abstract

Phthalidyl modified oligonucleotide thymidine-thymidine dimer building blocks were synthesized via the H-phosphonate-method. The compounds which are diastereomeric at the phosphorus atom were separated by chromatography and the absolute configuration at the phosphorus atoms was determined using ROE-experiments using the corresponding methyl-phosphonates.     

The immunostimulatory effect of IL-1β in vivo is blocked by antisense peptides complementary to the loop sequence 163-171

Antisense (AS) peptides complementary to the β-bulge surface loop VQGEESNDK (Boraschi loop) of the cytokine interleukin-1β (IL-1β) have been shown to bind IL-1β at the Boraschi loop position, and to inhibit some of the IL-1β-mediated biological effects in vitro. Here we show that primary AS peptide FVITFFSLY inhibits IL-1β-mediated immunostimulation in vivo in a dose-dependent fashion, while inactive on IL-1β-induced inflammation, an effect that takes place independently of the Boraschi loop. To the best of our knowledge, this is the first time that an AS peptide has been used successfully in vivo.     

Antisense Oligonucleotide-Induced Reduction in 5-Hydroxytryptamine7 Receptors in the Rat Hypothalamus Without Alteration in Exploratory Behaviour or Neuroendocrine Function

Abstract: The effect of a 5-hydroxytryptamine₇ (5-HT₇) receptor-directed antisense oligonucleotide on rat behaviour and neuroendocrine function was investigated. Six days of intracerebroventricular 5-HT₇ antisense oligonucleotide treatment significantly reduced [³H]5-HT binding to hypothalamic 5-HT₇ receptors, whereas cortical 5-HT_2C density remained unchanged. In rats on a food-restricted diet, both antisense and mismatch oligonucleotides reduced food intake and body weight compared with that in vehicle-treated controls by day 4 of administration. 5-HT₇ antisense oligonucleotide administration did not affect exploratory or locomotor activity in photocell activity monitors on day 4 or elevated plus-maze behaviour on day 6 of intracerebroventricular treatment. 5-HT₇ antisense oligonucleotide did not affect plasma corticosterone or prolactin levels or 5-HT turnover in either 5-HT cell body or terminal areas. These data demonstrate that intracerebroventricular 5-HT₇ antisense oligonucleotide administration selectively reduced rat hypothalamic 5-HT₇ receptor density without affecting any of the biochemical or behavioural measures. The results suggest that this antisense protocol could be a valuable tool to investigate central 5-HT₇ receptor functions, and that this receptor is not critical for the control of neuroendocrine function or food intake.