Discovery and analysis of antisense oligonucleotide activity in cell culture

In the past decade antisense oligonucleotides (ASOs) have proven to be a useful tool for dissection of gene function in molecular cell biology (Koller, E., Gaarde, W. A., and Monia, B. P. (2000) Trends Pharm. Sci., 21, 142-148), and validation of gene targets in animal models (Crooke, S. T. (1998) Biotechnol. Gen. Eng. Rev. 15, 121-157), as well as a means for therapeutic treatment of human diseases (Bennett, C. F. (1999) Exp. Opin. Invest. Drugs 8, 237-253). An important step toward usage of ASOs in the described applications is identification of an active ASO. This article describes the underlying basis and means for achieving this goal in cell culture.     

Phaeodactylum tricornutum: An established model species for diatom molecular research and an emerging chassis for algal synthetic biology

Diatoms are prominent and highly diverse microalgae in aquatic environments. Compared with other diatom species, Phaeodactylum tricornutum is an “atypical diatom” displaying three different morphotypes and lacking the usual silica shell. Despite being of limited ecological relevance, its ease of growth in the laboratory and well-known physiology, alongside the steady increase in genome-enabled information coupled with effective tools for manipulating gene expression, have meant it has gained increased recognition as a powerful experimental model for molecular research on diatoms. We here present a brief overview of how over the last 25 years P. tricornutum has contributed to the unveiling of fundamental aspects of diatom biology, while also emerging as a new tool for algal process engineering and synthetic biology.     

Antisense inhibition of membrane-bound human interleukin-5 receptor-alpha chain does not affect soluble receptor expression and induces apoptosis in TF-1 cells

Binding of human interleukin-5 (HuIL-5) to its membrane-anchored receptor (IL-5R) triggers multiple signaling pathways, cellular proliferation, and maturational responses, as well as protection from apoptosis. In contrast, soluble forms of the HuIL-5R have been shown to inhibit IL-5 signaling and, therefore, may represent naturally occurring negative regulators of IL-5 function. Because of the central role of IL-5 in promoting eosinophilia and airway hyperresponsiveness in animal models of asthma, antisense oligonucleotides specific either for the membrane form alone or for sequences shared between both the membrane and soluble forms of the HuIL-5Ralpha ligand binding chain were designed. The activities of these oligonucleotides were characterized in IL-5R-expressing erythroleukemic TF-1 cells. Herein we report that an antisense oligonucleotide targeted to a sequence unique to the alternatively spliced membrane-bound form of the HuIL-5Ralpha chain has been developed that selectively inhibits membrane, but not soluble, mRNA isoform expression. Both this membrane-specific oligonucleotide and an antisense oligonucleotide targeted to sequence common to both membrane and soluble isoforms were found to potently suppress cell surface IL-5Ralpha levels and IL-5-mediated cell survival by inducing apoptosis similar to IL-5 withdrawal. Thus, these oligonucleotides represent unique genetic agents with therapeutic potential for diseases with an eosinophilic component.     

Characterization of anti-coagulant properties of prenylated coumarin ferulenol

We investigated the mechanisms underlying severe bleeding occurring upon consumption of Ferula communis. The prenylated coumarin ferulenol extracted from this plant did not directly affect blood coagulation but showed hepatocyte cytotoxicity and, at non-cytotoxic concentrations (<100 nM), impaired factor X biosynthesis (40% reduction). Studies with ferulenol derivatives indicated the prenyl residue as major determinant of ferulenol activity.     

Peritoneal Tumor Carcinomatosis: Pharmacological Targeting with Hyaluronan-Based Bioconjugates Overcomes Therapeutic Indications of Current Drugs

Peritoneal carcinomatosis still lacks reliable therapeutic options. We aimed at testing a drug delivery strategy allowing a controlled release of cytotoxic molecules and selective targeting of tumor cells. We comparatively assessed the efficacy of a loco-regional intraperitoneal treatment in immunocompromised mice with bioconjugates formed by chemical linking of paclitaxel or SN-38 to hyaluronan, against three models of peritoneal carcinomatosis derived from human colorectal, gastric and esophageal tumor cell xenografts. In vitro, bioconjugates were selectively internalized through mechanisms largely dependent on interaction with the CD44 receptor and caveolin-mediated endocytosis, which led to accumulation of compounds into lysosomes of tumor cells. Moreover, they inhibited tumor growth comparably to free drugs. In vivo, efficacy of bioconjugates or free drugs against luciferase-transduced tumor cells was assessed by bioluminescence optical imaging, and by recording mice survival. The intraperitoneal administration of bioconjugates in tumor-bearing mice exerted overlapping or improved therapeutic efficacy compared with unconjugated drugs. Overall, drug conjugation to hyaluronan significantly improved the profiles of in vivo tolerability and widened the field of application of existing drugs, over their formal approval or current use. Therefore, this approach can be envisaged as a promising therapeutic strategy for loco-regional treatment of peritoneal carcinomatosis.     

Dose-dependent recruitment of CD25+ and CD26+ T cells in a novel F344 rat model of asthma

The ovalbumin (OVA)-induced airway inflammation in rats is a commonly used model to explore the pathobiology of asthma. However, its susceptibility varies greatly between rat strains, and presently Brown Norway (BN) rats are preferentially used. Since recruitment of T cells to the lungs depends on the CD26 (dipeptidyl peptidase IV, DPPIV) expression, Fischer 344 strain (F344) rats are a highly relevant rat strain, in particular because CD26-deficient substrains are available. To establish a F344 rat model of asthma, we challenged F344 rats using different doses of aerosolized antigen (0%, 1%, 2.5%, 5%, and 7.5% OVA) and compared these effects with intratracheal instillation of OVA (1.5 mg/0.3 ml). Asthmoid responsiveness was determined by analysis of early airway responsiveness (EAR), antigen-specific IgE levels, as well as airway inflammation including the composition of T cell subpopulations in the bronchoalveolar lavage (BAL) and lung tissue with special respect to the T cell activation markers CD25 and CD26. Even low allergen doses caused allergen-specific EAR and increases of antigen-specific IgE levels. However, EAR and IgE levels did not increase dose dependently. Higher concentrations of OVA led to a dose-dependent increase of several immunological markers of allergic asthma including an influx of eosinophils, T cells, and dendritic cells. Interestingly, a dose-dependent increase of CD4(+)/CD25(+)/CD26(+) T cells was found in the lungs. Summarizing, we established a novel F344 rat model of aerosolized OVA-induced asthma. Thereby, we found a dose-dependent recruitment of cellular markers of allergic asthma including the activated CD4(+)/CD25(+)/CD26(+) T cell subpopulation, which has not been described in asthma yet.     

Biophysical and antisense properties of oligodeoxynucleotides containing 7-propynyl-, 7-iodo- and 7-cyano-7-deaza-2-amino-2'-deoxyadenosines.

The synthesis of 7-propynyl-, 7-iodo- and 7-cyano-7-deaza-2-amino-2'-deoxyadenosines is described. The nucleosides were synthesized, functionalized into the phosphoramidites and incorporated into oligodeoxynucleotides. Spectroscopic melting experiments against complementary RNA showed increases of 3-4 degreesC per modification for single substitutions and smaller increases per incorporation for multiple substitutions relative to unmodified control sequences. The 7-propyne and 7-iodo nucleosides were incorporated into antisense sequences targeting the 3'-UTR of murine C- raf mRNA. Both nucleosides demonstrated substitution-dependent potency. The sequences with three and four substitutions of the 7-propyne-7-deaza-2-amino-2'-deoxyadenosine exhibited a 2-3-fold increase in potency over unmodifed controls.     

Antioxidant activity of blueberry fruit is impaired by association with milk

The antioxidant properties of dietary phenolics are believed to be reduced in vivo because of their affinity for proteins. In this study we assessed the bioavailability of phenolics and the in vivo plasma antioxidant capacity after the consumption of blueberries (Vaccinium corymbosum L.) with and without milk. In a crossover design, 11 healthy human volunteers consumed either (a) 200 g of blueberries plus 200 ml of water or (b) 200 g of blueberries plus 200 ml of whole milk. Venous samples were collected at baseline and at 1, 2, and 5 h postconsumption. Ingestion of blueberries increased plasma levels of reducing and chain-breaking potential (+ 6.1%, p < 0.001; + 11.1%, p < 0.05) and enhanced plasma concentrations of caffeic and ferulic acid. When blueberries and milk were ingested there was no increase in plasma antioxidant capacity. There was a reduction in the peak plasma concentrations of caffeic and ferulic acid (? 49.7%, p < 0.001, and ? 19.8%, p < 0.05, respectively) as well as the overall absorption (AUC) of caffeic acid (p < 0.001). The ingestion of blueberries in association with milk, thus, impairs the in vivo antioxidant properties of blueberries and reduces the absorption of caffeic acid.