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Oligodeoxynucleotides
Kinds of Oligodeoxynucleotides Selected AbstractsSynthesis and Structural Properties of New Oligodeoxynucleotide Analogues Containing a 2,,5,-Internucleotidic Squaryldiamide Linkage Capable of Formation of a Watson,Crick Base Pair with Adenine and a Wobble Base Pair with Guanine at the 3,-Downstream Junction SiteEUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 10 2004Kousuke Sato Abstract A TpT dimer analogue (U2,sq5,T), in which the 3,-5, phosphodiester linkage was replaced by a 2,-5, squaryldiamide linkage and the 5,-upstream T was replaced by a 3,-deoxyuridine, was synthesized in almost quantitative yield from diethyl squarate. This new dimer structural motif was designed to eliminate the squaryldiamide skeleton-induced overall strain in T3,sq5,T, previously incorporated into DNA fragments as a new TpT mimic, through the change in the connection mode from the 3,-5, linkage to a 2,-5, linkage. Spectral analyses of U2,sq5,T suggest that the overall structure of this dimer mimic is basically similar to that of TpT. A DNA 10mer 5,-d(CGCAU2,sq5,TAGCC)-3, incorporating this dimer was synthesized. From the CD analysis, it turned out that the overall structure of a DNA duplex of 5,-d(CGCAU2,sq5,TAGCC)-3,/3,-d(GCGTAATCGG)-5, is closer to that of the unmodified duplex than the DNA duplex of 5,-d(CGCAT3,sq5,TAGCC)-3,/3,-d(GCGTAATCGG)-5,. Interestingly, extensive Tm experiments suggest that d(CGCAU2,sq5,TAGCC)-3, exhibits intriguing inherent hybridization affinity not only for the completely complementary oligodeoxynucleotide 3,-d(GCGAATCGG)-5,, but also for 3,-d(GCGTAGTCGG)-5,, with a mismatched dG. The unique property of the 3,-downstream dT moiety of U2,sq5,T , the ability to recognize both dA and dG , was also supported by more detailed computational analysis of U2,sq5,T and TpT. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004) [source] Efficient killing of SW480 colon carcinoma cells by a signal transducer and activator of transcription (STAT) 3 hairpin decoy oligodeoxynucleotide , interference with interferon-,-STAT1-mediated killingFEBS JOURNAL, Issue 9 2009Ali Tadlaoui Hbibi The signal transducers and activators of transcription (STATs) convey signals from the membrane to the nucleus in response to cytokines or growth factors. STAT3 is activated in response to cytokines involved mostly in cell proliferation; STAT1 is activated by cytokines, including interferon-,, involved in defence against pathogens and the inhibition of cell proliferation. STAT3, which is frequently activated in tumour cells, is a valuable target with respect to achieving inhibition of tumour cell proliferation. Indeed, its inhibition results in cell death. We previously observed that inhibition of the transcription factor nuclear factor-,B, a key regulator of cell proliferation, with decoy oligodeoxynucleotides results in cell death. We used a similar approach for STAT3. A hairpin STAT3 oligodeoxynucleotide was added to a colon carcinoma cell line in which it induced cell death as efficiently as the STAT3 inhibitor stattic. The hairpin STAT3 oligodeoxynucleotide co-localized with STAT3 within the cytoplasm, prevented STAT3 localization to the nucleus, blocked a cyclin D1 reporter promoter and associated with STAT3 in pull-down assays. However, the same cells were efficiently killed by interferon-,. This effect was counteracted by the STAT3 oligodeoxynucleotide, which was found to efficiently inhibit STAT1. Thus, although it can inhibit STAT3, the hairpin STAT3 oligodeoxynucleotide appears also to inhibit STAT1-mediated interferon-, cell killing, highlighting the need to optimize STAT3-targeting oligodeoxynucleotides. [source] CpG oligodeoxynucleotides protect mice from lethal challenge with Candida albicans via a pathway involving tumor necrosis factor-,-dependent interleukin-12 inductionFEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 1 2007Jung-Hwa Choi Abstract In this study, we have attempted to determine whether the systemic administration of CpG oligodeoxynucleotide (CpG-ODN) 1826 would protect mice against systemic lethal Candida albicans infection. CpG-ODNs were found completely to protect mice from death and also reduced the growth of C. albicans in the kidneys. The administration of CpG-ODNs resulted in early interleukin (IL)-12 mRNA expression in the kidneys and an increase in serum IL-12 levels. The protective activity of CpG-ODN was abolished in IL-12-deficient (IL-12,/,) mice, thereby indicating the IL-12-dependency inherent to the effects of CpG-ODN. The protective effect of CpG-ODN was not associated with the activity of NF-,B. Interestingly, in tumor necrosis factor (TNF)-,-deficient (TNF,/,) mice CpG-ODN neither exerted protective effects nor induced IL-12 expression. These data indicate that CpG-ODN protects animals against lethal C. albicans challenge via a pathway that involves the TNF-,-dependent induction of IL-12. [source] Transforming growth factor-activated kinase 1 induced in spinal astrocytes contributes to mechanical hypersensitivity after nerve injuryGLIA, Issue 7 2008Hirokazu Katsura Abstract Mitogen-activated protein kinase (MAPK) plays an important role in the induction and maintenance of neuropathic pain. Transforming growth factor-activated kinase 1 (TAK1), a member of the MAPK kinase kinase family, is indispensable for the activation of c-Jun N-terminal kinase (JNK) and p38 MAPK. We now show that TAK1 induced in spinal cord astrocytes is crucial for mechanical hypersensitivity after peripheral nerve injury. Nerve injury induced a striking increase in the expression of TAK1 in the ipsilateral dorsal horn, and TAK1 was increased in hyperactive astrocytes, but not in neurons or microglia. Intrathecal administration of TAK1 antisense oligodeoxynucleotide (AS-ODN) prevented and reversed nerve injury-induced mechanical, but not heat hypersensitivity. Furthermore, TAK1 AS-ODN suppressed the activation of JNK1, but not p38 MAPK, in spinal astrocytes. In contrast, there was no change in TAK1 expression in primary sensory neurons, and TAK1 AS-ODN did not attenuate the induction of transient receptor potential ion channel TRPV1 in sensory neurons. Taken together, these results demonstrate that TAK1 upregulation in spinal astrocytes has a substantial role in the development and maintenance of mechanical hypersensitivity through the JNK1 pathway. Thus, preventing the TAK1/JNK1 signaling cascade in astrocytes might provide a fruitful strategy for treating intractable neuropathic pain. © 2008 Wiley-Liss, Inc. [source] Development and Evaluation of a DNA Vaccine Based on Helicobacter pylori urease B: Failure to Prevent Experimental Infection in the Mouse ModelHELICOBACTER, Issue 6 2006Livania Zavala-Spinetti Abstract Background:, The development of a vaccine against Helicobacter pylori has become a priority to prevent major morbidity and mortality associated with this infection. Our goal was to prepare and evaluate a DNA vaccine based on the urease B gene (ureB). Methods:, The ureB gene of H. pylori was amplified and cloned into the eukaryotic expression vector pcDNA3.1/TOPO. Plasmid DNA was purified from transformed Escherichia coli cells and used to immunize mice by the intragastric, intramuscular, intrarectal (40 µg each) and intranasal (16 µg) route, three doses every 2 weeks, with CpG oligodeoxynucleotide (ODN) as adjuvant. Four weeks after the third dose, animals were orally challenged with Helicobacter felis and were sacrificed 6 weeks later. The stomach was stained to detect the presence of infection. Results:, Despite in vitro confirmation of successful cloning and functionality of the ureB gene with expression of a protein morphologically and antigenically identical to urease B, the DNA vaccine did not perform well in vivo. Immunization of mice produced a weak immune response. Overall, intrarectal and intranasal administration seemed more immunogenic than other routes. Protection against challenge was modest and nonsignificant, and slightly better on animals immunized by the intramuscular and intranasal route. Conclusion:, A DNA vaccine based on H. pylori urease B was poorly immunogenic and nonprotective at the conditions evaluated. Higher doses, better adjuvants or a prime-boost approach may circumvent these limitations. [source] Photocleavage of Peptides and Oligodeoxynucleotides Carrying 2-Nitrobenzyl GroupsHELVETICA CHIMICA ACTA, Issue 4 2009Roger Ramos Abstract Peptides and oligodeoxynucleotides containing photolabile 2-nitrobenzyl groups as mid-sequences were prepared. Photocleavage of aqueous solutions of these compounds neared completion within 30,min to a few hours depending on the photolabile group used. A photolabile group was introduced in the loop of an intramolecular oligodeoxynucleotide hairpin. Melting curves of the hairpin with and without the complementary oligodeoxynucleotide showed a preference for the intramolecular hairpin form, but an intermolecular duplex was observed after photolysis. These results open the possibility of using photolabile DNA hairpins for the fabrication of patterned surfaces. [source] Rapid and reliable generation of invariant natural killer T-cell lines in vitroIMMUNOLOGY, Issue 3 2009Asako Chiba Summary Several tools have proved useful in the study of invariant natural killer T (iNKT) cells, including CD1d-deficient mice, J,281-deficient mice, synthetic lipid antigens and antigen-loaded CD1d tetramers. However, the generation and examination of long-term primary murine iNKT cell lines in vitro has been challenging. Here, we show the rapid generation of iNKT cell lines from splenic iNKT cells of V,14 T-cell receptor (TCR) transgenic (Tg) mice. These purified iNKT cells were stimulated by bone marrow-derived dendritic cells (BMDCs) loaded with ,-galactosylceramide (,GalCer) and cultured with interleukin (IL)-2 and IL-7. iNKT cells proliferated dramatically, and the cell number exhibited a 100-fold increase within 2 weeks and a 105 -fold increase in 8 weeks after repeated stimulation with ,GalCer. The iNKT cell lines consisted of iNKT cells expressing V, chains including V,8.1/8.2, V,14, V,10, V,6 and V,7, and responded to stimulation with ,GalCer presented both by BMDCs and by plate-bound CD1d. In addition, the iNKT cell lines produced interferon (IFN)-, when activated by lipopolysaccharide (LPS) or CpG oligodeoxynucleotide (ODN)-stimulated BMDCs. Further, we show that iNKT cell lines produced cytokines in response to microbial antigens. In summary, high-yield iNKT cell lines were generated very rapidly and robustly expanded, and these iNKT cells responded to both TCR and cytokine stimulation in vitro. Given the desire to study primary iNKT cells for many purposes, these iNKT cell lines should provide an important tool for the study of iNKT cell subsets, antigen and TCR specificity, activation, inactivation and effector functions. [source] H2-Dd -mediated upregulation of interleukin-4 production by natural killer T-cell and dendritic cell interactionIMMUNOLOGY, Issue 1 2008Kazuomi Mizuuchi Summary Natural killer T (NKT) cells are capable of subserving apparently opposite functions, the interferon-, (IFN-,)-mediated enhancement of host defence and interleukin-4 (IL-4) -mediated immune regulation. Although dendritic cells (DCs) potently activate NKT cells, DC regulation of the IL-4,IFN-, balance via NKT-cell activation is not well characterized. In the present study, we examined the effect of DC treatment with CpG oligodeoxynucleotide (ODN), a Toll-like receptor 9 ligand, on the induction of NKT-cell cytokine production. CpG-ODN-conditioned and ,-galactosylceramide (,-GalCer)-loaded myeloid DCs (CpG-DCs) from BALB/c mice showed enhanced ability to induce NKT-cell production of IL-4, but not IFN-,, compared to ,-GalCer-loaded control DCs (not treated with CpG-ODN). The CpG-DCs expressed significantly higher levels of H2-Dd than control DCs, and blocking of the H2-Dd and Ly49 receptor interaction during antigen presentation completely abolished the enhanced ability of the CpG-DCs to induce NKT-cell production of IL-4. These findings demonstrate that DC recognition of the CpG motif leads to induction of enhanced IL-4 production by NKT cells via interaction of the augmented H2-Dd with Ly49 receptors on NKT cells. [source] Interferon-,-dependent inhibition of late allergic airway responses and eosinophilia by CD8+,, T cellsIMMUNOLOGY, Issue 2 2007Susumu Isogai Summary We have previously shown that CD8+,, T cells decrease late allergic airway responses, airway eosinophilia, T helper 2 cytokine expression and increase interferon-, (IFN-,) expression. We hypothesized that the effects of CD8+,, T cells were IFN-, mediated. Brown Norway rats were sensitized to ovalbumin on day 1. Cervical lymph node CD8+,, T cells from sensitized animals were treated with antisense oligodeoxynucleotide (5 µmol/l) to inhibit IFN-, synthesis or control oligodeoxynucleotide and 3·5 × 104 CD8+,, T cells were injected intraperitoneally into sensitized recipients on day 13. Rats were challenged with aerosolized ovalbumin on day 15 and lung resistance was monitored over an 8 hr period, after which bronchoalveolar lavage was performed. Control oligodeoxynucleotide treated ,, T cells decreased late airway responses and eosinophilia in bronchoalveolar lavage. There was a complete recovery of late airway responses and a partial recovery of airway eosinophilia in recipients of antisense oligodeoxynucleotide treated cells. Macrophage ingestion of eosinophils was frequent in rats administered ,,T cells but reduced in recipients of antisense oligodeoxynucleotide treated cells. These results indicate that CD8+,, T cells inhibit late airway responses and airway eosinophilia through the secretion of IFN-,. Defective or altered ,, T-cell function may account for some forms of allergic asthma. [source] Innate immune responses induced by CpG oligodeoxyribonucleotide stimulation of ovine blood mononuclear cellsIMMUNOLOGY, Issue 2 2003Angelo Mena Summary Examples exist in the literature that demonstrate that treatment with immunostimulatory cytosine,phosphate,guanosine (CpG)-DNA can protect mice against infection by intracellular pathogens. There are, however, few studies reporting that CpG-DNA offers similar disease protection in other species. In this study, we assessed the potential of a class A and class B CpG oligodeoxynucleotide (ODN) to induce innate immune responses in sheep, an outbred species. Using peripheral blood mononuclear cells, we have for the first time demonstrated CpG-ODN-induced innate immune responses, including natural-killer-like activity [non-major histocompatibility complex (MHC)-restricted cytotoxicity], interferon-, secretion and 2,-5,A oligoadenylate synthetase activity, that could contribute to immune protection in sheep. The type and magnitude of these responses were dependent on ODN class and non-MHC-restricted killing was not associated with interferon-, production. The latter observation is in contrast with observations reported for mice and humans. These observations support the conclusion that differences in CpG-ODN-induced responses exist among species and that specific ODN sequences can significantly influence innate immune responses. [source] Antisense oligodeoxynucleotide therapy targeting clusterin gene for prostate cancer: Vancouver experience from discovery to clinicINTERNATIONAL JOURNAL OF UROLOGY, Issue 9 2005HIDEAKI MIYAKE Abstract Background The objective of this study was to review our experience in the development of antisense (AS) oligodeoxynucleotide (ODN) therapy for prostate cancer targeting antiapoptotic gene, clusterin. Methods We initially summarized our data demonstrating that clusterin could be an optimal therapeutic target for prostate cancer, then presented the process of developing AS ODN therapy using several preclinical animal models. Finally, the preliminary data of the recently completed phase I clinical trial using AS clusterin ODN as well as the future prospects of this therapy are discussed. Results Expression of clusterin was highly up-regulated after androgen withdrawal and during progression to androgen-independence, but low or absent in untreated tissues in both prostate cancer animal model systems and human clinical specimens. Introduction of the clusterin gene into human prostate cancer cells confers resistance to several therapeutic stimuli, including androgen ablation, chemotherapy and radiation. AS ODN targeting the translation initiation site of the clusterin gene markedly inhibited clusterin expression in prostate cancer cells in a dose-dependent and sequence-specific manner. Systemic treatment with AS clusterin ODN enhanced the effects of several conventional therapies through the effective induction of apoptosis in prostate cancer xenograft models. Based on these findings, a phase I clinical trial was completed using AS clusterin ODN incorporating 2,-O-(2-methoxy)ethyl-gapmer backbone (OGX-011), showing up to 90% suppression of clusterin in prostate cancer. Conclusions The data described above identified clusterin as an antiapoptotic gene up-regulated in an adaptive cell survival manner following various cell death triggers that helps confer a phenotype resistant to therapeutic stimuli. Inhibition of clusterin expression using AS ODN technology enhances apoptosis induced by several conventional treatments, resulting in the delay of AI progression and improved survival. Clinical trials using AS ODN confirm potent suppression of clusterin expression and phase II studies will begin in early 2005. [source] Antisense Oligodeoxynucleotide Evidence That a Unique Osteoclastic Protein-Tyrosine Phosphatase Is Essential for Osteoclastic ResorptionJOURNAL OF BONE AND MINERAL RESEARCH, Issue 10 2001Sung Min Suhr Abstract This study tested the hypothesis that a unique osteoclastic transmembrane protein tyrosine phosphatase (PTP-oc) is involved in osteoclastic resorption by determining whether suppression of PTP-oc expression with a specific phosphorothioated 20-mer PTP-oc antisense oligodeoxynucleotide (oligo) would inhibit basal, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]-stimulated, and PTH-stimulated osteoclastic resorption. Treatment of rabbit osteoclasts with 1 ,M of the antisense oligo for up to 4 days showed a time-dependent reduction in PTP-oc protein level, indicating that this PTP-oc antisense oligo was effective. To assess the effect of PTP-oc antisense oligo on osteoclastic resorption, rabbit osteoclasts were pretreated for 3 days with 1 ,M of the antisense, a scramble oligo, or vehicle, respectively, followed by a 3-day treatment with vehicle, 10 nM of 1,25(OH)2D3, or 10 nM of parathyroid hormone (PTH). 1,25(OH)2D3 and PTH each alone increased PTP-oc cellular level and stimulated resorptive activity of rabbit osteoclasts. The antisense oligo treatment, but not the scramble oligo, decreased the basal and the stimulated resorption activity and reduced the PTP-oc protein level. Treatment with the PTP-oc antisense oligo, but not the scramble oligo, also markedly increased the Y527 phosphorylation level of c- src in rabbit osteoclasts. In conclusion, these results provide the first antisense oligo evidence that PTP-oc plays an essential role in osteoclastic resorption. [source] Transforming growth factor-,1-dependent activation of Smad2/3 and up-regulation of PAI-1 expression is negatively regulated by Src in SKOV-3 human ovarian cancer cellsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 3 2004Kiyoshi Wakahara Abstract The net balance between urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type-1 (PAI-1) has been implicated in tumor cell invasion and metastasis. To elucidate the mechanism of the transforming growth factor-,1 (TGF-,1)-dependent up-regulation of PAI-1 expression, we investigated which signaling pathway transduced by TGF-,1 is responsible for this effect. Here, we show (1) nontoxic concentrations of TGF-,1 up-regulates uPA expression in HRA and SKOV-3 human ovarian cancer cells, (2) TGF-,1 activates Smads (phosphorylation of Smad2 and nuclear translocation of Smad3) and subsequently up-regulates PAI-1 expression in HRA cells, whereas TGF-,1 neither activates Smads nor up-regulates PAI-1 in SKOV-3 cells, (3) pharmacological Src inhibitor PP2 or antisense (AS) c-Src oligodeoxynucleotide (ODN) treatment significantly induces TGF-,1-dependent activation of Smads, leading to PAI-1 synthesis, compared with controls, in SKOV-3 cells, (4) combination of TGF-,1 and PP2, which activates PAI-1 expression and reduces uPA expression in SKOV-3, results in decreased invasiveness, (5) pharmacological inhibitors for mitogen-activated protein kinase (MAPK) (PD98059) and phosphoinositide-3-kinase (PI3K) (LY294002 and wortmannin) or AS-PI3K ODN transfection do not affect TGF-,1-induced Smad signaling and up-regulation of PAI-1 expression in SKOV-3 cells pr treated with PP2, and (6) the induction of PAI-1 protein was partially inhibited by an inhibitor of Sp1-DNA binding, mithramycin, implicating, at least in part, Sp1 in the regulation of this gene by TGF-,1. In conclusion, TGF-,1-dependent activation of Smad2/3, leading to PAI-1 synthesis, may be negatively regulated by Src, but not its downstream targets MAPK and PI3K in SKOV-3 cells. These data also reflect the complex biological effect of uPA-PAI-1 system. © 2004 Wiley-Liss, Inc. [source] Effects of antisense oligodeoxynucleotide to type I collagen gene on hypertrophic scars in the transplanted nude mouse modelJOURNAL OF CUTANEOUS PATHOLOGY, Issue 11 2009Julin Xie Background:, Antisense nucleic acids are effective in inhibiting harmful or uncontrolled gene expression. We had previously proved that the antisense DNA to type I collagen could effectively inhibit the synthesis of collagen type I in cultured hypertrophic scar fibroblasts, suggesting a potential role in anti-scarring, but there are no published reports of its effect on scar in the transplanted nude mouse model. Aims:, To investigate the effects of antisense oligodeoxynucleotide (ASODN) to type I collagen gene on hypertrophic scars in the transplanted nude mouse model and clarify the potential of ASODN for the treatment of scars. Methods:, The nude mouse model of hypertrophic scar was created and subjected to daily injections with ASODN and LipofectamineÔ for 2 ,4 or 6 weeks. We then examined the scars for changes in histopathological characteristics. The effects of ASODN on type I collagen gene expression were examined by reverse transcription-polymerase chain reaction and Western blots. Results:, The ASODN could remarkably alleviate the scar in the nude mouse model and consistently inhibit type I collagen gene expression at both the mRNA and protein levels. Conclusion:, ASODN was effective in downregulating type I collagen gene expression and could prove to be useful in the treatment of scars. [source] Effect of instrument tuning on the detectabilityof biopolymers in electrospray ionization mass spectrometryJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 1 2003Herbert Oberacher Abstract Electrospray ionization mass spectrometry of multiply charged biopolymer ions of different molecular size revealed a strong influence of tuning parameters on their detectability in quadrupole ion trap and triple quadrupole mass spectrometers. Hence, after optimizing the ion optical parameters with the signal of the 4, charge state of (dT)24 (low charge state tuning), a tenfold increase in the signal-to-noise ratio for a mixture of oligodeoxythymidylic acids (n = 12,18) was obtained compared with the results achieved with tune parameters optimized with a synthetic 80-mer oligodeoxynucleotide. By contrast, a detection limit in the upper femtomole region could only be reached for a 104-mer oligodeoxynucleotide utilizing the 24, charge state of the 80-mer (high charge state tuning). The same effect was observed for proteins investigated in the positive ion mode using low and high charge states of cytochrome c and carbonic anhydrase, respectively, for instrument tuning. By comparing the settings for low and high charge state tuning, it became obvious that the most significant difference was observed in the potential applied to the heated metal capillary used to transfer ions from the atmospheric pressure to the vacuum region of the ion source. Taking advantage of the optimized tuning procedure, the molecular mass of a 61 base pair product of polymerase chain reaction was accurately determined by electrospray ionization mass spectrometry on-line interfaced to ion-pair reversed-phase high-performance liquid chromatography. Copyright © 2003 John Wiley & Sons, Ltd. [source] Induction of hypoxia inducible factor-1 attenuates metabolic insults induced by 3-nitropropionic acid in rat C6 glioma cellsJOURNAL OF NEUROCHEMISTRY, Issue 3 2005Ya-Ting Yang Abstract Compromised mitochondrial function in neurons and glia has been observed in several neurodegenerative disorders, including Huntington's disease and Alzheimer's disease. Chemical/hypoxic preconditioning may afford protection against subsequently more severe oxidative damages. In this study, we tested whether induction of hypoxia inducible factor-1 (HIF-1) may exert cytoprotective effects against mitochondrial dysfunction caused by 3-nitropropionic acid (3-NP) in glial cells. Preconditioning of C6 astroglial cells with cobalt chloride, mimosine (MIM), and desferrioxamine (DFO), all of which known to activate HIF-1, significantly attenuated cytotoxicity induced by 3-NP, an irreversible inhibitor of mitochondrial complex II, and antimycin A, a mitochondrial complex III inhibitor. Application of cadmium chloride capable of neutralizing cobalt-induced HIF-1 activation, HIF-specific oligodeoxynucleotide (ODN) decoy, and antisense phosphorothioate ODN against HIF-1, abolished the protective effect mediated by preconditioning with cobalt chloride. Preloading of C6 cells with SN50, PD98059, or SB202190, the respective inhibitor of nuclear factor-,B (NF-,B), p44/p42 extracellular signal-regulated kinase (ERK), and p38 mitogen-activated protein kinase (MAPK), failed to affect the protection afforded by cobalt preconditioning. Taken together, these results suggest that HIF-1 induction secondary to preconditioning with cobalt chloride or iron chelators may mediate the protective effects against metabolic insult induced by the mitochondrial inhibitor 3-NP in C6 astroglial cells. [source] Phosphothioated oligodeoxynucleotides induce nonspecific effects on neuronal cell adhesion in a growth substrate-dependent manner,JOURNAL OF NEUROSCIENCE RESEARCH, Issue 8 2009Eitan Okun Abstract Synthetic phosphothioated (PTO) oligodeoxynucleotide (ODN) sequences are commonly used for a variety of applications that benefit from nuclease protection. The PTO modification is implemented mainly in antisense ODN, but also in ODN that were shown to activate members of the toll-like receptor (TLR) family such as TLR3 (poly-I:C), TLR8 (ssRNA), and TLR9 (CpG). Neurons are routinely plated on surfaces coated with either cationic substances such as poly-L-ornithine (PLO), polyethylenimine (PEI), poly-L-lysine or ECM components such as laminin, collagen, or fibronectin. We found that PTO-ODN aimed at activating TLR9 induces a non-TLR9-specific detachment phenotype in cortical neurons plated on either laminin or PEI, but not on PLO. This phenotype was correlated with decreased viability and was partially inhibited when caspase-3 was inhibited with Ac-DEVD-CMK. This finding suggests that the use of PTO-ODN can cause nonspecific effects on cell adhesion that could compromise interpretation of data from experiments using PTO-ODN. © 2009 Wiley-Liss, Inc. [source] Significant and prolonged antisense effect of a multifunctional envelope-type nano device encapsulating antisense oligodeoxynucleotideJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 4 2006Yoshio Nakamura A multifunctional envelope-type nano device (MEND) was developed for use as an efficient non-viral system for the delivery of plasmid DNA (pDNA) using octaarginine (R8) as an internalizing ligand. Three types of R8-MENDs were prepared, co-encapsulating luciferase-encoding pDNA and antiluciferase oligodeoxynucleotide (ODN) condensed by three polycations, stearyl octaarginine (STR-R8), poly- l -lysine (PLL) and protamine, and the antisense effects of the ODN-encapsulated R8-MENDs (ODN-MEND) were analysed in-vitro. The ODN-MEND packaged using protamine as a condenser showed a 90% antisense effect 16 h after the transfection, and a persistent antisense effect of over 75% for up to 48 h, which was much more effective than that of LipofectAmine2000. On the other hand, the ODN-MENDs prepared using PLL and STR-R8 as condensers did not show any significant inhibition of luciferase activity. Although there was no specific relation between the physicochemical characteristics of the ODN-MENDs and their antisense effect, the pattern of the antisense effect among the ODN-MENDs was similar to that of the silencing effect of R8-MEND encapsulating plasmid DNA encoding siRNA. These results suggest that R8-MENDs are able to deliver encapsulated DNA to the cytosol as well as to the nucleus, and that protamine can also function as an efficient decondenser, not only in the nucleus but also in the cytosol. In conclusion, we successfully developed an ODN-MEND with a high antisense effect using protamine as a DNA condensing as well as a decondensing agent. [source] Quantitative evaluation of the interaction between netropsin and double stranded oligodeoxynucleotides by microfabricated capillary array electrophoresisJOURNAL OF SEPARATION SCIENCE, JSS, Issue 10 2007Zheng Shen Abstract Microfabricated capillary array electrophoresis (,-CAE) was applied to study the interaction between minor groove binder netropsin and a non-selfcomplementary 12 mer double stranded oligodeoxynucleotide: d(CCCCTATACCGC)·d(GCGGTATAGGGG). ESI-MS was used to provide an independent verification of the microchip electrophoresis derived data. Simultaneous parallel quantitative assay of multiple samples was performed in a single run (<50 s) on the self-developed ,-CAE device. The binding constant and stoichiometry calculated from Scatchard plot were (2.88 ± 0.23)×105 M,1 and 1:1, respectively. The values showed a good quantitative agreement with the results determined by ESI-MS and those using other methods reported in the literature. [source] p53 cooperates berberine-induced growth inhibition and apoptosis of non-small cell human lung cancer cells in vitro and tumor xenograft growth in vivoMOLECULAR CARCINOGENESIS, Issue 1 2009Santosh K. Katiyar Abstract Berberine has been shown to have anti-carcinogenic effects. Since p53 is the most commonly mutated tumor suppressor gene, and a lack of functional p53 is associated with an increased risk of cancer development, we examined the effects of berberine on p53-positive and p53-deficient non-small cell human lung cancer cells in vitro and in vivo. Treatment of A549, which express wild-type p53, and H1299, which are p53-deficient, human lung cancer cells with berberine resulted in inhibition of cell proliferation and an increase in apoptotic cell death; however, A549 cells were more sensitive to the berberine-induced cytotoxic effects than H1299 cells. Further, the treatment of A549 cells with pifithrin-,, a specific inhibitor of p53, or transfection of A549 cells with a p53 antisense oligodeoxynucleotide resulted in a reduction in the berberine-induced inhibition of cell proliferation and apoptosis. The berberine-induced apoptosis of both the A549 and H1299 human lung cancer cells was associated with the disruption of mitochondrial membrane potential, reduction in the levels of Bcl-2, Bcl-xl while increase in Bax, Bak, and activation of caspase-3. Treatment of the cells with pan-caspase inhibitor (z-VAD-fmk) or caspase-3 inhibitor (z-DEVD-fmk) inhibited berberine-induced apoptosis, thus suggesting the role of caspase-3. Further, the administration of berberine by oral gavage inhibited the growth of s.c. A549 and H1299 lung tumor xenografts in athymic nude mice, however, the growth of tumor xenograft of H1299 cells was faster than A549 cells in mice and the chemotherapeutic effect of berberine was more pronounced in the p53-positive-A549 tumor xenograft than p53-deficient-H1299 tumor xenograft. © 2008 Wiley-Liss, Inc. [source] CpG-containing ODN has a limited role in the protection against Toxoplasma gondiiPARASITE IMMUNOLOGY, Issue 2 2004R. Saavedra SUMMARY Bacterial DNA containing immunostimulatory motifs (CpG) induces the development of a TH1 immune response. Since protection against Toxoplasma gondii is correlated with this type of response, the aim of this work was to determine if a synthetic oligodeoxynucleotide (ODN) containing CpG sequences could be useful as adjuvant for the induction of a long-lasting protective immune response against T. gondii. BALB/c mice immunized with a total soluble antigen of T. gondii (TSA2) mixed with ODN-containing CpG sequences developed a typical TH1 response, as determined by antibody isotypes and interferon-, (IFN-,) and interleukin-4 (IL-4) production by spleen cells. However, they did not resist a challenge with the virulent RH strain of the parasite. Absence of protection paralleled with lower levels of IFN-,, when compared with mice vaccinated with the live tachyzoites of the attenuated ts.4 strain of the parasite, which resisted this challenge. Intraperitoneal injection of ODN alone to mice induced a high degree of resistance to a lethal challenge inoculated by the same route. Nevertheless, this nonspecific protection was transient. Thus, the use of ODN containing CpG motifs as adjuvant is of limited value for the induction of a protective immune response against T. gondii. [source] Identification of a potent immunostimulatory oligodeoxynucleotide from Streptococcus thermophilus lacZANIMAL SCIENCE JOURNAL, Issue 5 2009Takeshi SHIMOSATO ABSTRACT Immunostimulatory sequences of oligodeoxynucleotides (ODNs), such as CpG ODNs, are potent stimulators of innate immunity. Here, we identified a strong immunostimulatory CpG ODN, which we named MsST, from the lac Z gene of Streptococcus (S.) thermophilus ATCC19258, and we evaluated its immune functions. In in vitro studies, MsST had a similar ability as the murine prototype CpG ODN 1555 to induce inflammatory cytokine production and cell proliferation. In mouse splenocytes, MsST increased the number of CD80+CD11c+and CD86+CD11c+ dendritic cells and CD4+CD25+ regulatory T cells. We also analyzed the effects of MsST on the expression of regulatory cytokines by real-time quantitative PCR. MsST was more potent at inducing interleukin-10 expression than the ODN control 1612, indicating that MsST can augment the regulatory T cell response via Toll-like receptor 9, which plays an important role in suppressing T helper type 2 responses. These results suggest that S. thermophilus, whose genes include a strong Immunostimulatory sequence-ODN, is a good candidate for a starter culture to develop new physiologically functional foods and feeds. [source] Evidence from immunoneutralization and antisense studies that the inhibitory actions of glucocorticoids on growth hormone release in vitro require annexin 1 (lipocortin 1)BRITISH JOURNAL OF PHARMACOLOGY, Issue 7 2000A D Taylor Our previous studies have identified a role for annexin 1 as a mediator of glucocorticoid action in the neuroendocrine system. The present study centred on growth hormone (GH) and exploited antisense and immunoneutralization strategies to examine in vitro the potential role of annexin 1 in effecting the regulatory actions of glucocorticoids on the secretion of this pituitary hormone. Rat anterior pituitary tissue responded in vitro to growth hormone releasing hormone, forskolin, 8-Bromo-cyclic adenosine 3,5,-monophosphate (8-Br-cyclic AMP) and an L-Ca2+ channel opener (BAY K8644) with concentration-dependent increases GH release which were readily inhibited by corticosterone and dexamethasone. The inhibitory actions of the steroids on GH release elicited by the above secretagogues were effectively reversed by an annexin 1 antisense oligodeoxynucleotide (ODN), but not by control (sense or scrambled) ODNs, as also were the glucocorticoid-induced increases in annexin 1. Similarly, a specific anti-annexin 1 monoclonal antibody quenched the corticosterone-induced suppression of secretagogue-evoked GH release while an isotype matched control antibody was without effect. Transmission electron micrographs showed that the integrity and ultrastructural morphology of the pituitary cells were well preserved at the end of the incubation and unaffected by exposure to the ODNs, antibodies, steroids or secretagogues. The results provide novel evidence for a role for annexin 1 as a mediator of the inhibitory actions of glucocorticoids on the secretion of GH by the anterior pituitary gland and suggest that its actions are effected at a point distal to the formation of cyclic AMP and Ca2+ entry. British Journal of Pharmacology (2000) 131, 1309,1316; doi:10.1038/sj.bjp.0703694 [source] The influence of STAT5 antisense oligonucleotides on the proliferation and apoptosis of selected human leukaemic cell linesCELL PROLIFERATION, Issue 5 2003M. Ba, kiewicz-Masiuk They influence the cell cycle, apoptosis and the proliferation of different types of cell lines. The STAT5 proteins are induced in response to multiple haematopoietic cytokines. Because they are constitutively active in certain haemato-oncologic diseases, it is also suggested that they play an important role in leukaemogenesis. However, function of these proteins in haematopoietic cell transformation and proliferation is not clear. The aim of this study was to evaluate the impact of perturbation of STAT5 expression [using oligodeoxynucleotide (ODN) against STAT5 mRNA], on the clonogenicity and survival of selected human leukaemic cell lines, HEL, HL-60, K562, TF-1. We analysed the effect of ODN pre-treatment on the cell clonogenicity in methylcellulose cultures according to the time and the temperature of exposure. Moreover, we attempted to estimate apoptosis induced in examined cells, by flow cytometry using combined Annexin V-PI staining and the TUNEL method. We also applied the RT-PCR method to analyse Bax and Bcl-xL gene expression. We found that the perturbation of STAT5 expression with antisense oligonucleotides caused a decrease in the proliferative potential of human K562 and TF-1 cell lines. Also, we observed higher induction of apoptotic cell death in the K562 and TF-1 cells incubated with the antisense STAT5A ODNs. We did not notice any impact of ODNs on the HL-60 and HEL cells. Our studies using STAT5 antisense oligonucleotides showed that these proteins may be critical in the regulation of growth and apoptosis of some types of leukaemic blasts. [source] Strong immunostimulatory activity of AT-oligodeoxynucleotide requires a six-base loop with a self-stabilized 5,-C,G-3, stem structureCELLULAR MICROBIOLOGY, Issue 3 2006Takeshi Shimosato Summary Lactobacillus gasseri OLL2716 has recently been discovered as a probiotic that suppresses the growth of Helicobacter pylori and reduces gastric mucosal inflammation in humans. This has resulted in the development of a new type of probiotic yoghurt ,LG21' in Japan. In our previous study, we found an immunostimulatory AT5ACL oligodeoxynucleotide (AT-ODN) containing a unique core sequence (5,-ATTTTTAC-3,) in L. gasseri JCM1131T. Interestingly, although the AT-ODN does not contain any CpG sequences, it exerts mitogenic activity in B cells and augments Th-1-type immune responses via Toll-like receptor 9. These findings prompted us to identify strong immunostimulatory non-CpG AT-ODNs that contain the 5,-ATTTTTAC-3, motif in the genomic sequence of L. gasseri OLL2716. We identified 280 kinds of AT-ODNs in the L. gasseri OLL2716 genome. Mitogenicity and NF-,B gene reporting assays showed that 13 of the 280 AT-ODNs were strongly immunostimulatory when in the TLR9 transfectant. Of these, AT-ODNs LGAT-145 and LGAT-243 were the most potent. With respect to the induction of Th-1-type cytokines, LGAT-243 had the greatest activity and was more potent than the swine prototype, ODN D25. We further found that a six-base secondary loop structure containing a self-stabilized 5,-C,G-3, stem sequence is important for potent immunostimulatory activity. These results show for the first time that AT-ODNs with a specific loop and stem structure are important factors for immunostimulatory activity. Finally, we found that novel strong immunostimulatory non-CpG AT-ODNs exist in the genome of probiotic lactic acid bacteria. [source] Strong immunostimulation in murine immune cells by Lactobacillus rhamnosus GG DNA containing novel oligodeoxynucleotide patternCELLULAR MICROBIOLOGY, Issue 3 2005Iliyan D. Iliev Summary Whole cells, cell wall components and some soluble factors from Lactobacillus rhamnosus GG (LGG) are known to invoke immune responses as they interact with animal and human immune cells. In the present study, we found that chromosomal DNA from LGG is a potent inducer of splenic B cell proliferation, CD86/CD69 expression and cytokine production in mice. In the genomic DNA of LGG we discovered TTTCGTTT oligodeoxynucleotide (ODN) ID35, which has a potent activity in a number of immunostimulatory assays. Phosphorothioate backbone is not required for the activity of ID35. The ODN ID35 showed levels of activity comparable with those induced by the murine prototype ODN 1826 in B cell proliferation, CD86/CD69 expression, interleukin (IL)-6, IL-12, IL-18, interferon gamma (IFN-,) and tumour necrosis factor alpha (TNF-,) mRNA expression and IFN-,/IL-12p70 protein production assays. Additionally, ID35 appeared to be equally active in both murine and human immune cells. These stimulatory effects are due to TTTCGTTT motif located in the 5, end of ID35. In this study we demonstrate for a first time that, DNA from LGG is a factor of immunobiotic activity. Furthermore, ODN ID35 is the first ODN, with such a strong immunostimulatory activity to be found in immunobiotic bacterial DNA. [source] Role of Environmental Factors on the Structure and Spectroscopic Response of 5,-DNA,Porphyrin Conjugates Caused by Changes in the Porphyrin,Porphyrin InteractionsCHEMISTRY - A EUROPEAN JOURNAL, Issue 44 2009Angela Mammana Dr. Abstract We have explored the utility, strength, and limitation of through-space exciton-coupled circular dichroism in determination of the secondary structure of optically active chromophoric nanoarrays using the example of end-capped porphyrin, and metalloporphyrin,oligodeoxynucleotide conjugates. We put special emphasis on the explanation of the origin and significance of the distinctive multiple bands in the CD spectra (trisignate and tetrasignate CD bands). Such CD profiles are often observed in chiral aggregates or multichromophoric arrays but have never before been studied in detail. We found that variation of temperature and ionic strength has a profound effect on the geometry of the porphyrin,DNA conjugates and thus the nature of electronic interactions. At lower temperatures and in the absence of NaCl all three 5,-DNA,porphyrin conjugates display negative bisignate CD exciton couplets of variable intensity in the Soret region resulting from through-space interaction between the electric transition dipole moments of the two end-capped porphyrins. As the temperature is raised these exciton couplets are transformed into single positive bands originating from the porphyrin,single-strand DNA interactions. At higher ionic strengths and low temperatures, multisignate CD bands are observed in the porphyrin Soret region. These CD signature bands originate from a combination of intermolecular, end-to-end porphyrin,porphyrin stacking between duplexes and porphyrin,DNA interactions. The intermolecular aggregation was confirmed by fluorescence and absorption spectroscopy and resonance light scattering. DeVoe theoretical CD calculations, in conjunction with molecular dynamics simulations and Monte Carlo conformational searches, were used to mimic the observed bisignate exciton-coupled CD spectra as well as multiple CD bands. Calculations correctly predicted the sign and shape of the experimentally observed CD spectra. These studies reveal that the exciton-coupled circular dichroism is a very useful technique for the determination of the structure of optically active arrays. [source] Synthesis and Hybridization Properties of Modified Oligodeoxynucleotides Carrying Non-Natural BasesCHEMISTRY & BIODIVERSITY, Issue 2 2009Anna Aviñó Abstract The impact of the presence of nonnatural bases on the properties of oligodeoxynucleotides has been studied. First, oligodeoxynucleotides carrying 2,-deoxyzebularine were prepared, and the stability of duplexes carrying this analogue was determined by DNA melting experiments. Melting temperatures and thermodynamic data indicated the preference of 2,-deoxyzebularine for 2,-deoxyguanosine, which behaves as a 2,-deoxycytidine analogue, forming a less stable base pair due to the absence of the amino group at position 4. Moreover, the duplex,hairpin equilibrium of a self-complementary oligodeoxynucleotide carrying several natural and nonnatural bases including 2,-deoxyzebularine as a central mispair, was studied. Depending on the base present in the middle of the sequence, it is possible to affect the stability of the bimolecular duplex modulating the duplex,hairpin equilibrium. Magnesium ions were shown to stabilize preferentially the bimolecular duplex form. The results indicate the importance of the modifications and the role of cations in shifting the structural equilibrium. [source] Protein phosphatase 1, is required for murine lung growth and morphogenesisDEVELOPMENTAL DYNAMICS, Issue 4 2004Kadija-Kathy Hormi-Carver Abstract Protein phosphatase 1 (PP1) plays important roles in cell cycle control and apoptosis, two processes that impinge on morphogenesis and differentiation. Following the precedent set by other molecules regulating the cell cycle and apoptosis, we hypothesized that PP1 may have context-specific roles in development. Therefore, we have studied the spatial and temporal expression of PP1, during murine lung development and determined the consequences of loss of PP1, function on branching morphogenesis. By using an immunohistochemical approach, we show here that PP1, was expressed throughout the epithelium and mesenchyme upon the emergence of the lung primordium on embryonic day 10, with immunostaining exclusively extranuclear. During the late pseudoglandular stage, PP1, was predominantly expressed in the distal lung epithelium, whereas the mesenchyme contained very little or no PP1, protein. Peri- and postnatally, PP1, immunostaining was mostly nuclear in apparently differentiated cells, as judged by colocalization with well-known markers for lung differentiation. Exposure of fetal lung explants to antisense oligodeoxynucleotides against PP1,, resulted in decreased overall size of the cultured lung, a defect in forming new airways, lack of expression of surfactant protein C, and histologic signs of poor differentiation. These data suggest that PP1, is required for branching morphogenesis and differentiation. Developmental Dynamics 229:791,801, 2004. © 2004 Wiley-Liss, Inc. [source] CpG ODN enhance antigen-specific NKT cell activation via plasmacytoid dendritic cellsEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 8 2005Anja Marschner Abstract Human V,24+ V,11+ natural killer T cells (NKT cells) are "natural memory" T cells that detect glycolipid antigens such as ,-galactosylceramide (,-GalCer) presented on CD1d. In the present study we found that highly purified V,24+ NKT cells lack TLR9 mRNA, and thus are not sensitive towards stimulation with CpG oligodeoxynucleotides (ODN). Within PBMC, however, CpG ODN synergistically activated NKT cells stimulated with their cognate antigen ,-GalCer. Depletion of plasmacytoid dendritic cells (PDC) or myeloid dendritic cells (MDC) revealed that both DC subsets were necessary for the synergistic activation of NKT cells by ,-GalCer and CpG ODN. While PDC were responsible for the stimulation of NKT cells with CpG ODN, MDC but not PDC presented ,-GalCer via CD1d. Partial activation of NKT cells was mediated by PDC-derived IFN-,, whereas full activation of NKT cells as indicated by IFN,, production required cell-to-cell contact of PDC and NKT cells in addition to IFN-,; OX40 was involved in this interaction. We conclude that CpG-activated PDC enhance ,-GalCer-specific NKT cell activation, and bias activated NKT cells towards a Th1 phenotype. Our results lead to a novel concept of PDC function: to regulate effector activity of antigen-stimulated T cells in a cell contact-dependent manner without the need of simultaneous presentation of the cognate T cell antigen. [source] |