Antisense Techniques Research Articles

1-Aminocyclopropane-1-Carboxylate Oxidase 2 Reduction Effects on Physical and Physiological Responses of Eksotika Papaya

The Malaysian Eksotika papaya (Carica papaya L.) has poor keeping quality due to its fast ripening attribute, which leads to post-harvest losses. This study is aimed at extending the shelf life of this perishable Eksotika fruit using antisense technology. A total of 6,000 Eksotika somatic embryogenic calli was transformed with the antisense 1-aminocyclopropane-1-carboxylic acid oxidase 2 gene (ACO2) construct, and 46 PCR-positive putative transformants were obtained. Gene expression analysis using real-time PCR on the 46 regenerated putative transgenic lines revealed that 42 showed down-regulation of the ACO2 gene with two- to five-fold differences among the lines. Out of 22 independently selected transformed lines grown under net house conditions, 16 harbored a single copy of the transgene. Physical stature of the transgenic plants was not significantly different from that of the non-transformed seed-derived papaya plants. Physiological evaluations of the transgenic fruits showed a 15-day delay in ripening compared with 4 days of the non-transformed seed-derived papaya fruits. The total soluble solid (TSS) of the transgenic fruits was comparable to that of the non-transformed seed-derived fruits with similar 11-15°Brix, implying the transgenes did not affect the TSS content. The transgenic fruits remained firm for additional 4 to 8 days at room temperature (25 ± 2°C) after achieving the full maturity index (index 6), whereas the non-transformed seed-derived fruits lost their firmness after 2 days. The outcomes of this study revealed that reduction of ethylene was successful in the Eksotika papaya by manipulating the ACO2 gene using the antisense technique.

Zebrafish Guanylate Cyclase Type 3 Signaling in Cone Photoreceptors

The zebrafish guanylate cyclase type 3 (zGC3) is specifically expressed in cone cells. A specifc antibody directed against zGC3 revealed expression at the protein level at 3.5 dpf in outer and inner retinal layers, which increased in intensity between 3.5 and 7 dpf. This expression pattern differed from sections of the adult retina showing strong immunostaining in outer segments of double cones and short single cones, less intense immunoreactivity in long single cones, but no staining in the inner retina. Although transcription and protein expression levels of zGC3 are similar to that of the cyclase regulator guanylate cyclase-activating protein 3 (zGCAP3), we surprisingly found that zGCAP3 is present in a 28-fold molar excess over zGC3 in zebrafish retinae. Further, zGCAP3 was an efficient regulator of guanylate cyclases activity in native zebrafish retinal membrane preparations. Therefore, we investigated the physiological function of zGCAP3 by two different behavioral assays. Using the morpholino antisense technique, we knocked down expression of zGCAP3 and recorded the optokinetic and optomotor responses of morphants, control morphants, and wild type fish at 5–6 dpf. No significant differences in behavioral responses among wild type, morphants and control morphants were found, indicating that a loss of zGCAP3 has no consequences in primary visual processing in the larval retina despite its prominent expression pattern. Its physiological function is therefore compensated by other zGCAP isoforms.

Molecular mechanisms underlying the anti‐proliferative and anti‐migratory effects of folate on homocysteine‐challenged rat aortic smooth muscle cells

Homocysteine is an intermediate product formed during the metabolism of methionine, and is increased in cells with folate deficiency. Patients with hyperhomocysteinemia tend to develop cardiovascular disease. Here, we have examined the molecular mechanisms underlying the anti-proliferative and anti-migratory effects of folate on homocysteine-challenged rat aortic smooth muscle cells (RASMCs). Cultures of RASMC were challenged with homocysteine and then incubated with folate added. Changes in p21/p27, AKT and RhoA were followed by RT-PCR, Western blotting and immunocytochemistry. Transfection and anti-sense techniques were also used. Cell viability, growth and migration were measured. Folate up-regulated p21/p27 through a Src/ERK-dependent mechanism that accounted for its anti-proliferative effects on RASMC. Folate protected RASMC from the effects of homocysteine by reducing AKT1, focal adhesion kinase (FAK), paxillin, and p190RhoGAP activation/phosphorylation, along with cytosolic levels of p21 and p27, and increasing RhoA activation. Overexpression of AKT1, but not of AKT2, induced p21/p27 phosphorylation and increased cytosolic p21/p27 levels, as did homocysteine treatment. By contrast, and similarly to folate treatment, transfection with dominant negative (DN) AKT1 counteracted these effects. Additionally, AKT was shown to be an upstream target of FAK activation. In RASMC overexpressing constitutively active RhoA, activation of RhoA mediated the anti-migratory effects of folate. Addition of Y27632 (a RhoA inhibitor) and DNRhoA counteracted the anti-migratory effects, confirming RhoA involvement. Folate was anti-proliferative and anti-migratory in homocysteine-challenged RASMC. Mechanisms underlying folate-mediated protection against the proatherosclerotic effects of homocysteine have been delineated.

Development of a highly sensitive single-tube nested PCR protocol directed toward the sequence of virion envelope proteins for detection of white spot syndrome virus infection: Improvement of PCR methods for detection of WSSV

White spot syndrome virus (WSSV) is a double-stranded DNA virus that causes severe mortality, leading to great economic loss for cultured shrimp farms. The Manual for Aquatic Animals published by the Office International des Epizooties (OIE, 2012) announced the nested PCR protocol, developed by Lo et al. in 1996 as the standard protocol for WSSV diagnosis. Because current detection tools are prone to carryover contamination and are labor-intensive, an accurate and convenient technique is required. Therefore, the aim of this study was to develop an improved diagnostic PCR method for WSSV that overcomes the known drawbacks of the conventional PCR method. We designed primers directed toward the nucleotide sequence encoding envelope proteins of WSSV, VP19 and VP28. By applying an antisense oligonucleotide technique, we converted this PCR assay into a non-stop one-tube system, subsequently enabling a faster and more sensible PCR assay. In practice, the newly developed protocol detected the WSSV genome from imported frozen shrimps, crabs and bivalves, with a shorter PCR running time and a higher sensitivity compared with other PCR methods.

IL-21 Enhances Phagocytosis in Mononuclear Phagocyte Cells: Identification of Spleen Tyrosine Kinase as a Novel Molecular Target of IL-21

The biological significance of the IL-21/IL-21R system in human monocytes/macrophages is not well documented, and the expression of IL-21R is unclear and has been disputed. In this study, we showed for the first time, to our knowledge, that human monocyte-like THP-1 cells expressed the two IL-21R components, CD132 (γc) and IL-21Rα, on their cell surface, as assessed by flow cytometry. Moreover, IL-21 was found to enhance FcR-mediated phagocytosis, but not endocytosis. The ability of IL-21 to enhance phagocytosis was not associated with an increased expression of both IL-21R components at the cell surface, and IL-21 did not act in synergy with IL-15. IL-21 activated spleen tyrosine kinase (Syk), as evidenced by its ability to increase Syk phosphorylation. Using a pharmacological approach to inhibit Syk activity, and an antisense technique to downregulate Syk protein expression, we demonstrated the importance of Syk in IL-21-induced phagocytosis. In addition, both CD132 and IL-21Rα were expressed on the cell surface of naive monocytes, as well as in GM-CSF-monocyte-derived macrophages. Moreover, IL-21 also induced phagocytosis in these cells. We conclude that IL-21 possesses important biological effects in mononuclear phagocyte cells and that Syk is a novel molecular target of IL-21 that was previously unknown. Therefore, future development of therapeutic strategies targeting the IL-21/IL-21R system should consider that monocyte and macrophage cell physiology may be affected by this system.

Isolation and identification of cell-specific microRNAs targeting a messenger RNA using a biotinylated anti-sense oligonucleotide capture affinity technique

MicroRNAs (miRNAs) are small non-coding RNAs that regulate expression by translational repression or messenger RNA (mRNA) degradation. Although numerous bioinformatic prediction models exist to identify miRNA–mRNA interactions, experimental validation of bona fide interactions can be difficult and laborious. Few methods can comprehensively identify miRNAs that target a single mRNA. We have developed an experimental approach to search for miRNAs targeting any mRNA using a capture affinity assay involving a biotinylated DNA anti-sense oligonucleotide. This method identifies miRNAs targeting the full length of the mRNA. The method was tested using three separate mRNA targets: alpha-1 antitrypsin (AAT) mRNA, interleukin-8 mRNA and secretory leucoprotease inhibitor mRNA. AAT mRNA-specific and total miRNAs from three different cell lines (monocytic THP-1, bronchial epithelial 16HBE14o− and liver HepG2 cells) were profiled, and validation studies revealed that AAT mRNA-specific miRNAs functionally target the AAT mRNA in a cell-specific manner, providing the first evidence of innate miRNAs selectively targeting and modulating AAT mRNA expression. Interleukin-8 and secretory leucoprotease inhibitor mRNAs and their cognate miRNAs were also successfully captured using this approach. This is a simple and an efficient method to potentially identify miRNAs targeting sequences within the full length of a given mRNA transcript.

Demethylation of miR-9-3 and miR-193a Genes Suppresses Proliferation and Promotes Apoptosis in Non-Small Cell Lung Cancer Cell Lines

Background: MicroRNAs miR-9-3 and miR-193a have recently been found to be hypermethylated in a variety of non-small cell lung cancer (NSCLC) cells and primary human tumors. The objectives of this study were to investigate the role of demethylation of miR-9-3 and miR-193a genes in regulating proliferation and apoptosis in NSCLCs, and to decipher the potential mechanisms underlying the properties. Methods: MTT and population doubling time by flow cytometry were used to assess cell proliferation. Enzyme-Linked Immunosorbent Assay and caspase-3 activity assay were employed to evaluate apoptosis. Real-time RT-PCR and Western blot were used to quantify gene expression at mRNA and protein levels, respectively. Methylation-specific PCR was utilized to assess methylation status. Results: We found that demethylation agent 5-Aza-2'-deoxycytidine (5-AzaC) reduced cell numbers and prolonged population doubling time (PDT), and promoted doxorubicin-induced apoptosis in seven NSCLC cell lines with different methylation statuses on miR-9-3 and miR-193a promoter regions: NCI-H1993/NCI-H1915 (miR-9-3⁺/miR-193a⁺), NCI-H1975/NCI-H200 (miR-9-3⁺/miR-193a^-), A427/NCI-H2073 (miR-9-3^-/miR-193a⁺), and NCI-H1703 (miR-9-3^-/miR-193a^-). Treatment with 5-AzaC concomitantly upregulated expression of miR-9-3 and miR-193a, and downregulated their respective target genes NF-κB and Mcl-1. The effects of 5-AzaC were abolished by concomitant knockdown of miR-9-3 and miR-193a using the complex antisense technique, whereas forced ectopic expression of miR-9-3 and miR-193a mimicked the effects of 5-AzaC. We further observed that the strength of proliferation inhibition and apoptosis promotion elicited by 5-AzaC was in the order of NCI-H1993/NCI-H1915 > A427/NCI-H2073 > NCI-H1975/NCI-H200 > NCI-H1703. Conclusions: Methylation-silencing of miR-9-3 and miR-193a may be an important epigenetic mechanisms favoring NSCLC cell growth and survival for carcinogenesis and cancer progression, and demethylation to reactivate expression of miR-9-3 and miR-193a genes contributes, at least partially, to the anti-cancer properties of 5-AzaC and thereby may be worthy of future studies for the possibility of being a new therapeutic strategy for the treatment of human NSCLCs.

Efficient down-regulation of PKC-α gene expression in A549 lung cancer cells mediated by antisense oligodeoxynucleotides in dendrosomes

The completion of human genome project has increased our knowledge of the molecular mechanisms of many diseases, including cancer, thus providing new opportunities for gene therapy. Antisense oligodeoxynucleotides (AsODN) possess great potential as sequence-specific therapeutic agents, which in contrast to classic treatments provide more efficient and target-specific approach to modulate disease-related genes. To be therapeutically effective, sufficient concentrations of intact AsODN must bypass membrane barriers and access the site of action. In this study, a dendrosome delivery strategy was designed to improve the encapsulation of AsODN in non-cationic liposomes to target PKC-α in lung cancer cells in vitro. Subcellular trafficking of fluorescently labeled AsODN was visualized using confocal microscopy. Uptake and expression of mRNA and target protein after AsODN delivery was measured by flow cytometry, qRT-PCR and Western blot analysis, respectively. Dendrosomes showed favorable physicochemical parameters: high encapsulation efficiency and uptake in serum-containing medium with no apparent cytotoxicity. AsODN encapsulated in dendrosome efficiently and specifically suppress the target gene at both mRNA and protein levels. Additional in vivo studies on the application of dendrosome as a delivery system for nucleic acid molecules may lead to improvement of this technology and facilitate the development of therapeutic antisense techniques.

Antisense-mediated depletion of GMPase gene expression in tobacco decreases plant tolerance to temperature stresses and alters plant development

In our previous work [1] we investigated the role of tomato GDP-mannose pyrophosphorylase (EC 2.7.7.22) in plants by overexpressing its gene in tobacco leaves and showed its function in AsA metabolism and detoxification of reactive oxygen species under temperature stresses. In this study, we use the antisense technique to block the endogenous GMPase gene expression in tobacco in order to further investigate its function. Northern and western blot analysis confirmed that the expression of endogenous tobacco GMPase mRNA and protein was inhibited by this antisense expression. Consequently, the activity of GMPase and the content of AsA in the leaves of antisense transgenic plants were markedly decreased. This was also the case for the activities of both chloroplastic SOD (superoxide dismutase EC 1.15.1.1), APX (ascorbate peroxidase EC 1.11.1.7) and the content of AsA in leaves of the transgenic plants. On the contrary, the contents of H(2)O(2) and O(2) (-•) were increased. Meanwhile, the net photosynthetic rate (Pn) and the maximal photochemical efficiency of PSII (Fv/Fm) also declined in the leaves of antisense plants. Under high or low temperature stresses, the seed germination rate of the antisense transgenic plants was significantly decreased in comparison with that of the wild-type tobacco. Interestingly, the antisense plants had smaller leaves and an earlier onset of flowering. In conclusion, the depletion of GMPase decreased the content of AsA, resulting in the plants susceptible to the oxidative damage caused by temperature stresses and subjected to developmental alternations.

P162 Ciliary neurotrophic factor protects mice against type 1 diabetes and interleukin-1B-induced beta-cell apoptosis through SOCS3 expression: Role of stat1/stat3 balance in cytokine- induced iNOS-dependent beta-cell death

Introduction Type 1 Diabetes (DM1) is characterized by a cytokine-dependent loss of pancreatic islet beta-cell mass with subsequent hyperglycaemia. Ciliary Neurotrophic Factor (CNTF) protects pancreatic islets against cytokine-induced apoptosis and increases SOCS3 expression. In addition, beta-cell apoptosis seems to be induced by STAT1 and inhibited by STAT3 activation. We evaluated if CNTF protects mice against Streptozotocin-induced Type 1 Diabetes, if this protection is dependent of SOCS3 and/or iNOS expression, and what the role of STAT1/STAT3 activation in CNTF effects on cytokine-induced iNOS-dependent beta-cell apoptosis. Methods We developed SOCS3 Knockdown mice (SOCS3Kd) by Antisense technique from a C57BL6 Wild-Type background. To evaluate the role of iNOS on the CNTF protective effects in vivo we used iNOS-knockout mice. For Streptozotocin-induced Type 1 Diabetes, 4–6 weeks-old mice were treated or not with 0.1 mg/kg of CNTF for 5 days, receiving, intraperitoneally, 80 mg/kg of Streptozotocin (STZ) on the 5th day. Glycaemia from non-fasted mice was measured at days 0, 1, 7, 14, 21, and 28 after STZ. DM1 was considered as blood glycaemia >11.2 mmol/l for 2 days. We evaluated mice glycaemia, DM1 incidence, time to DM1 onset and DM1 severity. Pancreatic islets were isolated by collagenase method for mRNA and protein extraction. SOCS3 was inhibited in MIN6 cells using an SOCS3 specific siRNA (SOCS3kd cells), and iNOS inhibition was obtained by treating MIN6 cells with 1 mmol/l of the iNOS inhibitor LMEM (iNOSkd cells). Wild-type (WT), SOCS3kd, and iNOSkd cells were cultured with 10 ug/ml of IL1beta, 1 nmol/l CNTF or both. mRNA levels were assessed by Real-Time RT-PCR and protein expression and phosphorylation by Western-Blot. ( n = 4 − 6) Data = Mean ± SEM. P Results CNTF reduced DM1 incidence by 80% in WT but only by 10% in SOCS3kd mice. In WT, CNTF increased STAT3 and reduced STAT1 phosphorylation, while in SOCS3kd mice CNTF further increased STAT3 but failed to reduce STAT1. STZ reduced STAT3 and increased STAT1 phosphorylation, while in SOCS3kd mice STZ failed to reduce STAT3 but further increased STAT1. CNTF prevented STZ-induced pancreatic islets apoptosis and IL1beta-induced MIN6 cells apoptosis in WT mice and cells, but not in SOCS3kd or iNOSkd cells. The incidence of STZ-induced DM1 was lower in iNOSkd mice (20% of Control), and CNTF failed to further protect these mice. Nevertheless, CNTF effects over upstream pathways, such as increased STAT3 and SOCS3, and reduced STAT1, were similar to those of WT mice. In iNOSkd cells, CNTF partially prevented IL-1beta-induced cell-death, even though its upstream effects were similar to the WT cells, as we observed in vivo . Conclusion CNTF protects against STZ-induced DM1 and against cytokine-induced beta-cell death. These effects are dependent on the increase in SOCS3 expression. The cytokine-induced apoptosis as well as the pro-survival effect of CNTF over beta-cells depend on the balance between STAT1/STAT3 activation.

Downregulation of miR-210 expression inhibits proliferation, induces apoptosis and enhances radiosensitivity in hypoxic human hepatoma cells in vitro

Hypoxia is a common feature of solid tumors and an important contributor to tumor radioresistance. miR-210 is the most consistently and robustly induced microRNA under hypoxia in different types of tumor cells and normal cells. In the present study, to explore the feasibility of miR-210 as an effective therapeutic target, lentiviral-mediated anti-sense miR-210 gene transfer technique was employed to downregulate miR-210 expression in hypoxic human hepatoma SMMC-7721, HepG2 and HuH7 cells, and phenotypic changes of which were analyzed. Hypoxia led to an increased hypoxia inducible factor-1α (HIF-1α) and miR-210 expression and cell arrest in the G0/G1 phase in all cell lines. miR-210 downregulation significantly suppressed cell viability, induced cell arrest in the G0/G1 phase, increased apoptotic rate and enhanced radiosensitivity in hypoxic human hepatoma cells. Moreover, apoptosis-inducing factor, mitochondrion-associated, 3 (AIFM3) was identified as a direct target gene of miR-210. AIFM3 downregulation by siRNA attenuated radiation induced apoptosis in miR-210 downregulated hypoxic human hepatoma cells. Taken together, these data suggest that miR-210 might be a potential therapeutic target and specific inhibition of miR-210 expression in combination with radiotherapy might be expected to exert strong anti-tumor effect on hypoxic human hepatoma cells.

Clinical significance of miR-155 expression in breast cancer and effects of miR-155 ASO on cell viability and apoptosis

Accumulating evidence shows that mircroRNAs (miRNAs) play a vital role in tumorigenesis. miR-155 is one of the most multifunctional miRNAs whose overexpression has been found to be associated with different types of cancer including breast cancer. To further determine the potential involvement of miR-155 in breast cancer, we evaluated the expression levels of miR-155 by real-time PCR and correlated the results with clinicopathological features. Matched non-tumor and tumor tissues of 42 infiltrating ductal carcinomas and 3 infiltrating lobular carcinomas were analyzed for miR-155 expression by real-time PCR. Further, we used an antisense technique to inhibit miR-155 expression in vitro. WST-8 test was performed to evaluate cell viability and apoptosis assay was used to investigate the effect of the miR-155 antisense oligonucleotide (miR-155 ASO) on HS578T cell death. The expression levels of miR-155 were significantly higher in tumor tissues than the levels in matched non-tumor tissues (P<0.001). Up-regulated miR-155 expression was associated with lymph node positivity (P=0.034), higher proliferation index (Ki-67 >10%) (P=0.019) and advanced breast cancer TNM clinical stage (P=0.002). Interestingly, we next found that miR-155 expression levels had close relations with ER status (P=0.041) and PR status (P=0.029). Transfection efficiency detected by flow cytometry was higher than 70%, the WST-8 test showed that viability of HS578T cells was greatly reduced after transfection with miR-155 ASO compared with the scramble (SCR) group or the liposome group. The Annexin V-FITC/PI assay also indicated that transfection with miR-155 ASO promoted apoptosis.

Specific expression of human interferon-gamma controls hepatitis B virus replication in vitro in secreting hepatitis B surface antigen hepatocytes

Interferon-gamma (IFN-γ) has been reported to have antiviral activity against Hepatitis B virus (HBV) and to suppress HBV replication noncytolytically in vivo. Since systemic administration of IFN-γ may cause severe adverse effects, studies of the effects of liver-specific IFN-γ expression from adenoviral vectors in vivo have been investigated. In this study, a novel strategy has been described that drives specific expression of human IFN-γ in HBsAg-secreting hepatocytes. A bicistronic expression vector has been developed, pcDNA3.1-HBV antisense S gene-HCV core protein gene-HCV internal ribosome entry sites (IRES)-IFN-γ (pcDNA-SCIγ), by inserting four DNA fragments into pcDNA3.1. Tight modulation of HCV IRES-dependent translation by the HCV core protein was achieved using an antisense RNA technique with a bicistronic expression vector. HepG2 cells and HepG2.2.15 cells stably expressing HBV were transduced with pcDNA-SCIγ to test the responsiveness of IFN-γ to HBsAg expression. Gene transfer resulted in a low background and a 30-fold induction of IFN-γ expression from pcDNA-SCIγ in a cell-specific fashion. Hepatocyte-specific IFN-γ expression controlled effectively HBV replication in HBsAg-secreting HepG2.2.15 cells without cell toxicity.

Steroid receptor coactivator 2 modulates steroid‐dependent male sexual behavior and neuroplasticity in Japanese quail (Coturnix japonica)

Steroid receptor coactivators are necessary for efficient transcriptional regulation by ligand-bound nuclear receptors, including estrogen and androgen receptors. Steroid receptor coactivator-2 (SRC-2) modulates estrogen- and progesterone-dependent sexual behavior in female rats but its implication in the control of male sexual behavior has not been studied to our knowledge. We cloned and sequenced the complete quail SRC-2 transcript and showed by semi-quantitative PCR that SRC-2 expression is nearly ubiquitous, with high levels of expression in the kidney, cerebellum and diencephalon. Real-time quantitative PCR did not reveal any differences between intact males and females the medial preoptic nucleus (POM), optic lobes and cerebellum. We next investigated the physiological and behavioral role of this coactivator using in vivo antisense oligonucleotide techniques. Daily injections in the third ventricle at the level of the POM of locked nucleic acid antisense targeting SRC-2 significantly reduced the expression of testosterone-dependent male-typical copulatory behavior but no inhibition of one aspect of the appetitive sexual behavior was observed. The volume of POM, defined by aromatase-immunoreactive cells, was markedly decreased in animals treated with antisense as compared with controls. These results demonstrate that SRC-2 plays a prominent role in the control of steroid-dependent male sexual behavior and its associated neuroplasticity in Japanese quail.

Antisense Strategies in Therapy of Gliomas

The malignant brain tumour, glioblastoma, is invariably fatal with an average survival of 8-11 months (recent pharmacology has increased the median survival to 15, and rarely to 18 months). Different growth factors participate in the malignant development of the brain, especially IGF-1, EGF, TGF-beta and VEGF. Among the strategies applied to target and arrest the expression of growth factors, their receptors (tyrosine kinase) and down stream signalling pathway elements (i.e. PKC, Bcl-2, glycogen synthase) involved in cancer processes, the use of different inhibitors and especially of antisense technique has been among the leading technology proposed. Recent clinical results of glioblastomas are especially promising in situations in which antisense was used either as gene therapy (antisense anti TGF beta oligodeoxynucleotides), or as cellular gene therapy (transfection with antisense anti IGFI expression vector), following a classical surgery, radio- and chemotherapy. Using anti - IGF-I approach, median survival in glioblastoma patients has reached 21 months. The immune anti-tumour response in treated patients was confirmed by an increase of CD8+ T level in peripheral blood lymphocytes.

Chemical modification and design of anti-miRNA oligonucleotides

Antisense techniques have been employed for over 30 years to suppress expression of target RNAs. Recently, microRNAs (miRNAs) have emerged as a new class of small, non-coding, regulatory RNA molecules that widely impact gene regulation, differentiation and disease states in both plants and animals. Antisense techniques that employ synthetic oligonucleotides have been used to study miRNA function and some of these compounds may have potential as novel drug candidates to intervene in diseases where miRNAs contribute to the underlying pathophysiology. Anti-miRNA oligonucleotides (AMOs) appear to work primarily through a steric blocking mechanism of action; these compounds are synthetic reverse complements that tightly bind and inactivate the miRNA. A variety of chemical modifications can be used to improve the performance and potency of AMOs. In general, modifications that confer nuclease stability and increase binding affinity improve AMO performance. Chemical modifications and/or certain structural features of the AMO may also facilitate invasion into the miRNA-induced silencing complex. In particular, it is essential that the AMO binds with high affinity to the miRNA 'seed region', which spans bases 2-8 from the 5'-end of the miRNA.

Inhibition of keratin 17 expression with antisense and RNAi strategies: exploring novel therapy for psoriasis

Psoriasis is now considered to be a chronic, immune-mediated and inflammatory skin disease. As the precise cause of psoriasis remains unknown, its treatment is challenging for dermatologists. Keratin 17 (K17), an intermediate filament protein, is highly expressed in psoriatic lesions, while not normally expressed in healthy epidermis. Studies have suggested that K17 plays a role in the pathogenesis of psoriasis. However, no study has been performed to determine the potential application of K17 down-regulation as a treatment option for psoriatic lesions. We hypothesized that anti-K17 interference may suppress the development and progression of psoriasis and potentially serve as a novel strategy for the treatment of psoriasis. Therefore, we down-regulated and silenced K17 gene expression in keratinocytes (KCs) using antisense and RNA interference (RNAi) techniques. We found that K17-specific antisense oligonucleotides (ASODN) or siRNAs inhibited proliferation and induced apoptosis in KCs as well as down-regulated K17 expression at both mRNA and protein levels. For our in vivo study, we constructed the SCID-hu xenogeneic transplantation psoriasis mouse model by grafting psoriatic lesions onto SCID mice and topically applied K17-specific ASODN and liposome-encapsulated siRNA to the grafts. We observed morphological and histological improvement in the treated psoriatic grafts. As a result, K17 mRNA and protein expression was significantly decreased in the grafts of the mouse model. Taken together, we conclude that anti-K17 therapy is an effective treatment option for psoriasis, and the K17 molecule, as a new target, may hold tremendous potential for the treatment of psoriasis in the future.

Cyanobacterial flavodoxin complements ferredoxin deficiency in knocked‐down transgenic tobacco plants

Ferredoxins are the main electron shuttles in chloroplasts, accepting electrons from photosystem I and delivering them to essential oxido-reductive pathways in the stroma. Ferredoxin levels decrease under adverse environmental conditions in both plants and photosynthetic micro-organisms. In cyanobacteria and some algae, this decrease is compensated for by induction of flavodoxin, an isofunctional flavoprotein that can replace ferredoxin in many reactions. Flavodoxin is not present in plants, but tobacco lines expressing a plastid-targeted cyanobacterial flavodoxin developed increased tolerance to environmental stress. Chloroplast-located flavodoxin interacts productively with endogenous ferredoxin-dependent pathways, suggesting that its protective role results from replacement of stress-labile ferredoxin. We tested this hypothesis by using RNA antisense and interference techniques to decrease ferredoxin levels in transgenic tobacco. Ferredoxin-deficient lines showed growth arrest, leaf chlorosis and decreased CO(2) assimilation. Chlorophyll fluorescence measurements indicated impaired photochemistry, over-reduction of the photosynthetic electron transport chain and enhanced non-photochemical quenching. Expression of flavodoxin from the nuclear or plastid genome restored growth, pigment contents and photosynthetic capacity, and relieved the electron pressure on the electron transport chain. Tolerance to oxidative stress also recovered. In the absence of flavodoxin, ferredoxin could not be decreased below 45% of physiological content without fatally compromising plant survival, but in its presence, lines with only 12% remaining ferredoxin could grow autotrophically, with almost wild-type phenotypes. The results indicate that the stress tolerance conferred by flavodoxin expression in plants stems largely from functional complementation of endogenous ferredoxin by the cyanobacterial flavoprotein.

Construction and transformation for the antisense expression vector of the polyphenol oxidase gene in Yali pear

To inhibit the browning process in fruits of Yali pear, in this paper, antisense gene techniques were used to reduce the expression of PPO gene. A cDNA fragment of 450 bp, which is located at the 3′ terminal of the polyphenol oxidase (PPO) gene, was amplified from Yali pear using the RT-PCR method, then the antisense expression vector was constructed by inserting the fragment of the Yali pear PPO gene between the CaMV promoter and NOS terminator of the expression vector pBI121 in a reverse orientation. After that, with the agrobacterium-mediated method, the PPO antisense gene was transformed into Yali pear shoots. Northern blot analysis and enzyme activity assay showed that the PPO activities in the transgenic Yali pear shoots were significantly decreased, compared with the non-transformed Yali pear shoots. This lays a good foundation for breeding new varieties of pears with browning resistance in the future.

Phosphatidylinositol 3‐kinase‐δ up‐regulates L‐type Ca2+ currents and increases vascular contractility in a mouse model of type 1 diabetes

Vasculopathies represent the main cause of morbidity and mortality in diabetes. Vascular malfunctioning in diabetes is associated with abnormal vasoconstriction and Ca(2+) handling by smooth muscle cells (SMC). Phosphatidylinositol 3-kinases (PI3K) are key mediators of insulin action and have been shown to modulate the function of voltage-dependent L-type Ca(2+) channels (Ca(V) 1.2). In the present work, we investigated the involvement of PI3K signalling in regulating Ca(2+) current through Ca(V) 1.2 (I(Ca,L) ) and vascular dysfunction in a mouse model of type I diabetes. Changes in isometric tension were recorded on myograph. Ca(2+) currents in freshly dissociated mice aortic SMCs were measured using the whole-cell patch-clamp technique. Antisense techniques were used to knock-down the PI3Kδ isoform. KEY RESULTS Contractile responses to phenylephrine and KCl were strongly enhanced in diabetic aorta independent of a functional endothelium. The magnitude of phenylephrine-induced I(Ca,L) was also greatly augmented. PI3Kδ expression, but not PI3Kα, PI3Kβ, PI3Kγ, was increased in diabetic aortas and treatment of vessels with a selective PI3Kδ inhibitor normalized I(Ca,L) and contractile response of diabetic vessels. Moreover, knock-down of PI3Kδin vivo decreased PI3Kδ expression and normalized I(Ca,L) and contractile response of diabetic vessels ex vivo. Phosphatidylinositol 3-kinase δ was essential to the increased vascular contractile response in our model of type I diabetes. PI3Kδ signalling was up-regulated and most likely accounted for the increased I(Ca,L,) leading to increased vascular contractility. Blockade of PI3Kδ may represent a novel therapeutic approach to treat vascular dysfunction in diabetic patients.