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Overexpression and aberrant activation of signal transducer and activator of transcription 3 (STAT3) contribute to tumorigenesis, drug resistance, and tumor-immune evasion, making it a potential cancer therapeutic target. BP1003 is a neutral liposome incorporated with a nuclease-resistant P-ethoxy antisense oligodeoxynucleotide (ASO) targeting the STAT3 mRNA. Its unique design enhances BP1003 stability, cellular uptake, and target affinity. BP1003 efficiently reduces STAT3 expression and enhances the sensitivity of breast cancer cells (HER2+, triple negative) and ovarian cancer cells (late stage, invasive ovarian cancer) to paclitaxel and 5-fluorouracil (5-FU) in both 2D and 3D cell cultures. Similarly, ex vivo and in vivo patient-derived models of pancreatic ductal adenocarcinoma (PDAC) show reduced tissue viability and tumor volume with BP1003 and gemcitabine combination treatments. In addition to directly affecting tumor cells, BP1003 can modulate the tumor microenvironment. Unlike M1 differentiation, monocyte differentiation into anti-inflammatory M2 macrophages is suppressed by BP1003, indicating its potential contribution to immunotherapy. The broad anti-tumor effect of BP1003 in numerous preclinical solid tumor models, such as breast, ovarian, and pancreatic cancer models shown in this work, makes it a promising cancer therapeutic. [ABSTRACT FROM AUTHOR]

Overexpression and aberrant activation of signal transducer and activator of transcription 3 (STAT3) contribute to tumorigenesis, drug resistance, and tumor-immune evasion, making it a potential cancer therapeutic target. BP1003 is a neutral liposome incorporated with a nuclease-resistant P-ethoxy antisense oligodeoxynucleotide (ASO) targeting the STAT3 mRNA. Its unique design enhances BP1003 stability, cellular uptake, and target affinity. BP1003 efficiently reduces STAT3 expression and enhances the sensitivity of breast cancer cells (HER2+, triple negative) and ovarian cancer cells (late stage, invasive ovarian cancer) to paclitaxel and 5-fluorouracil (5-FU) in both 2D and 3D cell cultures. Similarly, ex vivo and in vivo patient-derived models of pancreatic ductal adenocarcinoma (PDAC) show reduced tissue viability and tumor volume with BP1003 and gemcitabine combination treatments. In addition to directly affecting tumor cells, BP1003 can modulate the tumor microenvironment. Unlike M1 differentiation, monocyte differentiation into anti-inflammatory M2 macrophages is suppressed by BP1003, indicating its potential contribution to immunotherapy. The broad anti-tumor effect of BP1003 in numerous preclinical solid tumor models, such as breast, ovarian, and pancreatic cancer models shown in this work, makes it a promising cancer therapeutic.

Xiaomin An,1,2,* Guohua Li,1,* Shu Wang,1 Tianrun Xie,1 Xiaoxiao Ren,1 Yongli Zhao1 1Department of Pediatrics, The Second Hospital of Dalian Medical University, Dalian, 116027, People’s Republic of China; 2Department of Nephrology, Xi’an Children’s Hospital, Xi’an, 710003, People’s Republic of China*These authors contributed equally to this workCorrespondence: Yongli Zhao, Department of Pediatrics, The Second Hospital of Dalian Medical University, Dalian, 116027, People’s Republic of China, Tel +86 0411 84671291, Email zhaoyongli79@163.comIntroduction: Our previous study conducted in an obesity-related renal injury rat model have established a connection between increased connexin 43 (Cx43) expression and renal injury. In this study, we investigated whether inhibiting Cx43 expression could provide renoprotection in a mouse model of obesity-related renal injury.Methods: Five-week-old C57BL/6J mice were fed with a high-fat diet for 12 weeks to establish an obesity-related renal injury model, then they were treated with Cx43 antisense oligodeoxynucleotide (AS) or scrambled oligodeoxynucleotide (SCR) by an implanted osmotic pump for 4 weeks. Finally, the glomerular filtration function, the histological change in the glomeruli, and the markers of podocyte injury (WT-1, Nephrin) and inflammatory infiltration of renal tissue (CD68, F4/80 and VCAM-1) were examined respectively.Results: The results showed that inhibiting Cx43 expression by AS in this mouse model of obesity-related renal injury can effectively improve glomerular filtration function, alleviate glomerular expansion and podocyte injury, and attenuate the inflammatory infiltration of renal tissue.Conclusion: Our results demonstrated that inhibiting Cx43 expression by AS could provide renoprotection for the mouse model of obesity-related renal injury.Keywords: childhood obesity, obesity-related renal injury, connexin 43, antisense oligodeoxynucleotide

Antisense oligodeoxynucleotide (ASODN) can directly interfere a series of biological events of the target RNA derived from tumor cells through Watson-Crick base pairing, in turn, plays antitumor therapeutic roles. In the study, a novel HIF-1 a ASODN-loaded nanocomposite was formulated to efficiently deliver gene to the target RNA. The physicochemical properties of nanocomposite were characterized using TEM, FTIR, DLS and zeta potentials. The mean diameter of resulting GEL-DGL-FA-ASODN-DCA nanocomposite was about 170- 192 nm, and according to the agarose gel retardation assay, the loading amount of ASODN accounted for 166.7 mg/g. The results of cellular uptake showed that the nanocomposite could specifically target to HepG2 and Hela cells. The cytotoxicity assay demonstrated that the toxicity of vectors was greatly reduced by using DCA to reversibly block the cationic DGL. The subcellular distribution images clearly displayed the lysosomal escape ability of the DCA-modified nanocomposite. In vitro exploration of molecular mechanism indicated that the nanocomposite could inhibit mRNA expression and HIF-1 a protein translation at different levels. In vivo optical images and quantitative assay testified that the formulation accumulated preferentially in the tumor tissue. In vivo antitumor efficacy research confirmed that this nanocomposite had significant antitumor activity and the tumor inhibitory rate was 77.99%. These results manifested that the GEL-DGL-FA-ASODNDCA nanocomposite was promising in gene therapeutics for antitumor by interacting directly with target RNA. [ABSTRACT FROM AUTHOR]

While vaccination is highly effective for the prevention of many infectious diseases, the number of adjuvants licensed for human use is currently very limited. The aim of this study was to evaluate the safety, efficacy, and to clarify the mechanism of a phosphorothioated interleukin (IL)-10-targeted antisense oligonucleotide (ASO) as an immune adjuvant in intradermal vaccination. The cytotoxicity of IL-10 ASO and its ability to promote T cell proliferation were assessed by Cell Counting Kit-8 (CCK-8) assay. The contents of IL-6, IL-8, TNF-α, IL-1β, and IL-10 in inoculated local tissue and the antigen-specific antibody titers in mouse serum samples were determined by ELISA. The target cells of IL-10 ASO were observed using immunofluorescent staining. The results showed that the specific antibody titer of ovalbumin (OVA), a model antigen, was increased 100-fold upon addition of IL-10 ASO as an adjuvant compared to that of OVA alone. IL-10 ASO showed an immunopotentiation efficacy similar to that of Freund's incomplete adjuvant, with no detectable cell or tissue toxicity. In vitro and in vivo experiments confirmed that IL-10 ASO enhances immune responses by temporarily suppressing IL-10 expression from local dendritic cells and consequently promoting T cell proliferation. In conclusion, IL-10 ASO significantly enhances immune responses against co-delivered vaccine antigens with high efficacy and low toxicity. It has the potential to be developed into a safe and efficient immune adjuvant. [ABSTRACT FROM AUTHOR]

METHODS: NF-κB p65 ASODN and NF-κB p65 missense oligodeoxynucleotide (MSODN) were designed and synthesized. Human lens epithelial cell line (HLE B-3) cells were prepared for study and divided into 7 groups. Control group was HLE B-3 cells cultured in vitro in dulbecco's modified eagle medium (DMEM). T1, T2, and T3 group were HLE B-3 cells cultured in vitro in DMEM with 10 ng/mL TGF-β2 for 6h, 12h, 24h respectively. A+T group was HLE B-3 cells cultured with 10 ng/mL TGF-β2 for 24h after transfected by NF-κB p65 ASODN for 24h. M+T group was HLE B-3 cells cultured with 10 ng/mL TGF-β2 for 24h after transfected by NF-κB p65 MSODN for 24h. The negative control group was HLE B-3 cells cultured with 10 ng/mL TGF-β2 for 24h after cultured with transfer agent (HiPerFect) for 24h. Cell morphology was observed at different time points using an inverted microscope. The expression of NF-κB p65 mRNA was detected with reverse transcription-polymerase chain reaction (RT-PCR), and the expression of α-smooth muscle actin (α-SMA) protein was assayed with ELISA. RESULTS: With the TGF-β2 stimulation prolongation, the expression of NF-κB p65 mRNA and α-SMA protein increased in T1, T2, T3 groups compared with the control group, and the difference was statistically significant (P0.05), but the difference among A+T group and other groups was statistically significant (P

OBJECTIVE: This study was purposed to investigate the effects of hTERT anti-sense oligodeoxynucleotide (ASODN) on cell apoptosis and expression of hTERT and bcl-2 mRNA in keloid fibroblasts and to explore its anti-keloid effect. MATERIALS AND METHODS: Primary cultures of dermal fibroblasts derived from 12 keloid samples were established, strains of fibroblasts at passages 3 to 4 were used in this study. After treated by hTERT ASODN the proliferation of the fibroblasts was measured by cell count and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay method, the apoptosis was analyzed by flow cytometry (FCM), and the expression of hTERT and bcl-2 mRNA were observed by semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). The data was analyzed by statistical software (SPSS11.5). RESULTS: The results showed that after sealing hTERT gene with ASODN for 72 h, the fibroblasts growth was repressed and the ability of proliferation decreased as the fibroblasts were treated with 1.0 mol/L ASODN for 72 h, the fibroblasts apoptosis was induced and the expression of hTERT and bcl-2 mRNA was lower than that of controlled group. The result was significantly different between control group and treatment group and was related to the treatment time of ASODN (p<0.01), but the difference was no significant when compared 1.0 μmol/L SODN group with untreated group (p>0.05). CONCLUSIONS: As a negative modutory factor, hTERT-ASODN can suppress growth and proliferation of keloid fibroblasts. Decreasing the telomerase activity of keloid fibroblasts may be one of the most important mechanisms. That hTERT-ASODN inhibited telomerase activity in keloid fibroblasts is an important pathway that may play a key role in the anti- keloid therapy. [ABSTRACT FROM AUTHOR]

Fermented brown rice with Aspergillus oryzae, designated as FBRA, is known to be commercially available dietary fiber-rich food, which is appreciated as prebiotics to improve intestinal microflora, and also shown to contain various biologically active substances including polyphenolic compounds. On the other hand, polyphenolic compounds have been suggested to stimulate the expression of brain-derived neurotrophic factor (BDNF) gene in connection with the expression of heme oxidase-1 (HO-1) gene in glial cells, thus resulting in the augmentation of BDNF production in the brain, thereby being anticipated to have a putative effect on the brain function. Then, the effect of FBRA extract on HO-1 and BDNF messenger ribonucleic acid (mRNA) levels in C6 glioma cells was examined, and the extract was shown to stimulate both HO-1 and BDNF gene transcription in the glioma cells. Further studies showed that the stimulatory effect of FBRA extract on BDNF gene transcription was almost completely suppressed by silencing HO-1 gene expression with an HO-1 antisense oligodeoxynucleotide and also inhibiting HO-1 activity with an inhibitor zinc protoporphyrin, thus suggesting that FBRA might have a potential ability to induce BDNF gene expression through HO-1 activity in glial cells. [ABSTRACT FROM AUTHOR]

Background Kirsten rat sarcoma ( KRAS) mutations are widespread in lung adenocarcinoma patients. The combined utilization of KRAS antisense oligodeoxynucleotide ( ASODN) and insulin-like growth factor- I receptor ( IGF-IR) may inhibit the proliferation of A549 cell lines of lung adenocarcinoma. Methods Point mutations of the KRAS gene in A549 cells were detected by polymerase chain reaction with special sequence primers ( PCR-SSP) and gene sequence analysis; ASODN was designed and synthesized according to the mutation specialty of KRAS; and the correlation of gene mutations and clinicopathological features were analyzed. Inhibition on the proliferation and morphostructure change were measured by methyl thiazolyl tetrazolium and colony-forming unit assays. Flow cytometry was used to evaluate the expression of KRAS and IGF-IR proteins and cell apoptosis and reverse transcriptase-polymerase chain reaction were used to detect the expression of KRAS and IGF-IR messenger ribonucleic acid ( mRNA). Male nude mice were used to form the mice-human lung cancer model and show the inhibition of KRAS ASODN on A549 cells. Results PCR-SSP and gene sequence analysis results showed that the codon 12 of KRAS had changed from GGT to GTT. KRAS ASODN or IGF-IR ASODN could inhibit cell proliferation and promote apoptosis of A549 cells. However, the combined utilization of KRAS ASODN and IGF-IR ASODN could inhibit cell proliferation and promote apoptosis more powerfully than exclusive use of KRAS ASODN or IGF-IR ASODN. Conclusion The two ASODNs can inhibit the proliferation of lung adenocarcinoma cells through decreasing the expression of KRAS and IGF-IR mRNA and protein. [ABSTRACT FROM AUTHOR]

Introduction: The aim of the study was to investigate the efficiency of delivery and targeted binding of c-myc antisense oligodeoxynucleotide (ASODN) and find a novel therapy for hepatic carcinoma. Material and methods: A targeted ultrasound microbubble compound was synthesized to deliver the c-myc ASODN by ultrasound-targeted microbubble destruction (UTMD) and applied in hepatocellular carcinoma cells (HCC) and cancer bearing mice. Lipid microbubbles were conjugated with biotinylated galactosylated poly-L-lysine (G-PLL) and SonoVue to target the hepatocellular carcinoma SMMC7721 cells with asialoglycoprotein receptors. There were four groups in both in vitro and in vivo studies: control group (group A); c-myc ASODN + G-PLL (CG group, group B); c-myc ASODN + SonoVue (CUS group, group C); c-myc ASODN + G-PLL + SonoVue (CGUS group, group D). The expression of c-myc mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR), and proliferation investigations of the SMMC7721 cells were also performed. In addition, the tumor volume was calculated and compared among different groups. Results: The level of c-myc mRNA in the three experimental groups was significantly lower than that in the control group in vitro (p < 0.05). Furthermore, c-myc gene expression was suppressed more strongly in the CGUS group compared with other groups in both in vitro and in vivo studies (p < 0.05). In addition, ultrasound mediation of targeted microbubbles yielded the highest inhibition of tumor growth and cell proliferation among the four groups. Conclusions: The use of a G-PLL targeted microbubble contrast agent combined with ultrasound exposure could be a potential method for increasing gene delivery efficiency. This technique is a promising nonviral approach that can be used in liver cancer. [ABSTRACT FROM AUTHOR]

To study the inhibitory effect of nuclear transcription factor-kappa B(NF-κB) antisense oligodeoxynucleotide(ASODN) on the growth and tumorgenesis of human gastric cancer. We synthesized and transfected the ASODN of NF-κB/P65 to gastric cancer cell line. The effect of ASODN of NF-κB/P65 on the proliferation of gastric cancer cells was measured by MTT method. The subcutaneous xenograft model of human gastric cancer was established in nude mice, and the tumor growth curve was observed. The cell proliferation was significantly inhibited in P65 ASODN-transfected group in vitro ( P < 0.05). In vivo, tumor formation test showed that the tumor volume in nude mice in ASODN group was obviously smaller than in other groups ( P < 0.05); the apoptosis index (AI) was significantly higher ( P < 0.001). Simultaneously, MVD in ASODN group was markedly lower than in other groups ( P < 0.01). NF-κB could be used as a new biological therapeutic target of gastric cancer. [ABSTRACT FROM AUTHOR]

Background Liposomes are among the most widely used carriers for the delivery of antisense oligonucleotides (AsODNs) to intracellular targets. Although different strategies have been employed, the question of how to improve liposomal uptake and enhance the release of AsODN into cytoplasm still remains to be answered with respect to the use of a safe, easy and economic method. In the present study, the possibility of enhancing such processes at cellular and animal levels using urea as a penetration enhancer was investigated. Methods To perform this investigation, a cationic liposome containing an AsODN against protein kinase (PKC)-α was prepared, and the effect of urea on its cellular internalization and the related sequence-specific inhibition of gene expression in human lung adenocarcinoma A549 cells were investigated by flow cytometry and the reverse transcriptase-polymerase chain reaction, respectively. In in vivo studies, a xenograft lung tumor was established in nude mice by A549 cells and the enhancement effect of urea toward the effects of liposomal AsODN on tumor growth was investigated. Results Cellular studies revealed that urea treatment increases liposomal uptake and the release of AsODN into the cytoplasm by approximately 40%. Sequence-specific inhibition of target gene PKC-α expression was also increased by approximately two-fold by urea at 200-300 nM AsODN. In animal studies, urea significantly decreased the tumor volume (approximately 40%) and increased its doubling time from approximately 13 days to 17 days. Conclusions Urea, and possibly other membrane fluidizers, could be regarded as penetration enhancers for liposomal AsODN delivery and may improve the therapeutic effect of these gene-therapy vectors at both cellular and animal levels. Copyright © 2012 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Abstract: Moderate reduction in the protein content of the mother’s diet calorically compensated by carbohydrates (the so-called “hidden” prenatal malnutrition) leads to increased neocortical expression of the α2C-adrenoceptor subtype, together with decreased cortical release of noradrenaline and impaired long-term potentiation (LTP) and visuospatial memory performance during the rat postnatal life. Ín order to study whether overexpression of the α2C-adrenoceptor subtype is causally related to the decreased indices of neocortical plasticity found in prenatally malnourished rats, we evaluated the effect of intracortical (occipital cortex) administration of an antisense oligodeoxynucleotide (ODN) raised against the α2C-adrenoceptor mRNA on the LTP elicited in vivo in the occipital cortex of hidden prenatally malnourished rats. In addition, we compare the effect of the antisense ODN to that produced by systemical administration of the subtype-nonselective α2-adrenoceptor antagonist atipamezole. Prenatal protein malnutrition led to impaired occipital cortex LTP together with increased expression of α2C-adrenoceptors (about twice Bmax) in the same cortical region. [3H]-rauwolscine binding assay showed that a 7-day intracortical antisense ODN treatment in the malnourished rats resulted in 50% knockdown of α2C-adrenoceptor expression and, in addition, completely rescued the ability of the occipital cortex to develop and maintain long-term potentiation. Atipamezole (0.3mg/kg i.p.) also led to full recovery of neocortical LTP in malnourished rats. The present results argue in favor of our original hypothesis that the deleterious effect of prenatal malnutrition on neocortical plasticity in the adult progeny is in part consequence of increased neocortical α2C-adrenoceptor expression. This receptor subtype is known to be involved in the presynaptic control of noradrenaline release from central neurons, a neurotransmitter that critically influences LTP and memory formation. [Copyright &y& Elsevier]

Background and Aims: Glucose transporter protein-1 (Glut-1) is correlated with biological behaviors of malignant tumors. However, there was no evidence that overexpression of Glut-1 mechanistically lead to invasion or metastasis of cancer cells. We hypothesized that Glut-1 regulates the expression of membrane type 1-MMP (MT1-MMP) and matrix metalloproteinase-2 (MMP-2). Methods: Analysis of the expression of Glut-1, MMP-2, β-actin, and MT1-MMP was performed using RT-PCR. Expression of Glut-1 protein, MMP-2, and MT1-MMP was detected by Western blotting. Results: At mRNA and protein levels, Glut-1 and MMP-2 were co-expressed in the Hep-2 laryngeal carcinoma cell line. After transfection, Glut-1 antisense oligodeoxynucleotide (AS-ODN) decreased the expression of MMP-2 mRNA and protein as well as Glut-1 mRNA and protein. Glut-1 AS-ODN also decreased the expression of MT1-MMP mRNA. Conclusions: Co-expression of Glut-1 and MMP-2 in Hep-2 laryngeal carcinoma cells and Glut-1 may regulate MMP-2 and MT1-MMP expression. [Copyright &y& Elsevier]

NELL2, a protein containing epidermal growth factor-like repeat domains, is predominantly expressed in the nervous system. In the mammalian brain, NELL2 expression is mostly neuronal. Previously we found that NELL2 is involved in the onset of female puberty by regulating the release of gonadotropin-releasing hormone (GnRH), and in normal male sexual behavior by controlling the development of the sexually dimorphic nucleus of the preoptic area (POA). In this study we investigated the effect of NELL2 on the female rat estrous cycle. NELL2 expression in the POA was highest during the proestrous phase. NELL2 mRNA levels in the POA were increased by estrogen treatment in ovariectomized female rats. Blocking NELL2 synthesis in the female rat hypothalamus decreased the expression of kisspeptin 1, an important regulator of the GnRH neuronal apparatus, and resulted in disruption of the estrous cycle at the diestrous phase. These results indicate that NELL2 is involved in the maintenance of the normal female reproductive cycle in mammals. [ABSTRACT FROM AUTHOR]

Abstract: Syntaxin 1A is a membrane protein playing an integral role in exocytosis and membrane trafficking. The superficial dorsal horn (SDH) of the spinal cord, where nociceptive synaptic transmission is modulated, is rich in this protein. We recently reported that peripheral nerve ligation-induced nociceptive responses are considerably enhanced in syntaxin 1A-knockout mice [Takasusuki T, Fujiwara T, Yamaguchi S, Fukushima T, Akagawa K, Hori Y (2007) Eur J Neurosci 26:2179–2187]. On the basis of this earlier finding, we hypothesized that syntaxin 1A is involved in peripheral nerve injury-induced nociceptive plasticity. In this study, we examined this hypothesis by using nociceptive behavioral studies and tight-seal whole-cell recordings from neurons in the SDH of adult mouse spinal cord slices. Partial sciatic nerve ligation (PSNL) in adult male Institute of Cancer Research (ICR) mice increased the frequency of spontaneous miniature excitatory postsynaptic currents (mEPSCs). The amplitude of the mEPSCs did not exhibit any changes, suggesting that peripheral nerve injury is associated with increased synaptic release of excitatory neurotransmitters. Western blot and real-time quantitative reverse transcription-polymerase chain reaction analyses revealed that PSNL gradually decreased the expression level of syntaxin 1A in the spinal SDH. This downregulation of syntaxin 1A took several days to develop, whereas behavioral allodynia developed within one day after PSNL. Syntaxin 1A knockdown by intrathecal injection of an antisense oligodeoxynucleotide against the syntaxin 1A gene led to the gradual development of allodynia. These results indicate a possible involvement of syntaxin 1A downregulation in the late maintenance phase of peripheral nerve injury-induced allodynia. In addition, syntaxin 1A knockdown by ribonucleic acid interference enhanced the axonal elongation and sprouting of spinal dorsal horn neurons in culture, suggesting that PSNL-induced syntaxin 1A downregulation may result in the rearrangement of the synaptic connections between neurons in the spinal dorsal horn. Taken together, it is possible to conclude that syntaxin 1A might be involved in spinal nociceptive plasticity induced by peripheral nerve injury. [Copyright &y& Elsevier]

Purpose of work: Mutation of the p53 gene is the most common genetic alteration in human cancers. Our study proposes to rationally design a p53 antisense oligonucleotide (ASO) repository, which contains a series of ASOs containing single nucleotide differences to discriminate between each mutant and wild type (WT) p53. The Sfold software was used to predict target-accessibility and we designed an initial series of antisense oligonucleotides (ASO) that target the p53 mutants A161T, R175H and R249S. Western-blot analysis indicated that ASOs strongly inhibited the expression of p53 mutants in a panel of human tumor cell lines (SNU-449, SK-BR-3 and PLC/PRF/5) while having little effect on the expression of WT p53 (HepG2 cells). In three cancer lines harboring each of the p53 mutations, mutant-specific ASO treatment led to a dose-dependent inhibition of cell growth, cell viability, colony formation and invasion, and expression of mutant p53-dependent survival proteins. Our preliminary results indicate that a single nucleotide difference in ASOs can discriminate between mutant and WT p53. These observations support the hypothesis that a p53 ASO repository can be a potentially valuable tool to knock down oncogenic mutant p53 and warrant the testing of a p53 ASO repository in in vivo settings. [ABSTRACT FROM AUTHOR]

Abstract: Organotypic slice cultures obtained from immature brain tissue represent a well-established model system for neuroscience research. Current culture methods, however, do not allow long-term culture of mature brain slices. Slice cultures from mature animals would provide an in vitro experimental environment suitable for investigation of neuropathologies, which in human, predominate in aged individuals. We hypothesized that damage, incurred by slicing of the brain, is propagated through intercellular connexin43 (Cx43) gap junction channels and that this damage is not easily repaired in mature central nervous system (CNS) tissue that lacks the pluripotency of immature tissue. We investigated the role of Cx43 gap junctions in long-term survival of mature brain tissue using antisense oligodeoxynucleotide (AsODN) technology. The application of Cx43 AsODN immediately after slicing of the mature brain led to a significant but transient knockdown of Cx43 protein. This treatment was associated with the long-term survival of hippocampal neurons with normal morphology within whole brain slices taken from 14 and 40-day-old adult rats. [Copyright &y& Elsevier]

Aims: The role of antisense oligodeoxynucleotide against tissue factor (aODN/TF) in cultured human umbilical vein endothelial cells (HUVECs) subjected to anoxia-reoxygenation (A/R) was investigated. Methods: HUVECs were divided randomly into control group, A/R group, aODN/TF+A/R group, sense oligodeoxynucleotide (sODN/TF) + A/R group and mismatched oligodeoxynucleotide (mODN/TF) + A/R group, in the latter 3 groups, HUVECs were transfected with aODN/TF, sODN/TF and mODN/TF respectively. HUVECs in all A/R groups underwent 3 hrs of anoxia and followed by 2 hrs of reoxygenation. In order to investigate the potential mechanisms of how increased TF may contribute to A/R injury in HUVECs, another set of HUVECs were incubated with human recombinant active site blocked factor VII (FVIIai) during A/R. Results: After A/R, TF expression at both mRNA and protein level was increased, furthermore, cell viability and the concentrations of SOD, GSH-PX and NO were declined, while LDH, MDA and ET-1 were overproduced (P<0.05 to 0.001 versus control group). In HUVECs of aODN/TF+A/R group, however, TF expression was inhibited, while the declined cell viability and the concentrations of SOD, GSH-PX, NO as well as the enhanced LDH, MDA and ET-1 levels occurred during A/R were ameliorated and reversed effectively (P<0.05 to 0.01 versus those in other A/R groups). The results also showed that ROS was increased and PAR-1, PAR-2, p38 MAP kinase and p42/44 MAP kinase were all activated after A/R (P<0.001 versus HUVECs under normoxia), while FVIIai inhibited the increment of ROS, PAR-1, PAR-2, p38 MAP kinase and p42/44 MAP kinase, and improved the changes of TF:C, MDA, SOD, GSH-PX, cell viability and LDH occurred during A/R (P<0.05 to 0.001 versus HUVECs without FVIIai treatment). Conclusion: Tissue factor plays an important role in the development of HUVECs injury induced by anoxia-reoxygenation, inhibition of TF with antisense oligodeoxynucleotide is an effective approach to ameliorate the damage. Copyright © 2010 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The neuropeptide arginine vasopressin (AVP) is arguably among the most potent regulators of social behaviors in mammals identified to date. However, only the related neuropeptide oxytocin (OXT) has been shown to promote maternal behavior. Here, we assess the role of AVP in maternal care, in particular in arched back nursing, pup retrieval, and pup contact by using complementary pharmacological and genetic approaches. Also, experiments were performed in rat dams with differences in trait anxiety. i.e., rats bred for either high (HAB) or low (LAB) anxiety-related behavior as well as nonselected (NAB) dams. Viral vector-mediated up-regulation of AVP Via receptors (AVP-Rs) within the medial preoptic area of lactating NAB rats and chronic central AVP treatment of NAB and LAB dams improved, whereas local blockade of AVP-R expression by means of antisense oligodeoxynucleotides-or central AVP-R antagonism impaired, maternal care in NAB dams. Also, in HAB rats with a genetically determined elevated brain AVP activity, intrinsically high levels of maternal care were reversed by blockade of AVP-R actions. Treatment-induced impairment of AVP-mediated maternal behavior increased adult emotionality and impaired social interactions in ma!e offspring of NAB dams. These findings provide direct evidence for an essential and highly potent role of brain AVP in promoting maternal behavior, which seems to be independent of the dam's trait anxiety. [ABSTRACT FROM AUTHOR]

Abstract: Fabrication of polymeric multilayered films based on the electrostatic self-assembly of polycations and polyanions is a promising approach for controlled loading and release in gene delivery. In this study, we have fabricated a series of multilayered films based on alternate deposition between positively-charged cationic phosphorylcholine copolymer (PC copolymer) and negatively-charged c-myc anti-sense oligodeoxynucleotide (AS-ODN). The growth of film thickness and increase of ODN loading capacity were monitored by spectroscopic ellipsometry (SE) and confocal laser scanning microscopy (CLSM). After elution into PBS buffer under physiological conditions, the elution profile was monitored by UV spectrometry and gel electrophoresis. Employing a secondary transgenic vector, the cellular uptake of the eluted AS-ODN into HeLa cells was evaluated by fluorescent microscopy and FACS analysis. The biological effect of eluted AS-ODN was evaluated by cell growth inhibition. The results showed that AS-ODN loading capacity increased almost linearly with the number of PC polymer/ODN bilayers and was also strongly dependent upon the cationic charge density. Through swelling, a non-degradable release mechanism, the AS-ODN release was characterized by two distinguishable release regimes: a fast release regime during the first 6 hour period and a slow release regime from 6 hour to the 8th day, both of which were characterized by zero-order kinetics. Gel electrophoresis showed excellent DNA integrity and strong transfection was observed when the eluted ODN was transfected into HeLa cells. Cell growth was significantly inhibited by eluted AS-ODN, indicating its full bioactivity. These results demonstrate that PC multilayered polymer films are capable of delivering a prescribed amount of anti-sense ODN with a controllable kinetic profile and that the multilayer process is more efficient and reliable than most other existing coating approaches largely based on single-layer fabrication. [Copyright &y& Elsevier]

Abstract: Transient receptor potential ankyrin-1 (TRPA1) is an important receptor that contributes to inflammatory pain. However, previous studies were mainly concerned with its function in somatic hyperalgesia while few referred to visceral, especially colonic inflammatory hyperalgesia. The present study was aimed to investigate the role of TRPA1 in visceral hyperalgesia after trinitrobenzene sulfonic acid (TNBS)-induced colitis. Results indicate that TNBS induced a significant increase in visceral sensitivity to colonic distension and chemical irritation accompanied by up-regulation of TRPA1 in colonic afferent dorsal root ganglia (DRG). Intrathecal administration of TRPA1 antisense (AS) oligodeoxynucleotide (ODN) reduced the TRPA1 expression in DRG as well as suppressed the colitis-induced hyperalgesia to nociceptive colonic distension and intracolonic allyl isothiocyanate (AITC). Meanwhile the TRPA1 antisense ODN had no effect on transient receptor potential vanilloid-1 (TRPV1) expression, which was proposed to highly co-express with TRPA1, and no effect on the response to TRPV1 agonist, capsaicin. These data suggest an apparent role of TRPA1 in visceral hyperalgesia following colitis that might provide a novel therapeutic target for the relief of pain. [Copyright &y& Elsevier]

Implantation is a complex developmental event that is initiated by recognition and adhesion of the embryo to the endometrial epithelium. sLeX is an oligosaccharide antigen acting as the ligand of L-selectin, and is stage-specifically expressed in the endometrial epithelium. The adhesion system mediated by L-selectin and sLeX oligosaccharide plays an important role in this process. FUT7 is a key enzyme for sLeX synthesis, and the regulation of sLeX through FUT7 may influence maternal–fetal recognition. In this study, we observed the effect of FUT7 antisense oligodeoxynucleotide on the expression of FUT7 and sLeX, as well as adhesion in an in vitro implantation model consisting of the human uterine epithelial cell line RL95-2 and the human embryonic cell line JAR. Results showed that the expression of FUT7 was significantly decreased, compared with controls, after FUT7 antisense oligodeoxynucleotide transfection into RL95-2 cells, as determined by RT-PCR, Western blotting, and indirect immunofluorescence. Synthesis of sLeX was also decreased, consistent with the FUT7 decrease, as shown by indirect immunofluorescence. The adhesion of embryonic cells to uterine epithelial cells was significantly reduced (P < 0.01) compared with the control. These data indicate that the use of a FUT7 antisense oligodeoxynucleotide can cause a significant reduction of both FUT7 and sLeX antigen, and thereby inhibit the adhesion of embryo cells to endometrium. This approach may provide a new way to regulate reproduction. © 2008 IUBMB IUBMB Life, 60(7): 461–466, 2008 [ABSTRACT FROM AUTHOR]

Abstract: To investigate the effect of specific antisense oligodeoxynucleotide (ASODN) inhibition of Fas expression on T cell apoptosis induced by hepatocarcinoma cells. Fas receptor (Fas) and Fas ligand (FasL) expressed by the hepatocarcinoma cell line HepG2.2.15 and Jurkat T cells were detected by flow cytometry (FCM) and the ability of FasL-inducing T cell apoptosis was tested by co-culture assay in vitro with HepG2.2.15 cells and Jurkat T cells. The Jurkat cells were transfected with Fas-ASODN using lipofectin, and the effects of Fas-ASODN on Fas mRNA level, Fas expression on T cells surface, and apoptosis were investigated by RT–PCR, FCM and co-culture assay, respectively. It was found that HepG2.2.15 cells expressing functional FasL could induce the apoptosis of Jurkat cells as demonstrated by co-culture assays. After the Jurkat cells were transfected with Fas ASODN, the level of Fas mRNA, the expression rate of Fas and the apoptotic rate induced by hepatocarcinoma cells were all decreased. As a conclusion, it is evident that hepatocarcinoma cells expressing FasL can induce apoptosis in Fas-expressing T cells, indicating that transfection of Fas ASODN can partially convert the immune inhibitory condition induced by hepatocarcinoma cells. [Copyright &y& Elsevier]

Abstract: Objective. : In this report, we studied the role of Hpa in metastatic capability of human choriocarcinoma. At the same time, we investigated the effect of Hpa antisense oligodeoxynucleotide (ASODN) on inhibition of invasiveness of human choriocarcinoma. Methods. : The different invasion ability between JEG-3 and JAR cell lines was proved by Matrigel invasion assay in vitro. Reverse transcriptase–polymerase chain reaction (RT–PCR) and Western blot analyses were carried out respectively to determine Hpa gene and protein expression; the localization of this molecule was demonstrated by immunohistochemistry. Finally, Hpa antisense oligodeoxynucleotide (ASODN) was transfected into JEG-3 cells and Hpa mRNA and protein were quantified by RT–PCR and Western blot. The effect of ASODN on the metastatic capability of JEG-3 was evaluated by Matrigel invasion assay. Results. : (1) We proved that the invasion ability of JEG-3 cell line was stronger than that of JAR cell line (P <0.05). (2) We found that the Hpa gene and protein in JEG-3 and JAR cell lines were significantly higher than those in normal chorion (P <0.05). On the other hand, we detected that JEG-3 expressed much more Hpa than JAR (P <0.05). (3) Both in JEG-3 cell and in JAR cell, we found that Hpa protein express in cytoplasm. (4) After transfection of Hpa ASODN, Hpa mRNA and protein expression in JEG-3 cell decreased 4- and 5-fold. At the same time, we also observed that the invasion ability of JEG-3 cell was weakened than before (P <0.05). Conclusion. : The current study demonstrated that the expression of Hpa plays an important role in metastatic capability of human choriocarcinoma and reducing the expression of Hpa can help weaken the invasion ability of human choriocarcinoma. [Copyright &y& Elsevier]

Abstract: Interleukin (IL)-5 produced by allergen specific T cells is a major cytokine in the allergic inflammation such as allergic rhinitis (AR). To inhibit the production of IL-5, water-soluble chitosan (WSC)-based IL-5 antisense oligodeoxynucleotide (AS-ODN) complex was generated. WSC, a biocompatible cationic polymer, was used as a non-viral vector for the improvement of stability and transfection efficiency. After condensation IL-5 AS-ODN with WSC, the size, morphology and zeta potential analysis of IL-5 AS-ODN/WSC complexes were performed. The protective effect of complex was also observed against the enzymatic degradation. In vitro transfection efficiency into H1299 epithelial cells was investigated by flow cytometer and inhibition effect of IL-5 levels was also evaluated in D10.G4.1 cells. In the murine model with AR, the IL-5 and IgE levels closely related to the allergic inflammation were significantly reduced after the intranasal administration of IL-5 AS-ODN/WSC complexes. Based on these results, the condensation with WSC improved the physicochemical stability and transfection efficiency of IL-5 AS-ODN/WSC complex. Our results suggest that AS therapy using IL-5 AS-ODN/WSC complex can be an effective strategy in regulating IL-5 and may be applied to the treatment of allergic disorder related to IL-5. [Copyright &y& Elsevier]

Abstract: Invasion and metastasis are key features of human hepatocellular carcinoma (HCC). Heparanase is an endoglycosidase that can degrade extracellular matrix by cleaving heparan sulfate chains of heparan sulfate proteoglycan, thus playing important roles in the invasion and metastasis of human cancers. Heparanase has been detected in various human cancers and regarded as a prospective target in human cancer treatments. However, the effects of inhibiting the expression of heparanase on human HCC have not been fully evaluated. In this article we show that downregulating the expression of heparanase either by antisense oligodeoxynucleotide or by RNA interferencing can significantly reduce the expression of heparanase in SMMC7721 human HCC cells, leading to inhibition of the invasiveness, metastasis, and angiogenesis of HCC cells both in vitro and in vivo. Our results suggest that genetic downregulation of the expression of heparanase may serve as an efficient cancer therapeutic for human HCC. [Copyright &y& Elsevier]

Abstract: Transfection activity of antisense oligodeoxynucleotides (ODN)-loaded cationic liposomes is mainly restricted by uptake and ODN release into cytoplasm, which is difficult to evaluate in cell culture studies. Well-designed models of cellular membranes, aim of the present study, might facilitate investigation of such processes. In this investigation, a phosphorothioate ODN was actively encapsulated in a DODAP-containing cationic liposome by ethanol injection with 73% efficiency. ODN release was determined by fluorescence dequenching of FITC-ODN upon incubation of liposomes with early endosomal (EE), late endosomal (LE) and plasma membranes (PM) models. LE provided the highest release (up to 76%) in a temperature-dependent manner. Release by EE (<16%), total PM (<11%) and PM external layer (≈0) were not temperature sensitive. These differences are attributed to lipid charge, chain mobility, critical packing parameter and cholesterol content of the models. Intracellular distribution of FITC-ODN, determined by fluorescence microscopy and flowcytometry in the presence and absence of sodium azide, confirmed that liposomes were internalized mainly via endocytosis; hence inability of our PL models to simulate such active processes. Instead, release of ODN from endosomes into cytoplasm was pH-sensitive and in good agreement with model membrane studies in terms of amount and mechanism. [Copyright &y& Elsevier]

Abstract: Transcription factors of the nuclear factor-kappa B (NF-κB) p65/RelA may be involved in neuronal cell death. We examined the involvement of NF-κB p65 in N-methyl-d-aspartate (NMDA)-induced upregulation of interleukin (IL)-1β, a proinflammatory cytokine, and subsequent neurotoxicity in the rat retina. Immunohistochemistry showed that IL-1β is localized not only in glial cells, but also in neurons, especially retinal ganglion cells (RGCs) after intravitreal injection of NMDA. Semi-quantitative real-time PCR showed that NMDA induces an increase in IL-1β mRNA levels. Preinjection of NF-κB p65 antisense oligodeoxynucleotide (AS ODN) ameliorated the NMDA-induced increase in IL-1β mRNA expression. Western blot analysis showed elevated levels of retinal IL-1β protein 12 h after intravitreal NMDA injection and this elevation was significantly inhibited by NF-κB p65 AS ODN. Neurotracer labeling showed that the inhibition of NF-κB p65 by AS ODN or siRNA exerted a protective effect against NMDA-induced RGC loss. IL-1β siRNA also had a protective effect on RGC number in NMDA-treated eyes. Penetration of AS ODN and siRNA to cells in the RGC layer and inner nuclear layer was confirmed after labeling with rhodamine or Cy3. These results suggest that NF-κB p65 may participate in the induction of IL-1β expression in NMDA-induced retinal neuronal cell death and that the inhibition of NF-κB p65 and IL-1β with the use of AS ODN or siRNA may be a viable neuroprotective strategy for RGC survival. [Copyright &y& Elsevier]

The human ovarian mucinous cystadenocarcinoma (hOMC) cells were co-cultured with antisense oligodeoxynucleotide (antisense ODN), nonsense ODN, and follicle-stimulating hormone (FSH) at different concentrations for the purpose of observing the effects of antisense ODN to FSH receptor (FSHR) on the proliferation and apoptosis of cultured hOMC cells in vitro. The inhibitory rates of growth were measured by using MTT method on the 2nd, 4th, 6th, 8th and 10th days after the interference of antisense ODN, nonsense ODN, and FSH, respectively. The apoptotic rates and the cell cycles were determined by means of flow cytometry, the apoptosis indexes were detected by using TUNEL, and the expression of caspase-3 was measured by using SP immunohistochemistry. Compared with that in the control group, the proliferative activity of hOMC cells was increased obviously in FSH groups ( P<0.05 or P<0.01), decreased distinctly in antisense ODN groups ( P<0.05 or P<0.01), and unchanged in nonsense ODN groups, respectively. Meanwhile, antisense ODN could significantly antagonize the FSH-promoted cell proliferative activity ( P<0.01). Compared with those in the control group, the apoptotic rates and the expression of caspase-3 were dramatically increased in the mid-and high-dose antisense ODN groups ( P<0.05 or P<0.01), while the number of cells in G1/G0 phase was significantly decreased and that in S phase distinctly increased ( P<0.01). There was no change in nonsense ODN groups ( P>0.05). It was suggested that FSH may improve the development of hOMC cells. However, antisense ODN could inhibit proliferative activity and the FSH-promoted proliferative activity in hOMC cells, at the same time, antisense ODN could inhibit hOMC cell growth by inducing apoptosis. [ABSTRACT FROM AUTHOR]

In order to investigate the effects of connective tissue growth factor (CTGF) antisense oligodeoxynucleotide (ODN) on plasminogen activator inhibitor-1 (PAI-1) expression in renal tubular cells induced by transforming growth factor β1 (TGF-β1) and to explore the role of CTGF in the degradation of renal extracellular matrix (ECM), a human proximal tubular epithelial cell line (HKC) was cultured in vitro. Cationic lipid-mediated CTGF antisense ODN was transfected into HKC. After HKC were stimulated with TGF-β1 (5 μg/L), the mRNA level of PAI-1 was detected by RT-PCR. Intracellular PAI-1 protein synthesis was assessed by flow cytometry. The secreted PAI-1 in the media was determined by Western blot. The results showed that TGF-β1 could induce tubular CTGF and PAI-1 mRNA expression. The PAI-1 mRNA expression induced by TGF-β1 was significantly inhibited by CTGF antisense ODN. CTGF antisense ODN also inhibited intracellular PAI-1 protein synthesis and lowered the levels of PAI-1 protein secreted into the media. It was concluded that CTGF might play a crucial role in the degradation of excessive ECM during tubulointerstitial fibrosis, and blocking the biological effect of CTGF may be a novel way in preventing renal fibrosis. [ABSTRACT FROM AUTHOR]

Munc18, a mammalian homolog of C. elegans Unc, is essential for neurotransmitter release. The aim of this study was to identify estrogen-dependent expression of Munc18-1 and its role in the regulation of glutamate release for puberty onset. Hypothalamic munc18-1 mRNA levels were significantly increased by estrogen treatment in ovariectomized, immature female rats. During pubertal development, the munc18-1 mRNA levels dramatically increased between the juvenile period and the anestrous phase of puberty. Intracerebroventricular administration of an antisense oligodeoxynucleotide against munc18-1 mRNA significantly decreased glutamate release and delayed the day of puberty onset. These results suggest that Munc18-1, expressed in an estrogen-dependent manner, plays an important role in the onset of female puberty via the regulation of glutamate release. [ABSTRACT FROM AUTHOR]

Aim: To design and screen antisense oligodeoxynucleotides (ASODNs), which inhibit type-1 plasminogen activator inhibitor (PAI-1) expression in human umbilical vein endothelial cells (HUVEC) in vitro. Methods: Twenty seven ASODNs against different sites of PAI-1 mRNA were designed and transfected to HUVEC by lipofectin in vitro. The effects of ASODNs on PAI-1 antigen, PAI-1 activity and PAI-1 mRNA expression were detected by ELISA, amidolytical assay and RT-PCR, respectively. Results: Transforming growth factor β1(TGF-β1)-treated HUVEC increased the expression of PAI-1 compared with the normal HUVEC. Five among twenty seven designed ASODNs were effective in inhibiting the increase in PAI-1 antigen and PAI-1 activity in a dose-dependent manner after 48-h transfection. In particular, ASODN 14 (AO14) exhibited the best inhibitory effect. The control sequences of AO14, including sense, scramble, and mismatch sequences, did not significantly inhibit PAI-1 activity. It was revealed that the inhibitory efficacy of AO14 was in a sequence-specific manner. RT-PCR showed that ASODN 1, 7, 8, 14, and 15 decreased PAI-1 mRNA expression induced by TGF-β1 and AO14 showed the best inhibitory effect. Conclusion: ASODN 1, 7, 8, 14, and 15, among twenty seven designed ASODNs against PAI-1 mRNA, significantly decreased PAI-1 antigen and PAI-1 activity induced by TGF-β1 in a dose-dependent manner in HUVEC in vitro. AO14 showed the best inhibitory effect on PAI-1 expression in a sequence-specific manner. The results of RT-PCR indicated that inhibitory effects of ASODNs on PAI-1 biosynthesis occurred at the mRNA level. Four among five effective target sites of ASODNs located at the translation initiation site or within the translation area of PAI-1 mRNA, suggesting that these sites may be promising sites for the design of effective ASODNs. [ABSTRACT FROM AUTHOR]

Several lines of evidence have indicated that the establishment of long-term memory requires protein synthesis, including the synthesis of immediate-early gene products. Although the anatomical expression patterns of the c-fos gene, a transcription factor-encoding immediate-early gene, in conditioned taste aversion (CTA) are well documented, the functional roles of c-fos gene expression and Fos-mediated transcription remain to be clarified. Using the antisense oligodeoxynucleotide (AS-ODN) method in rats and gene-targeting knockout techniques in mice (c-fos-/- mice), we examined the roles of c-fos gene expression in the acquisition, retrieval, and retention of CTA. Preconditioning microinfusion of AS-ODN directed against c-fos mRNA (c-fos AS-ODN) into the parabrachial nucleus (PBN) impaired the acquisition, whereas infusion of control ODNs consisting of a randomized or inverted base order had no effect. Microinfusion of c-fos AS-ODN into either the amygdala or insular cortex did not impair the acquisition, whereas it attenuated the retention. Retrieval and subsequent retention of an acquired CTA were not disrupted by c-fos AS-ODN infusion into the PBN or amygdala. Microinfusion of another AS-ODN directed against zif268 (egr-1, krox-24, NGFI-A) mRNA into the PBN or amygdala did not affect the acquisition and retention. The genetic deficiency in c-fos-/- mice caused normal acquisition and retention. The present results suggest that the Fos-mediated gene transcription in the PBN, amygdala, or insular cortex plays critical roles in the acquisition and/or consolidation, but not the retrieval, of long-term taste memory; nevertheless, some other factors could compensate CTA mechanism when Fos-mediated transcription is not available. [ABSTRACT FROM AUTHOR]

Abstract: Hyperalgesia following unilateral complete Freund’s adjuvant-induced inflammation was characterized by paw withdrawal latency to thermal stimulus. Paw withdrawal latencies were significantly shorter on the complete Freund’s adjuvant-treated paw than on the contralateral paw of the complete Freund’s adjuvant- and the sham-treated rats. Total cytosolic protein kinase C activity in the lumbar enlargement was unchanged on the sides of the spinal cord ipsi- and contra-lateral to the inflamed paw. Membrane-associated activities of protein kinase Cα, protein kinase CβI and protein kinase Cγ did not change significantly on the sides of the cord ipsi- and contra-lateral to the inflammation. However, membrane-associated activity of protein kinase CβII was increased in the cord section ipsilateral to the inflammation, suggesting that increased translocation/activation of protein kinase CβII is related to thermal hyperalgesia. Dextrorphan (an N-methyl-d-aspartate receptor antagonist), L-703,606 (an NK-1 receptor antagonist) and an antisense oligodeoxynucleotide for a selective knockdown of protein kinase Cβ, reduced complete Freund’s adjuvant-induced hyperalgesia, and reversed significant changes in the membrane activity of protein kinase CβII on the spinal cord section ipsilateral to the inflamed paw. Dextrorphan and protein kinase Cβ antisense oligodeoxynucleotide were effective in reversing complete Freund’s adjuvant-induced increase in the activity of protein kinase CβII ipsilateral to the inflammation at all the doses tested, but L-703,606 was effective only at the highest dose. Furthermore, in the presence of inflammatory stimulus, dextrorphan and L-703,606 did not alter the activities of membrane-associated protein kinase Cα, protein kinase CβI, and protein kinase Cγ in the section of the spinal cord ipsi- and contra-lateral to the inflammation. Protein kinase Cβ antisense oligodeoxynucleotide had no significant effect on the membrane-associated activities of protein kinase Cα and protein kinase Cγ, but decreased the activities of both protein kinase CβI and protein kinase CβII and the expression of protein kinase Cβ isozyme in the spinal cord. The data provide evidence that a common molecular event that converges to initiate and maintain hyperalgesia may include the translocation and activation of protein kinase CβII in the spinal dorsal horn. [Copyright &y& Elsevier]

Abstract: Glial cell line-derived neurotrophic factor (GDNF) has been hypothesized to play an important role in the modulation of nociceptive signals especially during neuropathic pain. The present study examined the expression of GDNF and GFRα-1 (the high-affinity receptor of GDNF) in dorsal root ganglions (DRG) in a rat model of neuropathic pain induced by chronic constriction injury (CCI) to the sciatic nerve. In order to address the role of GDNF and GFRα-1 in neuropathic pain, antisense oligodeoxynucleotide (ODN) specifically against GFRα-1 was intrathecally administered to result in down-regulation of GFRα-1 expression. The results showed that both the protein and mRNA levels of GDNF and GFRα-1 were significantly increased after CCI, while the thermal hyperalgesia of neuropathic pain rats could be significantly aggravated by antisense ODN treatment, but not by normal saline (NS) or mismatch ODN treatment. The present study demonstrated that endogenous GDNF and GFRα-1 might play an anti-hyperalgesic role in neuropathic pain of rats. In addition, we found a down-regulation of somatostatin (SOM) in DRG and spinal dorsal horn after expression of GFRα-1 was knocked down, which suggested the possible relationship between the anti-hyperalgesic effect of GDNF and GFRα-1 on neuropathic pain and endogenous SOM. [Copyright &y& Elsevier]

The effects of antisense oligodeoxynucleotide (antisense ODN) to follicle-stimulating hormone receptor (FSHR) and follicle-stimulating bormone (FSH) on the expression of proliferating cell nuclear antigen (PCNA) and vascular endothelial growth factor (VEGF) were studied in primary culture cells derived from human ovarian mucinous cystadenocarcinoma (OMC). The primary OMC cells were cultured with the enzyme digestion method, and the expression of pan Keratin protein and FSHR mRNA was detected for identification of the cells. OMC cells were co-cultured with antisense ODN, nonsense ODN and FSH with different concentrations for 48 h and 72 h. The expression of PCNA and VEGF was detected by using SP immunohistochemistry. Compared with that in the control group, the PCNA and VEGF expression was increased obviously in FSH groups ( P<0.05 or P<0.01). while decreased significantly in antisense ODN groups ( P<0.05 or P<0.01) and unchanged in nonsense ODN groups, respectively. Meanwhile, antisense ODN could antagonize the increased expression of PCNA and VEGF caused by FSH significantly ( P<0.01). It was suggested that FSH might promote the development of OMC to some extent. Antisense ODN could inhibit the proliferative activity of OMC cells and the promoting proliferative activity enhanced by FSH. [ABSTRACT FROM AUTHOR]

Abstract: To examine whether the selected antisense oligodeoxynucleotides (AS-ODN) targeting against human brain acetylcholinesterase (AChE) mRNA could improve the cognitive deficit in the Alzheimer''s disease (AD) model mice induced by amyloid-β peptide (Aβ), we determined the time–effect relationship of AChE activity and the learning and memory after AS-ODN delivery. The results showed that the AChE activity decreased gradually along with time, initiating at 8 h and lasting 42 h. The time–effect curves of acetylcholine (ACh) behaved consistency with that of AChE activity. The animal cognition studies showed that in step-through test, the error number of the AS-ODN-treated AD model mice was significantly decreased, and the memory retention was increased. In the water maze performance, the swimming time obviously shortened. Our results indicated that antisense therapy is of potential use in the treatment of cognitive deficit in the Aβ model mice. [Copyright &y& Elsevier]

A novel cationic liposome modified with soybean sterylglucoside (SG) and polyethylene glycol-distearoylphosphatidylethanolamine (PEG-DSPE) as a carrier of antisense oligodeoxynucleotide (ODN) for hepatitis B virus (HBV) therapy was constructed. Characteristics of the cationic liposomes modified with SG and PEG (SG/PEG-CL) and their complexes with 15-mer phosphorothioate ODN (SG/PEG-CL-ODN complex) were investigated by incorporation efficiency, morphology, electrophoresis, zeta potentials, and size analysis. Antisense activity of the liposomes and ODN complexes was determined as hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) in HepG 2 2.2.15 cells by ELISA. Their tissue and intrahepatic distribution were evaluated following intravenous injection in mice. The complexes gained high incorporation efficiency and intact vesicular structure with mean size at &nvsim;200 nm. The SG/PEG-CL-ODN complexes enhanced the inhibition of both HBsAg and HBeAg expression in the cultured HepG 2 2.2.15 cells relative to free ODN. The uptake of SG/PEG-CL and nonmodified cationic liposomes (CL) was primarily by liver, spleen, and lung. Furthermore, the concentration of SG/PEG-CL was significant higher than that of CL in hepatoctyes at 0.5 hr postinjection. The biodistribution of SG/DSPE-CL-ODN complex compare with free ODN showed that liposomes enhanced the accumulation of ODN in the liver and spleen, while decreasing its blood concentration. SG/PEG-CL–mediated ODN transfer to the liver is an effective gene delivery method for cell-specific targeting, which has a potential for gene therapy of HBV infections. SG and PEG-modified cationic liposomes have proven to be an alternative carrier for hepatocyte-selective drug targeting. [ABSTRACT FROM AUTHOR]

Abstract: Antisense oligodeoxynucleotide against the splicing enhancer sequence (SES) in exon 19 of the dystrophin gene have been shown to induce exon 19 skipping and promote the expression of internally deleted dystrophin by correcting the translational reading frame in the cultured Duchenne muscular dystrophy (DMD) myocytes with the deletion of exon 20. Transfection of the antisense oligodeoxynucleotide, therefore, has been proposed as a promising means for therapeutic modification of dystrophin mRNA of DMD, a fatal disorder caused by defects in the dystrophin gene. A systemic delivery method targeting the large number of diseased muscles remains to be established for clinical application of antisense oligodeoxynucleotide. In this study, we investigated capability of oligodeoxynucleotide transfer into the skeletal muscles of mdx mouse, a mouse model of DMD. Thirty-one mer phosphorothioate oligodeoxynucleotide complementary to the SES of dystrophin exon 19 was intraperitoneally administered to mdx mice without any carrier. Histochemical study disclosed that fluorescence-labeled oligodeoxynucleotide appeared in the nuclei of femoral skeletal muscle cell at the second day after injection of 20mg/kg BW oligodeoxynucleotide, and still visible at 14th day. Reverse transcription (RT)-PCR analysis of dystrophin transcript in these cells disclosed that a proportion of it showed skipping of exon 19 from second to seventh day after injection. These results showed that the intraperitoneally administered oligodeoxynucleotide could be transfected to nucleus of mdx skeletal muscle without any carrier and was able to induce exon skipping in vivo. [Copyright &y& Elsevier]

Abstract: Although the serine/threonine protein kinases involved in the pharmacological action of morphine are well recognized, the critical contribution of serine/threonine protein phosphatase (PP) has been appreciated on to a slight degree. We examined the involvement of subtypes of serine/threonine protein phosphatase (PP) in the antinociceptive effect of morphine in mice. The antinociceptive effect of subcutaneously administered morphine was attenuated by simultaneously intracerebroventricular (i.c.v.) or intrathecal (i.t.) injection of okadaic acid (OA), a PP inhibitor. To reveal which subtypes of PPs participated in the antinociceptive effect of morphine, mice received i.c.v. or i.t. injections of antisense oligodeoxynucleotide (AS-ODN) directed against either the PP2A or PP5 subtypes of PPs before assessment of morphine-induced antinociception. Pretreatment with AS-ODN against PP2A or PP5 via each route weakened the antinociceptive effect of morphine, accompanied by reduction of expression levels of PP in the periaqueductal gray (PAG) and the spinal cord. Subcutaneously administered morphine increased activity of OA-sensitive PPs in the PAG and the spinal cord in a dose-dependent manner; this was prevented by concurrent administration of naloxone. These results suggest that PP2A and PP5 are involved in the antinociceptive effect of morphine in mice. [Copyright &y& Elsevier]