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Antisense
Terms modified by Antisense Selected AbstractsAntisense oligodeoxynucleotide-induced suppression of basal forebrain NMDA-NR1 subunits selectively impairs visual attentional performance in ratsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 1 2001Janita Turchi Abstract It is generally agreed that basal forebrain neuronal circuits contribute to the mediation of the ability to detect, select and discriminate signals, to suppress the processing of irrelevant information, and to allocate processing resources to competing tasks. Rats were trained in a task designed to assess sustained attention, or in a cued discrimination task that did not tax attentional processes. Animals were equipped with guide cannula to infuse bilaterally antisense oligodeoxynucleotides (ODNs) against the N -methyl- d -aspartate (NMDA) NR1 subunits, or missense ODNs, into the substantia innominata of the basal forebrain. Infusions of antisense or missense ODNs did not affect cued visual discrimination performance. Infusions of antisense ODNs dose-dependently impaired sustained attention performance by selectively decreasing the animals' ability to detect signals while their ability to reject nonsignal trials remained unchanged. The detrimental attentional effects of antisense infusions were maximal 24 h after the third and final infusion, and performance returned to baseline 24 h later. Missense infusions did not affect attentional performance. Separate experiments demonstrated extensive suppression of NR1 subunit immunoreactivity in the substantia innominata. Furthermore, infusions of antisense did not produce neurotoxic effects in that region as demonstrated by the Fluoro-Jade method. The present data support the hypothesis that NMDA receptor (NMDAR) stimulation in the basal forebrain, largely via glutamatergic inputs originating in the prefrontal cortex, represents a necessary mechanism to activate the basal forebrain corticopetal system for mediation of attentional performance. [source] APC-dependent regulation of ornithine decarboxylase in human colon tumor cellsMOLECULAR CARCINOGENESIS, Issue 1 2002Kimberly E. Fultz Abstract Mutation/deletion of the adenomatous polyposis coli (APC) tumor suppressor gene in germline cells of rodents and humans is associated with increased intestinal activity of ornithine decarboxylase (ODC), the first enzyme in polyamine synthesis, and intestinal neoplasia. To study the role of APC in signaling ODC expression, we used the human colon tumor cell line HT29 (wtAPC,/,), which has been stably transfected with a zinc-inducible wild-type APC gene. The addition of ZnCl2 to HT29-APC cells increased wild-type APC protein and Mad1 RNA and protein and decreased levels of c- myc and ODC RNA and protein, relative to these parameters in HT29 cells transfected with the same plasmid containing the ,-galactosidase gene in place of APC. Upon induction of APC expression, ODC promoter activity and RNA levels were suppressed. When the e-box domain in the 5, flanking region of the ODC gene was mutated, ODC promoter activity was unaffected by wild-type APC expression. Antisense, but not missense, c- myc oligonucleotides decreased ODC activity in HT29 cells expressing mutant APC. These results demonstrated that wild-type APC suppressed c-myc and activated Mad1 expression in HT29 colon-derived cells. These proteins, in turn, regulated the transcription of target genes, including ODC. The data presented indicate that ODC is a modifier of APC-dependent signaling in intestinal cells and tissues. © 2002 Wiley-Liss, Inc. [source] Probing Pineal-specific Gene Expression with Transgenic Zebrafish,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2008Daisuke Kojima The pineal gland of zebrafish (Danio rerio) contains light-sensitive photoreceptor cells and plays an important role in the neuroendocrine system. The zebrafish exorhodopsin gene encodes a pineal-specific photoreceptive protein, whose promoter region harbors a cis -acting element, pineal expression-promoting element (PIPE), directing pineal-specific gene expression. For in vivo genetic studies on PIPE-binding proteins and their regulatory mechanisms, we generated a transgenic zebrafish line, Tg(P20 -rh/P:gfp), that expresses green fluorescent protein (GFP) under the control of the zebrafish rhodopsin promoter fused with 20 PIPE repeats. In Tg(P20 -rh/P:gfp) fish, PIPE-dependent gene expression is visualized by GFP fluorescence in the pineal gland along with PIPE-independent GFP signals in the retinal rod photoreceptors. The transgenic fish exhibit detectable and reproducible GFP fluorescence in the larval pineal gland by 5 days postfertilization. Antisense morpholino-mediated knock-down of a pineal transcription factor gene, otx5, suppresses pineal GFP expression in the transgenic line. In a pilot screen of N -ethyl- N -nitrosourea-treated fish of the GFP transgenic line, we isolated potential dominant mutations that cause attenuation of pineal GFP fluorescence with a marginal effect on the retinal GFP signal. The results suggest that the Tg(P20 -rh/P:gfp) line will be useful for detecting deficits in PIPE-dependent gene expression in the pineal gland. [source] Proteomic profiling of antisense-induced exon skipping reveals reversal of pathobiochemical abnormalities in dystrophic mdx diaphragmPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 3 2009Philip Doran Abstract The disintegration of the dystrophin,glycoprotein complex represents the initial pathobiochemical insult in Duchenne muscular dystrophy. However, secondary changes in signalling, energy metabolism and ion homeostasis are probably the main factors that eventually cause progressive muscle wasting. Thus, for the proper evaluation of novel therapeutic approaches, it is essential to analyse the reversal of both primary and secondary abnormalities in treated muscles. Antisense oligomer-mediated exon skipping promises functional restoration of the primary deficiency in dystrophin. In this study, an established phosphorodiamidate morpholino oligomer coupled to a cell-penetrating peptide was employed for the specific removal of exon 23 in the mutated mouse dystrophin gene transcript. Using DIGE analysis, we could show the reversal of secondary pathobiochemical abnormalities in the dystrophic diaphragm following exon-23 skipping. In analogy to the restoration of dystrophin, ,-dystroglycan and neuronal nitric oxide synthase, the muscular dystrophy-associated differential expression of calsequestrin, adenylate kinase, aldolase, mitochondrial creatine kinase and cvHsp was reversed in treated muscle fibres. Hence, the re-establishment of Dp427 coded by the transcript missing exon 23 has counter-acted dystrophic alterations in Ca2+ -handling, nucleotide metabolism, bioenergetic pathways and cellular stress response. This clearly establishes the exon-skipping approach as a realistic treatment strategy for diminishing diverse downstream alterations in dystrophinopathy. [source] An Activatable siRNA Probe: Trigger-RNA-Dependent Activation of RNAi Function,ANGEWANDTE CHEMIE, Issue 50 2009Hiroki Masu Einfangen und Freisetzen: Eine Haarnadel-siRNA-Sonde aus einem ,Sense"- und einem ,Antisense"-Strang (rot bzw. blau) wurde entwickelt, die mit einer Auslöser-RNA aktiviert werden kann. In Gegenwart der Auslöser-RNA wurde ein stark sequenzabhängiges An- und Ausschalten der RNAi-Aktivität beobachtet (siehe Schema). [source] Early requirement for fgf8 function during hindbrain pattern formation in zebrafishDEVELOPMENTAL DYNAMICS, Issue 2 2004Elizabeth L. Wiellette Abstract Fibroblast growth factor (FGF) signaling is required for normal development of the vertebrate brain, including the isthmus and caudal regions of the hindbrain. Recent work in zebrafish has identified a requirement for the combination of fgf3 and fgf8 functions in specification of rhombomeres 5 and 6 (r5, r6), when evaluated at mid- and late somitogenesis stages. However, when examined earlier in development, during early somitogenesis stages, FGF8 alone is required to initiate r5 and r6 development. Both a mutation in fgf8 and injection of fgf8 -targeted antisense morpholino-modified oligonucleotides result in suppression of genes normally expressed in r5 and r6 by the one- to two-somite stage. This expression recovers by the six-somite stage, and we propose that this recovery is a response to activation of fgf3 and to delayed accumulation of fgf8. These data demonstrate an early, nonredundant requirement for fgf8 function in hindbrain patterning. Developmental Dynamics 229:393,399, 2004. © 2004 Wiley-Liss, Inc. [source] GDNF hyperalgesia is mediated by PLC,, MAPK/ERK, PI3K, CDK5 and Src family kinase signaling and dependent on the IB4-binding protein versicanEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 1 2008Oliver Bogen Abstract The function of the isolectin B4 (IB4+)-binding and GDNF-dependent Ret (Ret+)-expressing non-peptidergic subpopulation of nociceptors remain poorly understood. We demonstrate that acute administration of GDNF sensitizes nociceptors and produces mechanical hyperalgesia in the rat. Intrathecal IB4,saporin, a selective toxin for IB4+/Ret+ -nociceptors, attenuates GDNF but not NGF hyperalgesia. Conversely, intrathecal antisense to Trk A attenuated NGF but not GDNF hyperalgesia. Intrathecal administration of antisense oligodeoxynucleotides targeting mRNA for versican, the molecule that renders the Ret-expressing nociceptors IB4-positive (+), also attenuated GDNF but not NGF hyperalgesia, as did ADAMTS-4, a matrix metalloprotease known to degrade versican. Finally, inhibitors for all five signaling pathways known to be activated by GDNF at GFR,1/Ret: PLC,, CDK5, PI3K, MAPK/ERK and Src family kinases, attenuated GDNF hyperalgesia. Our results demonstrate a role of the non-peptidergic nociceptors in pain produced by the neurotrophin GDNF and suggest that the IB4-binding protein versican functions in the expression of this phenotype. [source] Inhibition of tyrosine kinase receptor type B synthesis blocks axogenic effect of estradiol on rat hypothalamic neurones in vitroEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 2 2004V. I. Brito Abstract 17-,-estradiol (E2) increases axonal growth and tyrosine kinase receptor (Trk)B levels of male-derived hypothalamic neurones in vitro. To investigate whether the axogenic response depends on the upregulation of TrkB, we analysed neuritic growth and neuronal polarization in cultures treated with an antisense oligonucleotide against TrkB mRNA. In cultures without E2, treatment with 7.5 or 10 µm antisense reduced TrkB levels and the percentage of neurones showing an identifiable axon; the number and length of minor processes were increased. In cultures treated with 5 µm antisense, morphometric parameters were normal although total TrkB levels were reduced. The same dose prevented the E2-dependent increase of TrkB levels and suppressed the axogenic effect of E2. These results indicate that TrkB is necessary for normal neuronal growth and maturation and further suggest that an increase in TrkB is necessary for E2 to exert its axogenic effect in male-derived neurones. [source] Antisense oligodeoxynucleotide-induced suppression of basal forebrain NMDA-NR1 subunits selectively impairs visual attentional performance in ratsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 1 2001Janita Turchi Abstract It is generally agreed that basal forebrain neuronal circuits contribute to the mediation of the ability to detect, select and discriminate signals, to suppress the processing of irrelevant information, and to allocate processing resources to competing tasks. Rats were trained in a task designed to assess sustained attention, or in a cued discrimination task that did not tax attentional processes. Animals were equipped with guide cannula to infuse bilaterally antisense oligodeoxynucleotides (ODNs) against the N -methyl- d -aspartate (NMDA) NR1 subunits, or missense ODNs, into the substantia innominata of the basal forebrain. Infusions of antisense or missense ODNs did not affect cued visual discrimination performance. Infusions of antisense ODNs dose-dependently impaired sustained attention performance by selectively decreasing the animals' ability to detect signals while their ability to reject nonsignal trials remained unchanged. The detrimental attentional effects of antisense infusions were maximal 24 h after the third and final infusion, and performance returned to baseline 24 h later. Missense infusions did not affect attentional performance. Separate experiments demonstrated extensive suppression of NR1 subunit immunoreactivity in the substantia innominata. Furthermore, infusions of antisense did not produce neurotoxic effects in that region as demonstrated by the Fluoro-Jade method. The present data support the hypothesis that NMDA receptor (NMDAR) stimulation in the basal forebrain, largely via glutamatergic inputs originating in the prefrontal cortex, represents a necessary mechanism to activate the basal forebrain corticopetal system for mediation of attentional performance. [source] APP is required during an early phase of memory formationEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 12 2000R. Mileusnic Abstract The amyloid ,/A4 protein precursor (APP) has been shown to be implicated in age-associated plastic changes at synapses that might contribute to memory loss in Alzheimer's disease. As APP has previously been reported to have multiple functions during normal development, we have employed a one-trial passive avoidance task in day-old chicks to study its role in the process of memory formation. Administration of anti-APP antibodies, injected 30 min pretraining, prevented memory for a one-trial passive avoidance task in day-old chicks without effects on general behaviour or initial acquisition. Amnesia was apparent by 30 min post-training and lasted for at least 24 h. The same result was obtained by down-regulation of APP expression by APP-antisense, injected 8,12 h pretraining. However, injections of anti-APP antibodies or APP antisense at later post-training time did not cause amnesia for the task. Unlike antibodies and antisense, injection of the APP328,332 pentapeptide, in either orientation, 30 min pretraining, rescued the memory and prevented antisense-induced amnesia. The post-training time within which the antibody- and antisense-induced amnesia, and within which the APP peptides prevent amnesia, correspond to that during which memory formation is vulnerable to disruption of the putative signal transduction functions of APP. These results suggest that: (i) APP is required during an early phase of memory formation, and (ii) the memory enhancing effect of secretory APP is localized within a 5-mer sequence of growth-promoting domain. [source] Inhibition of brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor expression reduces dopaminergic sprouting in the injured striatumEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 10 2000P. E. Batchelor Abstract After striatal injury, sprouting dopaminergic fibres grow towards and intimately surround wound macrophages which, together with microglia, express the dopaminergic neurotrophic factors glial cell line-derived neurotrophic factor (GDNF) and brain derived neurotrophic factor (BDNF). To evaluate the importance of these endogenously secreted neurotrophic factors in generating striatal peri-wound dopaminergic sprouting, the peri-wound expression of BDNF or GDNF was inhibited by intrastriatal infusion of antisense oligonucleotides for 2 weeks in mice. Knock-down of both BDNF and GDNF mRNA and protein levels in the wounded striatum were confirmed by in situ hybridization and enzyme-linked immunosorbent assay, respectively. Dopamine transporter immunohisto-chemistry revealed that inhibition of either BDNF or GDNF expression resulted in a marked decrease in the intensity of peri-wound sprouting. Quantification of this effect using [H3]-mazindol autoradiography confirmed that peri-wound sprouting was significantly reduced in mice receiving BDNF or GDNF antisense infusions whilst control infusions of buffered saline or sense oligonucleotides resulted in the pronounced peri-wound sprouting response normally associated with striatal injury. BDNF and GDNF thus appear to be important neurotrophic factors inducing dopaminergic sprouting after striatal injury. [source] Endothelial-Independent Prevention of High Blood Pressure in L-Name-Treated Rats by Angiotensin II type I Receptor Antisense Gene TherapyEXPERIMENTAL PHYSIOLOGY, Issue 4 2003Phyllis Y. Reaves It has previously been established that a single systemic administration of retroviral vector containing angiotensin II type I receptor antisense (AT1R-AS) in the neonatal spontaneously hypertensive rat (SHR) prevents development of hypertension, and in addition cardiac hypertrophy and endothelial dysfunction. However, these studies could not determine whether the effects of AT1R-AS on high blood pressure (BP) and endothelial function were independent. Angiotensin receptor blockers have been shown to reduce BP in the L-NAME (N , -nitro-L-arginine methyl ester hydrochloride)-induced rat model of hypertension. Our objective in the present study was to use the L-NAME model of hypertension to determine whether AT1R-AS treatment would lower high BP and attenuate cardiac hypertrophy under conditions of permanent endothelial damage. A single bolus of LNSV-AT1R-AS viral particles in neonatal Wistar-Kyoto (WKY) rats was without affect on basal BP. Efficacy of the transgene incorporation was assessed by observing a significant reduction in angiotensin-induced dipsogenic response in the AT1R-AS-treated animals. Introduction of L-NAME in the drinking water for 10 weeks resulted in the establishment of hypertension only in the WKY rats treated with vector alone. These hypertensive (BP, 179 ± 4 mmHg) animals showed a 17% increase in heart weight/body weight ratio and a 60% reduction in ACh-induced vasorelaxation in phenylephrine-preconstricted arteries. The L-NAME-induced high BP and cardiac hypertrophy were attenuated in rats expressing AT1R-AS. However, endothelial dysfunction could not be prevented with the antisense therapy. These observations demonstrate that attenuation of endothelial dysfunction is not a prerequisite for the antihypertensive effects of AT1R-AS treatment. [source] The A-subunit of surface-bound Shiga toxin stimulates clathrin-dependent uptake of the toxinFEBS JOURNAL, Issue 16 2005Maria L. Torgersen Shiga toxin can be internalized by clathrin-dependent endocytosis in different cell lines, although it binds specifically to the glycosphingolipid Gb3. It has been demonstrated previously that the toxin can induce recruitment of the toxin,receptor complex to clathrin-coated pits, but whether this process is concentration-dependent or which part of the toxin molecule is involved in this process, have so far been unresolved issues. In this article, we show that the rate of Shiga toxin uptake is dependent on the toxin concentration in several cell lines [HEp-2, HeLa, Vero and baby hamster kidney (BHK)], and that the increased rate observed at higher concentrations is strictly dependent on the presence of the A-subunit of cell surface-bound toxin. Surface-bound B-subunit has no stimulatory effect. Furthermore, this increase in toxin endocytosis is dependent on functional clathrin, as it did not occur in BHK cells after induction of antisense to clathrin heavy chain, thereby blocking clathrin-dependent endocytosis. By immunofluorescence, we show that there is an increased colocalization between Alexa-labeled Shiga toxin and Cy5-labeled transferrin in HeLa cells upon addition of unlabeled toxin. In conclusion, the data indicate that the Shiga toxin A-subunit of cell surface-bound toxin stimulates clathrin-dependent uptake of the toxin. Possible explanations for this phenomenon are discussed. [source] Inhibition of Proinflammatory Cytokine Expression by NF-,B (p65) Antisense Oligonucleotide in Helicobacter pylori -Infected MiceHELICOBACTER, Issue 6 2005Sang Gyun Kim ABSTRACT Background.,Helicobacter pylori induces the expression of proinflammatory cytokines in vitro by activating nuclear factor-,B, a transcriptional regulator. However, it has not been clarified whether H. pylori -induced proinflammatory cytokines are also mediated through nuclear factor-,B in vivo. The aim of this study was to evaluate the role of nuclear factor-,B on the expressions of proinflammatory cytokines in H. pylori -infected mice. Materials and Methods., We evaluated nuclear factor-,B (p65) activation in the H. pylori -infected gastric mucosa of mice by immunofluorescent staining using antip65 polyclonal antibody, and the expressions of proinflammatory cytokines with inhibition of nuclear factor-,B pathway by using phosphorothioate antisense and sense oligonucleotide against the nuclear factor-,B (p65). Results., In the H. pylori -infected gastric mucosa of mice, immunofluorescent staining using antip65 polyclonal antibody showed nuclear factor-,B (p65) activation, which was particularly localized to epithelial cells. Tumor necrosis factor-, and interleukin-1, concentrations in gastric mucosa by enzyme-linked immunosorbent assay (ELISA) were elevated in the infected group versus the uninfected group. Pretreatment with nuclear factor-,B (p65) antisense oligonucleotide inhibited the activation of nuclear factor-,B and the expressions of tumor necrosis factor-, and interleukin-1, in H. pylori -infected gastric mucosa. Sense oligonucleotide did not influence on the expression of proinflammatory cytokines. Conclusions.,H. pylori infection was found to activate the expressions of proinflammatory cytokines via nuclear factor-,B in vivo, and this may play an important role in the initiation of H. pylori- induced gastric inflammation. [source] Overexpression and significance of prion protein in gastric cancer and multidrug-resistant gastric carcinoma cell line SGC7901/ADRINTERNATIONAL JOURNAL OF CANCER, Issue 2 2005Jingping Du Abstract In our previous work, cellular prion protein (PrPc) was identified as an upregulated gene in adriamycin-resistant gastric carcinoma cell line SGC7901/ADR compared to its parental cell line SGC7901. Here we investigate the expression of PrPc in gastric cancer and whether it was involved in multidrug resistance (MDR) of gastric cancer. We demonstrated that PrPc was ubiquitously expressed in gastric cancer cell lines and tissues. PrPc conferred resistance of both P-glycoprotein (P-gp)-related and P-gp-nonrelated drugs on SGC7901, which was accompanied by decreased accumulation and increased releasing amount of adriamycin in PrPc-overexpressing cell line. Inhibition of PrPc expression by antisense or RNAi technology could partially reverse multidrug-resistant phenotype of SGC7901/ADR. PrPc significantly upregulated the expression of the classical MDR-related molecule P-gp but not multidrug resistance associated protein and glutathione S-transferase pi. The PrPc-induced MDR could be partially reversed by P-gp inhibitor verapamil. PrPc could also suppress adriamycin-induced apoptosis and alter the expression of Bcl-2 and Bax, which might be another pathway contributing to PrPc-related MDR. The further study of the biological functions of PrPc may be helpful for understanding the mechanisms of occurrence and development of clinical gastric carcinoma and PrPc-related MDR and developing possible strategies to treat gastric cancer. [source] Antisense oligodeoxynucleotide therapy targeting clusterin gene for prostate cancer: Vancouver experience from discovery to clinicINTERNATIONAL JOURNAL OF UROLOGY, Issue 9 2005HIDEAKI MIYAKE Abstract Background The objective of this study was to review our experience in the development of antisense (AS) oligodeoxynucleotide (ODN) therapy for prostate cancer targeting antiapoptotic gene, clusterin. Methods We initially summarized our data demonstrating that clusterin could be an optimal therapeutic target for prostate cancer, then presented the process of developing AS ODN therapy using several preclinical animal models. Finally, the preliminary data of the recently completed phase I clinical trial using AS clusterin ODN as well as the future prospects of this therapy are discussed. Results Expression of clusterin was highly up-regulated after androgen withdrawal and during progression to androgen-independence, but low or absent in untreated tissues in both prostate cancer animal model systems and human clinical specimens. Introduction of the clusterin gene into human prostate cancer cells confers resistance to several therapeutic stimuli, including androgen ablation, chemotherapy and radiation. AS ODN targeting the translation initiation site of the clusterin gene markedly inhibited clusterin expression in prostate cancer cells in a dose-dependent and sequence-specific manner. Systemic treatment with AS clusterin ODN enhanced the effects of several conventional therapies through the effective induction of apoptosis in prostate cancer xenograft models. Based on these findings, a phase I clinical trial was completed using AS clusterin ODN incorporating 2,-O-(2-methoxy)ethyl-gapmer backbone (OGX-011), showing up to 90% suppression of clusterin in prostate cancer. Conclusions The data described above identified clusterin as an antiapoptotic gene up-regulated in an adaptive cell survival manner following various cell death triggers that helps confer a phenotype resistant to therapeutic stimuli. Inhibition of clusterin expression using AS ODN technology enhances apoptosis induced by several conventional treatments, resulting in the delay of AI progression and improved survival. Clinical trials using AS ODN confirm potent suppression of clusterin expression and phase II studies will begin in early 2005. [source] Maspin Is Involved in Bone Matrix Maturation by Enhancing the Accumulation of Latent TGF-,,,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 10 2007Reiko Tokuyama Abstract Maspin, a serine protease inhibitor, is expressed by formative osteoblasts. The repression of maspin expression in osteoblastic cells decreased the level of latent TGF-, in the extracellular matrix, whereas the overexpression of maspin increased latent TGF-,. These findings suggest that maspin plays an important role in bone matrix formation, particularly in the accumulation of latent TGF-,. Introduction: Maspin is a serine protease inhibitor that exhibits tumor suppressive and anti-angiogenic activities. This study was performed to elucidate a possible role for maspin in bone formation. Materials and Methods: We performed immunohistochemical analysis of the expression of maspin during endochondral ossification. We evaluated the expression of maspin mRNA and protein in ROS 17/2.8 cells and primary rat osteoblastic cells by RT-PCR, immunocytochemistry, and Western blot analysis. We also examined the accumulation of TGF-, in the extracellular matrix of cultured ROS 17/2.8 cells after transfection with vectors expressing either maspin or maspin antisense. Results: We observed expression of maspin by active osteoblasts in vivo. Rat osteoblastic cells also expressed maspin mRNA and protein in vitro. Moreover, the accumulation of latent TGF-, in the extracellular matrix significantly decreased in cultures exposed to an anti-maspin antibody and when cells were transfected with a maspin antisense-expressing vector. In contrast, accumulation of latent TGF-, in the extracellular matrix increased after transfection of cells with a vector expressing maspin. Conclusions: These findings suggest that maspin expressed in active osteoblasts plays an important physiological role during maturation of the bone matrix, and in particular, during the process of accumulation of latent TGF-, in the extracellular matrix. [source] Lysyl Hydroxylase-2b Directs Collagen Cross-Linking Pathways in MC3T3-E1 Cells,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 8 2004Suchaya Pornprasertsuk Abstract To elucidate the roles of LH2b in collagen cross-linking, MC3T3-E1 cell clones expressing higher (S) or lower (AS) levels of LH2b were established. Compared with controls, the collagen cross-linking pattern was shifted toward hydroxylysine-aldehyde (S clones)- or lysine-aldehyde (AS clones)-derived pathways. The data indicate that LH2b directs collagen cross-linking pathways through its action on telopeptidyl lysine residues. Introduction: Lysine (Lys) hydroxylation is a post-translational modification of collagen critical for cross-linking and glycosylation. Currently, three isoforms of lysyl hydroxylase (LH) have been identified, but their specific functions are still not well defined. Recently, we proposed that LH2 might modulate collagen cross-linking pattern through its action on Lys residues located in the telopeptide domains of collagen. Materials and Methods: To directly test this hypothesis, several MC3T3-E1 cell-derived clones expressing higher (sense [S]) or lower (antisense [AS]) levels of LH2b, the predominant form of LH2 in this cell line, were established and cultured for 2 weeks, and collagen cross-links and precursor aldehydes in the matrices were analyzed. Results: In S clones tested, the ratio of dihydroxylysinonorleucine (DHLNL) to hydroxylysinonorleucine (HLNL) was significantly higher than the average of controls (76% and 140% increase, respectively), and the level of pyridinoline (Pyr) was elevated (100% and 150% increase, respectively). In contrast, when MC3T3-E1 cells were transfected with a LH2b antisense construct (AS clones), the DHLNL/HLNL ratios were significantly lower than that of controls (56% and 73% decrease, respectively), and Pyr was not detected. Furthermore, significant amounts of an aldol-derived cross-link, dehydrohistidinohydroxymerodesmosine, were produced (,0.3 mol/mol of collagen) in AS clones. Conclusions: The data clearly show a critical role of LH2b in determining collagen cross-linking pathways, most likely through its action on telopeptidyl Lys residues. [source] Antisense Oligodeoxynucleotide Evidence That a Unique Osteoclastic Protein-Tyrosine Phosphatase Is Essential for Osteoclastic ResorptionJOURNAL OF BONE AND MINERAL RESEARCH, Issue 10 2001Sung Min Suhr Abstract This study tested the hypothesis that a unique osteoclastic transmembrane protein tyrosine phosphatase (PTP-oc) is involved in osteoclastic resorption by determining whether suppression of PTP-oc expression with a specific phosphorothioated 20-mer PTP-oc antisense oligodeoxynucleotide (oligo) would inhibit basal, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]-stimulated, and PTH-stimulated osteoclastic resorption. Treatment of rabbit osteoclasts with 1 ,M of the antisense oligo for up to 4 days showed a time-dependent reduction in PTP-oc protein level, indicating that this PTP-oc antisense oligo was effective. To assess the effect of PTP-oc antisense oligo on osteoclastic resorption, rabbit osteoclasts were pretreated for 3 days with 1 ,M of the antisense, a scramble oligo, or vehicle, respectively, followed by a 3-day treatment with vehicle, 10 nM of 1,25(OH)2D3, or 10 nM of parathyroid hormone (PTH). 1,25(OH)2D3 and PTH each alone increased PTP-oc cellular level and stimulated resorptive activity of rabbit osteoclasts. The antisense oligo treatment, but not the scramble oligo, decreased the basal and the stimulated resorption activity and reduced the PTP-oc protein level. Treatment with the PTP-oc antisense oligo, but not the scramble oligo, also markedly increased the Y527 phosphorylation level of c- src in rabbit osteoclasts. In conclusion, these results provide the first antisense oligo evidence that PTP-oc plays an essential role in osteoclastic resorption. [source] Transforming growth factor-,1-dependent activation of Smad2/3 and up-regulation of PAI-1 expression is negatively regulated by Src in SKOV-3 human ovarian cancer cellsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 3 2004Kiyoshi Wakahara Abstract The net balance between urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type-1 (PAI-1) has been implicated in tumor cell invasion and metastasis. To elucidate the mechanism of the transforming growth factor-,1 (TGF-,1)-dependent up-regulation of PAI-1 expression, we investigated which signaling pathway transduced by TGF-,1 is responsible for this effect. Here, we show (1) nontoxic concentrations of TGF-,1 up-regulates uPA expression in HRA and SKOV-3 human ovarian cancer cells, (2) TGF-,1 activates Smads (phosphorylation of Smad2 and nuclear translocation of Smad3) and subsequently up-regulates PAI-1 expression in HRA cells, whereas TGF-,1 neither activates Smads nor up-regulates PAI-1 in SKOV-3 cells, (3) pharmacological Src inhibitor PP2 or antisense (AS) c-Src oligodeoxynucleotide (ODN) treatment significantly induces TGF-,1-dependent activation of Smads, leading to PAI-1 synthesis, compared with controls, in SKOV-3 cells, (4) combination of TGF-,1 and PP2, which activates PAI-1 expression and reduces uPA expression in SKOV-3, results in decreased invasiveness, (5) pharmacological inhibitors for mitogen-activated protein kinase (MAPK) (PD98059) and phosphoinositide-3-kinase (PI3K) (LY294002 and wortmannin) or AS-PI3K ODN transfection do not affect TGF-,1-induced Smad signaling and up-regulation of PAI-1 expression in SKOV-3 cells pr treated with PP2, and (6) the induction of PAI-1 protein was partially inhibited by an inhibitor of Sp1-DNA binding, mithramycin, implicating, at least in part, Sp1 in the regulation of this gene by TGF-,1. In conclusion, TGF-,1-dependent activation of Smad2/3, leading to PAI-1 synthesis, may be negatively regulated by Src, but not its downstream targets MAPK and PI3K in SKOV-3 cells. These data also reflect the complex biological effect of uPA-PAI-1 system. © 2004 Wiley-Liss, Inc. [source] Constitutive expression of thrombospondin 1 in MC3T3-E1 osteoblastic cells inhibits mineralizationJOURNAL OF CELLULAR PHYSIOLOGY, Issue 2 2006Akemichi Ueno Thrombospondin 1 (TSP1) is a multifunctional extracellular glycoprotein present mainly in the fetal and adult skeleton. Although an inhibitory effect of TSP1 against pathological mineralization in cultured vascular pericytes has been shown, its involvement in physiological mineralization by osteoblasts is still unknown. To determine the role of TSP1 in biomineralization, mouse osteoblastic MC3T3-E1 cells were cultured in the presence of antisense phosphorothioate oligodeoxynucleotides complementary to the TSP1 sequence. The 18- and 24-mer antisense oligonucleotides caused concentration-dependent increases in the number of mineralized nodules, acid-soluble calcium deposition in the cell/matrix layer, and alkaline phosphatase activity within 9 days, without affecting cell proliferation. The corresponding sense or scrambled oligonucleotides did not affect these parameters. In the antisense oligonucleotide-treated MC3T3-E1 cells, thickened extracellular matrix, well-developed cell processes, increased intracellular organelles, and collagen fibril bundles were observed. On the other hand, the addition of TSP1 to the culture decreased the production of a mineralized matrix by MC3T3-E1 cells. Furthermore, MC3T3-E1 clones overexpressing mouse TSP1 were established and assayed for TSP1 protein and their capacity to mineralize. TSP1 dose-dependently inhibited mineralization by these cells both in vitro and in vivo. These results indicate that TSP1 functions as an inhibitory regulator of bone mineralization and matrix production by osteoblasts to sustain bone homeostasis. J. Cell. Physiol. 209: 322,332, 2006. © 2006 Wiley-Liss, Inc. [source] Antisense suppression of tau in cultured rat oligodendrocytes inhibits process formationJOURNAL OF NEUROSCIENCE RESEARCH, Issue 12 2008David Gordon Abstract The microtubule-associated protein tau is integral to neuronal process development and has a role in the pathogenesis of several neurodegenerative conditions. We examined possible roles for tau in cultured oligodendrocyte process formation by using antisense oligonucleotide treatment. Inhibition of tau synthesis with single oligonucleotides resulted in decreased tau protein levels and significantly shorter cellular processes. Simultaneous use of two nonoverlapping oligonucleotides caused a major reduction in tau levels and severely inhibited process outgrowth. The timing of oligonucleotide addition to oligodendrocyte cultures was important, with addition of antisense at the time of plating into culture having the most significant effect on morphology through reduction of tau expression. © 2008 Wiley-Liss, Inc. [source] Altered Cbfal expression and biomineralization in an osteosarcoma cell lineJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 2 2004H. Perinpanayagam Abstract Osteoblast differentiation and expression are regulated by Cbfal transcription factors. Recent evidence suggests that Cbfal may also regulate bone mineralization. The purpose of this study was to characterize Cbfal expression in relation to mineralization in rat UMR106-01 osteoblastic cell cultures. UMR106-01 BSP cultures consistently form bone-like mineral, whereas the UI subclone mineralize gradually. BSP and UI cultures were grown for 48 h and then treated with ,-glycerophosphate. BSP cultures had alizarin red stained calcifications and mineral-like deposits within 24 h of phosphate. Atomic absorption spectroscopy measured significantly (P < 0.0001) more calcium in the phosphate-treated BSP cultures than in the UI. Cbfal message was detected in the BSP and UI cultures, but the Cbfal N-terminal isoform was deficient in UI and appeared to be up-regulated in the phosphate-treated BSP cultures. Cbfal protein levels were also reduced in the UI. DNA sequence from the RT-PCR products was utilized to design Taqman Real-time RT-PCR reagents. Quantitative Real-time RT-PCR analysis showed that Cbfal mRNA levels relative to endogenous 18 s rRNA were lower in the slower mineralizing UI cultures. Furthermore, the Cbfal N-terminal isoform mRNA levels were significantly (P < 0.001) lower in the slower mineralizing cultures. Transfection with Cbfal or isoform antisense caused a significant (P < 0.001) reduction in mineralization. Therefore, Cbfal expression may be associated with bone-like mineral formation in rat UMR106-01 osteoblastic cell cultures. © 2003 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved. [source] Bioavailability and efficacy of antisense morpholino oligomers targeted to c- myc and cytochrome P-450 3A2 following oral administration in ratsJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 4 2002Vikram Arora Abstract Antisense phosphorodiamidate Morpholino oligomers (PMO) are resistant to degradation by cellular hydrolases, DNases, RNases, and phosphodiesterases, but remain sensitive to prolonged exposure to low pH. The present studies evaluate the oral fractional bioavailability, stability, and efficacy of two distinct PMO sequences targeted to c- myc and cytochrome P-450 (CYP) 3A2. The c- myc antisense 20-mer, AVI-4126 (5,-ACGTTGAGGGGCATCGTCGC-3,), slowed the regenerative process in the rat liver after a 70% partial hepatectomy (PH). Rats were administered 3.0 mg/kg AVI-4126 in 0.1 mL saline via a bolus intravenous injection or in 0.5 mL sterile phosphate-buffered saline via gavage immediately following PH. The areas under the plasma concentration versus time curves revealed a fractional oral availability of 78.8% over a period of 10 min through 24 h. Immunoblot analysis of liver tissue from rats treated orally with AVI-4126 demonstrated a sequence-specific reduction in the target protein c-Myc, as well as secondary proliferation markers: proliferating cell nuclear antigen (PCNA), cyclin D1, and p53. The CYP3A2 antisense 22-mer AVI-4472 (5,-GAGCTGAAAGCAGGTCCATCCC-3,) caused a sequence-dependent reduction of approximately five-fold in the rat liver CYP3A2 protein levels and erythromycin demethylation activity in 24 h following oral administration at a dose of 2 mg/kg. It is concluded that oral administration of PMOs can inhibit c- myc and CYP3A2 gene expression in rat liver by an antisense-based mechanism of action. These studies highlight the potential for development of PMOs as orally administered therapeutic agents. © 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 91:1009,1018, 2002 [source] Defective Translocation of PKC, in EtOH-Induced Inhibition of Mg2+ Accumulation in Rat HepatocytesALCOHOLISM, Issue 9 2010Lisa M. Torres Background:, Rats chronically fed ethanol for 3 weeks presented a marked decreased in total hepatic Mg2+ content and required approximately 12 days to restore Mg2+ homeostasis upon ethanol withdrawal. This study was aimed at investigating the mechanisms responsible for the EtOH-induced delay. Methods:, Hepatocytes from rats fed ethanol for 3 weeks (Lieber-De Carli diet,chronic model), rats re-fed a control diet for varying periods of time following ethanol withdrawal, and age-matched control rats fed a liquid or a pellet diet were used. As acute models, hepatocytes from control animals or HepG2 cells were exposed to varying doses of ethanol in vitro for 8 minutes. Results:, Hepatocytes from ethanol-fed rats presented a marked inhibition of Mg2+ accumulation and a defective translocation of PKC, to the cell membrane. Upon ethanol withdrawal, 12 days were necessary for PKC, translocation and Mg2+ accumulation to return to normal levels. Exposure of control hepatocytes or HepG2 cells to a dose of ethanol as low as 0.01% for 8 minutes was already sufficient to inhibit Mg2+ accumulation and PKC, translocation for more than 60 minutes. Also in this model, recovery of Mg2+ accumulation was associated with restoration of PKC, translocation. The use of specific antisense in HepG2 cells confirmed the involvement of PKC, in modulating Mg2+ accumulation. Conclusions:, Translocation of PKC, isoform to the hepatocyte membrane is essential for Mg2+ accumulation to occur. Both acute and chronic ethanol administrations inhibit Mg2+ accumulation by specifically altering PKC, translocation to the cell membrane. [source] Structural compatibility of novel nucleotide modifications with shortened linkages designed for antigene/antisense therapyJOURNAL OF RAMAN SPECTROSCOPY, Issue 5 2004J. Hanu Abstract The impact of isopolar shortened internucleotide linkage modification on the hybridization properties of potential antisense or antigene oligonucleotides was studied by using a model molecular system consisting of polyuridylic acid (PolyU) and analogs of diadenosine monophosphate with the modified linkage. Raman spectra of mixed aqueous solutions were measured at various temperatures and compositions of the mixtures for four types of modified linkage and natural ApA (3,,5,) as a reference. Analysis of the spectral sets provided amounts of formed complexes and their Raman spectra. It has been found that as in the case of ApA (3,,5,), the studied ApA analogs form with PolyU triplex-like complexes containing a central pseudo-chain of closely arranged adenosine dimers. In the case of S - and R -configured 2,,5, and 3,,5, ApCOH A analogs, respectively, the amounts of complexes formed even exceed the ApA. This hybridization effectiveness is reached, however, by a more feasible prolongation of the pseudo-chain, while the stability constant for the initiation step is significantly lower. Raman spectra of the complexes showed that for both of the above analogs the structural compatibility with the natural nucleic acid chain is decreased, because of the distorted position of one adenine residue. This distortion influences the Watson,Crick hydrogen bonds. The two types of linkages may be suitable just for construction of longer antigene or antisense oligonucleotides with alternating lengthened and shortened linkages. There is, however, a warning of possible decreased binding specificity. Copyright © 2004 John Wiley & Sons, Ltd. [source] Study of Cytochrome P4502E1 mRNA Level of Mononuclear Cells in Patients With Alcoholic Liver DiseaseALCOHOLISM, Issue 2001Hirokazu Yano Background: Cytochrome P-4502E1 (CYP2E1) is an important enzyme because of its unique ability to convert many substrates to cytotoxins. The increased production of reactive intermediates by elevated enzyme concentrations leads to various pathological conditions. Therefore, it is important to detect induced CYP2E1 levels in alcoholic individuals to avoid xenobiotic-promoted liver injury. In the present investigation, we detected CYP2E1 mRNA levels of mononuclear cells obtained from 10 ml of blood by using competitive polymerase chain reaction (PCR) method. Methods: Mononuclear cells were obtained from healthy individuals who did and did not drink habitually and patients with alcoholic liver disease (ALD). Complementary DNA synthesis was performed with RNA obtained from mononuclear cells by reverse transcription-PCR. Competitive PCR of CYP2E1 was performed with the sense (5,-CTGCAACGTCATA-GCCGACA-3,) and antisense (5,-TCCATTTCCACGAGCAGGCA-3,) primer and competitor DNA. Competitive PCR of ,-actin also was performed. Electrophoresis was scanned, and each band was digitized. The concentration of CYP2E1 and ,-actin mRNA was calculated from the ratio of competitor DNA. Results: In healthy individuals who did and did not drink habitually, CYP2E1 mRNA levels were 103.3 copies/,l RNA and 101.7 copies/,l RNA, respectively. In actively drinking patients with ALD, CYP2E1 mRNA levels were 103.5 copies/,l RNA, but those levels decreased to 101.7 copies/,l RNA after 4 days of abstinence. No significant difference was observed in CYP2E1 mRNA levels between alcoholic fibrosis and cirrhosis. As control, we measured ,-actin mRNA levels in mononuclear cells in all samples. The mean value of ,-actin mRNA was 104.3 copies/,l RNA in all cases, which included patients with ALD. Conclusions: The results demonstrated that it is possible to measure the CYP2E1 mRNA levels of mononuclear cells in a 10 ml blood sample. The CYP2E1 mRNA level in mononuclear cells increases during drinking and decreases in abstinence for a short period of 3 to 4 days. It is concluded that CYP2E1 mRNA level may be used as an effective marker for alcoholic intake. [source] Inhibition of hepatitis B virus by lentiviral vector delivered antisense RNA and hammerhead ribozymesJOURNAL OF VIRAL HEPATITIS, Issue 4 2005K. L. Nash Summary., Chronic hepatitis B virus (HBV) infection is an important cause of cirrhosis and hepatocellular carcinoma. Current treatments are limited and may be ineffective. Nucleic acid-mediated targeting of viral mRNA is an attractive and specific approach for viral infection and lentiviral vectors provide a means to express antisense sequences or ribozymes stably in target cells permitting continuous production within that cell and its progeny. To demonstrate long-term gene expression by lentiviral vectors in hepatocytes and to introduce lentiviral vectors expressing anti-HBV genes to assess their effect against HBV, lentiviral vectors expressing a reporter gene were assessed for longevity of gene expression in hepatocytes in vitro. Hammerhead ribozymes and antisense sequences targeting the HBV encapsidation signal (,), X or surface antigen on mRNAs were cloned into lentiviral vectors and used to transduce HBV expressing hepatocytes where the effect on HBV mRNA level was assessed using ribonuclease protection. Gene expression in hepatocytes from integrated vectors continued for over 4 months without selection. Antisense RNA targeting HBs mRNA reduced this transcript, whilst antisense RNA to HBX mRNA was ineffective. Sense RNAs corresponding to , and HBX mRNA also reduced HBV mRNA levels. Ribozymes targeting HBs and HBX mRNA effectively reduced HBV mRNA levels compared with inactive constructs indicating their effect to be enzymatic rather than antisense. Lentiviral vectors can produce long-term gene expression in hepatocytes and thus permit prolonged expression of antiviral genes targeting the HBV encapsidation signal, surface and X mRNAs as treatments for chronic HBV infection. [source] Gene therapy as an alternative to liver transplantationLIVER TRANSPLANTATION, Issue 12 2002Betsy T. Kren Liver transplantation has become a well-recognized therapy for hepatic failure resulting from acute or chronic liver disease. It also plays a role in the treatment of certain inborn errors of metabolism that do not directly injure the liver. In fact, the liver maintains a central role in many inherited and acquired genetic disorders. There has been a considerable effort to develop new and more effective gene therapy approaches, in part, to overcome the need for transplantation as well as the shortage of donor livers. Traditional gene therapy involves the delivery of a piece of DNA to replace the faulty gene. More recently, there has been a growing interest in the use of gene repair to correct certain genetic defects. In fact, targeted gene repair has many advantages over conventional replacement strategies. In this review, we will describe a variety of viral and nonviral strategies that are now available to the liver. The ever-growing list includes viral vectors, antisense and ribozyme technology, and the Sleeping Beauty transposon system. In addition, targeted gene repair with RNA/DNA oligonucleotides, small-fragment homologous replacement, and triplex-forming and single-stranded oligonucleotides is a long-awaited and potentially exciting approach. Although each method uses different mechanisms for gene repair and therapy, they all share a basic requirement for the efficient delivery of DNA. [source] Spermatozoal RNAs: What about their functions?MICROSCOPY RESEARCH AND TECHNIQUE, Issue 8 2009Jean-Pierre Dadoune Abstract The profound architectural changes that transform spermatids into spermatozoa result in a high degree of DNA packaging within the sperm head. However, the mature sperm chromatin that harbors imprinted genes exhibits a dual nucleoprotamine/nucleohistone structure with DNase-sensitive regions, which could be implicated in the establishment of efficient epigenetic information in the developing embryo. Despite its apparent transcriptionally inert state, the sperm nucleus contains diverse RNA populations, mRNAs, antisense and miRNAs, that have been transcribed throughout spermatogenesis. There is also an endogenous reverse transcriptase that may be activated under certain circumstances. It is now commonly accepted that sperm can deliver some RNAs to the ovocyte at fertilization. This review presents potential links between male-specific genomic imprinting, chromatin organization, and the presence of diverse RNA populations within the sperm nucleus and discusses the functional significance of these RNAs in the spermatozoon itself and in the early embryo following fertilization. Some recent data are provided, supporting the view that analyzing the profile of spermatozoal RNAs could be useful for assessment of male fertility. Microsc. Res. Tech. 2009. © 2009 Wiley-Liss, Inc. [source] | ||||||||||||||||||||||||||||